RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of vascular endothelial growth factor (VEGF) and/or vascular endothelial growth factor receptor (e.g., VEGFR1, VEGFR2 and/or VEGFR3) gene expression and/or activity. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in vascular endothelial growth factor (VEGF) and/or vascular endothelial growth factor receptor (VEGFR) gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against VEGF and VEGFR gene expression.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). The corresponding process in plants (Heifetz et al., International PCT Publication No. WO 99/61631) is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized. This mechanism appears to be different from other known mechanisms involving double stranded RNA-specific ribonucleases, such as the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094; 5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17, 503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000, Nature, 404, 293). Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283 and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., International PCT Publication No. WO 01/75164, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end of the guide sequence (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither application postulates to what extent such modifications would be tolerated in siRNA molecules, nor provides any further guidance or examples of such modified siRNA. Kreutzer et al., Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge. However, Kreutzer et al. similarly fails to provide examples' or guidance as to what extent these modifications would be tolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that RNAs with two phosphorothioate modified bases also had substantial decreases in effectiveness as RNAi. Further, Parrish et al. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed. Id. at 1081. The authors also tested certain modifications at the 2′-position of the nucleotide sugar in the long siRNA transcripts and found that substituting deoxynucleotides for ribonucleotides produced a substantial decrease in interference activity, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine, for guanosine. Whereas 4-thiouracil and 5-bromouracil substitution appeared to be tolerated, Parrish reported that inosine produced a substantial decrease in interference activity when incorporated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al, International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously-derived dsRNA. Tuschl et al, International PCT Publication No. WO 01/75164, describe a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due to the danger of activating interferon response. Li et al., International PCT Publication No. WO 00/44914, describe the use of specific long (141 bp-488 bp) enzymatically synthesized or vector expressed dsRNAs for attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain long (550 bp-714 bp), enzymatically synthesized or vector expressed dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describe particular methods for introducing certain long dsRNA molecules into cells for use in inhibiting gene expression in nematodes. Plaetinck et al., International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific long dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Pachuck et al., International PCT Publication No. WO 00/63364, describe certain long (at least 200 nucleotide) dsRNA constructs. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al, International PCT Publication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. Driscoll et al., International PCT Publication No. WO 01/49844, describe specific DNA expression constructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. For example, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describe specific chemically-modified dsRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al., International PCT Publication No. WO 01/42443, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al, International PCT Publication No. WO 01/53475, describe certain methods for isolating a Neurospora silencing gene and uses thereof. Reed et al., International PCT Publication No. WO 01/68836, describe certain methods for gene silencing in plants. Honer et al., International PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe certain Drosophila-derived gene products that may be related to RNAi in Drosophila. Arndt et al., International PCT Publication No. WO 01/92513 describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and Satishchandran et al., International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain long (over 250 bp), vector expressed dsRNAs. Echeverri et al., International PCT Publication No. WO 02/38805, describe certain C. elegans genes identified via RNAi. Kreutzer et al., International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describes certain methods for inhibiting gene expression using dsRNA. Graham et al., International PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559, describe certain methods for inhibiting gene expression in vitro using certain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi. Martinez et al., 2002, Cell, 110, 563-574, describe certain single stranded siRNA constructs, including certain 5′-phosphorylated single stranded siRNAs that mediate RNA interference in Hela cells. Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105, describe certain chemically and structurally modified siRNA molecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically and structurally modified siRNA molecules. Woolf et al., International PCT Publication Nos. WO 03/064626 and WO 03/064625 describe certain chemically modified dsRNA constructs.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods useful for modulating the expression of genes, such as those genes associated with angiogenesis and proliferation, using short interfering nucleic acid (siNA) molecules. This invention further relates to compounds, compositions, and methods useful for modulating the expression and activity of vascular endothelial growth factor (VEGF) and/or vascular endothelial growth factor receptor (e.g., VEGFR1, VEGFR2, VEGFR3) genes, or genes involved in VEGF and/or VEGFR pathways of gene expression and/or VEGF activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of VEGF and/or VEGFR genes and/or other genes involved in VEGF and/or VEGFR mediated angiogenesis in a subject or organism.

A siNA of the invention can be unmodified or chemically-modified. A siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating VEGF and/or VEGFR gene expression or activity in cells by RNA interference (RNAi). The use of chemically-modified siNA improves various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, contrary to earlier published studies, siNA having multiple chemical modifications retains its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, veterinary, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of gene(s) encoding proteins, such as vascular endothelial growth factor (VEGF) and/or vascular endothelial growth factor receptors (e.g., VEGFR1, VEGFR2, VEGFR3), associated with the maintenance and/or development of cancer and other proliferative diseases, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as VEGF and/or VEGFR. The description below of the various aspects and embodiments of the invention is provided with reference to the exemplary VEGF and VEGFR (e.g., VEGFR1, VEGFR2, VEGFR3) genes referred to herein as VEGF and VEGFR respectively. However, the various aspects and embodiments are also directed to other VEGF and/or VEGFR genes, such as mutant VEGF and/or VEGFR genes, splice variants of VEGF and/or VEGFR genes, other VEGF and/or VEGFR ligands and receptors. The various aspects and embodiments are also directed to other genes that are involved in VEGF and/or VEGFR mediated pathways of signal transduction or gene expression that are involved in the progression, development, and/or maintenance of disease (e.g., cancer). These additional genes can be analyzed for target sites using the methods described for VEGF and/or VEGFR genes herein. Thus, the modulation of other genes and the effects of such modulation of the other genes can be performed, determined, and measured as described herein.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a vascular endothelial growth factor (e.g., VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D) gene, wherein said siNA molecule comprises about 15 to about 28 base pairs.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a vascular endothelial growth factor receptor (e.g., VEGFR1, VEGFR2, and/or VEGFR3) gene, wherein said siNA molecule comprises about 15 to about 28 base pairs.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a vascular endothelial growth factor (VEGF, e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D) RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 28 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the VEGF RNA for the siNA molecule to direct cleavage of the VEGF RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a vascular endothelial growth factor receptor (VEGFR, e.g., VEGFR1, VEGFR2, and/or VEGFR3) RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 28 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the VEGFR RNA for the siNA molecule to direct cleavage of the VEGFR RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a VEGF and/or VEGFR RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 28 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the VEGF and/or VEGFR RNA for the siNA molecule to direct cleavage of the VEGF and/or VEGFR RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a VEGF and/or VEGFR RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 23 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the VEGF and/or VEGFR RNA for the siNA molecule to direct cleavage of the VEGF and/or VEGFR RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a VEGF and/or VEGFR RNA via RNA interference (RNAi), wherein each strand of the siNA molecule is about 18 to about 28 nucleotides in length; and one strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the VEGF and/or VEGFR RNA for the siNA molecule to direct cleavage of the VEGF and/or VEGFR RNA via RNA interference.

In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a VEGF and/or VEGFR RNA via RNA interference (RNAi), wherein each strand of the siNA molecule is about 18 to about 23 nucleotides in length; and one strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the VEGF and/or VEGFR RNA for the siNA molecule to direct cleavage of the VEGF and/or VEGFR RNA via RNA interference.

In one embodiment, the invention features a siNA molecule that down-regulates expression of a VEGF and/or VEGFR gene or that directs cleavage of a VEGF and/or VEGFR RNA, for example, wherein the VEGF and/or VEGFR gene or RNA comprises VEGF and/or VEGFR encoding sequence. In one embodiment, the invention features a siNA molecule that down-regulates expression of a VEGF and/or VEGFR gene or that directs cleavage of a VEGF and/or VEGFR RNA, for example, wherein the VEGF and/or VEGFR gene of RNA comprises VEGF and/or VEGFR non-coding sequence or regulatory elements involved in VEGF and/or VEGFR gene expression.

In one embodiment, a siNA of the invention is used to inhibit the expression of VEGF and/or VEGFR genes or a VEGF and/or VEGFR gene family (e.g., one or more VEGF and/or VEGFR isoforms), wherein the genes or gene family sequences share sequence homology. Such homologous sequences can be identified as is known in the art, for example using sequence alignments. siNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences. In instances where mismatches are identified, non-canonical base pairs (for example, mismatches and/or wobble bases) can be used to generate siNA molecules that target more than one gene sequence. In a non-limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting sequences for differing VEGF and/or VEGFR targets that share sequence homology. As such, one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the homologous genes. In this approach, a single siNA can be used to inhibit expression of more than one gene instead of using more than one siNA molecule to target the different genes.

In one embodiment, the invention features a siNA molecule having RNAi activity against VEGF and/or VEGFR RNA, wherein the siNA molecule comprises a sequence complementary to any RNA having VEGF and/or VEGFR encoding sequence, such as those sequences having GenBank Accession Nos. shown in Table I. In another embodiment, the invention features a siNA molecule having RNAi activity against VEGF and/or VEGFR RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having variant VEGF and/or VEGFR encoding sequence, for example other mutant VEGF and/or VEGFR genes not shown in Table I but known in the art to be associated with, for example, the maintenance and/or development of, for example, angiogenesis, cancer, proliferative disease, ocular disease, and/or renal disease. Chemical modifications as shown in Tables III and IV or otherwise described herein can be applied to any siNA construct of the invention. In another embodiment, a siNA molecule of the invention includes a nucleotide sequence that can interact with nucleotide sequence of a VEGF and/or VEGFR gene and thereby mediate silencing of VEGF and/or VEGFR gene expression, for example, wherein the siNA mediates regulation of VEGF and/or VEGFR gene expression by cellular processes that modulate the transcription or translation of the VEGF and/or VEGFR gene and prevent expression of the VEGF and/or VEGFR gene.

In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of proteins arising from VEGF and/or VEGFR haplotype polymorphisms that are associated with a trait, disease or condition. Analysis of genes, or protein or RNA levels can be used to identify subjects with such polymorphisms or those subjects who are at risk of developing traits, conditions, or diseases described herein (see for example Silvestri et al., 2003, Int J Cancer., 104, 310-7). These subjects are amenable to treatment, for example, treatment with siNA molecules of the invention and any other composition useful in treating diseases related to VEGF and/or VEGFR gene expression. As such, analysis of VEGF and/or VEGFR protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of VEGF and/or VEGFR protein or RNA levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of certain VEGF and/or VEGFR proteins associated with a trait, condition, or disease.

In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of soluble VEGF receptors (e.g. sVEGFR1 or sVEGFR2). Analysis of soluble VEGF receptor levels can be used to identify subjects with certain cancer types. These cancers can be amenable to treatment, for example, treatment with siNA molecules of the invention and any other chemotherapeutic composition. As such, analysis of soluble VEGF receptor levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of soluble VEGF receptor levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of VEGF receptors (see for example Pavco U.S. Ser. No. 10/438,493, incorporated by reference herein in its entirety including the drawings).

In one embodiment of the invention a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a VEGF and/or VEGFR protein. The siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a VEGF and/or VEGFR gene or a portion thereof.

In another embodiment, a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a VEGF and/or VEGFR protein or a portion thereof. The siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a VEGF and/or VEGFR gene or a portion thereof.

In another embodiment, the invention features a siNA molecule comprising a nucleotide sequence in the antisense region of the siNA molecule that is complementary to a nucleotide sequence or portion of sequence of a VEGF and/or VEGFR gene. In another embodiment, the invention features a siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a VEGF and/or VEGFR gene sequence or a portion thereof.

In another embodiment, the invention features a siNA molecule comprising nucleotide sequence, for example, nucleotide sequence in the antisense region of the siNA molecule that is complementary to a nucleotide sequence or portion of sequence of a VEGF and/or VEGFR gene. In another embodiment, the invention features a siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a VEGF and/or VEGFR gene sequence or a portion thereof.

In one embodiment, the antisense region of siNA constructs comprises a sequence complementary to sequence having any of target SEQ ID NOs. shown in Tables II and III. In one embodiment, the antisense region of siNA constructs of the invention constructs comprises sequence having any of antisense SEQ ID NOs. in Tables II and III and FIGS. 4 and 5. In another embodiment, the sense region of siNA constructs of the invention comprises sequence having any of sense SEQ ID NOs. in Tables II and III and FIGS. 4 and 5.

In one embodiment, a siNA molecule of the invention comprises any of SEQ ID NOs. 1-4248. The sequences shown in SEQ ID NOs: 1-4248 are not limiting. A siNA molecule of the invention can comprise any contiguous VEGF and/or VEGFR sequence (e.g., about 15 to about 25 or more, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more contiguous VEGF and/or VEGFR nucleotides).

In yet another embodiment, the invention features a siNA molecule comprising a sequence, for example, the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table I. Chemical modifications in Tables m and IV and described herein can be applied to any siNA construct of the invention.

In one embodiment of the invention a siNA molecule comprises an antisense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense strand is complementary to a RNA sequence or a portion thereof encoding VEGF and/or VEGFR, and wherein said siNA further comprises a sense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences where at least about 15 nucleotides in each strand are complementary to the other strand.

In another embodiment of the invention a siNA molecule of the invention comprises an antisense region having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region is complementary to a RNA sequence encoding VEGF and/or VEGFR, and wherein said siNA further comprises a sense region having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein said sense region and said antisense region are comprised in a linear molecule where the sense region comprises at least about 15 nucleotides that are complementary to the antisense region.

In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a VEGF and/or VEGFR gene. Because VEGF and/or VEGFR genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of VEGF and/or VEGFR genes or alternately specific VEGF and/or VEGFR genes (e.g., polymorphic variants) by selecting sequences that are either shared amongst different VEGF and/or VEGFR targets or alternatively that are unique for a specific VEGF and/or VEGFR target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of VEGF and/or VEGFR RNA sequence having homology between several VEGF and/or VEGFR gene variants so as to target a class of VEGF and/or VEGFR genes with one siNA molecule. Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of one or both VEGF and/or VEGFR alleles in a subject. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific VEGF and/or VEGFR RNA sequence (e.g., a single VEGF and/or VEGFR allele or VEGF and/or VEGFR single nucleotide polymorphism (SNP)) due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a VEGFR gene. Because VEGFR genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of VEGFR genes (and associated receptor or ligand genes) or alternately specific VEGFR genes by selecting sequences that are either shared amongst different VEGFR targets or alternatively that are unique for a specific VEGFR target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of VEGFR RNA sequence having homology between several VEGFR genes so as to target several VEGFR genes (e.g., VEGFR1, VEGFR2 and/or VEGFR3, different VEGFR isoforms, splice variants, mutant genes etc.) with one siNA molecule. In one embodiment, the siNA molecule can be designed to target conserved regions of VEGFR1 and VEGFR2 RNA sequence having shared sequence homology (see for example Table III). Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of more than one VEGFR gene, i.e., VEGFR1, VEGFR2, and VEGFR3, or any combination thereof. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific VEGFR RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a VEGF gene. Because VEGF genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of VEGF genes (and associated receptor or ligand genes) or alternately specific VEGF genes by selecting sequences that are either shared amongst different VEGF targets or alternatively that are unique for a specific VEGF target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of VEGF RNA sequence having homology between several VEGF genes so as to target several VEGF genes (e.g., VEGF-A, VEGF-B, VEGF-C and/or VEGF-D, different VEGF isoforms, splice variants, mutant genes etc.) with one siNA molecule. Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of more than one VEGF gene, i.e., VEGF-A, VEGF-B, VRGF-C, and VEGF-D or any combination thereof. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific VEGF RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, a siNA molecule of the invention targeting one or more VEGF receptor genes (e.g., VEGFR1, VEGFR2, and/or VEGFR3) is used in combination with a siNA molecule of the invention targeting a VEGF gene (e.g., VEGF-A, VEGF-B, VEGF-C and/or VEGF-D) according to a use described herein, such as treating a subject with an angiogenesis or neovascularization related disease, such as tumor angiogenesis and cancer, including but not limited to breast cancer, lung cancer (including non-small cell lung carcinoma), prostate cancer, colorectal cancer, brain cancer, esophageal cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid adenocarcinoma, ovarian cancer, melanoma, lymphoma, glioma, endometrial sarcoma, multidrug resistant cancers, diabetic retinopathy, macular degeneration, neovascular glaucoma, myopic degeneration, arthritis, psoriasis, endometriosis, female reproduction, verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, Osler-Weber-Rendu syndrome, renal disease such as Autosomal dominant polycystic kidney disease (ADPKD), and any other diseases or conditions that are related to or will respond to the levels of VEGF, VEGFR1, and VEGFR2 in a cell or tissue, alone or in combination with other therapies.

In another embodiment, a siNA molecule of the invention that targets homologous VEGFR1 and VEGFR2 sequence is used in combination with a siNA molecule that targets VEGF-A according to a use described herein, such as treating a subject with an angiogenesis or neovascularization related disease such as tumor angiogenesis and cancer, including but not limited to breast cancer, lung cancer (including non-small cell lung carcinoma), prostate cancer, colorectal cancer, brain cancer, esophageal cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid adenocarcinoma, ovarian cancer, melanoma, lymphoma, glioma, endometrial sarcoma, multidrug resistant cancers, diabetic retinopathy, macular degeneration, neovascular glaucoma, myopic degeneration, arthritis, psoriasis, endometriosis, female reproduction, verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, Osler-Weber-Rendu syndrome, renal disease such as Autosomal dominant polycystic kidney disease (ADPKD), and any other diseases or conditions that are related to or will respond to the levels of VEGF, VEGFR1, and VEGFR2 in a cell or tissue, alone or in combination with other therapies.

In one embodiment, a siNA of the invention is used to inhibit the expression of VEGFR1, VEGFR2, and/or VEGFR3 genes, wherein the VEGFR1, VEGFR2, and/or VEGFR3 sequences share sequence homology. Such homologous sequences can be identified as is known in the art, for example using sequence alignments. siNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences. Non limiting examples of sequence alignments between VEGFR1 and VEGFR2 are shown in Table III. In instances where mismatches are shown, non-canonical base pairs, for example mismatches and/or wobble bases, can be used to generate siNA molecules that target both VEGFR1 and VEGFR2 RNA sequences. In a non-limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting differing VEGF and/or VEGFR sequences (e.g. VEGFR1 and VEGFR2). As such, one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the VEGF receptors (i.e., VEGFR1, VEGFR2, and/or VEGFR3) such that the siNA can interact with RNAs of the receptors and mediate RNAi to achieve inhibition of expression of the VEGF receptors. In this approach, a single siNA can be used to inhibit expression of more than one VEGF receptor instead of using more than one siNA molecule to target the different receptors.

In one embodiment, the invention features a method of designing a single siNA to inhibit the expression of both VEGFR1 and VEGFR2 genes comprising designing an siNA having nucleotide sequence that is complementary to nucleotide sequence encoded by or present in both VEGFR1 and VEGFR2 genes or a portion thereof, wherein the siNA mediates RNAi to inhibit the expression of both VEGFR1 and VEGFR2 genes. For example, a single siNA can inhibit the expression of two genes by binding to conserved or homologous sequence present in RNA encoded by VEGFR1 and VEGFR2 genes or a portion thereof.

In one embodiment, the invention features a method of designing a single siNA to inhibit the expression of both VEGFR1 and VEGFR3 genes comprising designing an siNA having nucleotide sequence that is complementary to nucleotide sequence encoded by or present in both VEGFR1 and VEGFR3 genes or a portion thereof, wherein the siNA mediates RNAi to inhibit the expression of both VEGFR1 and VEGFR3 genes. For example, a single siNA can inhibit the expression of two genes by binding to conserved or homologous sequence present in RNA encoded by VEGFR1 and VEGFR3 genes or a portion thereof.

In one embodiment, the invention features a method of designing a single siNA to inhibit the expression of both VEGFR2 and VEGFR3 genes comprising designing an siNA having nucleotide sequence that is complementary to nucleotide sequence encoded by or present in both VEGFR2 and VEGFR3 genes or a portion thereof, wherein the siNA mediates RNAi to inhibit the expression of both VEGFR2 and VEGFR3 genes. For example, a single siNA can inhibit the expression of two genes by binding to conserved or homologous sequence present in RNA encoded by VEGFR2 and VEGFR3 genes or a portion thereof.

In one embodiment, the invention features a method of designing a single siNA to inhibit the expression of VEGFR1, VEGFR2 and VEGFR3 genes comprising designing an siNA having nucleotide sequence that is complementary to nucleotide sequence encoded by or present in VEGFR1, VEGFR2 and VEGFR3 genes or a portion thereof, wherein the siNA mediates RNAi to inhibit the expression of VEGFR1, VEGFR2 and VEGFR3 genes. For example, a single siNA can inhibit the expression of two genes by binding to conserved or homologous sequence present in RNA encoded by VEGFR1, VEGFR2 and VEGFR3 genes or a portion thereof.

In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules. In another embodiment, the siNA molecules of the invention consist of duplex nucleic acid molecules containing about 15 to about 30 base pairs between oligonucleotides comprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3) nucleotides, for example, about 21-nucleotide duplexes with about 19 base pairs and 3′-terminal mononucleotide, dinucleotide, or trinucleotide overhangs. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with blunt ends, where both ends are blunt, or alternatively, where one of the ends is blunt.

In one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for VEGF and/or VEGFR expressing nucleic acid molecules, such as RNA encoding a VEGF and/or VEGFR protein or non-coding RNA associated with the expression of VEGF and/or VEGFR genes. In one embodiment, the invention features a RNA based siNA molecule (e.g., a siNA comprising 2′-OH nucleotides) having specificity for VEGF and/or VEGFR expressing nucleic acid molecules that includes one or more chemical modifications described herein. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA constructs, (e.g., RNA based siNA constructs), are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.

In one embodiment, a siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi. The modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, and/or bioavailability. For example, a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. As such, a siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the percent modification can be based upon the total number of nucleotides present in the single stranded siNA molecules. Likewise, if the siNA molecule is double stranded, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.

One aspect of the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a VEGF and/or VEGFR gene or that directs cleavage of a VEGF and/or VEGFR RNA. In one embodiment, the double stranded siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long. In one embodiment, the double-stranded siNA molecule does not contain any ribonucleotides. In another embodiment, the double-stranded siNA molecule comprises one or more ribonucleotides. In one embodiment, each strand of the double-stranded siNA molecule independently comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein each strand comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of the VEGF and/or VEGFR gene, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the VEGF and/or VEGFR gene or a portion thereof.

In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a VEGF and/or VEGFR gene or that directs cleavage of a VEGF and/or VEGFR RNA, comprising an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the VEGF and/or VEGFR gene or a portion thereof, and a sense region, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the VEGF and/or VEGFR gene or a portion thereof. In one embodiment, the antisense region and the sense region independently comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the sense region.

In one embodiment, a siNA molecule of the invention comprises blunt ends, i.e., ends that do not include any overhanging nucleotides. For example, a siNA molecule comprising modifications described herein (e.g., comprising nucleotides having Formulae I-VII or siNA constructs comprising “Stab 00”-“Stab 33” (Table 1V) or any combination thereof (see Table IV)) and/or any length described herein can comprise blunt ends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise one or more blunt ends, i.e. where a blunt end does not have any overhanging nucleotides. In one embodiment, the blunt ended siNA molecule has a number of base pairs equal to the number of nucleotides present in each strand of the siNA molecule. In another embodiment, the siNA molecule comprises one blunt end, for example wherein the 5′-end of the antisense strand and the 3′-end of the sense strand do not have any overhanging nucleotides. In another example, the siNA molecule comprises one blunt end, for example wherein the 3′-end of the antisense strand and the 5′-end of the sense strand do not have any overhanging nucleotides. In another example, a siNA molecule comprises two blunt ends, for example wherein the 3′-end of the antisense strand and the 5′-end of the sense strand as well as the 5′-end of the antisense strand and 3′-end of the sense strand do not have any overhanging nucleotides. A blunt ended siNA molecule can comprise, for example, from about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). Other nucleotides present in a blunt ended siNA molecule can comprise, for example, mismatches, bulges, loops, or wobble base pairs to modulate the activity of the siNA molecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of a double stranded siNA molecule having no overhanging nucleotides. The two strands of a double stranded siNA molecule align with each other without over-hanging nucleotides at the termini. For example, a blunt ended siNA construct comprises terminal nucleotides that are complementary between the sense and antisense regions of the siNA molecule.

In one embodiment, the invention features double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a VEGF and/or VEGFR gene of that directs cleavage of a VEGF and/or VEGFR RNA, wherein the siNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein each strand of the siNA molecule comprises one or more chemical modifications. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a VEGF and/or VEGFR gene or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the VEGF and/or VEGFR gene. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a VEGF and/or VEGFR gene or portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or portion thereof of the VEGF and/or VEGFR gene. In another embodiment, each strand of the siNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and each strand comprises at least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of the other strand. The VEGF and/or VEGFR gene can comprise, for example, sequences referred to in Table I.

In one embodiment, a siNA molecule of the invention comprises no ribonucleotides. In another embodiment, a siNA molecule of the invention comprises ribonucleotides.

In one embodiment, a siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a VEGF and/or VEGFR gene or a portion thereof, and the siNA further comprises a sense region comprising a nucleotide sequence substantially similar to the nucleotide sequence of the VEGF and/or VEGFR gene or a portion thereof. In another embodiment, the antisense region and the sense region each comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides and the antisense region comprises at least about 15 to about 30, (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the sense region. The VEGF and/or VEGFR gene can comprise, for example, sequences referred to in Table I. In another embodiment, the siNA is a double stranded nucleic acid molecule, where each of the two strands of the siNA molecule independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides, and where one of the strands of the siNA molecule comprises at least about 15 (e.g. about 15, 30-16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more) nucleotides that are complementary to the nucleic acid sequence of the VEGF and/or VEGFR gene or a portion thereof.

In one embodiment, a siNA molecule of the invention comprises a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by a VEGF and/or VEGFR gene, or a portion thereof, and the sense region comprises a nucleotide sequence that is complementary to the antisense region. In one embodiment, the siNA molecule is assembled from two separate oligonucleotide fragments, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule, such as a nucleotide or non-nucleotide linker. The VEGF and/or VEGFR gene can comprise, for example, sequences referred in to Table I.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a VEGF and/or VEGFR gene or that directs cleavage of a VEGF and/or VEGFR RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the VEGF and/or VEGFR gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the siNA molecule has one or more modified pyrimidine and/or purine nucleotides. In one embodiment, the pyrimidine nucleotides in the sense region are 2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In one embodiment, the pyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the sense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a siNA molecule comprising at least one modified nucleotide, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide, 2′-O-trifluoromethyl nucleotide, 2′-O-ethyl-trifluoromethoxy nucleotide, or 2′-O-difluoromethoxy-ethoxy nucleotide. The siNA can be, for example, about 15 to about 40 nucleotides in length. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy, pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing the stability of a siNA molecule against cleavage by ribonucleases comprising introducing at least one modified nucleotide into the siNA molecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a VEGF and/or VEGFR gene or that directs cleavage of a VEGF and/or VEGFR RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the VEGF and/or VEGFR gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the purine nucleotides present in the antisense region comprise 2′-deoxy-purine nucleotides. In an alternative embodiment, the purine nucleotides present in the antisense region comprise 2′-O-methyl purine nucleotides. In either of the above embodiments, the antisense region can comprise a phosphorothioate internucleotide linkage at the 3′ end of the antisense region. Alternatively, in either of the above embodiments, the antisense region can comprise a glyceryl modification at the 3′ end of the antisense region. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the antisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the antisense region of a siNA molecule of the invention comprises sequence complementary to a portion of an endogenous transcript having sequence unique to a particular VEGF and/or VEGFR disease related allele in a subject or organism, such as sequence comprising a single nucleotide polymorphism (SNP) associated with the disease specific allele. As such, the antisense region of a siNA molecule of the invention can comprise sequence complementary to sequences that are unique to a particular allele to provide specificity in mediating selective RNAi against the disease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a VEGF and/or VEGFR gene or that directs cleavage of a VEGF and/or VEGFR RNA, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule, where each strand is about 21 nucleotides long and where about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule, wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In another embodiment, all nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the VEGF and/or VEGFR gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the VEGF and/or VEGFR gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a VEGF and/or VEGFR RNA sequence (e.g., wherein said target RNA sequence is encoded by a VEGF and/or VEGFR gene involved in the VEGF and/or VEGFR pathway), wherein the siNA molecule does not contain any ribonucleotides and wherein each strand of the double-stranded siNA molecule is about 15 to about 30 nucleotides. In one embodiment, the siNA molecule is 21 nucleotides in length. Examples of non-ribonucleotide containing siNA constructs are combinations of stabilization chemistries shown in Table IV in any combination of Sense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab 8/32, or Stab 18/32 (e.g., any siNA having Stab 7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense or antisense strands or any combination thereof).

In one embodiment, the invention features a chemically synthesized double stranded RNA molecule that directs cleavage of a VEGF and/or VEGFR RNA via RNA interference, wherein each strand of said RNA molecule is about 15 to about 30 nucleotides in length; one strand of the RNA molecule comprises nucleotide sequence having sufficient complementarity to the VEGF and/or VEGFR RNA for the RNA molecule to direct cleavage of the VEGF and/or VEGFR RNA via RNA interference; and wherein at least one strand of the RNA molecule optionally comprises one or more chemically modified nucleotides described herein, such: as without limitation deoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-O-methoxyethyl nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, etc.

In one embodiment, the invention features a medicament comprising a siNA molecule of the invention.

In one embodiment, the invention features an active ingredient comprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule to inhibit, down-regulate, or reduce expression of a VEGF and/or VEGFR gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is independently about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more) nucleotides long. In one embodiment, the siNA molecule of the invention is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each of the two fragments of the siNA molecule independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and where one of the strands comprises at least 15 nucleotides that are complementary to nucleotide sequence of VEGF and/or VEGFR encoding RNA or a portion thereof. In a non-limiting example, each of the two fragments of the siNA molecule comprise about 21 nucleotides. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 21 nucleotide long and where about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule, wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In another embodiment, all nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region and comprising one or more chemical modifications, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the VEGF and/or VEGFR gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the VEGF and/or VEGFR gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of VEGF and/or VEGFR RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of VEGF and/or VEGFR RNA that encodes a protein or portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, each strand of the siNA molecule comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides, wherein each strand comprises at least about 15 nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, the siNA molecule is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siNA molecule and a second fragment comprises nucleotide sequence of the sense region of the siNA molecule. In one embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. In a further embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In still another embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-deoxy purine nucleotides. In another embodiment, the antisense strand comprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-O-methyl purine nucleotides. In a further embodiment the sense strand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety (e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotide moiety such as inverted thymidine) is present at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sense strand. In another embodiment, the antisense strand comprises a phosphorothioate internucleotide linkage at the 3′ end of the antisense strand. In another embodiment, the antisense strand comprises a glyceryl modification at the 3′ end. In another embodiment, the 5′-end of the antisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a VEGF and/or VEGFR gene, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, each of the two strands of the siNA molecule can comprise about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides. In one embodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule. In another embodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule, wherein at least two 3′ terminal nucleotides of each strand of the siNA molecule are not base-paired to the nucleotides of the other strand of the siNA molecule. In another embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine. In one embodiment, each strand of the siNA molecule is base-paired to the complementary nucleotides of the other strand of the siNA molecule. In one embodiment, about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of the antisense strand are base-paired to the nucleotide sequence of the VEGF and/or VEGFR RNA or a portion thereof. In one embodiment, about 18 to about 25 (e.g., about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of the antisense strand are base-paired to the nucleotide sequence of the VEGF and/or VEGFR RNA or a portion thereof.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of VEGF and/or VEGFR RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the 5′-end of the antisense strand optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of VEGF and/or VEGFR RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence or a portion thereof of the antisense strand is complementary to a nucleotide sequence of the untranslated region or a portion thereof of the VEGF and/or VEGFR RNA.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of VEGF and/or VEGFR RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence of the antisense strand is complementary to a nucleotide sequence of the VEGF and/or VEGFR RNA or a portion thereof that is present in the VEGF and/or VEGFR RNA.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention in a pharmaceutically acceptable carrier or diluent.

In a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example, when compared to an all-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, the antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.

One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to a RNA or DNA sequence encoding VEGF and/or VEGFR and the sense region can comprise sequence complementary to the antisense region. The siNA molecule can comprise two distinct strands having complementary sense and antisense regions. The siNA molecule can comprise a single strand having complementary sense and antisense regions.

- wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Z are optionally not all O. In another embodiment, a backbone modification of the invention comprises a phosphonoacetate and/or thiophosphonoacetate internucleotide linkage (see for example Sheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, for example, wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified internucleotide linkages having Formula I at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In another embodiment, a siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against VEGF and/or VEGFR inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula II:
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wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, b-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotides or non-nucleotides of Formula II at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 5′-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 3′-end of the sense strand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against VEGF and/or VEGFR inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula III:
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wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO₂, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotides or non-nucleotides of Formula III at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or non-nucleotide(s) of Formula III at the 5′-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end of the sense strand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand, for example, a strand complementary to a target RNA, wherein the siNA molecule comprises an all RNA siNA molecule. In another embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand wherein the siNA molecule also comprises about 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminal nucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3′-end of one or both strands. In another embodiment, a 5′-terminal phosphate group having Formula IV is present on the target-complementary strand of a siNA molecule of the invention, for example a siNA molecule having chemical modifications having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against VEGF and/or VEGFR inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the invention features a chemically-modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand. In yet another embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.

In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or one or more (e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein the antisense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having about 1 to about 5 or more (specifically about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one or both siNA sequence strands. In addition, the 2′-5′ internucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified, wherein each strand is independently about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex has about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemical modification comprises a structure having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein each strand consists of about 21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotide overhang, and wherein the duplex has about 19 base pairs. In another embodiment, a siNA molecule of the invention comprises a single stranded hairpin structure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA can include a chemical modification comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a 2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear hairpin siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises a hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In one embodiment, a linear hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises an asymmetric hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms an asymmetric hairpin structure having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In one embodiment, an asymmetric hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In another embodiment, an asymmetric hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, wherein the sense region and the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) nucleotides in length and wherein the sense region is about 3 to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides in length, wherein the sense region the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. In another embodiment, the asymmetric double stranded siNA molecule can also have a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV).

In another embodiment, a siNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA can include a chemical modification, which comprises a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable. For example, a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a compound having Formula V:
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wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for example a compound having Formula VI:
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wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3, R8 or R13 serve as points of attachment to the siNA molecule of the invention.

In another embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties, for example a compound having Formula VII:
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wherein each n is independently an integer from 1 to 12, each R1, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO₂, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having Formula I, and R1, R2 or R3 serves as points of attachment to the siNA molecule of the invention.

In another embodiment, the invention features a compound having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0 and is the point of attachment to the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both strands of a double-stranded siNA molecule of the invention or to a single-stranded siNA molecule of the invention. This modification is referred to herein as “glyceryl” (for example modification 6 in FIG. 10).

In another embodiment, a chemically modified nucleoside or non-nucleoside (e.g. a moiety having any of Formula V, VI or VII) of the invention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of a siNA molecule of the invention. For example, chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) can be present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense strand, the sense strand, or both antisense and sense strands of the siNA molecule. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the terminal position of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the two terminal positions of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the penultimate position of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In addition, a moiety having Formula VII can be present at the 3′-end or the 5′-end of a hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Formula VI or VI is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g. wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g. one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2°-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein any nucleotides comprising a. 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g. one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against VEGF and/or VEGFR inside a cell or reconstituted in vitro system comprising a sense region, wherein one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or more purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and an antisense region, wherein one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or more purine nucleotides present in the antisense region are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). The sense region and/or the antisense region can have a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/or antisense sequence. The sense and/or antisense region can optionally further comprise a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhang nucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III and IV herein. In any of these described embodiments, the purine nucleotides present in the sense region are alternatively 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides) and one or more purine nucleotides present in the antisense region are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Also, in any of these embodiments, one or more purine nucleotides present in the sense region are alternatively purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides) and any purine nucleotides present in the antisense region are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Additionally, in any of these embodiments, one or more purine nucleotides present in the sense region and/or present in the antisense region are alternatively selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA molecule of the invention comprises a terminal cap moiety, (see for example FIG. 10) such as an inverted deoxyabaisc moiety, at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against VEGF and/or VEGFR inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule. Non-limiting examples of conjugates contemplated by the invention include conjugates and ligands described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein in its entirety, including the drawings. In another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3′-end and 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system, such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified siNA molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002 incorporated by reference herein. The type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity. As such, one skilled in the art can screen siNA constructs that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide linker of the invention can be a linker of ≧2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. (See, for example, Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the C1 position of the sugar.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides. For example, a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA comprise separate oligonucleotides that do not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotides. In another example, a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA are linked or circularized by a nucleotide or non-nucleotide linker as described herein, wherein the oligonucleotide does not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotide. Applicant has surprisingly found that the presense of ribonucleotides (e.g., nucleotides having a 2′-hydroxyl group) within the siNA molecule is not required or essential to support RNAi activity. As such, in one embodiment, all positions within the siNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system comprising a single stranded polynucleotide having complementarity to a target nucleic acid sequence, wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence. The siNA optionally further comprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group. In any of these embodiments, any purine nucleotides present in the antisense region are alternatively 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA (i.e., purine nucleotides present in the sense and/or antisense region) can alternatively be locked nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA are alternatively 2′-methoxyethyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-methoxyethyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-methoxyethyl purine nucleotides). In another embodiment, any modified nucleotides present in the single stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.

In one embodiment, a siNA molecule of the invention comprises chemically modified nucleotides or non-nucleotides (e.g., having any of Formulae I-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides) at alternating positions within one or more strands or regions of the siNA molecule. For example, such chemical modifications can be introduced at every other position of a RNA based siNA molecule, starting at either the first or second nucleotide from the 3′-end or 5′-end of the siNA. In a non-limiting example, a double stranded siNA molecule of the invention in which each strand of the siNA is 21 nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 of each strand are chemically modified (e.g., with compounds having any of Formulae 1-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In another non-limiting example, a double stranded siNA molecule of the invention in which each strand of the siNA is 21 nucleotides in length is featured wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand are chemically modified (e.g., with compounds having any of Formulae 1-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). Such siNA molecules can further comprise terminal cap moieties and/or backbone modifications as described herein.

In one embodiment, the invention features a method for modulating the expression of a VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the VEGF and/or VEGFR gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in the cell.

In one embodiment, the invention features a method for modulating the expression of a VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the VEGF and/or VEGFR gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the VEGF and/or VEGFR genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in the cell.

In another embodiment, the invention features a method for modulating the expression of two or more VEGF and/or VEGFR genes within a cell comprising: (a) synthesizing one or more siNA molecules of the invention, which can be chemically-modified, wherein the siNA strands comprise sequences complementary to RNA of the VEGF and/or VEGFR genes and wherein the sense strand sequences of the siNAs comprise sequences identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the VEGF and/or VEGFR gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagents in ex vivo applications. For example, siNA reagents are introduced into tissue or cells that are transplanted into a subject for therapeutic effect. The cells and/or tissue can be derived from an organism or subject that later receives the explant, or can be derived from another organism or subject prior to transplantation. The siNA molecules can be used to modulate the expression of one or more genes in the cells or tissue, such that the cells or tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo. In one embodiment, certain target cells from a patient are extracted. These extracted cells are contacted with siNAs targeting a specific nucleotide sequence within the cells under conditions suitable for uptake of the siNAs by these cells (e.g. using delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells). The cells are then reintroduced back into the same patient or other patients. In one embodiment, the invention features a method of modulating the expression of a VEGF and/or VEGFR gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the VEGF and/or VEGFR gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in that organism.

In one embodiment, the invention features a method of modulating the expression of a VEGF and/or VEGFR gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the VEGF and/or VEGFR gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one VEGF and/or VEGFR gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the VEGF and/or VEGFR genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a VEGF and/or VEGFR gene in a subject or organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the VEGF and/or VEGFR gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in the subject or organism. The level of VEGF and/or VEGFR protein or RNA can be determined using various methods well-known in the art.

In another embodiment, the invention features a method of modulating the expression of more than one VEGF and/or VEGFR gene in a subject or organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the VEGF and/or VEGFR genes; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in the subject or organism. The level of VEGF and/or VEGFR protein or RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating the expression of a VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the VEGF and/or VEGFR gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the VEGF and/or VEGFR gene; and (b) contacting the cell in vitro or in vivo with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in the cell.

In one embodiment, the invention features a method of modulating the expression of a VEGF and/or VEGFR gene in a tissue explant (e.g., a liver transplant) comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the VEGF and/or VEGFR gene; and (b) contacting a cell of the tissue explant derived from a particular subject or organism with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in that subject or organism.

In another embodiment, the invention features a method of modulating the expression of more than one VEGF and/or VEGFR gene in a tissue explant (e.g., a liver transplant) comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the VEGF and/or VEGFR gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in that subject or organism.

In one embodiment, the invention features a method of modulating the expression of a VEGF and/or VEGFR gene in a subject or organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the VEGF and/or VEGFR gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in the subject or organism.

In another embodiment, the invention features a method of modulating the expression of more than one VEGF and/or VEGFR gene in a subject or organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the VEGF and/or VEGFR gene; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in the subject or organism.

In one embodiment, the invention features a method of modulating the expression of a VEGF and/or VEGFR gene in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR gene in the subject or organism.

In one embodiment, the invention features a method for treating or preventing ocular disease in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of an inhibitor of VEGF and/or VEGFR gene expression in the subject or organism. In one embodiment, the ocular disease is age related macular degeneration (e.g., wet or dry AMD). In one embodiment, the ocular disease is diabetic retinopathy.

In one embodiment, the invention features a method for treating or preventing cancer in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of an inhibitor of VEGF and/or VEGFR gene expression in the subject or organism. In one embodiment, the cancer is selected from the group consisting of colorectal cancer, breast cancer, uterine cancer, ovarian cancer, or tumor angiogenesis.

In one embodiment, the invention features a method for treating or preventing a proliferative disease in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of an inhibitor of VEGF and/or VEGFR gene expression in the subject or organism.

In one embodiment, the invention features a method for treating or preventing renal disease in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of an inhibitor of VEGF and/or VEGFR gene expression in the subject or organism. In one embodiment, the renal disease is polycystic kidney disease.

In one embodiment, the invention features a method for inhibiting or preventing angiogenesis in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of an inhibitor of VEGF and/or VEGFR gene expression in the subject or organism.

In another embodiment, the invention features a method of modulating the expression of more than one VEGF and/or VEGFR gene in a subject or organism comprising contacting the subject or organism with one or more siNA molecules of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the VEGF and/or VEGFR genes in the subject or organism.

The siNA molecules of the invention can be designed to down regulate or inhibit target (e.g., VEGF and/or VEGFR) gene expression through RNAi targeting of a variety of RNA molecules. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target gene. Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcripts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as VEGF and/or VEGFR family genes. As such, siNA molecules targeting multiple VEGF and/or VEGFR targets can provide increased therapeutic effect. In addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, the progression and/or maintenance of cancer.

In one embodiment, siNA molecule(s) and/or methods of the invention are used to down regulate the expression of gene(s) that encode RNA referred to by Genbank Accession, for example, VEGF and/or VEGFR genes encoding RNA sequence(s) referred to herein by Genbank Accession number, for example, Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a) generating a library of siNA constructs having a predetermined complexity; and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence. In one embodiment, the siNA molecules of (a) have strands of a fixed length, for example, about 23 nucleotides in length. In another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a) generating a randomized library of siNA constructs having a predetermined complexity, such as of 4N, where N represents the number of base paired nucleotides in each of the siNA construct strands (eg. for a siNA construct having 21 nucleotide sense and antisense strands with 19 base pairs, the complexity would be 4¹⁹); and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target VEGF and/or VEGFR RNA sequence. In another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 6 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of VEGF and/or VEGFR RNA are analyzed for detectable levels of cleavage, for example, by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target VEGF and/or VEGFR RNA sequence. The target VEGF and/or VEGFR RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In another embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a target gene; (b) synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In one embodiment, the siNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length. In another embodiment, the siNA molecules of (b) are of differing length, for example having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for diagnosing a disease or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease or condition in the subject. In another embodiment, the invention features a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds. In yet another embodiment, the invention features a method for inhibiting, reducing or preventing ocular disease, cancer, proliferative disease, angiogenesis, and/or renal disease in a subject or organism comprising administering to the subject a composition of the invention under conditions suitable for inhibiting, reducing or preventing ocular disease, cancer, proliferative disease, angiogenesis, and/or renal disease in the subject or organism.

In another embodiment, the invention features a method for validating a VEGF and/or VEGFR gene target, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a VEGF and/or VEGFR target gene; (b) introducing the siNA molecule into a cell, tissue, subject, or organism under conditions suitable for modulating expression of the VEGF and/or VEGFR target gene in the cell, tissue, subject, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, subject, or organism.

In another embodiment, the invention features a method for validating a VEGF and/or VEGFR target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a VEGF and/or VEGFR target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the VEGF and/or VEGFR target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human or animal, wherein the system comprises the components required for RNAi activity. The term “biological system” includes, for example, a cell, tissue, subject, or organism, or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.

By “phenotypic change” is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA). Such detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art. The detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a VEGF and/or VEGFR target gene in a biological system, including, for example, in a cell, tissue, subject, or organism. In another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one VEGF and/or VEGFR target gene in a biological system, including, for example, in a cell, tissue, subject, or organism.

In one embodiment, the invention features a cell containing one or more siNA molecules of the invention, which can be chemically-modified. In another embodiment, the cell containing a siNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention, which can be chemically-modified, comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions using an alkylamine base such as methylamine. In one embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker and under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In one embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.

In another embodiment, the invention features a method for making a double-stranded siNA molecule in a single synthetic process comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5′-protecting group, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-stranded siNA molecule, for example using a trityl-on synthesis strategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of the invention comprises the teachings of Scaringe et al., U.S. Pat. Nos. 5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein in their entirety.

In one embodiment, the invention features siNA constructs that mediate RNAi against VEGF and/or VEGFR, wherein the siNA construct comprises one or more chemical modifications, for example, one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generating siNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased nuclease resistance.

In another embodiment, the invention features a method for generating siNA molecules with improved toxicologic profiles (e.g., have attenuated or no immunstimulatory properties) comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved toxicologic profiles.

In another embodiment, the invention features a method for generating siNA molecules that do not stimulate an interferon response (e.g., no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate an interferon response.

By “improved toxicologic profile”, is meant that the chemically modified siNA construct exhibits decreased toxicity in a cell, subject, or organism compared to an unmodified siNA or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology. In a non-limiting example, siNA molecules with improved toxicologic profiles are associated with a decreased or attenuated immunostimulatory response in a cell, subject, or organism compared to an unmodified siNA or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology. In one embodiment, a siNA molecule with an improved toxicological profile comprises no ribonucleotides. In one embodiment, a siNA molecule with an improved toxicological profile comprises less than 5 ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides). In one embodiment, a siNA molecule with an improved toxicological profile comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab 25, Stab 26, Stab 27, Stab 28, Stab 29, Stab 30, Stab 31, Stab 32, Stab 33 or any combination thereof (see Table IV). In one embodiment, the level of immunostimulatory response associated with a given siNA molecule can be measured as is known in the art, for example by determining the level of PKR/interferon response, proliferation, B-cell activation, and/or cytokine production in assays to quantitate the immunostimulatory response of particular siNA molecules (see, for example, Leifer et al., 2003, J Immunother. 26, 313-9; and U.S. Pat. No. 5,968,909, incorporated in its entirety by reference).

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the sense and antisense strands of the siNA molecule.

In one embodiment, the invention features siNA constructs that mediate RNAi against VEGF and/or VEGFR, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediate RNAi against VEGF and/or VEGFR, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediate RNAi against VEGF and/or VEGFR, wherein the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.

In another embodiment, the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to a chemically-modified siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.

In one embodiment, the invention features chemically-modified siNA constructs that mediate RNAi against VEGF and/or VEGFR in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a target RNA molecule, DNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against VEGF and/or VEGFR comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against VEGF and/or VEGFR target RNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against VEGF and/or VEGFR target DNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target DNA.

In another embodiment, the invention features a method for generating siNA molecules against VEGF and/or VEGFR with improved cellular uptake comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediate RNAi against VEGF and/or VEGFR, wherein the siNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siNA construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing a conjugate into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; polyamines, such as spermine or spermidine; and others.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein the second sequence is designed or modified in a manner that prevents its entry into the RNAi pathway as a guide sequence or as a sequence that is complementary to a target nucleic acid (e.g., RNA) sequence. Such design or modifications are expected to enhance the activity of siNA and/or improve the specificity of siNA molecules of the invention. These modifications are also expected to minimize any off-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end of said second sequence. In one embodiment, the terminal cap moiety comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end and 3′-end of said second sequence. In one embodiment, each terminal cap moiety individually comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a target nucleic acid (e.g., a DNA or RNA such as a gene or its corresponding RNA), comprising (a) introducing one or more chemical modifications into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved specificity. In another embodiment, the chemical modification used to improve specificity comprises terminal cap modifications at the 5′-end, 3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal cap modifications can comprise, for example, structures shown in FIG. 10 (e.g. inverted deoxyabasic moieties) or any other chemical modification that renders a portion of the siNA molecule (e.g. the sense strand) incapable of mediating RNA interference against an off target nucleic acid sequence. In a non-limiting example, a siNA molecule is designed such that only the antisense sequence of the siNA molecule can serve as a guide sequence for RISC mediated degradation of a corresponding target RNA sequence. This can be accomplished by rendering the sense sequence of the siNA inactive by introducing chemical modifications to the sense strand that preclude recognition of the sense strand as a guide sequence by RNAi machinery. In one embodiment, such chemical modifications comprise any chemical group at the 5′-end of the sense strand of the siNA, or any other group that serves to render the sense strand inactive as a guide sequence for mediating RNA interference. These modifications, for example, can result in a molecule where the 5′-end of the sense strand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphate group (e.g., phosphate, diphosphate, triphosphate, cyclic phosphate etc.). Non-limiting examples of such siNA constructs are described herein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”, “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (see Table IV) wherein the 5′-end and 3′-end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of unmodified siNA molecules, (b) screening the siNA molecules of step (a) under conditions suitable for isolating siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence, and (c) introducing chemical modifications (e.g. chemical modifications as described herein or as otherwise known in the art) into the active siNA molecules of (b). In one embodiment, the method further comprises re-screening the chemically modified siNA molecules of step (c) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

In one embodiment, the invention features a method for screening chemically modified siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of chemically modified siNA molecules (e.g. siNA molecules as described herein or as otherwise known in the art), and (b) screening the siNA molecules of step (a) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly. The receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercellular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing an excipient formulation to a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands, and others.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing nucleotides having any of Formulae I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 100 to about 50,000 daltons (Da).

The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include a siNA molecule of the invention and a vehicle that promotes introduction of the siNA into cells of interest as described herein (e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, U.S. Pat. No. 6,395,713). The kit Can be used for target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., U.S. Ser. No. 60/402,996). Such a kit can also include instructions to allow a user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “short interfering oligonucleotide molecule”, or “chemically-modified short interfering nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner; see for example Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples of siNA molecules of the invention are shown in FIGS. 4-6, and Tables II and III herein. For example the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g. about 15 to about 25 or more nucleotides of the siNA molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5′-phosphate (see for example Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certain embodiments, the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2′-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides “siMON.” As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

In one embodiment, a siNA molecule of the invention is a duplex forming oligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al., U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCT Application No. US04/16390, filed May 24, 2004).

In one embodiment, a siNA molecule of the invention is a multifunctional siNA, (see for example FIGS. 16-21 and Jadhav et al., U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and International PCT Application No. US04/16390, filed May 24, 2004). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting, for example, two or more regions of VEGF and/or VEGFR RNA (see for example target sequences in Tables II and III). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more VEGF isoforms (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more VEGF receptors (e.g., VEGFR1, VEGFR2, and/or VEGFR3). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more VEGF isoforms (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) and one or more VEGF receptors, (e.g., VEGFR1, VEGFR2, and/or VEGFR3).

By “asymmetric hairpin” as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprising about 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12) nucleotides, and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also comprise a 5′-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence. In one embodiment, inhibition, down regulation, or reduction of gene expression is associated with post transcriptional silencing, such as RNAi mediated cleavage of a target nucleic acid molecule (e.g. RNA) or inhibition of translation. In one embodiment, inhibition, down regulation, or reduction of gene expression is associated with pretranscriptional silencing.

By “gene”, or “target gene”, is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. A gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (mRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve as target nucleic acid molecules for siNA mediated RNA interference in modulating the activity of FRNA or ncRNA involved in functional or regulatory cellular processes. Abberant FRNA or ncRNA activity leading to disease can therefore be modulated by siNA molecules of the invention. siNA molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.). The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts. For a review, see for example Snyder and Gerstein, 2003, Science, 300, 258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(H1)-N-3-amino(H2), GA sheared, UC 4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-Ni amino-carbonyl, GA+ carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3, GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “VEGF” as used herein is meant, any vascular endothelial growth factor (e.g., VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D) protein, peptide, or polypeptide having vascular endothelial growth factor activity, such as encoded by VEGF Genbank Accession Nos. shown in Table I. The term VEGF also refers to nucleic acid sequences encloding any vascular endothelial growth factor protein, peptide, or polypeptide having vascular endothelial growth factor activity.

By “VEGF-B” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM_—003377, having vascular endothelial growth factor type B activity. The term VEGF-B also refers to nucleic acid sequences encloding any VEGF-B protein, peptide, or polypeptide having VEGF-B activity.

By “VEGF-C” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM_—005429, having vascular endothelial growth factor type C activity. The term VEGF-C also refers to nucleic acid sequences encloding any VEGF-C protein, peptide, or polypeptide having VEGF-C activity.

By “VEGF-D” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM_—004469, having vascular endothelial growth factor type D activity. The term VEGF-D also refers to nucleic acid sequences encloding any VEGF-D protein, peptide, or polypeptide having VEGF-D activity.

By “VEGFR” as used herein is meant, any vascular endothelial growth factor receptor protein, peptide, or polypeptide (e.g., VEGFR1, VEGFR2, or VEGFR3, including both membrane bound and/or soluble forms thereof) having vascular endothelial growth factor receptor activity, such as encoded by VEGFR Genbank Accession Nos. shown in Table 1. The term VEGFR also refers to nucleic acid sequences encloding any vascular endothelial growth factor receptor protein, peptide, or polypeptide having vascular endothelial growth factor receptor activity.

By “VEGFR1” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM_—002019, having vascular endothelial growth factor receptor type 1 (flt) activity, for example, having the ability to bind a vascular endothelial growth factor. The term VEGF1 also refers to nucleic acid sequences encloding any VEGFR1 protein, peptide, or polypeptide having VEGFR1 activity.

By “VEGFR2” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM_—002253, having vascular endothelial growth factor receptor type 2 (kdr) activity, for example, having the ability to bind a vascular endothelial growth factor. The term VEGF2 also refers to nucleic acid sequences encloding any VEGFR2 protein, peptide, or polypeptide having VEGFR2 activity.

By “VEGFR3” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM_—002020 having vascular endothelial growth factor receptor type 3 (kdr) activity, for example, having the ability to bind a vascular endothelial growth factor. The term VEGFR3 also refers to nucleic acid sequences encloding any VEGFR3 protein, peptide, or polypeptide having VEGFR3 activity.

By “homologous sequence” is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism. The polynucleotide can include both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA. In one embodiment, a target nucleic acid of the invention is VEGF RNA or DNA. In another embodiment, a target nucleic acid of the invention is a VEGFR RNA or DNA.

By “complementarity” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, a siNA molecule of the invention comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.

In one embodiment, siNA molecules of the invention that down regulate or reduce VEGF and/or VEGFR gene expression are used for treating, preventing or reducing ocular disease, cancer, proliferative disease, renal disease, or angiogenesis in a subject or organism.

By “proliferative disease” or “cancer” as used herein is meant, any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art; including AIDS related cancers such as Kaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas; Brain cancers such as Meningiomas, Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, and Metastatic brain cancers; cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct cancers, cancers of the retina such as retinoblastoma, cancers of the esophagus, gastric cancers, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, colorectal cancer, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrug resistant cancers; and proliferative diseases and conditions, such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet/dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration and other proliferative diseases and conditions such as restenosis and renal disease such as polycystic kidney disease, and any other cancer or proliferative disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.

By “ocular disease” as used herein is meant, any disease, condition, trait, genotype or phenotype of the eye and related structures, such as Cystoid Macular Edema, Asteroid Hyalosis, Pathological Myopia and Posterior Staphyloma, Toxocariasis (Ocular Larva Migrans), Retinal Vein Occlusion, Posterior Vitreous Detachment, Tractional Retinal Tears, Epiretinal Membrane, Diabetic Retinopathy, Lattice Degeneration, Retinal Vein Occlusion, Retinal Artery Occlusion, Macular Degeneration (e.g., age related macular degeneration such as wet AMD or dry AMD), Toxoplasmosis, Choroidal Melanoma, Acquired Retinoschisis, Hollenhorst Plaque, Idiopathic Central Serous Chorioretinopathy, Macular Hole, Presumed Ocular Histoplasmosis Syndrome, Retinal Macroaneursym, Retinitis Pigmentosa, Retinal Detachment, Hypertensive Retinopathy, Retinal Pigment Epithelium (RPE) Detachment, Papillophlebitis, Ocular Ischemic Syndrome, Coats' Disease, Leber's Miliary Aneurysm, Conjunctival Neoplasms, Allergic Conjunctivitis, Vernal Conjunctivitis, Acute Bacterial Conjunctivitis, Allergic Conjunctivitis & Vernal Keratoconjunctivitis, Viral Conjunctivitis, Bacterial Conjunctivitis, Chlamydial & Gonococcal Conjunctivitis, Conjunctival Laceration, Episcleritis, Scleritis, Pingueculitis, Pterygium, Superior Limbic Keratoconjunctivitis (SLK of Theodore), Toxic Conjunctivitis, Conjunctivitis with Pseudomembrane, Giant Papillary Conjunctivitis, Terrien's Marginal Degeneration, Acanthamoeba Keratitis, Fungal Keratitis, Filamentary Keratitis, Bacterial Keratitis, Keratitis Sicca/Dry Eye Syndrome, Bacterial Keratitis, Herpes Simplex Keratitis, Sterile Corneal Infiltrates, Phlyctenulosis, Corneal Abrasion & Recurrent Corneal Erosion, Corneal Foreign Body, Chemical Burs, Epithelial Basement Membrane Dystrophy (EBMD), Thygeson's Superficial Punctate Keratopathy, Corneal Laceration, Salzmann's Nodular Degeneration, Fuchs' Endothelial Dystrophy, Crystalline Lens Subluxation, Ciliary-Block Glaucoma, Primary Open-Angle Glaucoma, Pigment Dispersion Syndrome and Pigmentary Glaucoma, Pseudoexfoliation Syndrom and Pseudoexfoliative Glaucoma, Anterior Uveitis, Primary Open Angle Glaucoma, Uveitic Glaucoma & Glaucomatocyclitic Crisis, Pigment Dispersion Syndrome & Pigmentary Glaucoma, Acute Angle Closure Glaucoma, Anterior Uveitis, Hyphema, Angle Recession Glaucoma, Lens Induced Glaucoma, Pseudoexfoliation Syndrome and Pseudoexfoliative Glaucoma, Axenfeld-Rieger Syndrome, Neovascular Glaucoma, Pars Planitis, Choroidal Rupture, Duane's Retraction Syndrome, Toxic/Nutritional Optic Neuropathy, Aberrant Regeneration of Cranial Nerve III, Intracranial Mass Lesions, Carotid-Cavernous Sinus Fistula, Anterior Ischemic Optic Neuropathy, Optic Disc Edema & Papilledema, Cranial Nerve III Palsy, Cranial Nerve IV Palsy, Cranial Nerve VI Palsy, Cranial Nerve VII (Facial Nerve) Palsy, Homer's Syndrome, Internuclear Ophthalmoplegia, Optic Nerve Head Hypoplasia, Optic Pit, Tonic Pupil, Optic Nerve Head Drusen, Demyelinafing Optic Neuropathy (Optic Neuritis, Retrobulbar Optic Neuritis), Amaurosis Fugax and Transient Ischemic Attack, Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum, Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis, Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal Cell Carcinoma, Herpes Zoster Ophthalmicus, Pediculosis & Phthiriasis, Blow-out Fracture, Chronic Epiphora, Dacryocystitis, Herpes Simplex Blepharitis, Orbital Cellulitis, Senile Entropion, and Squamous Cell Carcinoma.

In one embodiment of the present invention, each sequence of a siNA molecule of the invention is independently about 15 to about 30 nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In another embodiment, one or more strands of the siNA molecule of the invention independently comprises about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) that are complementary to a target nucleic acid molecule. In yet another embodiment, siNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15 to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs. Exemplary siNA molecules of the invention are shown in Table II. Exemplary synthetic siNA molecules of the invention are shown in Table III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures. Furthermore, the chemically modified constructs described in Table IV can be applied to any siNA sequence of the invention.

In another aspect, the invention provides mammalian cells containing one or more siNA molecules of this invention. The one or more siNA molecules can independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

The term “phosphorothioate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise an acetyl or protected acetyl group.

The term “thiophosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z comprises an acetyl or protected acetyl group and W comprises a sulfur atom or alternately W comprises an acetyl or protected acetyl group and Z comprises a sulfur atom.

The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat, inhibit, reduce, or prevent ocular disease, cancer, proliferative disease, renal disease, or angiogenesis in a subject or organism. For example, the siNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combination with other known treatments to treat, inhibit, reduce, or prevent ocular disease, cancer, proliferative disease, renal disease, or angiogenesis in a subject or organism. For example, the described molecules could be used in combination with one or more known compounds, treatments, or procedures to treat, inhibit, reduce, or prevent ocular disease, cancer, proliferative disease, renal disease, or angiogenesis in a subject or organism as are known in the art.

In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner which allows expression of the siNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725.

In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example Genbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.

In another aspect of the invention, siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siNA strands spontaneously hybridize to form a siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.

FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directly into a cell by appropriate means. An active siNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siNA constructs.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-deoxy nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand. The antisense strand of constructs A-F comprise sequence complementary to any target nucleic acid sequence of the invention. Furthermore, when a glyceryl moiety (L) is present at the 3′-end of the antisense strand for any construct shown in FIG. 4A-F, the modified internucleotide linkage is optional.

FIG. 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described in FIG. 4A-F to a VEGFR1 siNA sequence. Such chemical modifications can be applied to any VEGF and/or VEGFR sequence and/or cellular target sequence.

FIG. 6 shows non-limiting examples of different siNA constructs of the invention. The examples shown (constructs 1, 2, and 3) have 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example, comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined VEGF and/or VEGFR target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended-by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in a siNA transcript having specificity for a VEGF and/or VEGFR target sequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3′-restriction sequence of the first strand. The double-stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3′-terminal nucleotide overhang results from the transcription, for example, by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al., 2002, Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined VEGF and/or VEGFR target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3′-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific to R1 and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted into vectors such that (FIG. 9C) transfection of a vector into cells results in the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.

FIG. 10 shows non-limiting examples of different stabilization chemistries (1-10) that can be used, for example, to stabilize the 3′-end of siNA sequences of the invention, including (1) [3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3) [5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5) [5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7) [3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9) [5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistance while preserving the ability to mediate RNAi activity. Chemical modifications are introduced into the siNA construct based on educated design parameters (e.g. introducing. 2′-mofications, base modifications, backbone modifications, terminal cap modifications etc). The modified construct in tested in an appropriate system (e.g. human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters). In parallel, the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay). Lead siNA constructs are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and duplex constructs and asymmetric derivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminal phosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are identified in a target nucleic acid sequence. (i) A palindrome or repeat sequence is identified in a nucleic acid target sequence. (ii) A sequence is designed that is complementary to the target nucleic acid sequence and the palindrome sequence. (iii) An inverse repeat sequence of the non-palindrome/repeat portion of the complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO molecule comprising sequence complementary to the nucleic acid target. (iv) The DFO molecule can self-assemble to form a double stranded oligonucleotide. FIG. 14B shows a non-limiting representative example of a duplex forming oligonucleotide sequence. FIG. 14C shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence. FIG. 14D shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence followed by interaction with a target nucleic acid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are incorporated into the DFO constructs that have sequence complementary to any target nucleic acid sequence of interest. Incorporation of these palindrome/repeat sequences allow the design of DFO constructs that form duplexes in which each strand is capable of mediating modulation of target gene expression, for example by RNAi. First, the target sequence is identified. A complementary sequence is then generated in which nucleotide or non-nucleotide modifications (shown as X or Y) are introduced into the complementary sequence that generate an artificial palindrome (shown as XYXYXY in the Figure). An inverse repeat of the non-palindrome/repeat complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO comprising sequence complementary to the nucleic acid target. The DFO can self-assemble to form a double stranded oligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 16A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 16B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 17A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 17B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifuctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 18A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 18B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional sNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifuctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 19A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 19B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid molecules, such as separate RNA molecules encoding differing proteins, for example, a cytokine and its corresponding receptor, differing viral strains, a virus and a cellular protein involved in viral infection or replication, or differing proteins involved in a common or divergent biologic pathway that is implicated in the maintenance of progression of disease. Each strand of the multifunctional siNA construct comprises a region having complementarity to separate target nucleic acid molecules. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid sequences within the same target nucleic acid molecule, such as alternate coding regions of a RNA, coding and non-coding regions of a RNA, or alternate splice variant regions of a RNA. Each strand of the multifunctional siNA construct comprises a region having complementarity to the separate regions of the target nucleic acid molecule. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target region. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

FIG. 22 shows a non-limiting example of reduction of VEGFR1 mRNA in A375 cells mediated by chemically-modified siNAs that target VEGFR1 mRNA. A549 cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA. A screen of siNA constructs (Stabilization “Stab” chemistries are shown in Table IV, constructs are referred to by Compound number, see Table III) comprising Stab 4/5 chemistry (Compound 31190/31193), Stab 1/2 chemistry (Compound 31183/31186 and Compound 31184/31187), and unmodified RNA (Compound 30075/30076) were compared to untreated cells, matched chemistry inverted control siNA constructs, (Compound 31208/31211, Compound 31201/31204, Compound 31202/31205, and Compound 30077/30078) scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). All of the siNA constructs show significant reduction of VEGFR1 RNA expression.

FIG. 23 shows a non-limiting example of reduction of VEGFR1 mRNA levels in HAEC cell culture using Stab 9/10 directed against eight sites in VEGFR1 mRNA compared to matched chemistry inverted controls siNA constructs. Controls UNT and LF2K refer to untreated cells and cells treated with LF2K transfection reagent alone, respectively.

FIG. 24 shows a non-limiting example of reduction of VEGFR2 mRNA in HAEC cells mediated by chemically-modified siNAs that target VEGFR2 mRNA. HAEC cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA. A screen of siNA constructs (Stabilization “Stab” chemistries are shown in Table IV, constructs are referred to by Compound No., see Table III) in site 3854 comprising Stab 4/5 chemistry (Compound No. 30786/30790), Stab 7/8 chemistry (Compound No. 31858/31860), and Stab 9/10 chemistry (Compound No. 31862/31864) and in site 3948 comprising Stab 4/5 chemistry (Compound No. 31856/31857), Stab 7/8 chemistry (Compound No. 31859/31861), and Stab 9/10 chemistry (Compound No. 31863/31865) were compared to untreated cells, matched chemistry inverted control siNA constructs in site 3854 (Compound No. 31878/31880, Compound No. 31882/31884, and Compound No. 31886/31888), and in site 3948 (Compound No. 31879/31881, Compound No. 31883/31885, and Compound No. 31887/31889), cells transfected with LF2K (transfection reagent), and an all RNA control (Compound No. 31435/31439 in site 3854 and Compound No. 31437/31441 in site 3948). All of the siNA constructs show significant reduction of VEGFR2 RNA expression.

FIG. 25 shows a non-limiting example of reduction of VEGFR2 mRNA levels in HAEC cell culture using Stab 0/0 directed against four sites in VEGFR2 mRNA compared to irrelevant control siNA constructs (IC1, IC2). Controls UNT and LF2K refer to untreated cells and cells treated with LF2K transfection reagent alone, respectively.

FIG. 26 shows non-limiting examples of reduction of VEGFR1 (Flt-1) mRNA levels in HAEC cells (15,000 cells/well) 24 hours after treatment with siNA molecules targeting sequences having VEGFR1 (Flt-1) and VEGFR2 (KDR) homology. HAEC cells were transfected with 1.5 ug/well of lipid complexed with 25 nM siNA. Activity of the siNA moleclues is shown compared to matched chemistry inverted siNA controls, untreated cells, and cells treated with lipid only (transfection control). siNA molecules and controls are referred to by compound numbers (sense/antisense), see Table m for sequences. FIG. 26A shows data for Stab 9/10 siNA constructs. FIG. 26B shows data for Stab 7/8 siNA constructs. The FIG. 26B study includes a construct that targets only VEGFR1 (32748/32755) and a matched chemistry inverted control thereof (32772/32779) as additional controls. As shown in the figures, the siNA constructs that target both VEGFR1 and VEGFR2 sequences demonstrate potent efficacy in inhibiting VEGFR1 expression in cell cuture experiments.

FIG. 27 shows non-limiting examples of reduction of VEGFR2 (KDR) mRNA levels in HAEC cells (15,000 cells/well) 24 hours after treatment with siNA molecules targeting sequences having VEGFR1 and VEGFR2 homology. HAEC cells were transfected with 1.5 ug/well of lipid complexed with 25 nM siNA. Activity of the siNA moleclues is shown compared to matched chemistry inverted siNA controls, untreated cells, and cells treated with lipid only (transfection control). siNA molecules and controls are referred to by compound numbers (sense/antisense), see Table III for sequences. FIG. 27A shows data for Stab 9/10 siNA constructs. FIG. 237 shows data for Stab 7/8 siNA constructs. The FIG. 27B study includes a construct that targets only VEGFR1 (32748/32755) and a matched chemistry inverted control thereof (32772/32779) as additional controls. As shown in the figures, the siNA constructs that target both VEGFR1 and VEGFR2 sequences demonstrate potent efficacy in inhibiting VEGFR2 expression in cell cuture experiments.

FIG. 28 shows a non-limiting example of siNA mediated inhibition of VEGF-induced angiogenesis using the rat corneal model of angiogenesis. siNA targeting site 2340 of VEGFR1 RNA (shown as Compound No. 29695/29699 sense strand/antisense strand) was compared to an inverted control siNA (shown as Compound No. 29983/29984 sense strand/antisense strand) at three different concentrations (1ug, 3ug, and 10ug) and compared to a VEGF control in which no siNA was administered. As shown in the Figure, siNA constructs targeting VEGFR1 RNA can provide significant inhibition of angiogenesis in the rat corneal model.

FIG. 29 shows a non-limiting example of inhibition of VEGF induced neovascularization in the rat corneal model. VEGFR1 site 349 active siNA having “Stab 9/10” chemistry (Compound No. 31270/31273) was tested for inhibition of VEGF-induced angiogenesis at three different concentrations (2.0 ug, 1.0 ug, and 0.1 ug dose response) as compared to a matched chemistry inverted control siNA construct (Compound No. 31276/31279) at each concentration and a VEGF control in which no siNA was administered. As shown in the figure, the active siNA construct having “Stab 9/10” chemistry (Compound No. 31270/31273) is highly effective in inhibiting VEGF-induced angiogenesis in the rat corneal model compared to the matched chemistry inverted control siNA at concentrations from 0.1 ug to 2.0 ug.

FIG. 30 shows a non-limiting example of a study in which sites adjacent to VEGFR1 site 349 were evaluated for efficacy using two different siNA stabilization chemistries. Chemistry C=Stab 9/10 whereas Chemistry D=Stab 7/8.

FIG. 31 shows a non-limiting example of inhibition of VEGF induced ocular angiogenesis using siNA constructs that target homologous sequences shared by VEGFR1 and VEGFR2 via subconjuctival administration of the siNA after VEGF disk implantation. siNA constructs were administered intraocularly on days 1 and 7 following laser induced injury to the choroid, and choroidal neovascularization assessed on day 14.

FIG. 32 shows a non-limiting example of inhibition of VEGF induced neovascularization in a mouse model of coroidal neovascularization via intraocular administration of siNA. VEGFR1 site 349 active siNA having “Stab 9/10” chemistry (Compound No. 31270/31273) was tested for inhibition of neovascularization at two different concentrations (1.5 ug, and 0.5 ug) as compared to a matched chemistry inverted control siNA construct (Compound No. 31276/31279) and phosphate buffered saline (PBS). siNA constructs were administered intraocularly on days 1 and 7 following laser induced injury to the choroid, and choroidal neovascularization assessed on day 14. As shown in the figure, the active siNA construct having “Stab 9/10” chemistry (Compound No. 31270/31273) is highly effective in inhibiting neovascularization via intraocular administration in this model.

FIG. 33 shows a non-limiting example of inhibition of VEGF induced neovascularization in a mouse model of coroidal neovascularization via periocular administration of siNA. VEGFR1 site 349 active siNA having “Stab 9/10” chemistry (Compound No. 31270/31273) was tested for inhibition of neovascularization at two different concentrations (1.5 ug with a saline control, and 0.5 ug with an inverted siNA control, Compound No. 31276/31279). Eight mice were used in each arm of the study with one eye receiving the active siNA and the other eye receiving the saline or inverted control. siNA constructs and controls were adminitered daily up to 14 days, and neovascularization was assessed at day 17 following laser induced injury to the choroid. As shown in the figure, the active siNA construct having “Stab 9/10” chemistry (Compound No. 31270/31273) is highly effective in inhibiting neovascularization via periocular administration in this model.

FIG. 34 shows another non-limiting example of inhibition of VEGF induced neovascularization in a mouse model of coroidal neovascularization via periocular administration of siNA. VEGFR1 site 349 active siNA having “Stab 9/10” chemistry (Compound No. 31270/31273) was tested for inhibition of neovascularization at two different concentrations (1.5 ug with an inverted siNA control, Compound No. 31276/31279 and 0.5 ug with a saline control). Nine mice were used in the active versus inverted arm of the study with one eye receiving the active siNA and the other eye receiving the inverted control. Eight mice were used in the active versus saline arm of the study with one eye receiving the active siNA and the other eye receiving the saline control. siNA constructs and controls were administered daily up to 14 days, and neovascularization was assessed at day 17 following laser induced injury to the choroid. As shown in the figure, the active siNA construct having “Stab 9/10” chemistry (Compound No. 31270/31273) is highly effective in inhibiting neovascularization via periocular administration in this model.

FIG. 35 shows a non-limiting example of siNA mediated inhibition of choroidal neovascularization (CNV) in mice injected with active siNA (31270/31273) targeting site 349 of VEGFR1 mRNA compared to mice injected with a matched chemistry inverted control siNA construct (31276/31279) in a mouse model of ocular neovascularization. Periocular injections were performed every three days after rupture of Bruch's membrane. Eyes treated with active siNA had significantly smaller areas of CNV than eyes treated with inverted control siNA constructs (n=13, p=0.0002).

FIG. 36 shows a non-limiting example of siNA mediated inhibition of VEGFR1 mRNA levels in mice injected with active siNA (31270/31273) targeting site 349 of VEGFR1 mRNA compared to mice injected with a matched chemistry inverted control siNA construct (31276/31279) in a mouse model of oxygen induced retinopathy (OIR). Periocular injections of VEGFR1 siNA (31270/31273) (5 μl; 1.5 μg/III) on P12, P14, and P16 significantly reduced VEGFR1 mRNA expression compared to injections with a matched chemistry inverted control siNA construct (31276/31279), (40% inhibition; n=9, p=0.0121).

FIG. 37 shows a non-limiting example of siNA mediated inhibition of VEGFR1 protein levels in mice injected with active siNA (31270/31273) targeting site 349 of VEGFR1 mRNA compared to mice injected with a matched chemistry inverted control siNA construct (31276/31279) in a mouse model of oxygen induced retinopathy (OIR). Intraocular injections of VEGFR1 siNA (31270/31273) (5 μg), significantly reduced. VEGFR1 protein levels compared to injections with a matched chemistry inverted control siNA construct (31276/31279), (30% inhibition; n=7, p=0.0103).

FIG. 38 shows a non-limiting example of the reduction of primary tumor volume in a mouse 4T1-luciferase mammary carcinoma syngeneic tumor model using active Stab 9/10 siNA targeting site 349 of VEGFR1 RNA (Compound #31270/31273) compared to a matched chemistry inactive inverted control siNA (Compound #31276/31279) and saline. As shown in the figure, the active siNA construct is effective in reducing tumor volume in this model.

FIG. 39 shows a non-limiting example of the reduction of soluble VEGFR1 serum levels in a mouse 4T1-luciferase mammary carcinoma syngeneic tumor model using active Stab 9/10 siNA targeting site 349 of VEGFR1 RNA (Compound #31270/31273) compared to a matched chemistry inactive inverted control siNA (Compound #31276/31279). As shown in the figure, the active siNA construct is effective in reducing soluble VEGFR1 serum levels in this model.

FIG. 40 shows the results of a study in which multifunctional siNAs targeting VEGF site 1420 and VEGFR1/VEGFR2 conserved site 3646/3718 (MF 34702/34703), VEGF site 1423 and VEGFR1/VEGFR2 conserved site 3646/3718 (MF 34706/34707), VEGF site 1421 and VEGFR1/VEGFR2 conserved site 3646/3718 (MF 34708/34709) and VEGF site 1562 and VEGFR1/VEGFR2 conserved site 3646/3718 (MF 34695/34700) were evaluated at 25 nM with irrelevant multifunctional siNA controls having differing lengths corresponding to the differing multifunctional lengths (IC-1, IC-2, IC-3, and IC-4) and individual siNA constructs targeting VEGF sites 1420 (32530/32548), 1421 (32531/32549), and 1562 (34682/34690) along with untreated cells. Compound numbers for the siNA constructs are shown in Table III. (A) Data is shown as the ratio of Renilla/Firefly luminescence for VEGF expression. (B) Data is shown as the ratio of Renilla/Firefly luminescence for VEGFR1 expression. (C) Data is shown as the ratio of Renilla/Firefly luminescence for VEGFR2 expression. As shown in the figures, the multifunctional siNA constructs show selective inhibition of VEGF, VEGFR1, and VEGFR2 compared to untreated cells and irrelevant matched chemistry and matched length controls.

FIG. 41 shows the results of a dose response study in which stabilized multifunctional siNAs targeting VEGF site 1562 and VEGFR1/VEGFR2 conserved site 3646/3718 (MF 37538/37579) was evaluated at 0.02 to 10 nM compared to individual siNA constructs targeting VEGF site 1562 (37575/37577) and VEGFR1/VEGFR2 conserved site 3646/3718 (33726/37576) and pooled individual siNA constructs targeting VEGF site 1562 (37575/37577) and VEGFR1/VEGFR2 conserved site 3646/3718 (33726/37576). Compound numbers for the siNA constructs are shown in Table III. (A) Data is shown as the ratio of Renilla/Firefly luminescence for VEGF expression. (B) Data is shown as the ratio of Renilla/Firefly luminescence for VEGFR1 expression. (C) Data is shown as the ratio of Renilla/Firefly luminescence for VEGFR2 expression. As shown in the figures, the stabilized multifunctional siNA constructs show selective inhibition of VEGF, VEGFR1, and VEGFR2 that is similar to the corresponding individual and pooled siNA constructs.

FIG. 42 shows the results of a study in which various non-nucleotide tethered multifunctional siNAs targeting VEGF site 1421 and VEGFR1/VEGFR2 conserved site 3646/3718 were evaluated at 25 nM compared to untreated cells (no siRNA), irrelevant siNA controls targeting HCV RNA site 327 (HCV 327, 34585/36447), individual active siNA constructs targeting VEGF site 1421 (32531/32549) and VEGFR1/VEGFR2 conserved site 3646/3718 (32236/32551), an irrelevant matched length multifunctional siNA construct (35414/36447/36446). Each construct was evaluated for VEGF, VEGFR1 (Flt), or VEGFR2 (KDR) expression levels as determined by the ratio of renilla to firefly luciferase signal. Data is shown for active tethered multifunctional siNA having a hexaethylene glycol tether (36425/32251/32549), C12 tether (36426/32251/32549), tetraethylene glycol tether (36427/32251/32549), C3 tether (36428/32251/32549) and double hexaethylene glycol tether (36429/32251/32549). Compound numbers for the siNA constructs are shown in Table III. As shown in the figure, the non-nucleotide tethered multifunctional siNA constructs show similar activity to the corresponding individual siNA constructs targeting VEGF, VEGFR1, and VEGFR2.

FIG. 43(A-H) shows non-limiting examples of tethered multiifunctional siNA constructs of the invention. In the examples shown, a linker (e.g., nucleotide or non-nucleotide linker) connects two siNA regions (e.g., two sense, two antisense, or alternately a sense and an antisense region together. Separate sense (or sense and antisense) sequences corresponding to a first target sequence and second target sequence are hybridized to their corresponding sense and/or antisense sequences in the multifunctional siNA. In addition, various conjugates, ligands, aptamers, polymers or reporter molecules can be attached to the linker region for selective or improved delivery and/or pharmacokinetic properties.

FIG. 44 shows a non-limiting example of various dendrimer based multifunctional siNA designs.

FIG. 45 shows a non-limiting example of various supramolecular multifunctional siNA designs.

FIG. 46 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 30 nucleotide precursor siNA construct. A 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown). For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2′-OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs.

FIG. 47 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 40 nucleotide precursor siNA construct. A 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end. For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Four targeting sequences are shown in four colors, blue, light-blue and red and orange. The required sequence identity overlapped is indicated by grey boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multiifunctional designs. For example cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.

FIG. 48 shows a non-limiting example of inhibition of HBV RNA by dicer enabled multifunctional siNA constructs targeting HBV site 263. When the first 17 nucleotides of a siNA antisense strand (e.g., 21 nucleotide strands in a duplex with 3′-TT overhangs) are complementary to a target RNA, robust silencing was observed at 25 nM. 80% silencing was observed with only 16 nucleotide complementarity in the same format.

FIG. 49 shows a non-limiting example of additional multifunctional siNA construct designs of the invention. In one example, a conjugate, ligand, aptamer, label, or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.

FIG. 50 shows a non-limiting example of additional multifunctional siNA construct designs of the invention. In one example, a conjugate, ligand, aptamer, label, or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.

DETAILED DESCRIPTION OF THE INVENTION

Mechanism of Action of Nucleic Acid Molecules of the Invention The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limiting only to siRNA and can be applied to siNA as a whole. By “improved capacity to mediate RNAi” or “improved RNAi activity” is meant to include RNAi activity measured in vitro and/or in vivo where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention. In this invention, the product of these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or a siNA containing a plurality of ribonucleotides. In some cases, the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), but the overall activity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188). In addition, RNA interference can also involve small RNA (e.g., micro-RNA or mRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). As such, siNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two 2-nucleotide 3′-terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.

Duplex Foming Oligonucleotides (DFO) of the Invention

In one embodiment, the invention features siNA molecules comprising duplex forming oligonucleotides (DFO) that can self-assemble into double stranded oligonucleotides. The duplex forming oligonucleotides of the invention can be chemically synthesized or expressed from transcription units and/or vectors. The DFO molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, refered to herein for convenience but not limitation as duplex forming oligonucleotides or DFO molecules, are potent mediators of sequence specific regulation of gene expression. The oligonucleotides of the invention are distinct from other nucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides etc.) in that they represent a class of linear polynucleotide sequences that are designed to self-assemble into double stranded oligonucleotides, where each strand in the double stranded oligonucleotides comprises a nucleotide sequence that is complementary to a target nucleic acid molecule. Nucleic acid molecules of the invention can thus self assemble into functional duplexes in which each strand of the duplex comprises the same polynucleotide sequence and each strand comprises a nucleotide sequence that is complementary to a target nucleic acid molecule.

Generally, double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotide sequences where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are assembled from two separate oligonucleotides, or from a single molecule that folds on itself to form a double stranded structure, often referred to in the field as hairpin stem-loop structure (e.g., shRNA or short hairpin RNA). These double stranded oligonucleotides known in the art all have a common feature in that each strand of the duplex has a distict nucleotide sequence.

Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of forming a double stranded nucleic acid molecule starting from a single stranded or linear oligonucleotide. The two strands of the double stranded oligonucleotide formed according to the instant invention have the same nucleotide sequence and are not covalently linked to each other. Such double-stranded oligonucleotides molecules can be readily linked post-synthetically by methods and reagents known in the art and are within the scope of the invention. In one embodiment, the single stranded oligonucleotide of the invention (the duplex forming oligonucleotide) that forms a double stranded oligonucleotide comprises a first region and a second region, where the second region includes a nucleotide sequence that is an inverted repeat of the nucleotide sequence in the first region, or a portion thereof, such that the single stranded oligonucleotide self assembles to form a duplex oligonucleotide in which the nucleotide sequence of one strand of the duplex is the same as the nucleotide sequence of the second strand. Non-limiting examples of such duplex forming oligonucleotides are illustrated in FIGS. 14 and 15. These duplex forming oligonucleotides (DFOs) can optionally include certain palindrome or repeat sequences where such palindrome or repeat sequences are present in between the first region and the second region of the DFO.

In one embodiment, the invention features a duplex forming oligonucleotide (DFO) molecule, wherein the DFO comprises a duplex forming self complementary nucleic acid sequence that has nucleotide sequence complementary to a VEGF and/or VEGFR target nucleic acid sequence. The DFO molecule can comprise a single self complementary sequence or a duplex resulting from assembly of such self complementary sequences.

In one embodiment, a duplex forming oligonucleotide (DFO) of the invention comprises a first region and a second region, wherein the second region comprises a nucleotide sequence comprising an inverted repeat of nucleotide sequence of the first region such that the DFO molecule can assemble into a double stranded oligonucleotide. Such double stranded oligonucleotides can act as a short interfering nucleic acid (siNA) to modulate gene expression. Each strand of the double stranded oligonucleotide duplex formed by DFO molecules of the invention can comprise a nucleotide sequence region that is complementary to the same nucleotide sequence in a target nucleic acid molecule (e.g., target VEGF and/or VEGFR RNA).

In one embodiment, the invention features a single stranded DFO that can assemble into a double stranded oligonucleotide. The applicant has surprisingly found that a single stranded oligonucleotide with nucleotide regions of self complementarity can readily assemble into duplex oligonucleotide constructs. Such DFOs can assemble into duplexes that can inhibit gene expression in a sequence specific manner. The DFO moleucles of the invention comprise a first region with nucleotide sequence that is complementary to the nucleotide sequence of a second region and where the sequence of the first region is complementary to a target nucleic acid (e.g., RNA). The DFO can form a double stranded oligonucleotide wherein a portion of each strand of the double stranded oligonucleotide comprises a sequence complementary to a target nucleic acid sequence.

In one embodiment, the invention features a double stranded oligonucleotide, wherein the two strands of the double stranded oligonucleotide are not covalently linked to each other, and wherein each strand of the double stranded oligonucleotide comprises a nucleotide sequence that is complementary to the same nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., VEGF and/or VEGFR RNA target). In another embodiment, the two strands of the double stranded oligonucleotide share an identical nucleotide sequence of at least about 15, preferably at least about 16, 17, 18, 19, 20, or 21 nucleotides.

In one embodiment, a DFO molecule of the invention comprises a structure having Formula DFO-I:

5′-p-X Z X′-3′

wherein Z comprises a palindromic or repeat nucleic acid sequence optionally with one or more modified nucleotides (e.g., nucleotide with a modified base, such as 2-amino purine, 2-amino-1,6-dihydro purine or a universal base), for example of length about 2 to about 24 nucleotides in even numbers (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 or 24 nucleotides), X represents a nucleic acid sequence, for example of length of about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 1 and about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein sequence X and Z, either independently or together, comprise nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof and is of length sufficient to interact (e.g., base pair) with the target nucleic acid sequence or a portion thereof (e.g., VEGF and/or VEGFR RNA target). For example, X independently can comprise a sequence from about 12 to about 21 or more (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) nucleotides in length that is complementary to nucleotide sequence in a target VEGF and/or VEGFR RNA or a portion thereof. In another non-limiting example, the length of the nucleotide sequence of X and Z together, when X is present, that is complementary to the target RNA or a portion thereof (e.g., VEGF and/or VEGFR RNA target) is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In yet another non-limiting example, when X is absent, the length of the nucleotide sequence of Z that is complementary to the target VEGF and/or VEGFR RNA or a portion thereof is from about 12 to about 24 or more nucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24, or more). In one embodiment X, Z and X′ are independently oligonucleotides, where X and/or Z comprises a nucleotide sequence of length sufficient to interact (e.g., base pair) with a nucleotide sequence in the target RNA or a portion thereof (e.g., VEGF and/or VEGFR RNA target). In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In another embodiment, the lengths of oligonucleotides X and Z, or Z and X′, or X, Z and X′ are either identical or different.

When a sequence is described in this specification as being of “sufficient” length to interact (i.e., base pair) with another sequence, it is meant that the the length is such that the number of bonds (e.g., hydrogen bonds) formed between the two sequences is enough to enable the two sequence to form a duplex under the conditions of interest. Such conditions can be in vitro (e.g., for diagnostic or assay purposes) or in vivo (e.g., for therapeutic purposes). It is a simple and routine matter to determine such lengths.

In one embodiment, the invention features a double stranded oligonucleotide construct having Formula DFO-I(a):

5′-p-X Z X′-3′
3′-X′ Z X-p-5′

wherein Z comprises a palindromic or repeat nucleic acid sequence or palindromic or repeat-like nucleic acid sequence with one or more modified nucleotides (e.g., nucleotides with a modified base, such as 2-amino purine, 2-amino-1,6-dihydro purine or a universal base), for example of length about 2 to about 24 nucleotides in even numbers (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 nucleotides), X represents a nucleic acid sequence, for example of length about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein each X and Z independently comprises a nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof (e.g., VEGF and/or VEGFR RNA target) and is of length sufficient to interact with the target nucleic acid sequence of a portion thereof (e.g., VEGF and/or VEGFR RNA target). For example, sequence X independently can comprise a sequence from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) in length that is complementary to a nucleotide sequence in a target RNA or a portion thereof (e.g., VEGF and/or VEGFR RNA target). In another non-limiting example, the length of the nucleotide sequence of X and Z together (when X is present) that is complementary to the target VEGF and/or VEGFR RNA or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In yet another non-limiting example, when X is absent, the length of the nucleotide sequence of Z that is complementary to the target VEGF and/or VEGFR RNA or a portion thereof is from about 12 to about 24 or more nucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24 or more). In one embodiment X, Z and X′ are independently oligonucleotides, where X and/or Z comprises a nucleotide sequence of length sufficient to interact (e.g., base pair) with nucleotide sequence in the target RNA or a portion thereof (e.g., VEGF and/or VEGFR RNA target). In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In another embodiment, the lengths of oligonucleotides X and Z or Z and X′ or X, Z and X′ are either identical or different. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a DFO molecule of the invention comprises structure having Formula DFO-II:

5′-p-X X′-3′

wherein each X and X′ are independently oligonucleotides of length about 12 nucleotides to about 21 nucleotides, wherein X comprises, for example, a nucleic acid sequence of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides. (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein X comprises a nucleotide sequence that is complementary to a target nucleic acid sequence (e.g., VEGF and/or VEGFR RNA) or a portion thereof and is of length sufficient to interact (e.g., base pair) with the target nucleic acid sequence of a portion thereof. In one embodiment, the length of oligonucleotides X and X′ are identical. In another embodiment the length of oligonucleotides X and X′ are not identical. In one embodiment, length of the oligonucleotides X and X′ are sufficint to form a relatively stable double stranded oligonucleotide.

In one embodiment, the invention features a double stranded oligonucleotide construct having Formula DFO-II(a):

5′-p-X X′-3′
3′-X′X′-p-5′

wherein each X and X′ are independently oligonucleotides of length about 12 nucleotides to about 21 nucleotides, wherein X comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein X comprises nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof (e.g., VEGF and/or VEGFR RNA target) and is of length sufficient to interact (e.g., base pair) with the target nucleic acid sequence (e.g., VEGF and/or VEGFR RNA) or a portion thereof. In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In one embodiment, the lengths of the oligonucleotides X and X′ are sufficint to form a relatively stable double stranded oligonucleotide. In one embodiment, the double stranded oligonucleotide construct of Formula II(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, the invention features a DFO molecule having Formula DFO-I(b):

5′-p-Z-3′

where Z comprises a palindromic or repeat nucleic acid sequence optionally including one or more non-standard or modified nucleotides (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) that can facilitate base-pairing with other nucleotides. Z can be, for example, of length sufficient to interact (e.g., base pair) with nucleotide sequence of a target nucleic acid (e.g., VEGF and/or VEGFR RNA) molecule, preferably of length of at least 12 nucleotides, specifically about 12 to about 24 nucleotides (e.g., about 12, 14, 16, 18, 20, 22 or 24 nucleotides). p represents a terminal phosphate group that can be present or absent.

In one embodiment, a DFO molecule having any of Formula DFO-I, DFO-I(a), DFO-I(b), DFO-II(a) or DFO-II can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I-VII, stabilization chemistries as described in Table IV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.

In one embodiment, the palidrome or repeat sequence or modified nucleotide (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of DFO constructs having Formula DFO-I, DFO-I(a) and DFO-I(b), comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence (e.g., modified base analogs that can form Watson Crick base pairs or non-Watson Crick base pairs).

In one embodiment, a DFO molecule of the invention, for example a DFO having Formula DFO-I or DFO-II, comprises about 15 to about 40 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, a DFO molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum or in cells or tissues. Furthermore, certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.

Multifunctional or Multi-Targeted siNA Molecules of the Invention

In one embodiment, the invention features siNA molecules comprising multifunctional short interfering nucleic acid (multifunctional siNA) molecules that modulate the expression of one or more genes in a biologic system, such as a cell, tissue, or organism. The multifunctional short interfering nucleic acid (multifunctional siNA) molecules of the invention can target more than one region a VEGF and/or VEGFR target nucleic acid sequence or can target sequences of more than one distinct target nucleic acid molecules (e.g., VEGF and/or VEGFR RNA targets). The multifunctional siNA molecules of the invention can be chemically synthesized or expressed from transcription units and/or vectors. The multifunctional siNA molecules of the instant invention provide useful reagents and methods for a variety of human applications, therapeutic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, refered to herein for convenience but not limitation as multifunctional short interfering nucleic acid or multifunctional siNA molecules, are potent mediators of sequence specific regulation of gene expression. The multifunctional siNA molecules of the invention are distinct from other nucleic acid sequences known in the art (e.g., siRNA, mRNA, stRNA, shRNA, antisense oligonucleotides, etc.) in that they represent a class of polynucleotide molecules that are designed such that each strand in the multifunctional siNA construct comprises a nucleotide sequence that is complementary to a distinct nucleic acid sequence in one or more target nucleic acid molecules. A single multifunctional siNA molecule (generally a double-stranded molecule) of the invention can thus target more than one (e.g., 2, 3, 4, 5, or more) differing target nucleic acid target molecules. Nucleic acid molecules of the invention can also target more than one (e.g., 2, 3, 4, 5, or more) region of the same target nucleic acid sequence. As such multifunctional siNA molecules of the invention are useful in down regulating or inhibiting the expression of one or more target nucleic acid molecules. For example, a multifunctional siNA molecule of the invention can target nucleic acid molecules encoding a cytokine and its corresponding receptor(s) (e.g., VEGF and VEGF receptors described herein). By reducing or inhibiting expression of more than one target nucleic acid molecule with one multifunctional siNA construct, multifunctional siNA molecules of the invention represent a class of potent therapeutic agents that can provide simultaneous inhibition of multiple targets within a disease or pathogen related pathway. Such simultaneous inhibition can provide synergistic therapeutic treatment strategies without the need for separate preclinical and clinical development efforts or complex regulatory approval process.

Use of multifunctional siNA molecules that target more then one region of a target nucleic acid molecule (e.g., messenger RNA) is expected to provide potent inhibition of gene expression. For example, a single multifunctional siNA construct of the invention can target both conserved and variable regions of a target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA), thereby allowing down regulation or inhibition of different splice variants encoded by a single gene, or allowing for targeting of both coding and non-coding regions of a target nucleic acid molecule.

Generally, double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotides where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are generally assembled from two separate oligonucleotides (e.g., siRNA). Alternately, a duplex can be formed from a single molecule that folds on itself (e.g., shRNA or short hairpin RNA). These double stranded oligonucleotides are known in the art to mediate RNA interference and all have a common feature wherein only one nucleotide sequence region (guide sequence or the antisense sequence) has complementarity to a target nucleic acid sequence (e.g., VEGF and/or VEGFR RNA) and the other strand (sense sequence) comprises nucleotide sequence that is homologous to the target nucleic acid sequence. Generally, the antisense sequence is retained in the active RISC complex and guides the RISC to the target nucleotide sequence by means of complementary base-pairing of the antisense sequence with the target seqeunce for mediating sequence-specific RNA interference. It is known in the art that in some cell culture systems, certain types of unmodified siRNAs can exhibit “off target” effects. It is hypothesized that this off-target effect involves the participation of the sense sequence instead of the antisense sequence of the siRNA in the RISC complex (see for example Schwarz et al., 2003, Cell, 115, 199-208). In this instance the sense sequence is believed to direct the RISC complex to a sequence (off-target sequence) that is distinct from the intended target sequence, resulting in the inhibition of the off-target sequence. In these double stranded nucleic acid molecules, each strand is complementary to a distinct target nucleic acid sequence. However, the off-targets that are affected by these dsRNAs are not entirely predictable and are non-specific.

Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of down regulating or inhibiting the expression of more than one target nucleic acid sequence using a single multifunctional siNA construct. The multifunctional siNA molecules of the invention are designed to be double-stranded or partially double stranded, such that a portion of each strand or region of the multifunctional siNA is complementary to a target nucleic acid sequence of choice. As such, the multifunctional siNA molecules of the invention are not limited to targeting sequences that are complementary to each other, but rather to any two differing target nucleic acid sequences. Multifunctional siNA molecules of the invention are designed such that each strand or region of the multifunctional siNA molecule, that is complementary to a given target nucleic acid sequence, is of suitable length (e.g., from about 16 to about 28 nucleotides in length, preferably from about 18 to about 28 nucleotides in length) for mediating RNA interference against the target nucleic acid sequence. The complementarity between the target nucleic acid sequence and a strand or region of the multifunctional siNA must be sufficient (at least about 8 base pairs) for cleavage of the target nucleic acid sequence by RNA interference multifunctional siNA of the invention is expected to minimize off-target effects seen with certain siRNA sequences, such as those described in (Schwarz et al., supra).

It has been reported that dsRNAs of length between 29 base pairs and 36 base pairs (Tuschl et al., International PCT Publication No. WO 02/44321) do not mediate RNAi. One reason these dsRNAs are inactive may be the lack of turnover or dissociation of the strand that interacts with the target RNA sequence, such that the RISC complex is not able to efficiently interact with multiple copies of the target RNA resulting in a significant decrease in the potency and efficiency of the RNAi process. Applicant has surprisingly found that the multifunctional siNAs of the invention can overcome this hurdle and are capable of enhancing the efficiency and potency of RNAi process. As such, in certain embodiments of the invention, multifunctional siNAs of length of about 29 to about 36 base pairs can be designed such that, a portion of each strand of the multifunctional siNA molecule comprises a nucleotide sequence region that is complementary to a target nucleic acid of length sufficient to mediate RNAi efficiently (e.g., about 15 to about 23 base pairs) and a nucleotide sequence region that is not complementary to the target nucleic acid. By having both complementary and non-complementary portions in each strand of the multifunctional siNA, the multifunctional siNA can mediate RNA interference against a target nucleic acid sequence without being prohibitive to turnover or dissociation (e.g., where the length of each strand is too long to mediate RNAi against the respective target nucleic acid sequence). Furthermore, design of multifunctional siNA molecules of the invention with internal overlapping regions allows the multifunctional siNA molecules to be of favorable (decreased) size for mediating RNA interference and of size that is well suited for use as a therapeutic agent (e.g., wherein each strand is independently from about 18 to about 28 nucleotides in length). Non-limiting examples are illustrated in the enclosed FIGS. 16-21 and 42.

In one embodiment, a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises a nucleotide sequence complementary to a nucleic acid sequence of a first target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleic acid sequence complementary to a nucleic acid sequence of a second target nucleic acid molecule. In one embodiment, a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of the first region of a target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of a second region of a the target nucleic acid molecule. In another embodiment, the first region and second region of the multifunctional siNA can comprise separate nucleic acid sequences that share some degree of complementarity (e.g., from about 1 to about 10 complementary nucleotides). In certain embodiments, multifunctional siNA constructs comprising separate nucleic acid seqeunces can be readily linked post-synthetically by methods and reagents known in the art and such linked constructs are within the scope of the invention. Alternately, the first region and second region of the multifunctional siNA can comprise a single nucleic acid sequence having some degree of self complementarity, such as in a hairpin or stem-loop structure. Non-limiting examples of such double stranded and hairpin multifunctional short interfering nucleic acids are illustrated in FIGS. 16 and 17 respectively. These multifunctional short interfering nucleic acids (multifunctional siNAs) can optionally include certain overlapping nucleotide sequence where such overlapping nucleotide sequence is present in between the first region and the second region of the multifunctional siNA (see for example FIGS. 18 and 19).

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein each strand of the the multifunctional siNA independently comprises a first region of nucleic acid sequence that is complementary to a distinct target nucleic acid sequence and the second region of nucleotide sequence that is not complementary to the target sequence. The target nucleic acid sequence of each strand is in the same target nucleic acid molecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence that is distinct from the target nucleotide sequence complementary to the first strand nucleotide sequence (complementary region 2), and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 1 of the first strand. The target nucleic acid sequence of complementary region 1 and complementary region 2 is in the same target nucleic acid molecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene (e.g., VEGF and/or VEGFR gene) (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene that is distinct from the gene of complementary region 1 (complementary region 2), and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 1 of the first strand.

In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene (e.g., VEGF and/or VEGFR gene) (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence distinct from the target nucleic acid sequence of complementary region 1 (complementary region 2), provided, however, that the target nucleic acid sequence for complementary region 1 and target nucleic acid sequence for complementary region 2 are both derived from the same gene, and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to nucleotide sequence in the non-complementary region 1 of the first strand.

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having nucleotide sequence complementary to nucleotide sequence within a target nucleic acid molecule, and in which the second seqeunce comprises a first region having nucleotide sequence complementary to a distinct nucleotide sequence within the same target nucleic acid molecule. Preferably, the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence,

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having a nucleotide sequence complementary to a nucleotide sequence within a first target nucleic acid molecule, and in which the second seqeunce comprises a first region having a nucleotide sequence complementary to a distinct nucleotide sequence within a second target nucleic acid molecule. Preferably, the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence,

In one embodiment, the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises a nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a first target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within a second target nucleic acid molecule.

In one embodiment, the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within the same target nucleic acid molecule.

In one embodiment, the invention features a double stranded multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein one strand of the multifunctional siNA comprises a first region having nucleotide sequence complementary to a first target nucleic acid sequence, and the second strand comprises a first region having a nucleotide sequence complementary to a second target nucleic acid sequence. The first and second target nucleic acid sequences can be present in separate target nucleic acid molecules or can be different regions within the same target nucleic acid molecule. As such, multifunctional siNA molecules of the invention can be used to target the expression of different genes, splice variants of the same gene, both mutant and conserved regions of one or more gene transcripts, or both coding and non-coding sequences of the same or differeing genes or gene transcripts.

In one embodiment, a target nucleic acid molecule of the invention encodes a single protein. In another embodiment, a target nucleic acid molecule encodes more than one protein (e.g., 1, 2, 3, 4, 5 or more proteins). As such, a multifunctional siNA construct of the invention can be used to down regulate or inhibit the expression of several proteins. For example, a multifunctional siNA molecule comprising a region in one strand having nucleotide sequence complementarity to a first target nucleic acid sequence derived from a gene encoding one protein (e.g., a cytokine, such as vascular endothelial growth factor or VEGF) and the second strand comprising a region with nucleotide sequence complementarity to a second target nucleic acid sequence present in target nucleic acid molecules derived from genes encoding two proteins (e.g., two differing receptors, such as VEGF receptor 1 and VEGF receptor 2, for a single cytokine, such as VEGF) can be used to down regulate, inhibit, or shut down a particular biologic pathway by targeting, for example, a cytokine and receptors for the cytokine, or a ligand and receptors for the ligand.

In one embodiment the invention takes advantage of conserved nucleotide sequences present in different isoforms of cytokines or ligands and receptors for the cytokines or ligands. By designing multifunctional siNAs in a manner where one strand includes a sequence that is complementary to a target nucleic acid sequence conserved among various isoforms of a cytokine and the other strand includes sequence that is complementary to a target nucleic acid sequence conserved among the receptors for the cytokine, it is possible to selectively and effectively modulate or inhibit a biological pathway or multiple genes in a biological pathway using a single multifunctional siNA.

In another nonlimiting example, a multifunctional siNA molecule comprising a region in one strand having a nucleotide sequence complementarity to a first target nucleic acid sequence present in target nucleic acid molecules encoding two proteins (e.g., two isoforms of a cytokine such as VEGF, inlcuding for example any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) and the second strand comprising a region with a nucleotide sequence complementarity to a second target nucleic acid sequence present in target nucleotide molecules encoding two additional proteins (e.g., two differing receptors for the cytokine, such as VEGFR1, VEGFR2, and/or VEGFR3) can be used to down regulate, inhibit, or shut down a particular biologic pathway by targeting different isoforms of a cytokine and receptors for such cytokines.

In one embodiment, a multifunctional short interfering nucleic acid (multifunctional siNA) of the invention comprises a region in each strand, wherein the region in one strand comprises nucleotide sequence complementary to a cytokine and the region in the second strand comprises nucleotide sequence complementary to a corresponding receptor for the cytokine. Non-limiting examples of cytokines include vascular endothelial growth factors (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D), and non-limiting examples of cytokine receptors include VEGFR1, VEGFR2, and VEGFR3.

In one embodiment, a double stranded multifunctional siNA molecule of the invention comprises a structure having Formula MF-I:

5′-p-X Z X′-3′
3′-Y′Z Y-p-5′

wherein each 5′-p-XZX′-3′ and 5′-p-YZY′-3′ are independently an oligonucleotide of length of about 20 nucleotides to about 300 nucleotides, preferably of about 20 to about 200 nucleotides, about 20 to about 100 nucleotides, about 20 to about 40 nucleotides, about 20 to about 40 nucleotides, about 24 to about 38 nucleotides, or about 26 to about 38 nucleotides; XZ comprises a nucleic acid sequence that is complementary to a first target nucleic acid sequence; YZ is an oligonucleotide comprising nucleic acid sequence that is complementary to a second target nucleic acid sequence; Z comprises nucleotide sequence of length about 1 to about 24 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides) that is self complimentary; X comprises nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) that is complementary to nucleotide sequence present in region Y′; Y comprises nucleotide sequence of length about 1 to about 100 nucleotides, prefereably about 1- about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) that is complementary to nucleotide sequence present in region X′; each p comprises a terminal phosphate group that is independently present or absent; each XZ and YZ is independently of length sufficient to stably interact (i.e., base pair) with the first and second target nucleic acid sequence, respectively, or a portion thereof. For example, each sequence X and Y can independently comprise sequence from about 12 to about 21 or more nucleotides in length (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to a target nucleotide sequence in different target nucleic acid molecules, such as target RNAs or a portion thereof. In another non-limiting example, the length of the nucleotide sequence of X and Z together that is complementary to the first target nucleic acid sequence or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In another non-limiting example, the length of the nucleotide sequence of Y and Z together, that is complementary to the second target nucleic acid sequence or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules (e.g., VEGF and/or VEGFR RNA). In one embodiment, Z comprises a palindrome or a repeat sequence. In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In one embodiment, the lengths of oligonucleotides Y and Y′ are identical. In another embodiment, the lengths of oligonucleotides Y and Y′ are not identical. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-II:

5′-p-X X′-3′
3′-Y′ Y-p-5′

wherein each 5′-p-XX′-3′ and 5′-p-YY′-3′ are independently an oligonucleotide of length of about 20 nucleotides to about 300 nucleotides, preferably about 20 to about 200 nucleotides, about 20 to about 100 nucleotides, about 20 to about 40 nucleotides, about 20 to about 40 nucleotides, about 24 to about 38 nucleotides, or about 26 to about 38 nucleotides; X comprises a nucleic acid sequence that is complementary to a first target nucleic acid sequence; Y is an oligonucleotide comprising nucleic acid sequence that is complementary to a second target nucleic acid sequence; X comprises a nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) that is complementary to nucleotide sequence present in region Y′; Y comprises nucleotide sequence of length about 1 to about 100 nucleotides, prefereably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) that is complementary to nucleotide sequence present in region X′; each p comprises a terminal phosphate group that is independently present or absent; each X and Y independently is of length sufficient to stably interact (i.e., base pair) with the first and second target nucleic acid sequence, respectively, or a portion thereof. For example, each sequence X and Y can independently comprise sequence from about 12 to about 21 or more nucleotides in length (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to a target nucleotide sequence in different target nucleic acid molecules, such as VEGF and/or VEGFR target RNAs or a portion thereof. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules (e.g., VEGF and/or VEGFR RNA). In one embodiment, Z comprises a palindrome or a repeat sequence. In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In one embodiment, the lengths of oligonucleotides Y and Y′ are identical. In another embodiment, the lengths of oligonucleotides Y and Y′ are not identical. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-III:
embedded image

wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y′; X′ comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each X and X′ is independently of length sufficient to stably interact (i.e., base pair) with a first and a second target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y; and the multifunctional siNA directs cleavage of the first and second target sequence via RNA interference. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules (e.g., VEGF and/or VEGFR RNA). In one embodiment, region W connects the 3′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, region W connects the 3′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X′. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y′. In one embodiment, W connects sequences Y and Y′ via a biodegradable linker. In one embodiment, W further comprises a conjugate, lable, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-IV:
embedded image

wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y′; X′ comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each Y and Y′ is independently of length sufficient to stably interact (i.e., base pair) with a first and a second target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y; and the multifunctional siNA directs cleavage of the first and second target sequence via RNA interference. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules (e.g., VEGF and/or VEGFR RNA). In one embodiment, region W connects the 3′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, region W connects the 3′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X′. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y′. In one embodiment, W connects sequences Y and Y′ via a biodegradable linker. In one embodiment, W further comprises a conjugate, lable, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-V:
embedded image

wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y′; X′ comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each X, X′, Y, or Y′ is independently of length sufficient to stably interact (i.e., base pair) with a first, second, third, or fourth target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y; and the multifunctional siNA directs cleavage of the first, second, third, and/or fourth target sequence via RNA interference. In one embodiment, the first, second, third and fourth target nucleic acid sequence are all present in the same target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA). In another embodiment, the first, second, third and fourth target nucleic acid sequence are independently present in different target nucleic acid molecules (e.g., VEGF and/or VEGFR RNA). In one embodiment, region W connects the 3′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, region W connects the 3′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X′. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y′. In one embodiment, W connects sequences Y and Y′ via a biodegradable linker. In one embodiment, W further comprises a conjugate, lable, aptamer, ligand, lipid, or polymer.

In one embodiment, regions X and Y of multifunctional siNA molecule of the invention (e.g., having any of Formula MF-I-MF-V), are complementary to different target nucleic acid sequences that are portions of the same target nucleic acid molecule. In one embodiment, such target nucleic acid sequences are at different locations within the coding region of a RNA transcript. In one embodiment, such target nucleic acid sequences comprise coding and non-coding regions of the same RNA transcript. In one embodiment, such target nucleic acid sequences comprise regions of alternately spliced transcripts or precursors of such alternately spliced transcripts.

In one embodiment, a multifunctional siNA molecule having any of Formula MF-I-MF-V can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I-VII described herein, stabilization chemistries as described in Table IV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.

In one embodiment, the palidrome or repeat sequence or modified nucleotide (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of multifunctional siNA constructs having Formula MF-I or MF-II comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence (e.g., modified base analogs that can form Watson Crick base pairs or non-Watson Crick base pairs).

In one embodiment, a multifunctional siNA molecule of the invention, for example each strand of a multifunctional siNA having MF-I-MF-V, independently comprises about 15 to about 40 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, a multifunctional siNA molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum or in cells or tissues. Furthermore, certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.

In another embodiment, the invention features multifunctional siNAs, wherein the multifunctional siNAs are assembled from two separate double-stranded siNAs, with one of the ends of each sense strand is tethered to the end of the sense strand of the other siNA molecule, such that the two antisense siNA strands are annealed to their corresponding sense strand that are tethered to each other at one end (see FIG. 43). The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5′-end of one antisense strand of the siNA is tethered to the 3′-end of the antisense strand of the other siNA molecule, such that the 5′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand (see FIG. 43(G-H)). In one embodiment, the linkage between the 5′-end of the first antisense strand and the 3′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interefence-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5′-end of one antisense strand of the siNA is tethered to the 5′-end of the antisense strand of the other siNA molecule, such that the 3′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand (see FIG. 43(E)). In one embodiment, the linkage between the 5′-end of the first antisense strand and the 5′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interefence-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 3′-end of one antisense strand of the siNA is tethered to the 3′-end of the antisense strand of the other siNA molecule, such that the 5′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand (see FIG. 43(F)). In one embodiment, the linkage between the 5′-end of the first antisense strand and the 5′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interefence-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In any of the above embodiments, a first target nucleic acid sequence or second target nucleic acid sequence can independently comprise VEGF and/or VEGFR RNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGF (e.g., any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) RNA or a portion thereof and the second target nucleic acid sequence is a VEGFR (e.g., any of VEGFR1, VEGFR2, and/or VEGFR3) RNA of a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGFR (e.g., any of VEGFR1, VEGFR2, and/or VEGFR3) RNA or a portion thereof and the second target nucleic acid sequence is a VEGF (e.g., any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) RNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGF (e.g., any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) RNA or a portion thereof and the second target nucleic acid sequence is a VEGF (e.g., any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) RNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGFR (e.g., any of VEGFR1, VEGFR2, and/or VEGFR3) RNA or a portion thereof and the second target nucleic acid sequence is a VEGFR (e.g., any of VEGFR1, VEGFR2, and/or VEGFR3) RNA or a portion thereof.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoro nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M 15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by calorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.

The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 mmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 0.40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA·3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 minutes. The vial is brought to room temperature TEA·3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 minutes. The sample is cooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.

In another aspect of the invention, siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.

Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al, 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).

In another embodiment, the invention features conjugates and/or complexes of siNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in a biological system, for example, enzymatic degradation or chemical degradation.

The term “biologically active molecule” as used herein refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siNA molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered exogenously optimally are stable within cells until reverse transcription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead to better treatments by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more 5′ and/or a 3′-cap structure, for example, on only the sense siNA strand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5′-terminus (5′-cap) or at the 3′-terminal (3′-cap) or may be present on both termini. In non-limiting examples, the 5′-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety. Non-limiting examples of cap moieties are shown in FIG. 10.

Non-limiting examples of the 3′-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

By the term “non-nucleotide” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino or SH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents.

In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, see for example Adamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1′ carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides are shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH₂or 2′-O—NH₂, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al, U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.

Various modifications to nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to treat, prevent, inhibit, or reduce cancer, ocular, proliferative, or angiogenesis related diseases, conditions, or disorders, and/or any other trait, disease or condition that is related to or will respond to the levels of VEGF and/or VEGFR in a cell or tissue, alone or in combination with other therapies.

For example, a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol; Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al, 2000, ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and U.S. patent application Publication No. U.S. 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acid molecules of the invention are formulated as described in U.S. Patent Application Publication No. 20030077829, incorporated by reference herein in its entirety.

In one embodiment, a siNA molecule of the invention is complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666, incorporated by reference herein in its entirety including the drawings. In another embodiment, the membrane disruptive agent or agents and the siNA molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Pat. No. 6,235,310, incorporated by reference herein in its entirety including the drawings.

In one embodiment, a siNA molecule of the invention is complexed with delivery systems as described in U.S. Patent Application Publication No. 2003077829 and International PCT Publication Nos. WO 00/03683 and WO 02/087541, all incorporated by reference herein in their entirety including the drawings.

In one embodiment, a compound, molecule, or composition for the treatment of ocular conditions (e.g., macular degeneration, diabetic retinopathy etc.) is administered to a subject intraocularly or by intraocular means. In another embodiment, a compound, molecule, or composition for the treatment of ocular conditions (e.g., macular degeneration, diabetic retinopathy etc.) is administered to a subject periocularly or by periocular means (see for example Ahlheim et al., International PCT publication No. WO 03/24420). In one embodiment, a siNA molecule and/or formulation or composition thereof is administered to a subject intraocularly or by intraocular means. In another embodiment, a siNA molecule and/or formualtion or composition thereof is administered to a subject periocularly or by periocular means. Periocular administration generally provides a less invasive approach to administering siNA molecules and formualtion or composition thereof to a subject (see for example Ahlheim et al., International PCT publication No. WO 03/24420). The use of periocular administraction also minimizes the risk of retinal detachment, allows for more frequent dosing or administraction, provides a clinically relevant route of administraction for macular degeneration and other optic conditions, and also provides the possiblilty of using resevoirs (e.g., implants, pumps or other devices) for drug delivery. In one embodiment, siNA compounds and compositions of the invention are administered locally, e.g., via intraocular or periocular means, such as injection, iontophoresis (see, for example, WO 03/043689 and WO 03/030989), or implant, about every 1-50 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 weeks), alone or in combination with other comounds and/or therapeis herein. In one embodiment, siNA compounds and compositions of the invention are administered systemically (e.g., via intravenous, subcutaneous, intramuscular, infusion, pump, implant etc.) about every 1-50 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 weeks), alone or in combination with other comounds and/or therapies described herein and/or otherwise known in the art.

In one embodiment, a siNA molecule of the invention is administered iontophoretically, for example to a particular organ or compartment (e.g., the eye, back of the eye, heart, liver, kidney, bladder, prostate, tumor, CNS etc.). Non-limiting examples of iontophoretic delivery are described in, for example, WO 03/043689 and WO 03/030989, which are incorporated by reference in their entireties herein.

In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to the liver as is generally known in the art (see for example Wen et al., 2004, World J Gastroenterol., 10, 244-9; Murao et al., 2002, Pharm Res., 19, 1808-14; Liu et al., 2003, Gene Ther., 10, 180-7; Hong et al., 2003, J Pharm Pharmacol., 54, 51-8; Herrmann et al., 2004, Arch Virol., 149, 1611-7; and Matsuno et al., 2003, Gene Ther., 10, 1559-66).

In one embodiment, the invention features the use of methods to deliver the nucleic acid molecules of the instant invention to hematopoietic cells, including monocytes and lymphocytes. These methods are described in detail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2), 920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion and Phillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei, 1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, Nucleic Acids Research, 22(22), 4681-8. Such methods, as described above, include the use of free oligonucleitide, cationic lipid formulations, liposome formulations including pH sensitive liposomes and immunoliposomes, and bioconjugates including oligonucleotides conjugated to fusogenic peptides, for the transfection of hematopoietic cells with oligonucleotides.

In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to the central nervous system and/or peripheral nervous system. Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a 15mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain. Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells. Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons. Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J. PharmocoL, 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express repeat expansion allelic variants for modulation of RE gene expression. The delivery of nucleic acid molecules of the invention, targeting RE is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.

In one embodiment, the nucleic acid molecules of the invention are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues. Solid particulate compositions containing respirable dry particles of micronized nucleic acid compositions can be prepared by grinding dried or lyophilized nucleic acid compositions, and then passing the micronized composition through, for example, a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprising the nucleic acid compositions of the invention can optionally contain a dispersant which serves to facilitate the formation of an aerosol as well as other therapeutic compounds. A suitable dispersant is lactose, which can be blended with the nucleic acid compound in any suitable ratio, such as a 1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition of the invention can be produced by any suitable means, such as with a nebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers are commercially available devices which transform solutions or suspensions of an active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers comprise the active ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride or other suitable salts. Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants. The aerosols of solid particles comprising the active composition and surfactant can likewise be produced with any solid particulate aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a therapeutic composition at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which can be delivered by means of an insufflator. In the insufflator, the powder, e.g., a metered dose thereof effective to carry out the treatments described herein, is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation can additionally contain one or more co-solvents, for example, ethanol, emulsifiers and other formulation surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents. Other methods for pulmonary delivery are described in, for example U.S. patent application No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728; 6,565,885.

In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered directly or topically (e.g., locally) to the dermis or follicles as is generally known in the art (see for example Brand, 2001, Curr. Opin. Mol. Ther., 3, 244-8; Regnier et al., 1998, J. Drug Target, 5, 275-89; Kanikkannan, 2002, BioDrugs, 16, 339-47; Wraight et al., 2001, Pharmacol. Ther., 90, 89-104; Preat and Dujardin, 2001, STP PharmaSciences, 11, 57-68; and Vogt et al., 2003, Hautarzt. 54, 692-8).

In one embodiment, delivery systems of the invention include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. Examples of liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL).

In one embodiment, delivery systems of the invention include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, transdermal delivery systems of the invention include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, siNA molecules of the invention are formulated or complexed with polyethylenimine (e.g., linear or branched PEI) and/or polyethylenimine derivatives, including for example grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, Phramaceutical Research, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999, PNAS USA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release, 60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274, 19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; and Sagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises a bioconjugate, for example a nucleic acid conjugate as described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S. Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886; U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced to a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as creams, gels, sprays, oils and other suitable compositions for topical, dermal, or transdermal administration as is known in the art.

The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to a subject by systemic administration in a pharmaceutically acceptable composition or formulation. By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceutically acceptable composition” is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate. Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of a composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) and nucleic acid molecules of the invention. These formulations offer a method for increasing the accumulation of drugs (e.g., siNA) in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). In another example, the folate receptor is overexpressed in many cancer cells. Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser. No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 143241; Weerasinghe et al., 1991, J. Virol., 65, 55314; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al, 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).

In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the instant invention. The expression vector can encode one or both strands of a siNA duplex, or a single self-complementary strand that self hybridizes into a siNA duplex. The nucleic acid sequences encoding the siNA molecules of the instant invention can be operably linked in a manner that allows expression of the siNA molecule (see for example Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725).

In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or m initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the siNA of the invention; and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L′Huillier et al, 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S. A, 90, 8000-4; Thompson et al, 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (TRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al, U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al, International PCT Publication No. WO 96/18736. The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention in a manner that allows expression of that siNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule, wherein the sequence is operably linked to the initiation region and the termination region in a manner that allows expression and/or delivery of the siNA molecule.

In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region in a manner that allows expression and/or delivery of the siNA molecule. In yet another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherein the sequence is operably linked to the initiation region, the intron and the termination region in a manner which allows expression and/or delivery of the nucleic acid molecule.

In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or delivery of the siNA molecule.

VEGF and/or VEGFR Biology and Biochemistry

The following discussion is adapted from R&D Systems, Cytokine Mini Reviews, Vascular Endothelial Growth Factor (VEGF), Copyright ®2002 R&D Systems. Angiogenesis is a process of new blood vessel development from pre-existing vasculature. It plays an essential role in embryonic development, normal growth of tissues, wound healing, the female reproductive cycle (i.e., ovulation, menstruation and placental development), as well as a major role in many diseases. Particular interest has focused on cancer, since tumors cannot grow beyond a few millimeters in size without developing a new blood supply. Angiogenesis is also necessary for the spread and growth of tumor cell metastases.

One of the most important growth and survival factors for endothelium is vascular endothelial growth factor (VEGF). VEGF induces angiogenesis and endothelial cell proliferation and plays an important role in regulating vasculogenesis. VEGF is a heparin-binding glycoprotein that is secreted as a homodimer of 45 kDa. Most types of cells, but usually not endothelial cells themselves, secrete VEGF. Since the initially discovered VEGF, VEGF-A, increases vascular permeability, it was known as vascular permeability factor. In addition, VEGF causes vasodilatation, partly through stimulation of nitric oxide synthase in endothelial cells. VEGF can also stimulate cell migration and inhibit apoptosis.

There are several splice variants of VEGF-A. The major ones include: 121, 165, 189 and 206 amino acids (aa), each one comprising a specific exon addition. VEGF165 is the most predominant protein, but transcripts of VEGF 121 may be more abundant. VEGF206 is rarely expressed and has been detected only in fetal liver. Recently, other splice variants of 145 and 183 aa have also been described. The 165, 189 and 206 aa splice variants have heparin-binding domains, which help anchor them in extracellular matrix and are involved in binding to heparin sulfate and presentation to VEGF receptors. Such presentation is a key factor for VEGF potency (i.e., the heparin-binding forms are more active). Several other members of the VEGF family have been cloned including VEGF-B, -C, and -D. Placenta growth factor (PlGF) is also closely related to VEGF-A. VEGF-A, -B, -C, -D, and PlGF are all distantly related to platelet-derived growth factors-A and -B. Less is known about the function and regulation of VEGF-B, -C, and -D, but they do not seem to be regulated by the major pathways that regulate VEGF-A.

VEGF-A transcription is potentiated in response to hypoxia and by activated oncogenes. The transcription factors, hypoxia inducible factor-1a (hif-1a) and -2a, are degraded by proteosomes in normoxia and stabilized in hypoxia. This pathway is dependent on the Von Hippel-Lindau gene product. Hif-1a and hif-2 a heterodimerize with the aryl hydrocarbon nuclear translocator in the nucleus and bind the VEGF promoter/enhancer. This is a key pathway expressed in most types of cells. Hypoxia inducibility, in particular, characterizes VEGF-A versus other members of the VEGF family and other angiogenic factors. VEGF transcription in normoxia is activated by many oncogenes, including H-ras and several transmembrane tyrosine kinases, such as the epidermal growth factor receptor and erbB2. These pathways together account for a marked upregulation of VEGF-A in tumors compared to normal tissues and are often of prognostic importance.

There are three receptors in the VEGF receptor family. They have the common properties of multiple IgG-like extracellular domains and tyrosine kinase activity. The enzyme domains of VEGF receptor 1 (VEGFR1, also known as Flt-1), VEGFR2 (also known as KDR or Flk-1), and VEGFR3 (also known as Flt4) are divided by an inserted sequence. Endothelial cells also express additional VEGF receptors, Neuropilin-1 and Neuropilin-2. VEGF-A binds to VEGFR1 and VEGFR2 and to Neuropilin-1 and Neuropilin-2. PlGF and VEGF-B bind VEGFR1 and Neuropilin-1. VEGF-C and -D bind VEGFR3 and VEGFR2.

The VEGF-C/VEGFR3 pathway is important for lymphatic proliferation. VEGFR3 is specifically expressed on lymphatic endothelium. A soluble form of Flt-1 can be detected in peripheral blood and is a high affinity ligand for VEGF. Soluble Flt-1 can be used to antagonize VEGF function. VEGFR1 and VEGFR2 are upregulated in tumor and proliferating endothelium, partly by hypoxia and also in response to VEGF-A itself. VEGFR1 and VEGFR2 can interact with multiple downstream signaling pathways via proteins such as PLC-g, Ras, Shc, Nck, PKC and PI3-kinase. VEGFR1 is of higher affinity than VEGFR2 and mediates motility and vascular permeability. VEGFR2 is necessary for proliferation.

VEGF can be detected in both plasma and serum samples of patients, with much higher levels in serum. Platelets release VEGF upon aggregation and may be a major source of VEGF delivery to tumors. Several studies have shown that association of high serum levels of VEGF with poor prognosis in cancer patients may be correlated with an elevated platelet count. Many tumors release cytokines that can stimulate the production of megakaryocytes in the marrow and elevate the platelet count. This can result in an indirect increase of VEGF delivery to tumors.

VEGF is implicated in several other pathological conditions associated with enhanced angiogenesis. For example, VEGF plays a role in both psoriasis and rheumatoid arthritis. Diabetic retinopathy is associated with high intraocular levels of VEGF. Inhibition of VEGF function may result in infertility by blockade of corpus luteum function. Direct demonstration of the importance of VEGF in tumor growth has been achieved using dominant negative VEGF receptors to block in vivo proliferation, as well as blocking antibodies to VEGF39 or to VEGFR2.

The use of small interfering nucleic acid molecules targeting VEGF and corresponding receptors and ligands therefore provides a class of novel therapeutic agents that can be used in the diagnosis of and the treatment of cancer, proliferative diseases, or any other disease or condition that responds to modulation of VEGF and/or VEGFR genes.

EXAMPLES

The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1
Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complement in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complementary strand comprises a terminal 5′-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (see FIG. 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5′-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example, using a Waters C18 SepPak 1 g cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H₂O, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H₂O or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50 nM NaOAc and 50 mM NaCl). The column is then washed, for example with 1 CV H₂O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes. The remaining TFA solution is removed and the column washed with H₂O followed by 1 CV 1M NaCl and additional H₂O. The siNA duplex product is then eluted, for example, using 1 CV 20% aqueous CAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of a purified siNA construct in which each peak corresponds to the calculated mass of an individual siNA strand of the siNA duplex. The same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA contract only shows a single peak. Testing of the purified siNA construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.

Example 2
Identification of Potential siNA Target Sites in any RNA Sequence

The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complementarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA construct to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.

Example 3
Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.
2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.
3. In some instances the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.
4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.
5. The ranked siNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.
6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic and can potentially interfere with RNAi activity, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.
7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end of the sequence (to yield 3′ UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.
8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Tables II and III). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3′ terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.
9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.
10. Other design considerations can be used when selecting target nucleic acid sequences, see, for example, Reynolds et al., 2004, Nature Biotechnology Advanced Online Publication, 1 Feb. 2004, doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32, doi: 10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a VEGF and/or VEGFR target sequence is used to screen for target sites in cells expressing VEGF and/or VEGFR RNA, such as HUVEC, HMVEC, or A375 cells. The general strategy used in this approach is shown in FIG. 9. A non-limiting example of such is a pool comprising sequences having any of SEQ ID NOS 1-4248. Cells expressing VEGF and/or VEGFR (e.g., HUVEC, HMVEC, or A375 cells) are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with VEGF and/or VEGFR inhibition are sorted. The pool of siNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for example FIG. 7 and FIG. 8). The siNA from cells demonstrating a positive phenotypic change (e.g., decreased proliferation, decreased VEGF and/or VEGFR mRNA levels or decreased VEGF and/or VEGFR protein expression), are sequenced to determine the most suitable target site(s) within the target VEGF and/or VEGFR RNA sequence.

Example 4
VEGF and/or VEGFR Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the VEGF and/or VEGFR RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein. siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siNA duplexes or varying length or base composition. By using such methodologies, siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constructs are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g. liver extracts). The synthetic siNA constructs are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity. Synthetic siNA constructs that possess both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. The chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for therapeutic development (see for example FIG. 11).

Example 5
Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above. The siNA molecules can be chemically synthesized using methods described herein. Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence. Generally, siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein in their entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl, 3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be used in conjunction with acid-labile 2′-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra. Differing 2′ chemistries can require different protecting groups, for example 2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′- to 5′-direction) to the solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support (e.g., controlled pore glass or polystyrene) using various linkers. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5′-end of the first nucleoside. The support is then washed and any unreacted 5′-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage. At the end of the nucleotide addition cycle, the 5′-O-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.

Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized. Deprotection and purification of the siNA can be performed as is generally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringe supra, incorporated by reference herein in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constructs. For example, applicant has observed that oligonucleotides comprising 2′-deoxy-2′-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35° C. for 30 minutes. If the 2′-deoxy-2′-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35° C. for 30 minutes, TEA-HF is added and the reaction maintained at about 65° C. for an additional 15 minutes.

Example 6
RNAi in Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting VEOF and/or VEGFR RNA targets. The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with VEGF and/or VEGFR target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate VEGF and/or VEGFR expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25×Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-³²P] CTP, passed over a G50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by PHOSPHOR IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites in the VEGF and/or VEGFR RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the VEGF and/or VEGFR RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.

Example 7
Nucleic Acid Inhibition of VEGF and/or VEGFR Target RNA in Vivo

siNA molecules targeted to the human VEGF and/or VEGFR RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the VEGF and/or VEGFR RNA are given in Table II and III.

Two formats are used to test the efficacy of siNAs targeting VEGF and/or VEGFR. First, the reagents are tested in cell culture using, for example, HUVEC, HMVEC, or A375 cells to determine the extent of RNA and protein inhibition. siNA reagents (e.g.; see Tables II and III) are selected against the VEGF and/or VEGFR target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, HUVEC, HMVEC, or A375 cells. Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (eg., ABI 7700 TAQMAN®). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but randomly substituted at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead siNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., HUVEC, HMVEC, or A375 cells) are seeded, for example, at 1×10⁵cells per well of a six-well dish in EGM-2 (BioWhittaker) the day before transfection. siNA (final concentration, for example 20 nM) and cationic lipid (e.g., final concentration 2 μg/ml) are complexed in EGM basal media (Biowhittaker) at 37° C. for 30 minutes in polystyrene tubes. Following vortexing, the complexed siNA is added to each well and incubated for the times indicated. For initial optimization experiments, cells are seeded, for example, at 1×10³in 96 well plates and siNA complex added as described. Efficiency of delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid. Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and Lightcycler Quantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example, using Qiagen RNA purification kits for 6-well or Rneasy extraction kits for 96-well assays. For TAQMAN® analysis (real-time PCR monitoring of amplification), dual-labeled probes are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5′-end and the quencher dye TAMRA conjugated to the 3′-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each DATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U AMPLITAQ GOLD® (DNA polymerase) (PE-Applied Biosystems) and 10U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 minutes at 48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/reaction) and normalizing to β-actin or GAPDH mRNA in parallel TAQMAN® reactions (real-time PCR monitoring of amplification). For each gene of interest an upper and lower primer and a fluorescently labeled probe are designed. Real time incorporation of SYBR Green I dye into a specific PCR product can be measured in glass capillary tubes using a lightcyler. A standard curve is generated for each primer pair using control cRNA. Values are represented as relative expression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4° C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).

Example 8
Animal Models Useful to Evaluate the Down-Regulation of VEGF and/or VEGFR Gene Expression

There are several animal models in which the anti-angiogenesis effect of nucleic acids of the present invention, such as siRNA, directed against VEGF, VEGFR1, VEGFR2 and/or VEGFR3 mRNAs can be tested. Typically a corneal model has been used to study angiogenesis in rat and rabbit since recruitment of vessels can easily be followed in this normally avascular tissue (Pandey et al., 1995 Science 268: 567-569). In these models, a small Teflon or Hydron disk pretreated with an angiogenesis factor (e.g. bFGF or VEGF) is inserted into a pocket surgically created in the cornea. Angiogenesis is monitored 3 to 5 days later. siRNA directed against VEGF, VEGFR1, VEGFR2 and/or VEGFR3 mRNAs are delivered in the disk as well, or dropwise to the eye over the time course of the experiment. In another eye model, hypoxia has been shown to cause both increased expression of VEGF and neovascularization in the retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92: 905-909; Shweiki et al., 1992 J. Clin. Invest. 91: 2235-2243).

In human glioblastomas, it has been shown that VEGF is at least partially responsible for tumor angiogenesis (Plate et al., 1992 Nature 359, 845). Animal models have been developed in which glioblastoma cells are implanted subcutaneously into nude mice and the progress of tumor growth and angiogenesism is studied (Kim et al., 1993 supra; Millauer et al., 1994 supra).

Another animal model that addresses neovascularization involves Matrigel, an extract of basement membrane that becomes a solid gel when injected subcutaneously (Passaniti et al., 1992 Lab. Invest. 67: 519-528). When the Matrigel is supplemented with angiogenesis factors such as VEGF, vessels grow into the Matrigel over a period of 3 to 5 days and angiogenesis can be assessed. Again, nucleic acids directed against VEGFR mRNAs are delivered in the Matrigel.

Several animal models exist for screening of anti-angiogenic agents. These include corneal vessel formation following corneal injury (Burger et al., 1985 Cornea 4: 3541; Lepri, et al., 1994 J. Ocular Pharmacol. 10: 273-280; Ormerod et al., 1990 Am. J. Pathol. 137: 1243-1252) or intracorneal growth factor implant (Grant et al., 1993 Diabetologia 36: 282-291; Pandey et al. 1995 supra; Zieche et al., 1992 Lab. Invest. 67: 711-715), vessel growth into Matrigel matrix containing growth factors (Passaniti et al., 1992 supra), female reproductive organ neovascularization following hormonal manipulation (Shweiki et al., 1993 Clin. Invest. 91: 2235-2243), several models involving inhibition of tumor growth in highly vascularized solid tumors (O'Reilly et al., 1994 Cell 79: 315-328; Senger et al., 1993 Cancer and Metas. Rev. 12: 303-324; Takahasi et al., 1994 Cancer Res. 54: 4233-4237; Kim et al., 1993 supra), and transient hypoxia-induced neovascularization in the mouse retina Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92: 905-909). Other model systems to study tumor angiogenesis are reviewed by Folkman, 1985 Adv. Cancer. Res. 43, 175.

Ocular Models of Angiogenesis

The cornea model, described in Pandey et al. supra, is the most common and well characterized model for screening anti-angiogenic agent efficacy. This model involves an avascular tissue into which vessels are recruited by a stimulating agent (growth factor, thermal or alkalai burn, endotoxin). The corneal model utilizes the intrastromal corneal implantation of a Teflon pellet soaked in a VEGF-Hydron solution to recruit blood vessels toward the pellet, which can be quantitated using standard microscopic and image analysis techniques. To evaluate their anti-angiogenic efficacy, nucleic acids are applied topically to the eye or bound within Hydron on the Teflon pellet itself. This avascular cornea as well as the Matrigel (see below) provide for low background assays. While the corneal model has been performed extensively in the rabbit, studies in the rat have also been conducted.

The mouse model (Passaniti et al., supra) is a non-tissue model that utilizes Matrigel, an extract of basement membrane (Kleinman et al., 1986) or Millipore® filter disk, which can be impregnated with growth factors and anti-angiogenic agents in a liquid form prior to injection. Upon subcutaneous administration at body temperature, the Matrigel or Millipore® filter disk forms a solid implant. VEGF embedded in the Matrigel or Millipore® filter disk is used to recruit vessels within the matrix of the Matrigel or Millipore® filter disk which can be processed histologically for endothelial cell specific vWF (factor VIII antigen) immunohistochemistry, Trichrome-Masson stain, or hemoglobin content. Like the cornea, the Matrigel or Millipore® filter disk is avascular; however, it is not tissue. In the Matrigel or Millipore® filter disk model, nucleic acids are administered within the matrix of the Matrigel or Millipore® filter disk to test their anti-angiogenic efficacy. Thus, delivery issues in this model, as with delivery of nucleic acids by Hydron-coated Teflon pellets in the rat cornea model, may be less problematic due to the homogeneous presence of the nucleic acid within the respective matrix.

Additionally, siNA molecules of the invention targeting VEGF and/or VEGFR (e.g. VEGFR1, VEGFR2, and/or VEGFR3) can be assesed for activity transgenic mice. to determine whether modulation of VEGF and/or VEGFR can inhibit optic neovasculariation. Animal models of choroidal neovascularization are described in, for exmaple, Mori et al., 2001, Journal of Cellular Physiology, 188, 253; Mori et al., 2001, American Journal of Pathology, 159, 313; Ohno-Matsui et al., 2002, American Journal of Pathology, 160, 711; and Kwak et al., 2000, Investigative Ophthalmology & Visual Science, 41, 3158. VEGF plays a central role in causing retinal neovascularization. Increased expression of VEGFR2 in retinal photoreceptors of transgenic mice stimulates neovascularization within the retina, and a blockade of VEGFR2 signaling has been shown to inhibit retinal choroidal neovascularization (CNV) (Mori et al., 2001, J. Cell. Physiol., 188,253).

CNV is laser induced in, for example, adult C57BL/6 mice. The mice are also given an intravitreous, periocular or a subretinal injection of VEGF and/or VEGFR (e.g., VEGFR2) siNA in each eye. Intravitreous injections are made using a Harvard pump microinjection apparatus and pulled glass micropipets. Then a micropipette is passed through the sclera just behind the limbus into the vitreous cavity. The subretinal injections are made using a condensing lens system on a dissecting microscope. The pipet tip is then passed through the sclera posterior to the limbus and positioned above the retina. Five days after the injection of the vector the mice are anesthetized with ketamine hydrochloride (100 mg/kg body weight), 1% tropicamide is also used to dilate the pupil, and a diode laser photocoagulation is used to rupture Bruch's membrane at three locations in each eye. A slit lamp delivery system and a hand-held cover slide are used for laser photocoagulation. Burns are made in the 9, 12, and 3 o'clock positions 2-3 disc diameters from the optic nerve (Mori et al., supra).

The mice typically develop subretinal neovasculariation due to the expression of VEGF in photoreceptors beginning at prenatal day 7. At prenatal day 21, the mice are anesthetized and perfused with 1 ml of phosphate-buffered saline containing 50 mg/ml of fluorescein-labeled dextran. Then the eyes are removed and placed for 1 hour in a 10% phosphate-buffered formalin. The retinas are removed and examined by fluorescence microscopy (Mori et al., supra).

Fourteen days after the laser induced rupture of Bruch's membrane, the eyes that received intravitreous and subretinal injection of siNA are evaluated for smaller appearing areas of CNV, while control eyes are evaluated for large areas of CNV. The eyes that receive intravitreous injections or a subretinal injection of siNA are also evaluated for fewer areas of neovasculariation on the outer surface of the retina and potenial abortive sprouts from deep retinal capillaries that do not reach the retinal surface compared to eyes that did not receive an injection of siNA.

Tumor Models of Angiogenesis

Use of Murine Models

For a typical systemic study involving 10 mice (20 g each) per dose group, 5 doses (1, 3, 10, 30 and 100 mg/kg daily over 14 days continuous administration), approximately 400 mg of siRNA, formulated in saline is used. A similar study in young adult rats (200 g) requires over 4 g. Parallel pharmacokinetic studies involve the use of similar quantities of siRNA further justifying the use of murine models.

Lewis Lung Carcinoma and B-16 Melanoma Murine Models

Identifying a common animal model for systemic efficacy testing of nucleic acids is an efficient way of screening siNA for systemic efficacy.

The Lewis lung carcinoma and B-16 murine melanoma models are well accepted models of primary and metastatic cancer and are used for initial screening of anti-cancer agents. These murine models are not dependent upon the use of immunodeficient mice, are relatively inexpensive, and minimize housing concerns. Both the Lewis lung and B-16 melanoma models involve subcutaneous implantation of approximately 10⁶tumor cells from metastatically aggressive tumor cell lines (Lewis lung lines 3LL or D122, LLc-LN7; B-16-BL6 melanoma) in C57BL/6J mice. Alternatively, the Lewis lung model can be produced by the surgical implantation of tumor spheres (approximately 0.8 mm in diameter). Metastasis also can be modeled by injecting the tumor cells directly intravenously. In the Lewis lung model, microscopic metastases can be observed approximately 14 days following implantation with quantifiable macroscopic metastatic tumors developing within 21-25 days. The B-16 melanoma exhibits a similar time course with tumor neovascularization beginning 4 days following implantation. Since both primary and metastatic tumors exist in these models after 21-25 days in the same animal, multiple measurements can be taken as indices of efficacy. Primary tumor volume and growth latency as well as the number of micro- and macroscopic metastatic lung foci or number of animals exhibiting metastases can be quantitated. The percent increase in lifespan can also be measured. Thus, these models provide suitable primary efficacy assays for screening systemically administered siRNA nucleic acids and siRNA nucleic acid formulations.

In the Lewis lung and B-16 melanoma models, systemic pharmacotherapy with a wide variety of agents usually begins 1-7 days following tumor implantation/inoculation with either continuous or multiple administration regimens. Concurrent pharmacokinetic studies can be performed to determine whether sufficient tissue levels of siRNA can be achieved for pharmacodynamic effect to be expected. Furthermore, primary tumors and secondary lung metastases can be removed and subjected to a variety of in vitro studies (i.e. target RNA reduction).

In addition, animal models are useful in screening compounds, eg. siNA molecules, for efficacy in treating renal failure, such as a result of autosomal dominant polycystic kidney disease (ADPKD). The Han:SPRD rat model, mice with a targeted mutation in the Pkd2 gene and congenital polycystic kidney (cpk) mice, closely resemble human ADPKD and provide animal models to evaluate the therapeutic effect of siRNA constructs that have the potential to interfere with one or more of the pathogenic elements of ADPKD mediated renal failure, such as angiogenesis. Angiogenesis may be necessary in the progression of ADPKD for growth of cyst cells as well as increased vascular permeability promoting fluid secretion into cysts. Proliferation of cystic epithelium is also a feature of ADPKD because cyst cells in culture produce soluble vascular endothelial growth factor (VEGF). VEGFR1 has also been detected in epithelial cells of cystic tubules but not in endothelial cells in the vasculature of cystic kidneys or normal kidneys. VEGFR2 expression is increased in endothelial cells of cyst vessels and in endothelial cells during renal ischemia-reperfusion. It is proposed that inhibition of VEGF receptors with anti-VEGFR1 and anti-VEGFR2 siRNA molecules would attenuate cyst formation, renal failure and mortality in ADPKD. Anti-VEGFR2 siRNA molecules would therefore be designed to inhibit angiogenesis involved in cyst formation. As VEGFR1 is present in cystic epithelium and not in vascular endothelium of cysts, it is proposed that anti-VEGFR1 siRNA molecules would attenuate cystic epithelial cell proliferation and apoptosis which would in turn lead to less cyst formation. Further, it is proposed that VEGF produced by cystic epithelial cells is one of the stimuli for angiogenesis as well as epithelial cell proliferation and apoptosis. The use of Han:SPRD rats (see for eaxmple Kaspareit-Rittinghausen et al., 1991, Am. J. Pathol. 139, 693-696), mice with a targeted mutation in the Pkd2 gene (Pkd2−/− mice, see for example Wu et al., 2000, Nat. Genet. 24, 75-78) and cpk mice (see for example Woo et al., 1994, Nature, 368, 750-753) all provide animal models to study the efficacy of siRNA molecles of the invention against VEGFR1 and VEGFR2 mediated renal failure.

VEGF, VEGFR1 VGFR2 and/or YEGFR3 protein levels can be measured clinically or experimentally by FACS analysis. VEGF, VEGFR1 VGFR2 and/or VEGFR3 encoded mRNA levels are assessed by Northern analysis, RNase-protection, primer extension analysis and/or quantitative RT-PCR. siRNA nucleic acids that block VEGF, VEGFR1 VGFR2 and/or VEGFR3 protein encoding mRNAs and therefore result in decreased levels of VEGF, VEGFR1 VGFR2 and/or VEGFR3 activity by more than 20% in vitro can be identified.

Example 9
RNAi Mediated Inhibition of VEGFR Expression in Cell Culture

Inhibition of VEGFR1 RNA Expression Using siNA Targeting, VEGFR1 RNA

siNA constructs (Table III) are tested for efficacy in reducing VEGF and/or VEGFR RNA expression in, for example, HUVEC, HMVEC, or A375 cells. Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells are 70-90% confluent. For transfection, annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 μl/well and incubated for 20 min. at room temperature. The siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 μl. Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24 h in the continued presence of the siNA transfection mixture. At 24 h, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs is determined.

FIG. 22 shows a non-limiting example of reduction of VEGFR1 mRNA in A375 cells mediated by chemically-modified siNAs that target VEGFR1 mRNA. A549 cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA. A screen of siNA constructs (Stabilization “Stab” chemistries are shown in Table IV, constructs are referred to by RPI number, see Table III) comprising Stab 4/5 chemistry (Sima/RPI 31190/31193), Stab 1/2 chemistry (Sima/RPI 31183/31186 and Sima/RPI 31184/31187), and unmodified RNA (Sima/RPI 30075/30076) were compared to untreated cells, matched chemistry inverted control siNA constructs (Sima/RPI 31208/31211, Sima/RPI 31201/31204, Sima/RPI 31202/31205, and Sima/RPI 30077/30078), scrambled siNA control constructs (Scram1 and Scram2), and cells transfected with lipid alone (transfection control). As shown in the figure, all of the siNA constructs significantly reduce VEGFR1 RNA expression. Additional stabilization chemistries as described in Table IV are similarly assayed for activity. These siNA constructs are compared to appropriate matched chemistry inverted controls. In addition, the siNA constructs are also compared to untreated cells, cells transfected with lipid and scrambled siNA constructs, and cells transfected with lipid alone (transfection control).

Inhibition of VEGFR2 RNA Expression Using siNA Targeting VEGFR2 RNA

FIG. 24 shows a non-limiting example of reduction of VEGFR2 mRNA in HAEC cells mediated by chemically-modified siNAs that target VEGFR2 mRNA. HAEC cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA. A screen of siNA constructs (Stabilization “Stab” chemistries are shown in Table IV, constructs are referred to by Compound No., see Table III) in site 3854 comprising Stab 4/5 chemistry (Compound No. 30786/30790), Stab 7/8 chemistry (Compound No. 31858/31860), and Stab 9/10 chemistry (Compound No. 31862/31864) and in site 3948 comprising Stab 4/5 chemistry (Compound No. 31856/31857), Stab 7/8 chemistry (Compound No. 31859/31861), and Stab 9/10 chemistry (Compound No. 31863/31865) were compared to untreated cells, matched chemistry inverted control siNA constructs in site 3854 (Compound No. 31878/31880, Compound No. 31882/31884, and Compound No. 31886/31888) and in site 3948 (Compound No. 31879/31881, Compound No. 31883/31885, and Compound No. 31887/31889), and cells transfected with LF2K (transfection reagent), and an all RNA control (Compound No. 31435/31439 in site 3854 and Compound No. 31437/31441 in site 3948). As shown in the figure, all of the siNA constructs significantly reduce VEGFR2 RNA expression. Additional stabilization chemistries as described in Table IV are similarly assayed for activity. These siNA constructs are compared to appropriate matched chemistry inverted controls. In addition, the siNA constructs are also compared to untreated cells, cells transfected with lipid and scrambled siNA constructs, and cells transfected with lipid alone (transfection control).

Inhibition of VEGFR1 and VEGFR2 RNA Expression Using siNA Targeting VEGFR1 and VEGFR2 Homologous RNA Sequences

VEGFR1 and VEGFR2 RNA levels were assessed in HAEC cells 24 hours after treatment with siNA molecules targeting sequences having VEGFR1 and VEGFR2 homology. HAEC cells were transfected with 1.5 ug/well of lipid complexed with 25 nM siNA. Activity of the siNA moleclues is shown compared to matched chemistry inverted siNA controls, untreated cells, and cells treated with lipid only (transfection control). siNA molecules and controls are referred to by compound numbers (sense/antisense), see Table III for sequences. As shown in FIGS. 26A and B, siNA constructs that target both VEGFR1 and VEGFR2 sequences demonstrate potent efficacy in inhibiting VEGFR1 expression in cell cuture experiments. As shown in FIGS. 27A and B, siNA constructs that target both VEGFR1 and VEGFR2 sequences demonstrate potent efficacy in inhibiting VEGFR2 expression in cell cuture experiments.

Example 10
siNA-Mediated Inhibition of Angiogenesis in Vivo

Evaluation of siNA molecules in the rat cornea model of VEGF induced angiogenesis The purpose of this study was to assess the anti-angiogenic activity of siNA targeted against VEGFR1, using the rat cornea model of VEGF induced angiogenesis. The siNA molecules referred to in FIG. 28 have matched inverted controls which are inactive since they are not able to interact with the RNA target. The siNA molecules and VEGF were co-delivered using the filter disk method. Nitrocellulose filter disks (Millipore®) of 0.057 diameter were immersed in appropriate solutions and were surgically implanted in rat cornea as described by Pandey et al., supra.

The stimulus for angiogenesis in this study was the treatment of the filter disk with 30 μM VEGF, which is implanted within the comea's stroma. This dose yields reproducible neovascularization stemming from the pericorneal vascular plexus growing toward the disk in a dose-response study 5 days following implant. Filter disks treated only with the vehicle for VEGF show no angiogenic response. The siNA were co-adminstered with VEGF on a disk in three different siNA concentrations. One concern with the simultaneous administration is that the siNA would not be able to inhibit angiogenesis since VEGF receptors can be stimulated. However, Applicant has observed that in low VEGF doses, the neovascular response reverts to normal suggesting that the VEGF stimulus is essential for maintaining the angiogenic response. Blocking the production of VEGF receptors using simultaneous administration of anti-VEGF-R mRNA siNA could attenuate the normal neovascularization induced by the filter disk treated with VEGF.

Materials and Methods:

Test Compounds and Controls

- R&D Systems VEGF, carrier free at 75 μM in 82 mM Tris-Cl, pH 6.9
- Active siNA constructs and inverted controls (Table III)
  
  Animals
- Harlan Sprague-Dawley Rats, Approximately 225-250 g
- 45 males, 5 animals per group.
  
  Husbandry

Animals are housed in groups of two. Feed, water, temperature and humidity are determined according to Pharmacology Testing Facility performance standards (SOP's) which are in accordance with the 1996 Guide for the Care and Use of Laboratory Animals (NRC). Animals are acclimated to the facility for at least 7 days prior to experimentation. During this time, animals are observed for overall health and sentinels are bled for baseline serology.

Experimental Groups

Each solution (VEGF and siNAs) was prepared as a 1× solution for final concentrations shown in the experimental groups described in Table III.

siNA Annealing Conditions

siNA sense and antisense strands are annealed for 1 minute in H₂O at 1.67 mg/mL/strand followed by a 1 hour incubation at 37° C. producing 3.34 mg/mL of duplexed siNA. For the 20 μg/eye treatment, 6 μLs of the 3.34 mg/mL duplex is injected into the eye (see below). The 3.34 mg/mL duplex siNA can then be serially diluted for dose response assays.

Preparation of VEGF Filter Disk

For corneal implantation, 0.57 mm diameter nitrocellulose disks, prepared from 0.45 μm pore diameter nitrocellulose filter membranes (Millipore Corporation), were soaked for 30 min in 1 μL of 75 μM VEGF in 82 mM Tris HCl (pH 6.9) in covered petri dishes on ice. Filter disks soaked only with the vehicle for VEGF (83 mM Tris-Cl pH 6.9) elicit no angiogenic response.

Corneal Surgery

The rat corneal model used in this study was a modified from Koch et al. Supra and Pandey et al., supra. Briefly, corneas were irrigated with 0.5% povidone iodine solution followed by normal saline and two drops of 2% lidocaine. Under a dissecting microscope (Leica MZ-6), a stromal pocket was created and a presoaked filter disk (see above) was inserted into the pocket such that its edge was 1 mm from the corneal limbus.

Intraconjunctival Injection of Test Solutions

Immediately after disk insertion, the tip of a 40-50 μm OD injector (constructed in our laboratory) was inserted within the conjunctival tissue 1 mm away from the edge of the corneal limbus that was directly adjacent to the VEGF-soaked filter disk. Six hundred nanoliters of test solution (siNA, inverted control or sterile water vehicle) were dispensed at a rate of 1.2 μL/min using a syringe pump (Kd Scientific). The injector was then removed, serially rinsed in 70% ethanol and sterile water and immersed in sterile water between each injection. Once the test solution was injected, closure of the eyelid was maintained using microaneurism clips until the animal began to recover gross motor activity. Following treatment, animals were warmed on a heating pad at 37° C.

Quantitation of Angiogenic Response

Five days after disk implantation, animals were euthanized following administration of 0.4 mg/kg atropine and corneas were digitally imaged. The neovascular surface area (NSA, expressed in pixels) was measured postmortem from blood-filled corneal vessels using computerized morphometry (Image Pro Plus, Media Cybernetics, v2.0). The individual mean NSA was determined in triplicate from three regions of identical size in the area of maximal neovascularization between the filter disk and the limbus. The number of pixels corresponding to the blood-filled corneal vessels in these regions was summated to produce an index of NSA. A group mean NSA was then calculated. Data from each treatment group were normalized to VEGF/siNA vehicle-treated control NSA and finally expressed as percent inhibition of VEGF-induced angiogenesis.

Statistics

After determining the normality of treatment group means, group mean percent inhibition of VEGF-induced angiogenesis was subjected to a one-way analysis of variance. This was followed by two post-hoc tests for significance including Dunnett's (comparison to VEGF control) and Tukey-Kramer (all other group mean comparisons) at alpha=0.05. Statistical analyses were performed using JMP v.3.1.6 (SAS Institute).

Results of the study are graphically represented in FIGS. 28 and 29. As shown in FIG. 28, VEGFR1 site 4229 active siNA (Sima/RPI 29695/29699) at three concentrations was effective at inhibiting angiogenesis compared to the inverted siNA control (Sima/RPI 29983/29984) and the VEGF control. A chemically modified version of the VEGFR1 site 4229 active siNA comprising a sense strand having 2′-deoxy-2′-fluoro pyrimidines and ribo purines with 5′ and 3′ terminal inverted deoxyabasic residues and an antisense strand having having 2′-deoxy-2′-fluoro pyrimidines and ribo purines with a terminal 3′-phosphorothioate internucleotide linkage (Sima/RPI 30196/30416), showed similar inhibition. Furthermore, VEGFR1 site 349 active siNA having “Stab 9/10” chemistry (Compound No. 31270/31273) was tested for inhibition of VEGF-induced angiogenesis at three different concentrations (2.0 ug, 1.0 ug, and 0.1 ug dose response) as compared to a matched chemistry inverted control siNA construct (Compound No. 31276/31279) at each concentration and a VEGF control in which no siNA was administered. As shown in FIG. 29, the active siNA construct having “Stab 9/10” chemistry (Compound No. 31270/31273) is highly effective in inhibiting VEGF-induced angiogenesis in the rat corneal model compared to the matched chemistry inverted control siNA at concentrations from 0.1 ug to 2.0 ug. These results demonstrate that siNA molecules having different chemically modified compositions, such as the modifications described herein, are capable of significantly inhibiting angiogenesis in vivo. Results of a follow study in which sites adjacent to VEGFR1 site 349 were evaluated for efficacy using two different siNA stabilization chemistries is shown in FIG. 30.

Evaluation of siNA Molecules Targeting Homologous VEGFR1 and VEGFR2 Sequences in the Rat Cornea Model of VEGF Induced Angiogenesis

The above model was utilized to evaluate the efficacy of siNA molecules targeting homologous VEGFR1 and VEGFR2 sequences in inibiting VEGF induced ocular angiogenesis. Test compounds and controls are referred to in Table VII, sequences are shown in Table H. The siNAs or other test articles were administered by subconjunctival injection after VEGF disk implantation. The siNAs were preannealed prior to administration. Subconjuctival injections were performed using polyimide coated fused silica glass catheter tubing (OD=148 μm, ID=74 μm). This tubing was inserted into a borosilicate glass micropipette that was pulled to a fine point of approximately 40-50 microns OD using a Flaming/Brown Micropipette Puller (Model P-87, Sutter Instrument Co.). The micropipette was inserted into the pericorneal conjunctiva in the vicinity of the implanted filter disc and a volume of 1.2 μL was delivered over 15 seconds using a Hamilton Gastight syringe (25 μL) and a syringe pump. The rat eye was prepared by trimming the whiskers around the eye and washing the eye with providone iodine following topical lidocaine anesthesia. The silver nitrate sticks were touched to the surface of the cornea to induce a wound healing response and concurrent neovascularization. On day five, animals were anesthetized using ketamine/xylazine/acepromazine and vessel growth scores obtained. Animals were euthanized by CO₂inhalation and digital images of each eye were obtained for quantitation of vessel growth using Image Pro Plus. Quantitated neovascular surface area was analyzed by ANOVA followed by two post-hoc tests including Dunnet's and Tukey-Kramer tests for significance at the 95% confidence level. Results are shown in FIG. 31 as percent inhibition of VEGF induced angiogenesis compared to VEGF control. As shown in the figure, several siNA constructs that target both VEGFR1 and VEGFR2 via homologous sequences (e.g., compound Nos. 33725/33731, 33737/33743, 33742/33748, and 33729/33735) provide inhibition of VEGF-induced angiogenesis in this model. These compounds appear to provide equal or greater inhibition than a siNA construct (Compound No. 31270/31273) targeting VEGFR1 only.

Evaluation of siNA Molecules in the Mouse Coroidal Model of Neovascularization.

Intraocular Administration of siNA

Female C57B/6 mice (4-5 weeks old) were anesthetized with a 0.2 ml of a mixture of ketamine/xylazine (8:1), and the pupils were dilated with a single drop of 1% tropicamide. Then a 532 nm diode laser photocoagulation (75 μm spot size, 0.1-second duration, 120 mW) was used to generate three laser spots in each eye surrounding the optic nerve by using a hand-held coverslip as a contact lens. A bubble formed at the laser spot indicating a rupture of the Bruch's membrane. Next, the laser spots were evaluated for the presence of CNV on day 17 after laser treatment.

After laser induction of multiple CNV lesions in mice, the siNA was administered by intraocular injections under a dissecting microscope. Intravitreous injections were performed with a Harvard pump microinjection apparatus and pulled glass micropipets. Each micropipet was calibrated to deliver 1 μL of vehicle containing 0.5 ug or 1.5 ug of siNA, inverted control siNA, or saline. The mice were anesthetized, pupils were dilated, and, the sharpened tip of the micropipet was passed through the sclera, just behind the limbus into the vitreous cavity, and the foot switch was depressed. The injection was repeated at day 7 after laser photocoagulation.

At the time of death, mice were anesthetized (ketamine/xylazine mixture, 8:1) and perfused through the heart with 1 ml PBS containing 50 mg/ml fluorescein-labeled dextran (FITC-Dextran, 2 million average molecular weight, Sigma). The eyes were removed and fixed for overnight in 1% phosphate-buffered 4% Formalin. The cornea and the lens were removed and the neurosensory retina was carefully dissected from the eyecup. Five radial cuts were made from the edge of the eyecup to the equator; the sclera-choroid-retinal pigment epithelium (RPE) complex was flat-mounted, with the sclera facing down, on a glass slide in Aquamount. Flat mounts were examined with a Nikon fluorescence microscope. A laser spot with green vessels was scored CNV-positive, and a laser spot lacking green vessels was scored CNV-negative. Flatmounts were examined by fluorescence microscopy (Axioskop; Carl Zeiss, Thornwood, N.Y.), and images were digitized with a three-color charge-coupled device (CCD) video camera and a frame grabber. Image-analysis software (Image-Pro Plus; Media Cybernetics, Silver Spring, Md.) was used to measure the total area of hyperfluorescence associated with each burn, corresponding to the total fibrovascular scar. The areas within each eye were averaged to give one experimental value per eye for plotting the areas.

Measurement of VEGFR1 expression was also determined using RT-PCR and/or real-time PCR. Retinal RNA was isolated by a Rnaeasy kit, and reverse transcription was performed with approximately 0.5 μg total RNA, reverse transcriptase (SuperScript II), and 5.0 μM oligo-d(T) primer. PCR amplification was performed using primers specific for VEGFR-1 (5′-AAGATGCCAGCCGAAGGAGA-3′, SEQ ID NO: 4253) and (5′-GGCTCGGCACCTATAGACA-3′, SEQ ID NO: 4254). Titrations were determined to ensure that PCR reactions were performed in the linear range of amplification. Mouse S16 ribosomal protein primers (5′-CACTGCAAACGGGGAAATGG-3′, SEQ ID NO: 4255 and 5′-TGAGATGGACTGTCGGATGG-3′, SEQ ID. NO: 4256) were used to provide an internal control for the amount of template in the PCR reactions.

VEGFR1 site 349 active siNA having “Stab 9/10” chemistry (Compound No. 31270/31273, Table III) was tested for inhibition of VEGF-induced neovascularization at two different concentrations (1.5 ug, and 0.5 ug dose response) as compared to a matched chemistry 1.5 ug inverted control siNA construct (Compound No. 31276/31279, Table III) and a saline control. As shown in FIG. 32, the active siNA construct having “Stab 9/10” chemistry is highly effective in inhibiting VEGFR1 induced neovascularization (57% inhibition) in the C57BL/6 mice intraocular delivery model compared to the matched chemistry inverted control siNA. The active siNA construct was also highly effective in inhibiting VEGFR1 induced neovascularization (66% inhibition) compared to the saline control. Additionally, RT-PCR analysis of VEGFR1 site 349 siNA having “Stab 9/10” chemistry (Compound No. 31270/31273, Table III) showed significant reduction in the level of VEGFR1 mRNA compared to the inverted siNA construct (Compound No. 31276/31279, Table III) and saline. Furthermore, ELISA analysis of VEGFR1 protein using the active siNA and inverted control siNA above showed significant reduction in the level of VEGFR1 protein expression using the active siNA compared to the inactive siNA construct. These results demonstrate that siNA molecules having different chemically modified compositions, such as the modifications described herein, are capable of significantly inhibiting neovascularization as shown in this model of intraocular administration.

Periocular Administration of siNA

Female C57BL/6 mice (4-5 weeks old) were anesthetized with a 0.2 ml of a mixture of ketamine/xylazine (8:1), and the pupils were dilated with a single drop of 1% tropicamide. Then a 532 nm diode laser photocoagulation (75 μm spot size, 0.1-s duration, 120 mW) was used to generate three laser spots in each eye surrounding the optic nerve by using a hand-held coverslip as a contact lens. A bubble formed at the laser spot indicating a rupture of the Bruch's membrane. Next, the laser spots were evaluated for the presence of CNV on day 17 after laser treatment.

After laser induction of multiple CNV lesions in mice, the siNA was administered via periocular injections under a dissecting microscope. Periocular injections were performed with a Harvard pump microinjection apparatus and pulled glass micropipets. Each micropipet was calibrated to deliver 5 μL of vehicle containing test siNA at concentrations of 0.5 ug or 1.5 ug of siNA. The mice were anesthetized, pupils were dilated, and, the sharpened tip of the micropipet was passed, and the foot switch was depressed. Periocular injections were given daily starting at day 1 through day 14 after laser photocoagulation. Alternately, periocular injections are given every 3 days after rupture of Bruch's membrane.

At the time of death, mice were anesthetized (ketamine/xylazine mixture, 8:1) and perfused through the heart with 1 mL PBS containing 50 mg/mL fluorescein-labeled dextran (FITC-Dextran, 2 million average molecular weight, Sigma). The eyes were removed and fixed overnight in 1% phosphate-buffered 4% Formalin. The cornea and the lens were removed and the neurosensory retina was carefully dissected from the eyecup. Five radial cuts were made from the edge of the eyecup to the equator; the sclera-choroid-retinal pigment epithelium (RPE) complex was flat-mounted, with the sclera facing down, on a glass slide in Aquamount. Flat mounts were examined with a Nikon fluorescence microscope. A laser spot with green vessels was scored CNV-positive, and a laser spot lacking green vessels was scored CNV-negative. Flatmounts were examined by fluorescence microscopy (Axioskop; Carl Zeiss, Thornwood, N.Y.) and images were digitized with a three-color charge-coupled device (CCD) video camera and a frame grabber. Image-analysis software (Image-Pro Plus; Media Cybernetics, Silver Spring, Md.) was used to measure the total area of hyperfluorescence associated with each burn, corresponding to the total fibrovascular scar. The areas within each eye were averaged to give one experimental value per eye.

VEGFR1 site 349 active siNA having “Stab 9/10” chemistry (Compound No. 31270/31273, Table III) was tested for inhibition of VEGF-induced neovascularization at two different concentrations (1.5 ug, and 0.5 ug dose response) as compared to a matched chemistry saline control and 0.5 ug inverted control siRNA construct (Compound No. 31276/31279, Table III). As shown in FIG. 33, the active siNA construct having “Stab 9/10” chemistry (Compound No. 31270/31273) is effective in inhibiting VEGFR1 induced neovascularization (20% inhibition) in the C57BL/6 mice periocular delivery model compared to the matched chemistry inverted control siNA. The active siNA construct was also highly effective in inhibiting VEGFR1 induced neovascularization (54% inhibition) compared to the saline control. In an additional assay shown in FIG. 34, VEGFR1 site 349 active siNA having “Stab 9/10” chemistry (Compound No. 31270/31273) at two concentrations was effective at inhibiting neovascularization in CNV lesions compared to the inverted siNA control and the saline control. As shown in FIG. 34, the active siNA construct having “Stab 9/10” chemistry (Compound No. 31270/31273) is effective in inhibiting VEGFR1 induced neovascularization (43% inhibition) in the C57BL/6 mice periocular delivery model compared to the matched chemistry inverted control siNA. The active siNA construct was also effective in inhibiting VEGFR1 induced neovascularization (45% inhibition) compared to the saline control with periocular injection treatment given every 3 days after rupture of Bruch's membrane (see FIG. 35). These results demonstrate that siNA molecules having different chemically modified compositions, such as the modifications described herein, are capable of significantly inhibiting neovascularization as shown in this model of periocular administration.

Evaluation of siNA Molecules in the Mouse Retinopathy of Prematurity Model

The following protocol was used to evaluate siNA molecules targeting VEGF receptor mRNA in an oxygen-induced ischemic retinopathy/retinopathy of prematurity model. Pups from female C57BL/6 mice were placed into a 75% oxygen (ROP) environment at P7 (seven days after birth). Mothers were changed quickly at P10. Mice were removed from 75% oxygen chamber at P12. Pups were injected on P12, three hours after being removed from the 75% oxygen environment. siNA was delivered via an intravitreal or periocular injection under a dissecting microscope. A Harvard pump microinjection apparatus and pulled glass micropipette were used for injection. Each micropipette was calibrated to deliver 1 μL of vehicle containing test siRNA. The mice were anesthetized, the pupils were dilated, and the sharpened tip of the micropipette was passed through the limbus and the foot of the microinjection apparatus was depressed. Mice were sacrificed by cervical dislocation for RNA and protein extraction on P15, three days after being removed from the high oxygen environment. The retinas were removed and placed in appropriate lysis buffer (see below for protein and RNA analysis methods).

Protein Analysis: Protein lysis buffer contained 50 μL 1M Tris-HCl (pH 7.4), 50 μL 10% SDS (Sodium Dodecyl Sulfate), 50 μL 100 nM PHSF (Phenylmethaneculfonyl) and 5 mL serilized, de-ionized water. 200 μL of lysis buffer was added to fresh tissue, and homogenized by pipeting. Tissue was sonicated at 4° C. for 25 minutes, and spun at 13K for 5 minutes at 4° C. The pellet was discarded, and supemate transferred to fresh tube. BioRad assay was used to measure protein concentration using BSA as a standard. Samples were stored at −80° C. ELISAs were carried out using VEGFR1 and R2 kits from R&D Systems (Quantikine® Immunoassay). The protocols provided in the manuals were followed exactly.

RNA analysis: RNA was extracted using Quiagen, RNeasy mini kit and following protocol for extraction from animal cells. RNA samples were treated with DNA-free™ by Ambion following company protocol. First Strand cDNA was then synthesized for real time PCR using Invitrogen, Superscript 1st Strand System for RT-PCR, and following protocol. Real-time PCR was then preformed in a Roche Lightcycler using Fast Start DNA Master SYBR Green I. Cyclophilin A was used as a control, and purified PCR products were used as standards.

Analysis of neovascularization: Mice were sacrificed on P17 by cervical dislocation. Eyes were removed and fresh frozen in OCT and stored at −80° C. Eyes were then sectioned and immunohistochemically stained for lectin. 10 μm frozen sections of eyes were histochemically stained with biotinylated Griffonia simplicifolia lectin B4 (GSA; Vector Laboratories, Burlingame, Calif.), which selectively binds to endothelial cells. Slides were dried and fixed with 4% PFA for 20 minutes, then incubated in methanol/H₂O₂for 10 minutes at room temperature. After washing with 0.05 M Tris-buffered saline, pH 7.6 (TBS), the slides were blocked with 10% swine serum for 30 minutes. Slides were first stained with biotinylated GSA for 2 hours at room temperature, followed by a thorough wash with 0.05 M TBS. The slides were further stained with avidin coupled to alkaline phosphatase (Vector Laboratories) for 45 minutes at room temperature. Slides were incubated with a red stain (Histomark Red; Kirkegaard and Perry, Gaithersburg, Md.) to give a red reaction product. A computer and image-analysis software (Image-Pro Plus software; Media Cybernetics, Silver Spring, Md.) was used to quantify GSA-stained cells on the surface of the retina, and their area was measured. The mean of the 15 measurements from each eye was used as a single experimental value.

Results of a representative study are shown in FIGS. 36 and 37. As shown in FIG. 36, in mice with oxygen induced retinopathy (OIR), periocular injections of VEGFR1 siNA (31270/31273) (5 μl; 1.5 μg/μl) on P12, P14, and P16 significantly reduced VEGFR1 mRNA expression compared to injections with a matched chemistry inverted control siNA construct (31276/31279), (40% inhibition; n=9, p=0.0121). As shown in FIG. 37, in mice with oxygen induced retinopathy (OIR), intraocular injections of VEGFR1 siNA (31270/31273) (5 μg), significantly reduced VEGFR1 protein levels compared to injections with a matched chemistry inverted control siNA construct (31276/31279), (30% inhibition; n=7, p=0.0103).

Evaluation of siNA Molecules in the Mouse 4T1-Luciferase Mammary Carcinoma Syngeneic Tumor Model

The current study was designed to determine if systemically administered siRNA directed against VEGFR-1 inhibits the growth of subcutaneous tumors. Test compounds included active Stab 9/10 siNA targeting site 349 of VEGFR-1 RNA (Compound #31270/31273), a matched chemistry inactive inverted control siNA (Compound #31276/31279) and saline. Animal subjects were female Balb/c mice approximately 20-25 g (5-7 weeks old). The number of subjects tested was 40 mice; treatment groups are described in Table VI. Mice were housed in groups of four. The feed, water, temperature and humidity conditions followed. Pharmacology Testing Facility performance standards (SOP's) which are in accordance with the 1996 Guide for the Care and Use of Laboratory Animals (NRC). Animals were acclimated to the facility for at least 3 days prior to experimentation. During this time, animals were observed for overall health and sentinels were bled for baseline serology. 4T1-luc mammary carcinoma tumor cells were maintained in cell culture until injection into animals used in the study. On day 0 of the study, animals were anesthetized with ketamine/xylazine and 1.0×10⁶cells in an injection volume of 100 μl were subcutaneously inoculated in the right flank. Primary tumor volume was measured using microcalipers. Length and width measurements were obtained from each tumor 3×/week (M,W,F) beginning 3 days after inoculation up through and including 21 days after inoculation. Tumor volumes were calculated from the length/width measurements according to the equation: Tumor volume=(a) (b)²/2 where a=the long axis of the tumor and b=the shorter axis of the tumor. Tumors were allowed to grow for a period of 3 days prior to dosing. Dosing consisted of a daily intravenous tail vein injection of the test compounds for 18 days. On day 21, animals were euthanized 24 hours following the last dose of test compound, or when the animals began to exhibit signs of moribundity (such as weight loss, lethargia, lack of grooming etc.) using CO₂inhalation and lungs were subsequently removed. Lung metastases were counted under a Leitz dissecting microscope at 25× magnification. Tumors were removed and flash frozen in LN₂for analysis of immunohistochemical endpoints or mRNA levels. Results are shown in FIG. 38. As shown in the Figure, the active siNA construct inhibited tumor growth by 50% compared to the inactive control siNA construct.

In addition, levels of soluble VEGFR1 in plasma were assessed in mice treated with the active and inverted control siNA constucts. FIG. 39 shows the reduction of soluble VEGFR1 serum levels in the mouse 4T1-luciferase mammary carcinoma syngeneic tumor model using active Stab 9/10 siNA targeting site 349 of VEGFR1 RNA (Compound #31270/31273) compared to a matched chemistry inactive inverted control siNA (Compound #31276/31279). As shown in FIG. 39, the active siNA construct is effective in reducing soluble VEGFR1 serum levels in this model.

Example 11
Multifunctional siNA Inhibition of VEGF and/or VEGFR RNA Expression

Multifunctional siNA Design

Once target sites have been identified for multifunctional siNA constructs, each strand of the siNA is designed with a complementary region of length, for example, of about 18 to about 28 nucleotides, that is complementary to a different target nucleic acid sequence. Each complementary region is designed with an adjacent flanking region of about 4 to about 22 nucleotides that is not complementary to the target sequence, but which comprises complementarity to the complementary region of the other sequence (see for example FIG. 16). Hairpin constructs can likewise be designed (see for example FIG. 17). Identification of complementary, palindrome or repeat sequences that are shared between the different target nucleic acid sequences can be used to shorten the overall length of the multifunctional siNA constructs (see for example FIGS. 18 and 19).

In a non-limiting example, a multifunctional siNA is designed to target two separate nucleic acid sequences. The goal is to combine two different siNAs together in one siNA that is active against two different targets. The siNAs are joined in a way that the 5′ of each strand starts with the “antisense” sequence of one of two siRNAs as shown in italics below.

SEQ ID NO: 42573′ TTAGAAACCAGACGUAAGUGU GGUACGACCUGACGACCGU 5′SEQ ID NO: 42585′ UCUUUGGUCUGCAUUCACAC CAUGCUGGACUGCUGGCATT3′

RISC is expected to incorporate either of the two strands from the 5′ end. This would lead to two types of active RISC populations carrying either strand. The 5′ 19 nt of each strand will act as guide sequence for degradation of separate target sequences.

In another example, the size of multifunctional siNA molecules is reduced by either finding overlaps or truncating the individual siNA length. The exemplary excercise described below indicates that for any given first target sequence, a shared complementary sequence in a second target sequence is likely to be found.

The number of spontaneous matches of short polynucleotide sequences (e.g., less than 14 nucleotides) that are expected to occur between two longer sequences generated independent of one another was investigated. A simulation using the uniform random generator SAS V8.1 utilized a 4,000 character string that was generated as a random repeating occurrence of the letters {ACGU}. This sequence was then broken into the nearly 4000 overlapping sets formed by taking S1 as the characters from positions (1,2 . . . n), S2 from positions (2,3 . . . , n+1) completely through the sequence to the last set, S 4000−n+1 from position (4000−n+1, . . . ,4000). This process was then repeated for a second 4000 character string. Occurrence of same sets (of size n) were then checked for sequence identity between the two strings by a sorting and match-merging routine. This procedure was repeated for sets of 9-11 characters. Results were an average of 55 matching sequences of length n=9 characters (range 39 to 72); 13 common sets (range 6 to 18) for size n=10, and 4 matches on average (range 0 to 6) for sets of 11 characters. The choice of 4000 for the original string length is approximately the length of the coding region of both VEGFR1 and VEGFR2. This simple simulation suggests that any two long coding regions formed independent of one-another will share common short sequences that can be used to shorten the length of multifunctional siNA constructs. In this example, common sequences of size 9 occurred by chance alone in >1% frequency.

Below is an example of a multifunctional siNA construct that targets VEGFR1 and VEGFR2 in which each strand has a total length of 24 nt with a 14 nt self complementary region (underline). The antisense region of each siNA ‘1’ targeting VEGFR1 and siNA ‘2’ targeting VEGFR2 (complementary regions are shown in italic) are used

siNA ‘1’5′CAAUUAGAGUGGCAGUGAG(SEQ ID NO: 4259)3′ GUUAAUCUCACCGUCACUC(SEQ ID NO: 4260)siNA ‘2’AGAGUGGCAGUGAGCAAAG 5′(SEQ ID NO: 4261)UCUCACCGUCACUCGUUUC 3′(SEQ ID NO: 4262)Multifunctional siNACAAUUAGAGUGGCAGUGAGCAAAG(SEQ ID NO: 4263)GUUAAUCUCACCGUCACUCGUUUC(SEQ ID NO: 4264)

In another example, the length of a multifunctional siNA construct is reduced by determining whether fewer base pairs of sequence homology to each target sequence can be tolerated for effective RNAi activity. If so, the overall length of multifunctional siNA can be reduced as shown below. In the following hypothetical example, 4 nucleotides (bold) are reduced from each 19 nucleotide siNA ‘1’ and siNA ‘2’ constructs. The resulting multifunctional siNA is 30 base pairs long.

siNA ‘1’5′CAAUUAGAGUGGCAG custom character

(SEQ ID NO: 4259)3′ GUUAAUCUCACCGUCACUC(SEQ ID NO: 4260)siNA ‘2’AGAGUGGCAGUGAGCAAAG 5′(SEQ ID NO: 4261) custom character

ACCGUCACUCGUUUC 3′(SEQ ID NO: 4262)Multifunctional siNACAAUUAGAGUGGCAGUGGGAGUGAGCAAAG(SEQ ID NO: 4265)GUUAAUCUCACCGUCACCGUCACUCGUUUC(SEQ ID NO: 4266)

Multifunctional siNA Constructs Targeting VEGF and VEGFR RNA in a Dual-Reporter Plasmid System

The dual reporter assay used to evaluate multifunctional siNA constructs targeting VEGF and VEGFR RNA targets uses a dual-reporter plasmid, psiCHECK-II (Promega) that contains firefly and renilla luciferase genes. The sequence of interest (target RNA for siNAs) is cloned downstream of renilla luciferase stop codon. The loss of renilla luciferase activity is directly correlated to message degradation by the multifunctional siNA. The firefly luciferase activity is used as transfection control.

Cell Culture Analysis of Multifunctional siNA Activity

RNAi activities were evaluated in HeLa cells grown in 75 μl Iscove's solution containing 10% fetal calf serum to 70-80% confluency in 96-well plates at 37° C., 5% CO₂. Transfection mixtures consisting of 175.5 μl Opti-MEM I (Gibco-BRL), 2 μl Lipofectamine 2000 (Invitrogen) and 10 μl siCHECK™-2 plasmid containing appropriate target RNA sequence at 50 ng/μl (Promega) were prepared in microtiter plates. A 12.5 μl siRNA (1 μM) solution was added to the above mixture to bring the siRNA concentration to 62.5 nM. The transfection mixture was incubated for 20-30 min at 25° C. 50 μl of the transfection mixture was then added to 75 μl medium containing HeLa cells to bring the final siRNA concentration to 25 nM. Cell were incubated for 20 hours at 37° C., 5% CO₂.

Quantification of Gene Knockdown

Firefly and renilla luciferase luminescence was measured according to manufacturer's instructions for experiments carried out in a 96 well plate format. In a typical procedure, after 20 h transfection, 50 μl medium was removed from the culture and 75 μl Dual Go Luciferase reagent was added, and gently rocked for 10 minutes at: room temperature. Firefly luminescence was measured on a 96 well plate reader. Subsequently 75 μl of freshly prepared Dual Glo Stop and Glow reagent was added, and plates were gently rocked for additional 10 minutes at room temperature. Renilla luminescence was measured on a 96 well plate reader. The ratio of firefly luminescence to renilla luminescence provided a normalized value of silencing activity. Results are shown in FIGS. 40-42. FIG. 40 shows RNA based multifunctional siNA mediated inhibition of (A) VEGF, (B) VEGFR1 and (C) VEGFR2 RNA. FIG. 41 shows stabilized multifunctional siNA mediated inhibition of (A) VEGF, (B) VEGFR1 and (C) VEGFR2 RNA. FIG. 42 shows non-nucleotide tethered multifunctional siNA mediated inhibition of VEGF, VEGFR1 and VEGFR2 RNA. These data demonstrate that the multifunctional siNA constructs are similarly effective in inhibition of VEGF and VEGFR RNA expression by targeting multiple sites as are individual siNA constructs that target each site.

Additional Multifunctional siNA Designs

Three categories of additional multifunctional siNA designs are presented that allow a single siNA molecule to silence multiple targets. The first method utilizes linkers to join siNAs (or multiunctional siNAs) in a direct manner. This can allow the most potent siNAs to be joined without creating a long, continuous stretch of RNA that has potential to trigger an interferon response. The second method is a dendrimeric extension of the overlapping or the linked multifunctional design; or alternatively the organization of siNA in a supramolecular format. The third method uses helix lengths greater than 30 base pairs. Processing of these siNAs by Dicer will reveal new, active 5′ antisense ends. Therefore, the long siNAs can target the sites defined by the original 5′ ends and those defined by the new ends that are created by Dicer processing. When used in combination with traditional multifunctional siNAs (where the sense and antisense strands each define a target) the approach can be used for example to target 4 or more sites.

I. Tethered Bifunctional siNAs

The basic idea is a novel approach to the design of multifunctional siNAs in which two antisense siNA strands are annealed to a single sense strand. The sense strand oligonucleotide contains a linker (e.g., non-nulcoetide linker as described herein) and two segments that anneal to the antisense siNA strands (see FIG. 43). The linkers can also optionally comprise nucleotide-based linkers. Several potential advantages and variations to this approach include, but are not limited to:

1. The two antisense siNAs are independent. Therefore, the choice of target sites is not constrained by a requirement for sequence conservation between two sites. Any two highly active sINAs can be combined to form a multifunctional siNA.
2. When used in combination with target sites having homology, siNAs that target a sequence present in two genes (e.g., different VEGF and/or VEGFR strains), the design can be used to target more than two sites. A single multifunctional siNA can be for example, used to target RNA of two different VEGF and/or VEGFR RNAs (using one antisense strand of the multifunctional siNA targeting of conserved sequence between to the two RNAs) and a host RNA (using the second antisense strand of the multifunctional siNA targeting host RNA (e.g., La antigen or FAS) This approach allows targeting of more than one VEGF and/or VEGFR strain and one or more host RNAs using a single multifunctional siNA.
3. Multifunctional siNAs that use both the sense and antisense strands to target a gene can also be incorporated into a tethered multifuctional design. This leaves open the possibility of targeting 6 4 or more sites with a single complex.
4. It can be possible to anneal more than two antisense strand siNAs to a single tethered sense strand.
5. The design avoids long continuous stretches of dsRNA. Therefore, it is less likely to initiate an interferon response.
6. The linker (or modifications attached to it, such as conjugates described herein) can improve the pharmacokinetic properties of the complex or improve its incorporation into liposomes. Modifications introduced to the linker should not impact siNA activity to the same extent that they would if directly attached to the siNA (see for example FIGS. 49 and 50).
7. The sense strand can extend beyond the annealed antisense strands to provide additional sites for the attachment of conjugates.
8. The polarity of the complex can be switched such that both of the antisense 3′ ends are adjacent to the linker and the 5′ ends are distal to the linker or combination thereof.

Dendrimer and Supramolecular siNAs

In the dendrimer siNA approach, the synthesis of siNA is initiated by first synthesizing the dendrimer template followed by attaching various functional siNAs. Various constructs are depicted in FIG. 44. The number of functional siNAs that can be attached is only limited by the dimensions of the dendrimer used.

Supramolecular Approach to Multifunctional siNA

The supramolecular format simplifies the challenges of dendrimer synthesis. In this format, the siNA strands are synthesized by standard RNA chemistry, followed by annealing of various complementary strands. The individual strand synthesis contains an antisense sense sequence of one siNA at the 5′-end followed by a nucleic acid or synthetic linker, such as hexaethyleneglyol, which in turn is followed by sense strand of another siNA in 5′ to 3′ direction. Thus, the synthesis of siNA strands can be carried out in a standard 3′ to 5′ direction. Representative examples of trifunctional and tetrafunctional siNAs are depicted in FIG. 45. Based on a similar principle, higher functionality siNA constucts can be designed as long as efficient annealing of various strands is achieved.

Dicer Enabled Multifunctional siNA

Using bioinformatic analysis of multiple targets, stretches of identical sequences shared between differeing target sequences can be identified ranging from about two to about fourteen nucleotides in length. These identical regions can be designed into extended siNA. helixes (e.g., >30 base pairs) such that the processing by Dicer reveals a secondary functional 5′-antisense site (see for example FIG. 46). For example, when the first 17 nucleotides of a siNA antisense strand (e.g., 21 nucleotide strands in a duplex with 3′-TT overhangs) are complementary to a target RNA, robust silencing was observed at 25 nM. 80% silencing was observed with only 16 nucleotide complementarity in the same format (see FIG. 48).

Incorporation of this property into the designs of siNAs of about 30 to 40 or more base pairs results in additional multifunctional siNA constructs. The example in FIG. 46 illustrates how a 30 base-pair duplex can target three distinct sequences after processing by Dicer-RNaseIII; these sequences can be on the same mRNA or separate RNAs, such as viral and host factor messages, or multiple points along a given pathway (e.g., inflammatory cascades). Furthermore, a 40 base-pair duplex can combine a bifunctional design in tandem, to provide a single duplex targeting four target sequences. An even more extensive approach can include use of homologous sequences (e.g. VEGFR-1/VEGFR-2) to enable five or six targets silenced for one multifunctional duplex. The example in FIG. 46 demonstrates how this can be achieved. A 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown). For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2′-OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs. Another non-limiting example is shown in FIG. 47. A 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end. For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Four targeting sequences are shown in four colors, blue, light-blue and red and orange. The required sequence identity overlapped is indicated by grey boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multiifunctional designs. For example cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.

Another important aspect of this approach is its ability to restrict escape mutants. Processing to reveal an internal target site can ensure that escape mutations complementary to the eight nucleotides at the antisense 5′ end will not reduce siNA effectiveness. If about 17 nucleotidest of complementarity are required for RISC-mediated target cleavage, this will restrict, for example 8/17 or 47% of potential escape mutants.

Example 12
Indications

The present body of knowledge in VEGF and/or VEGFR research indicates the need for methods to assay VEGF and/or VEGFR activity and for compounds that can regulate VEGF and/or VEGFR expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of VEGF and/or VEGFR levels. In addition, the nucleic acid molecules can be used to treat disease state related to VEGF and/or VEGFR levels.

Particular conditions and disease states that can be associated with VEGF and/or VEGFR expression modulation include, but are not limited to:

1) Tumor angiogenesis: Angiogenesis has been shown to be necessary for tumors to grow into pathological size (Folkman, 1971, PNAS 76, 5217-5221; Wellstein & Czubayko, 1996, Breast Cancer Res and Treatment 38, 109-119). In addition, it allows tumor cells to travel through the circulatory system during metastasis. Increased levels of gene expression of a number of angiogenic factors such as vascular endothelial growth factor (VEGF) have been reported in vascularized and edema-associated brain tumors (Berlman et al., 1993 J. Clini. Invest. 91, 153). A more direct demostration of the role of VEGF in tumor angiogenesis was demonstrated by Jim Kim et al., 1993 Nature 362,841 wherein, monoclonal antibodies against VEGF were successfully used to inhibit the growth of rhabdomyosarcoma, glioblastoma multiforme cells in nude mice. Similarly, expression of a dominant negative mutated form of the flt-1 VEGF receptor inhibits vascularization induced by human glioblastoma cells in nude mice (Millauer et al., 1994, Nature 367, 576). Specific tumor/cancer types that can be targeted using the nucleic acid molecules of the invention include but are not limited to the tumor/cancer types described herein.

2) Ocular diseases: Neovascularization has been shown to cause or exacerbate ocular diseases including, but not limited to, macular degeneration, including age related macular degeneration (AMD), dry AMD, wet AMD, predominantly classic AMD (PD AMD), minimally classic AMD (MC AMD), and occult AMD; neovascular glaucoma, diabetic retinopathy, including diabetic macular edema (DME) and proliferative diabetic retinopathy; myopic degeneration, uveitis, and trachoma (Norrby, 1997, APMIS 105, 417-437). Aiello et al., 1994 New Engl. J. Med. 331, 1480, showed that the ocular fluid of a majority of patients suffering from diabetic retinopathy and other retinal disorders contains a high concentration of VEGF. Miller et al., 1994 Am. J. Pathol. 145, 574, reported elevated levels of VEGF mRNA in patients suffering from retinal ischemia. These observations support a direct role for VEGF in ocular diseases. Other factors, including those that stimulate VEGF synthesis, may also contribute to these indications.

3) Dermatological Disorders: Many indications have been identified which may beangiogenesis dependent, including but not limited to, psoriasis, verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, and Osler-Weber-Rendu syndrome (Norrby, supra). Intradermal injection of the angiogenic factor b-FGF demonstrated angiogenesis in nude mice (Weckbecker et al., 1992, Angiogenesis: Key principles-Science-Technology-Medicine, ed R. Steiner). Detmar et al., 1994 J. Exp. Med. 180, 1141 reported that VEGF and its receptors were over-expressed in psoriatic skin and psoriatic dermal microvessels, suggesting that VEGF plays a significant role in psoriasis.

4) Rheumatoid arthritis: Immunohistochemistry and in situ hybridization studies on tissues from the joints of patients suffering from rheumatoid arthritis show an increased level of VEGF and its receptors (Fava et al., 1994 J. Exp. Med. 180, 341). Additionally, Koch et al., 1994 J. Immunol. 152, 4149, found that VEGF-specific antibodies were able to significantly reduce the mitogenic activity of synovial tissues from patients suffering from rheumatoid arthritis. These observations support a direct role for VEGF in rheumatoid arthritis. Other angiogenic factors including those of the present invention may also be involved in arthritis.

5) Endometriosis: Various studies indicate that VEGF is directly implicated in endometriosis. In one study, VEGF concentrations measured by ELISA in peritoneal fluid were found to be significantly higher in women with endometriosis than in women without endometriosis (24.1±15 ng/ml vs 13.3±7.2 ng/ml in normals). In patients with endometriosis, higher concentrations of VEGF were detected in the proliferative phase of the menstrual cycle (33±13 ng/ml) compared to the secretory phase (10.7±5 ng/ml). The cyclic variation was not noted in fluid from normal patients (McLaren et al., 1996, Human Reprod. 11, 220-223). In another study, women with moderate to severe endometriosis had significantly higher concentrations of peritoneal fluid VEGF than women without endometriosis. There was a positive correlation between the severity of endometriosis and the concentration of VEGF in peritoneal fluid. In human endometrial biopsies, VEGF expression increased relative to the early proliferative phase approximately 1.6-, 2-, and 3.6-fold in midproliferative, late proliferative, and secretory endometrium (Shifren et al., 1996, J. Clin. Endocrinol. Metab. 81, 3112-3118). In a third study, VEGF-positive staining of human ectopic endometrium was shown to be localized to macrophages (double immunofluorescent staining with CD14 marker). Peritoneal fluid macrophages demonstrated VEGF staining in women with and without endometriosis. However, increased activation of macrophages (acid phosphatatse activity) was demonstrated in fluid from women with endometriosis compared with controls. Peritoneal fluid macrophage conditioned media from patients with endometriosis resulted in significantly increased cell proliferation ([³H] thymidine incorporation) in HUVEC cells compared to controls. The percentage of peritoneal fluid macrophages with VEGFR2 mRNA was higher during the secretory phase, and significantly higher in fluid from women with endometriosis (80±15%) compared with controls (32±20%). Flt-mRNA was detected in peritoneal fluid macrophages from women with and without endometriosis, but there was no difference between the groups or any evidence of cyclic dependence (McLaren et al., 1996, J. Clin. Invest. 98, 482-489). In the early proliferative phase of the menstrual cycle, VEGF has been found to be expressed in secretory columnar epithelium (estrogen-responsive) lining both the oviducts and the uterus in female mice. During the secretory phase, VEGF expression was shown to have shifted to the underlying stroma composing the functional endometrium. In addition to examining the endometium, neovascularization of ovarian follicles and the corpus luteum, as well as angiogenesis in embryonic implantation sites have been analyzed. For these processes, VEGF was expressed in spatial and temporal proximity to forming vasculature (Shweiki et al., 1993, J. Clin. Invest. 91, 2235-2243).

6) Kidney disease: Autosomal dominant polycystic kidney disease (ADPKD) is the most common life threatening hereditary disease in the USA. It affects about 1:400 to 1:1000 people and approximately 50% of people with ADPKD develop renal failure. ADPKD accounts for about 5-10% of end-stage renal failure in the USA, requiring dialysis and renal transplantation. Angiogenesis is implicated in the progression of ADPKD for growth of cyst cells, as well as increased vascular permeability promoting fluid secretion into cysts. Proliferation of cystic epithelium is a feature of ADPKD because cyst cells in culture produce soluble vascular endothelial growth factor (VEGF). VEGFR1 has been detected in epithelial cells of cystic tubules but not in endothelial cells in the vasculature of cystic kidneys or normal kidneys. VEGFR2 expression is increased in endothelial cells of cyst vessels and in endothelial cells during renal ischemia-reperfusion.

The use of radiation treatments and chemotherapeutics, such as Gemcytabine and cyclophosphamide, are non-limiting examples of chemotherapeutic agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention. Those skilled in the art will recognize that other anti-cancer compounds and therapies can similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. siNA molecules) and are hence within the scope of the instant invention. Such compounds and therapies are well known in the art (see for example Cancer: Principles and Pranctice of Oncology, Volumes 1 and 2, eds Devita, V. T., Hellman, S., and Rosenberg, S. A., J. B. Lippincott Company, Philadelphia, USA; incorporated herein by reference) and include, without limitation, folates, antifolates, pyrimidine analogs, fluoropyrimidines, purine analogs, adenosine analogs, topoisomerase I inhibitors, anthrapyrazoles, retinoids, antibiotics, anthacyclins, platinum analogs, alkylating agents, nitrosoureas, plant derived compounds such as vinca alkaloids, epipodophyllotoxins, tyrosine kinase inhibitors, taxols, radiation therapy, surgery, nutritional supplements, gene therapy, radiotherapy, for example 3D-CRT, immunotoxin therapy, for example ricin, and monoclonal antibodies. Specific examples of chemotherapeutic compounds that can be combined with or used in conjuction with the nucleic acid molecules of the invention include, but are not limited to, Paclitaxel; Docetaxel; Methotrexate; Doxorubin; Edatrexate; Vinorelbine; Tomaxifen; Leucovorin; 5-fluoro uridine (5-FU); Ionotecan; Cisplatin; Carboplatin; Amsacrine; Cytarabine; Bleomycin; Mitomycin C; Dactinomycin; Mithramycin; Hexamethylmelamine; Dacarbazine; L-asperginase; Nitrogen mustard; Melphalan, Chlorambucil; Busulfan; Ifosfamide; 4-hydroperoxycyclophosphamide; Thiotepa; Irinotecan (CAMPTOSAR®, CPT-11, Camptothecin-11, Campto) Tamoxifen; Herceptin; IMC C225; ABX-EGF; and combinations thereof. Non-limiting examples of therapies and compounds that can be used in combination with siNA molecules of the invention for ocular based diseases and conditions include submacular surgery, focal laser retinal photocoagulation, limited macular translocation surgery, retina and retinal pigment epithelial transplantation, retinal microchip prosthesis, feeder vessel CNVM laser photocoagulation, interferon alpha treatment, intravitreal steroid therapy, transpupillary thermotherapy, membrane differential filtration therapy, aptamers targeting VEGF (e.g., Macugen™) and/or VEGF receptors, antibodies targeting VEGF (e.g., Lucentis™) and/or VEGF receptors, Visudyne™ (e.g. use in photodynamic therapy, PDT), anti-imflammatory compounds such as Celebrex™ or anecortave acetate (e.g., Retaane™), angiostatic steroids such as glucocorticoids, intravitreal implants such as Posurdex™, FGF2 modulators, antiangiogenic compounds such as squalamine, and/or VEGF traps and other cytokine traps (see for example Economides et al., 2003, Nature Medicine, 9, 47-52). The above list of compounds are non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA) of the instant invention. Those skilled in the art will recognize that other drug compounds and therapies can similarly be readily combined with the nucleic acid molecules of the instant invention (e.g., siNA molecules) are hence within the scope of the instant invention.

Example 13
Diagnostic Uses

The siNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates. siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siNA molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes, siNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siNA molecules and/or other chemical or biological molecules). Other in vitro uses of siNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).

In a specific example, siNA molecules that cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siNA molecules (i.e., those that cleave only wild-type forms of target RNA) are used to identify wild-type RNA present in the sample and the second siNA molecules (i.e., those that cleave only mutant forms of target RNA) are used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis requires two siNA molecules, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying siNA molecules with improved RNAi activity. The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

TABLE IVEGF and/or VEGFR Accession NumbersNM_005429Homo sapiens vascular endothelial growth factor C(VEGFC), mRNAgi|19924300|ref|NM_005429.2|[19924300]NM_003376Homo sapiens vascular endothelial growth factor(VEGF), mRNAgi|19923239|ref|NM_003376.2|[19923239]AF095785Homo sapiens vascular endothelial growth factor (VEGF)gene, promoter region andpartial cdsgi|4154290|gb|AF095785.1|[4154290]NM_003377Homo sapiens vascular endothelial growth factor B(VEGFB), mRNAgi|20070172|ref|NM_003377.2|[20070172]AF486837Homo sapiens vascular endothelial growth factorisoform VEGF165 (VEGF) mRNA,complete cdsgi|19909064|gb|AF486837.1|[19909064]AF468110Homo sapiens vascular endothelial growth factor Bisoform (VEGFB) gene, completecds, alternatively splicedgi|18766397|gb|AF468110.1|[18766397]AF437895Homo sapiens vascular endothelial growth factor (VEGF)gene, partial cdsgi|16660685|gb|AF437895.1|AF437895[16660685]AY047581Homo sapiens vascular endothelial growth factor (VEGF)mRNA, complete cdsgi|15422108|gb|AY047581.1|[15422108]AF063657Homo sapiens vascular endothelial growth factorreceptor (FLT1) mRNA, completecdsgi|3132830|gb|AF063657.1|AF063657[3132830]AF092127Homo sapiens vascular endothelial growth factor (VEGF)gene, partial sequencegi|4139168|gb|AF092127.1|AF092127[4139168]AF092126Homo sapiens vascular endothelial growth factor (VEGF)gene, 5′ UTRgi|4139167|gb|AF092126.1|AF092126[4139167]AF092125Homo sapiens vascular endothelial growth factor (VEGF)gene, partial cdsgi|4139165|gb|AF092125.1|AF092125[4139165]E15157Human VEGF mRNAgi|5709840|dbj|E15157.1| |pat|JP|1998052285|2[5709840]E15156Human VEGF mRNAgi|5709839|dbj|E15156.1| |pat|JP|1998052285|1[5709839]E14233Human mRNA for vascular endothelial growth factor(VEGF), complete cdsgi|5708916|dbj|E14233.1| |pat|JP|1997286795|1[5708916]AF024710Homo sapiens vascular endothelial growth factor (VEGF)mRNA, 3′UTRgi|2565322|gb|AF024710.1|AF024710[2565322]AJ010438Homo sapiens mRNA for vascular endothelial growthfactor, splicing variantVEGF183gi|3647280|emb|AJ010438.1|HSA010438[3647280]AF098331Homo sapiens vascular endothelial growth factor (VEGF)gene, promoter, partialsequencegi|4235431|gb|AF098331.1|AF098331[4235431]AF022375Homo sapiens vascular endothelial growth factor mRNA,complete cdsgi|3719220|gb|AF022375.1|AF022375[3719220]AH006909vascular endothelial growth factor {alternativesplicing} [human, Genomic, 414nt 5 segments]gi|1680143|gb|AH006909.1| |bbm|191843[1680143]U01134Human soluble vascular endothelial cell growth factorreceptor (sflt) mRNA,complete cdsgi|451321|gb|U01134.1|U01134[451321]E14000Human mRNA for FLTgi|3252767|dbj|E14000.1| |pat|JP|1997255700|1[3252767]E13332cDNA encoding vascular endodermal cell growth factorVEGFgi|3252137|dbj|E13332.1| |pat|JP|1997173075|1[3252137]E13256Human mRNA for FLT, complete cdsgi|3252061|dbj|E13256.1| |pat|JP|1997154588|1[3252061]AF063658Homo sapiens vascular endothelial growth factorreceptor 2 (KDR) mRNA, completecdsgi|3132832|gb|AF063658.1|AF063658[3132832]AJ000185Homo Sapiens mRNA for vascular endothelial growthfactor-Dgi|2879833|emb|AJ000185.1|HSAJ185[2879833]D89630Homo sapiens mRNA for VEGF-D, complete cdsgi|2780339|dbj|D89630.1|[2780339]AF035121Homo sapiens KDR/flk-1 protein mRNA, complete cdsgi|2655411|gb|AF035121.1|AF035121[2655411]AF020393Homo sapiens vascular endothelial growth factor Cgene, partial cds and 5′upstream regiongi|2582366|gb|AF020393.1|AF020393[2582366]Y08736H. sapiens vegf gene, 3′UTRgi|1619596|emb|Y08736.1|HSVEGF3UT[1619596]X62568H. sapiens vegf gene for vascular endothelial growthfactorgi|37658|emb|X62568.1|HSVEGF[37658]X94216H. sapiens mRNA for VEGF-C proteingi|1177488|emb|X94216.1|HSVEGFC[1177488]NM_002020Homo sapiens fms-related tyrosine kinase 4 (FLT4),mRNAgi|4503752|ref|NM_002020.1|[4503752]NM_002253Homo sapiens kinase insert domain receptor (a type IIIreceptor tyrosine kinase)(KDR), mRNAgi|11321596|ref|NM_002253.1|[11321596]

TABLE II

VEGF and/or VEGFR siNA AND TARGET SEQUENCES

Seq

Seq

Seq

Pos
Target Sequence
ID
UPos
Upper seq
ID
LPos
Lower seq
ID

VEGFR1/FLT1 NM_002019.1

1
GCGGACACUCCUCUCGGCU
1
1
GCGGACACUCCUCUCGGCU
1
19
AGCCGAGAGGAGUGUCCGC
428

19
UCCUCCCCGGCAGCGGCGG
2
19
UCCUCCCCGGCAGCGGCGG
2
37
CCGCCGCUGCCGGGGAGGA
429

37
GCGGCUCGGAGCGGGCUCC
3
37
GCGGCUCGGAGCGGGCUCC
3
55
GGAGCCCGCUCCGAGCCGC
430

55
CGGGGCUCGGGUGCAGCGG
4
55
CGGGGCUCGGGUGCAGCGG
4
73
CCGCUGCACCCGAGCCCCG
431

73
GCCAGCGGGCCUGGCGGCG
5
73
GCCAGCGGGCCUGGCGGCG
5
91
CGCCGCCAGGCCCGCUGGC
432

91
GAGGAUUACCCGGGGAAGU
6
91
GAGGAUUACCCGGGGAAGU
6
109
ACUUCCCCGGGUAAUCCUC
433

109
UGGUUGUCUCCUGGCUGGA
7
109
UGGUUGUCUCCUGGCUGGA
7
127
UCCAGCCAGGAGACAACCA
434

127
AGCCGCGAGACGGGCGCUC
8
127
AGCCGCGAGACGGGCGCUC
8
145
GAGCGCCCGUCUCGCGGCU
435

145
CAGGGCGCGGGGCCGGCGG
9
145
CAGGGCGCGGGGCCGGCGG
9
163
CCGCCGGCCCCGCGCCCUG
436

163
GCGGCGAACGAGAGGACGG
10
163
GCGGCGAACGAGAGGACGG
10
181
CCGUCCUCUCGUUCGCCGC
437

181
GACUCUGGCGGCCGGGUCG
11
181
GACUCUGGCGGCCGGGUCG
11
199
CGACCCGGCCGCCAGAGUC
438

199
GUUGGCCGGGGGAGCGCGG
12
199
GUUGGCCGGGGGAGCGCGG
12
217
CCGCGCUCCCCCGGCCAAC
439

217
GGCACCGGGCGAGCAGGCC
13
217
GGCACCGGGCGAGCAGGCC
13
235
GGCCUGCUCGCCCGGUGCC
440

235
CGCGUCGCGCUCACCAUGG
14
235
CGCGUCGCGCUCACCAUGG
14
253
CCAUGGUGAGCGCGACGCG
441

253
GUCAGCUACUGGGACACCG
15
253
GUCAGCUACUGGGACACCG
15
271
CGGUGUCCCAGUAGCUGAC
442

271
GGGGUCCUGCUGUGCGCGC
16
271
GGGGUCCUGCUGUGCGCGC
16
289
GCGCGCACAGCAGGACCCC
443

289
CUGCUCAGCUGUCUGCUUC
17
289
CUGCUCAGCUGUCUGCUUC
17
307
GAAGCAGACAGCUGAGCAG
444

307
CUCACAGGAUCUAGUUCAG
18
307
CUCACAGGAUCUAGUUCAG
18
325
CUGAACUAGAUCCUGUGAG
445

325
GGUUCAAAAUUAAAAGAUC
19
325
GGUUCAAAAUUAAAAGAUC
19
343
GAUCUUUUAAUUUUGAACC
446

343
CCUGAACUGAGUUUAAAAG
20
343
CCUGAACUGAGUUUAAAAG
20
361
CUUUUAAACUCAGUUCAGG
447

361
GGCACCCAGCACAUCAUGC
21
361
GGCACCCAGCACAUCAUGC
21
379
GCAUGAUGUGCUGGGUGCC
448

379
CAAGCAGGCCAGACACUGC
22
379
CAAGCAGGCCAGACACUGC
22
397
GCAGUGUCUGGCCUGCUUG
449

397
CAUCUCCAAUGCAGGGGGG
23
397
CAUCUCCAAUGCAGGGGGG
23
415
CCCCCCUGCAUUGGAGAUG
450

415
GAAGCAGCCCAUAAAUGGU
24
415
GAAGCAGCCCAUAAAUGGU
24
433
ACCAUUUAUGGGCUGCUUC
451

433
UCUUUGCCUGAAAUGGUGA
25
433
UCUUUGCCUGAAAUGGUGA
25
451
UCACCAUUUCAGGCAAAGA
452

451
AGUAAGGAAAGCGAAAGGC
26
451
AGUAAGGAAAGCGAAAGGC
26
469
GCCUUUCGCUUUCCUUACU
453

469
CUGAGCAUAACUAAAUCUG
27
469
CUGAGCAUAACUAAAUCUG
27
487
CAGAUUUAGUUAUGCUCAG
454

487
GCCUGUGGAAGAAAUGGCA
28
487
GCCUGUGGAAGAAAUGGCA
28
505
UGCCAUUUCUUCCACAGGC
455

505
AAACAAUUCUGCAGUACUU
29
505
AAACAAUUCUGCAGUACUU
29
523
AAGUACUGCAGAAUUGUUU
456

523
UUAACCUUGAACACAGCUC
30
523
UUAACCUUGAACACAGCUC
30
541
GAGCUGUGUUCAAGGUUAA
457

541
CAAGCAAACCACACUGGCU
31
541
CAAGCAAACCACACUGGCU
31
559
AGCCAGUGUGGUUUGCUUG
458

559
UUCUACAGCUGCAAAUAUC
32
559
UUCUACAGCUGCAAAUAUC
32
577
GAUAUUUGCAGCUGUAGAA
459

577
CUAGCUGUACCUACUUCAA
33
577
CUAGCUGUACCUACUUCAA
33
595
UUGAAGUAGGUACAGCUAG
460

595
AAGAAGAAGGAAACAGAAU
34
595
AAGAAGAAGGAAACAGAAU
34
613
AUUCUGUUUCCUUCUUCUU
461

613
UCUGCAAUCUAUAUAUUUA
35
613
UCUGCAAUCUAUAUAUUUA
35
631
UAAAUAUAUAGAUUGCAGA
462

631
AUUAGUGAUACAGGUAGAC
36
631
AUUAGUGAUACAGGUAGAC
36
649
GUCUACCUGUAUCACUAAU
463

649
CCUUUCGUAGAGAUGUACA
37
649
CCUUUCGUAGAGAUGUACA
37
667
UGUACAUCUCUACGAAAGG
464

667
AGUGAAAUCCCCGAAAUUA
38
667
AGUGAAAUCCCCGAAAUUA
38
685
UAAUUUCGGGGAUUUCACU
465

685
AUACACAUGACUGAAGGAA
39
685
AUACACAUGACUGAAGGAA
39
703
UUCCUUCAGUCAUGUGUAU
466

703
AGGGAGCUCGUCAUUCCCU
40
703
AGGGAGCUCGUCAUUCCCU
40
721
AGGGAAUGACGAGCUCCCU
467

721
UGCCGGGUUACGUCACCUA
41
721
UGCCGGGUUACGUCACCUA
41
739
UAGGUGACGUAACCCGGCA
468

739
AACAUCACUGUUACUUUAA
42
739
AACAUCACUGUUACUUUAA
42
757
UUAAAGUAACAGUGAUGUU
469

757
AAAAAGUUUCCACUUGACA
43
757
AAAAAGUUUCCACUUGACA
43
775
UGUCAAGUGGAAACUUUUU
470

775
ACUUUGAUCCCUGAUGGAA
44
775
ACUUUGAUCCCUGAUGGAA
44
793
UUCCAUCAGGGAUCAAAGU
471

793
AAACGCAUAAUCUGGGACA
45
793
AAACGCAUAAUCUGGGACA
45
811
UGUCCCAGAUUAUGCGUUU
472

811
AGUAGAAAGGGCUUCAUCA
46
811
AGUAGAAAGGGCUUCAUCA
46
829
UGAUGAAGCCCUUUCUACU
473

829
AUAUCAAAUGCAACGUACA
47
829
AUAUCAAAUGCAACGUACA
47
847
UGUACGUUGCAUUUGAUAU
474

847
AAAGAAAUAGGGCUUCUGA
48
847
AAAGAAAUAGGGCUUCUGA
48
865
UCAGAAGCCCUAUUUCUUU
475

865
ACCUGUGAAGCAACAGUCA
49
865
ACCUGUGAAGCAACAGUCA
49
883
UGACUGUUGCUUCACAGGU
476

883
AAUGGGCAUUUGUAUAAGA
50
883
AAUGGGCAUUUGUAUAAGA
50
901
UCUUAUACAAAUGCCCAUU
477

901
ACAAACUAUCUCACACAUC
51
901
ACAAACUAUCUCACACAUC
51
919
GAUGUGUGAGAUAGUUUGU
478

919
CGACAAACCAAUACAAUCA
52
919
CGACAAACCAAUACAAUCA
52
937
UGAUUGUAUUGGUUUGUCG
479

937
AUAGAUGUCCAAAUAAGCA
53
937
AUAGAUGUCCAAAUAAGCA
53
955
UGCUUAUUUGGACAUCUAU
480

955
ACACCACGCCCAGUCAAAU
54
955
ACACCACGCCCAGUCAAAU
54
973
AUUUGACUGGGCGUGGUGU
481

973
UUACUUAGAGGCCAUACUC
55
973
UUACUUAGAGGCCAUACUC
55
991
GAGUAUGGCCUCUAAGUAA
482

991
CUUGUCCUCAAUUGUACUG
56
991
CUUGUCCUCAAUUGUACUG
56
1009
CAGUACAAUUGAGGACAAG
483

1009
GCUACCACUCCCUUGAACA
57
1009
GCUACCACUCCCUUGAACA
57
1027
UGUUCAAGGGAGUGGUAGC
484

1027
ACGAGAGUUCAAAUGACCU
58
1027
ACGAGAGUUCAAAUGACCU
58
1045
AGGUCAUUUGAACUCUCGU
485

1045
UGGAGUUACCCUGAUGAAA
59
1045
UGGAGUUACCCUGAUGAAA
59
1063
UUUCAUCAGGGUAACUCCA
486

1063
AAAAAUAAGAGAGCUUCCG
60
1063
AAAAAUAAGAGAGCUUCCG
60
1081
CGGAAGCUCUCUUAUUUUU
487

1081
GUAAGGCGACGAAUUGACC
61
1081
GUAAGGCGACGAAUUGACC
61
1099
GGUCAAUUCGUCGCCUUAC
488

1099
CAAAGCAAUUCCCAUGCCA
62
1099
CAAAGCAAUUCCCAUGCCA
62
1117
UGGCAUGGGAAUUGCUUUG
489

1117
AACAUAUUCUACAGUGUUC
63
1117
AACAUAUUCUACAGUGUUC
63
1135
GAACACUGUAGAAUAUGUU
490

1135
CUUACUAUUGACAAAAUGC
64
1135
CUUACUAUUGACAAAAUGC
64
1153
GCAUUUUGUCAAUAGUAAG
491

1153
CAGAACAAAGACAAAGGAC
65
1153
CAGAACAAAGACAAAGGAC
65
1171
GUCCUUUGUCUUUGUUCUG
492

1171
CUUUAUACUUGUCGUGUAA
66
1171
CUUUAUACUUGUCGUGUAA
66
1189
UUACACGACAAGUAUAAAG
493

1189
AGGAGUGGACCAUCAUUCA
67
1189
AGGAGUGGACCAUCAUUCA
67
1207
UGAAUGAUGGUCCACUCCU
494

1207
AAAUCUGUUAACACCUCAG
68
1207
AAAUCUGUUAACACCUCAG
68
1225
CUGAGGUGUUAACAGAUUU
495

1225
GUGCAUAUAUAUGAUAAAG
69
1225
GUGCAUAUAUAUGAUAAAG
69
1243
CUUUAUCAUAUAUAUGCAC
496

1243
GCAUUCAUCACUGUGAAAC
70
1243
GCAUUCAUCACUGUGAAAC
70
1261
GUUUCACAGUGAUGAAUGC
497

1261
CAUCGAAAACAGCAGGUGC
71
1261
CAUCGAAAACAGCAGGUGC
71
1279
GCACCUGCUGUUUUCGAUG
498

1279
CUUGAAACCGUAGCUGGCA
72
1279
CUUGAAACCGUAGCUGGCA
72
1297
UGCCAGCUACGGUUUCUAG
499

1297
AAGCGGUCUUACCGGCUCU
73
1297
AAGCGGUCUUACCGGCUCU
73
1315
AGAGCCGGUAAGACCGCUU
500

1315
UCUAUGAAAGUGAAGGCAU
74
1315
UCUAUGAAAGUGAAGGCAU
74
1333
AUGCCUUCACUUUCAUAGA
501

1333
UUUCCCUCGCCGGAAGUUG
75
1333
UUUCCCUCGCCGGAAGUUG
75
1351
CAACUUCCGGCGAGGGAAA
502

1351
GUAUGGUUAAAAGAUGGGU
76
1351
GUAUGGUUAAAAGAUGGGU
76
1369
ACCCAUCUUUUAACCAUAC
503

1369
UUACCUGCGACUGAGAAAU
77
1369
UUACCUGCGACUGAGAAAU
77
1387
AUUUCUCAGUCGCAGGUAA
504

1387
UCUGCUCGCUAUUUGACUC
78
1387
UCUGCUCGCUAUUUGACUC
78
1405
GAGUCAAAUAGCGAGCAGA
505

1405
CGUGGCUACUCGUUAAUUA
79
1405
CGUGGCUACUCGUUAAUUA
79
1423
UAAUUAACGAGUAGCCACG
506

1423
AUCAAGGACGUAACUGAAG
80
1423
AUCAAGGACGUAACUGAAG
80
1441
CUUCAGUUACGUCCUUGAU
507

1441
GAGGAUGCAGGGAAUUAUA
81
1441
GAGGAUGCAGGGAAUUAUA
81
1459
UAUAAUUCCCUGCAUCCUC
508

1459
ACAAUCUUGCUGAGCAUAA
82
1459
ACAAUCUUGCUGAGCAUAA
82
1477
UUAUGCUCAGCAAGAUUGU
509

1477
AAACAGUCAAAUGUGUUUA
83
1477
AAACAGUCAAAUGUGUUUA
83
1495
UAAACACAUUUGACUGUUU
510

1495
AAAAACCUCACUGCCACUC
84
1495
AAAAACCUCACUGCCACUC
84
1513
GAGUGGCAGUGAGGUUUUU
511

1513
CUAAUUGUCAAUGUGAAAC
85
1513
CUAAUUGUCAAUGUGAAAC
85
1531
GUUUCACAUUGACAAUUAG
512

1531
CCCCAGAUUUACGAAAAGG
86
1531
CCCCAGAUUUACGAAAAGG
86
1549
CCUUUUCGUA8AUCUGGGG
513

1549
GCCGUGUCAUCGUUUCCAG
87
1549
GCCGUGUCAUCGUUUCCAG
87
1567
CUGGAAACGAUGACACGGC
514

1567
GACCCGGCUCUCUACCCAC
88
1567
GACCCGGCUCUCUACCCAC
88
1585
GUGGGUAGAGAGCCGGGUC
515

1585
CUGGGCAGCAGACAAAUCC
89
1585
CUGGGCAGCAGACAAAUCC
89
1603
GGAUUUGUCUGCUGCCCAG
516

1603
CUGACUUGUACCGCAUAUG
90
1603
CUGACUUGUACCGCAUAUG
90
1621
CAUAUGCGGUACAAGUCAG
517

1621
GGUAUCCCUCAACCUACAA
91
1621
GGUAUCCCUCAACCUACAA
91
1639
UUGUAGGUUGAGGGAUACC
518

1639
AUCAAGUGGUUCUGGCACC
92
1639
AUCAAGUGGUUCUGGCACC
92
1657
GGUGCCAGAACCACUUGAU
519

1657
CCCUGUAACCAUAAUCAUU
93
1657
CCCUGUAACCAUAAUCAUU
93
1675
AAUGAUUAUGGUUACAGGG
520

1675
UCCGAAGCAAGGUGUGACU
94
1675
UCCGAAGCAAGGUGUGACU
94
1693
AGUCACACCUUGCUUCGGA
521

1693
UUUUGUUCCAAUAAUGAAG
95
1693
UUUUGUUCCAAUAAUGAAG
95
1711
CUUCAUUAUUGGAACAAAA
522

1711
GAGUCCUUUAUCCUGGAUG
96
1711
GAGUCCUUUAUCCUGGAUG
96
1729
CAUCCAGGAUAAAGGACUC
523

1729
GCUGACAGCAACAUGGGAA
97
1729
GCUGACAGCAACAUGGGAA
97
1747
UUCCCAUGUUGCUGUCAGC
524

1747
AACAGAAUUGAGAGCAUCA
98
1747
AACAGAAUUGAGAGCAUCA
98
1765
UGAUGCUCUCPAUUCUGUU
525

1765
ACUCAGCGCAUGGCAAUAA
99
1765
ACUCAGCGCAUGGCAAUAA
99
1783
UUAUUGCCAUGCGCUGAGU
526

1783
AUAGAAGGAAAGAAUAAGA
100
1783
AUAGAAGGAAAGAAUAAGA
100
1801
UCUUAUUCUUUCCUUCUAU
527

1801
AUGGCUAGCACCUUGGUUG
101
1801
AUGGCUAGCACCUUGGUUG
101
1819
CAACCAAGGUGCUAGCCAU
528

1819
GUGGCUGACUCUAGAAUUU
102
1819
GUGGCUGACUCUAGAAUUU
102
1837
AAAUUCUAGAGUCAGCCAC
529

1837
UCUGGAAUCUACAUUUGCA
103
1837
UCUGGAAUCUACAUUUGCA
103
1855
UGCAAAUGUAGAUUCCAGA
530

1855
AUAGCUUCCAAUAAAGUUG
104
1855
AUAGCUUCCAAUAAAGUUG
104
1873
CAACUUUAUUGGAAGCUAU
531

1873
GGGACUGUGGGAAGAAACA
105
1873
GGGACUGUGGGAAGAAACA
105
1891
UGUUUCUUCCCACAGUCCC
532

1891
AUAAGCUUUUAUAUCACAG
106
1891
AUAAGCUUUUAUAUCACAG
106
1909
CUGUGAUAUAAAAGCUUAU
533

1909
GAUGUGCCAAAUGGGUUUC
107
1909
GAUGUGCCAAAUGGGUUUC
107
1927
GAAACCCAUUUGGCACAUC
534

1927
CAUGUUAACUUGGAAAAAA
108
1927
CAUGUUAACUUGGAAAAAA
108
1945
UUUUUUCCAAGUUAACAUG
535

1945
AUGCCGACGGAAGGAGAGG
109
1945
AUGCCGACGGAAGGAGAGG
109
1963
CCUCUCCUUCCGUCGGCAU
536

1963
GACCUGAAACUGUCUUGCA
110
1963
GACCUGAAACUGUCUUGCA
110
1981
UGCAAGACAGUUUCAGGUC
537

1981
ACAGUUAACAAGUUCUUAU
111
1981
ACAGUUAACAAGUUCUUAU
111
1999
AUAAGAACUUGUUAACUGU
538

1999
UACAGAGACGUUACUUGGA
112
1999
UACAGAGACGUUACUUGGA
112
2017
UCCAAGUAACGUCUCUGUA
539

2017
AUUUUACUGCGGACAGUUA
113
2017
AUUUUACUGCGGACAGUUA
113
2035
UAACUGUCCGCAGUAAAAU
540

2035
AAUAACAGAACAAUGCACU
114
2035
AAUAACAGAACAAUGCACU
114
2053
AGUGCAUUGUUCUGUUAUU
541

2053
UACAGUAUUAGCAAGCAAA
115
2053
UACAGUAUUAGCAAGCAAA
115
2071
UUUGCUUGCUAAUACUGUA
542

2071
AAAAUGGCCAUCACUAAGG
116
2071
AAAAUGGCCAUCACUAAGG
116
2089
CCUUAGUGAUGGCCAUUUU
543

2089
GAGCACUCCAUCACUCUUA
117
2089
GAGCACUCCAUCACUCUUA
117
2107
UAAGAGUGAUGGAGUGCUC
544

2107
AAUCUUACCAUCAUGAAUG
118
2107
AAUCUUACCAUCAUGAAUG
118
2125
CAUUCAUGAUGGUAAGAUU
545

2125
GUUUCCCUGCAAGAUUCAG
119
2125
GUUUCCCUGCAAGAUUCAG
119
2143
CUGAAUCUUGCAGGGAAAC
546

2143
GGCACCUAUGCCUGCAGAG
120
2143
GGCACCUAUGCCUGCAGAG
120
2161
CUCUGCAGGCAUAGGUGCC
547

2161
GCCAGGAAUGUAUACACAG
121
2161
GCCAGGAAUGUAUACACAG
121
2179
CUGUGUAUACAUUCCUGGC
548

2179
GGGGAAGAAAUCCUCCAGA
122
2179
GGGGAAGAAAUCCUCCAGA
122
2197
UCUGGAGGAUUUCUUCCCC
549

2197
AAGAAAGAAAUUACAAUCA
123
2197
AAGAAAGAAAUUACAAUCA
123
2215
UGAUUGUAAUUUCUUUCUU
550

2215
AGAGAUCAGGAAGCACCAU
124
2215
AGAGAUCAGGAAGCACCAU
124
2233
AUGGUGCUUCCUGAUCUCU
551

2233
UACCUCCUGCGAAACCUCA
125
2233
UACCUCCUGCGAAACCUCA
125
2251
UGAGGUUUCGCAGGAGGUA
552

2251
AGUGAUCACACAGUGGCCA
126
2251
AGUGAUCACACAGUGGCCA
126
2269
UGGCCACUGUGUGAUCACU
553

2269
AUCAGCAGUUCCACCACUU
127
2269
AUCAGCAGUUCCACCACUU
127
2287
AAGUGGUGGAACUGCUGAU
554

2287
UUAGACUGUCAUGCUAAUG
128
2287
UUAGACUGUCAUGCUAAUG
128
2305
CAUUAGCAUGACAGUCUAA
555

2305
GGUGUCCCCGAGCCUCAGA
129
2305
GGUGUCCCCGAGCCUCAGA
129
2323
UCUGAGGCUCGGGGACACC
556

2323
AUCACUUGGUUUAAAAACA
130
2323
AUCACUUGGUUUAAAAACA
130
2341
UGUUUUUAAACCAAGUGAU
557

2341
AACCACAAAAUACAACAAG
131
2341
AACCACAAAAUACAACAAG
131
2359
CUUGUUGUAUUUUGUGGUU
558

2359
GAGCCUGGAAUUAUUUUAG
132
2359
GAGCCUGGAAUUAUUUUAG
132
2377
CUAAAAUAAUUCCAGGCUC
559

2377
GGACCAGGAAGCAGCACGC
133
2377
GGACCAGGAAGCAGCACGC
133
2395
GCGUGCUGCUUCCUGGUCC
560

2395
CUGUUUAUUGAAAGAGUCA
134
2395
CUGUUUAUUGAAAGAGUCA
134
2413
UGACUCUUUCAAUAAACAG
561

2413
ACAGAAGAGGAUGAAGGUG
135
2413
ACAGAAGAGGAUGAAGGUG
135
2431
CACCUUCAUCCUCUUCUGU
562

2431
GUCUAUCACUGCAAAGCCA
136
2431
GUCUAUCACUGCAAAGCCA
136
2449
UGGCUUUGCAGUGAUAGAC
563

2449
ACCAACCAGAAGGGCUCUG
137
2449
ACCAACCAGAAGGGCUCUG
137
2467
CAGAGCCCUUCUGGUUGGU
564

2467
GUGGAAAGUUCAGCAUACC
138
2467
GUGGAAAGUUCAGCAUACC
138
2485
GGUAUGCUGAACUUUCCAC
565

2485
CUCACUGUUCAAGGAACCU
139
2485
CUCACUGUUCAAGGAACCU
139
2503
AGGUUCCUUGAACAGUGAG
566

2503
UCGGACAAGUCUAAUCUGG
140
2503
UCGGACAAGUCUAAUCUGG
140
2521
CCAGAUUAGACUUGUCCGA
567

2521
GAGCUGAUCACUCUAACAU
141
2521
GAGCUGAUCACUCUAACAU
141
2539
AUGUUAGAGUGAUCAGCUC
568

2539
UGCACCUGUGUGGCUGCGA
142
2539
UGCACCUGUGUGGCUGCGA
142
2557
UCGCAGCCACACAGGUGCA
569

2557
ACUCUCUUCUGGCUCCUAU
143
2557
ACUCUCUUCUGGCUCCUAU
143
2575
AUAGGAGCCAGAAGAGAGU
570

2575
UUAACCCUCCUUAUCCGAA
144
2575
UUAACCCUCCUUAUCCGAA
144
2593
UUCGGAUAAGGAGGGUUAA
571

2593
AAAAUGAAAAGGUCUUCUU
145
2593
AAAAUGAAAAGGUCUUCUU
145
2611
AAGAAGACCUUUUCAUUUU
572

2611
UCUGAAAUAAAGACUGACU
146
2611
UCUGAAAUAAAGACUGACU
146
2629
AGUCAGUCUUUAUUUCAGA
573

2629
UACCUAUCAAUUAUAAUGG
147
2629
UACCUAUCAAUUAUAAUGG
147
2647
CCAUUAUAAUUGAUAGGUA
574

2647
GACCCAGAUGAAGUUCCUU
148
2647
GACCCAGAUGAAGUUCCUU
148
2665
AAGGAACUUCAUCUGGGUC
575

2665
UUGGAUGAGCAGUGUGAGC
149
2665
UUGGAUGAGCAGUGUGAGC
149
2683
GCUCACACUGCUCAUCCAA
576

2683
CGGCUCCCUUAUGAUGCCA
150
2683
CGGCUCCCUUAUGAUGCCA
150
2701
UGGCAUCAUAAGGGAGCCG
577

2701
AGCAAGUGGGAGUUUGCCC
151
2701
AGCAAGUGGGAGUUUGCCC
151
2719
GGGCAAACUCCCACUUGCU
578

2719
CGGGAGAGACUUAAACUGG
152
2719
CGGGAGAGACUUAAACUGG
152
2737
CCAGUUUAAGUCUCUCCCG
579

2737
GGCAAAUCACUUGGAAGAG
153
2737
GGCAAAUCACUUGGAAGAG
153
2755
CUCUUCCAAGUGAUUUGCC
580

2755
GGGGCUUUUGGAAAAGUGG
154
2755
GGGGCUUUUGGAAAAGUGG
154
2773
CCACUUUUCCAAAAGCCCC
581

2773
GUUCAAGCAUCAGCAUUUG
155
2773
GUUCAAGCAUCAGCAUUUG
155
2791
CAAAUGCUGAUGCUUGAAC
582

2791
GGCAUUAAGAAAUCACCUA
156
2791
GGCAUUAAGAAAUCACCUA
156
2809
UAGGUGAUUUCUUAAUGCC
583

2809
ACGUGCCGGACUGUGGCUG
157
2809
ACGUGCCGGACUGUGGCUG
157
2827
CAGCCACAGUCCGGCACGU
584

2827
GUGAAAAUGCUGAAAGAGG
158
2827
GUGAAAAUGCUGAAAGAGG
158
2845
CCUCUUUCAGCAUUUUCAC
585

2845
GGGGCCACGGCCAGCGAGU
159
2845
GGGGCCACGGCCAGCGAGU
159
2863
ACUCGCUGGCCGUGGCCCC
586

2863
UACAAAGCUCUGAUGACUG
160
2863
UACAAAGCUCUGAUGACUG
160
2881
CAGUCAUCAGAGCUUUGUA
587

2881
GAGCUAAAAAUCUUGACCC
161
2881
GAGCUAAAAAUCUUGACCC
161
2899
GGGUCAAGAUUUUUAGCUC
588

2899
CACAUUGGCCACCAUCUGA
162
2899
CACAUUGGCCACCAUCUGA
162
2917
UCAGAUGGUGGCCAAUGUG
589

2917
AACGUGGUUAACCUGCUGG
163
2917
AACGUGGUUAACCUGCUGG
163
2935
CCAGCAGGUUAACCACGUU
590

2935
GGAGCCUGCACCAAGCAAG
164
2935
GGAGCCUGCACCAAGCAAG
164
2953
CUUGCUUGGUGCAGGCUCC
591

2953
GGAGGGCCUCUGAUGGUGA
165
2953
GGAGGGCCUCUGAUGGUGA
165
2971
UCACCAUCAGAGGCCCUCC
592

2971
AUUGUUGAAUACUGCAAAU
166
2971
AUUGUUGAAUACUGCAAAU
166
2989
AUUUGCAGUAUUCAACAAU
593

2989
UAUGGAAAUCUCUCCAACU
167
2989
UAUGGAAAUCUCUCCAACU
167
3007
AGUUGGAGAGAUUUCCAUA
594

3007
UACCUCAAGAGCAAACGUG
168
3007
UACCUCAAGAGCAAACGUG
168
3025
CACGUUUGCUCUUGAGGUA
595

3025
GACUUAUUUUUUCUCAACA
169
3025
GACUUAUUUUUUCUCAACA
169
3043
UGUUGAGAAAAAAUAAGUC
596

3043
AAGGAUGCAGCACUACACA
170
3043
AAGGAUGCAGCACUACACA
170
3061
UGUGUAGUGCUGCAUCCUU
597

3061
AUGGAGCCUAAGAAAGAAA
171
3061
AUGGAGCCUAAGAAAGAAA
171
3079
UUUCUUUCUUAGGCUCCAU
598

3079
AAAAUGGAGCCAGGCCUGG
172
3079
AAAAUGGAGCCAGGCCUGG
172
3097
CCAGGCCUGGCUCCAUUUU
599

3097
GAACAAGGCAAGAAACCAA
173
3097
GAACAAGGCAAGAAACCAA
173
3115
UUGGUUUCUUGCCUUGUUC
600

3115
AGACUAGAUAGCGUCACCA
174
3115
AGACUAGAUAGCGUCACCA
174
3133
UGGUGACGCUAUCUAGUCU
601

3133
AGCAGCGAAAGCUUUGCGA
175
3133
AGCAGCGAAAGCUUUGCGA
175
3151
UCGCAAAGCUUUCGCUGCU
602

3151
AGCUCCGGCUUUCAGGAAG
176
3151
AGCUCCGGCUUUCAGGAAG
176
3169
CUUCCUGAAAGCCGGAGCU
603

3169
GAUAAAAGUCUGAGUGAUG
177
3169
GAUAAAAGUCUGAGUGAUG
177
3187
CAUCACUCAGACUUUUAUC
604

3187
GUUGAGGAAGAGGAGGAUU
178
3187
GUUGAGGAAGAGGAGGAUU
178
3205
AAUCCUCCUCUUCCUCAAC
605

3205
UCUGACGGUUUCUACAAGG
179
3205
UCUGACGGUUUCUACAAGG
179
3223
CCUUGUAGAAACCGUCAGA
606

3223
GAGCCCAUCACUAUGGAAG
180
3223
GAGCCCAUCACUAUGGAAG
180
3241
CUUCCAUAGUGAUGGGCUC
607

3241
GAUCUGAUUUCUUACAGUU
181
3241
GAUCUGAUUUCUUACAGUU
181
3259
AACUGUAAGAAAUCAGAUC
608

3259
UUUCAAGUGGCCAGAGGCA
182
3259
UUUCAAGUGGCCAGAGGCA
182
3277
UGCCUCUGGCCACUUGAAA
609

3277
AUGGAGUUCCUGUCUUCCA
183
3277
AUGGAGUUCCUGUCUUCCA
183
3295
UGGAAGACAGGAACUCCAU
610

3295
AGAAAGUGCAUUCAUCGGG
184
3295
AGAAAGUGCAUUCAUCGGG
184
3313
CCCGAUGAAUGCACUUUCU
611

3313
GACCUGGCAGCGAGAAACA
185
3313
GACCUGGCAGCGAGAAACA
185
3331
UGUUUCUCGCUGCCAGGUC
612

3331
AUUCUUUUAUCUGAGAACA
186
3331
AUUCUUUUAUCUGAGAACA
186
3349
UGUUCUCAGAUAAAAGAAU
613

3349
AACGUGGUGAAGAUUUGUG
187
3349
AACGUGGUGAAGAUUUGUG
187
3367
CACAAAUCUUCACCACGUU
614

3367
GAUUUUGGCCUUGCCCGGG
188
3367
GAUUUUGGCCUUGCCCGGG
188
3385
CCCGGGCAAGGCCAAAAUC
615

3385
GAUAUUUAUAAGAACCCCG
189
3385
GAUAUUUAUAAGAACCCCG
189
3403
CGGGGUUCUUAUAAAUAUC
616

3403
GAUUAUGUGAGAAAAGGAG
190
3403
GAUUAUGUGAGAAAAGGAG
190
3421
CUCCUUUUCUCACAUAAUC
617

3421
GAUACUCGACUUCCUCUGA
191
3421
GAUACUCGACUUCCUCUGA
191
3439
UCAGAGGAAGUCGAGUAUC
618

3439
AAAUGGAUGGCUCCCGAAU
192
3439
AAAUGGAUGGCUCCCGAAU
192
3457
AUUCGGGAGCCAUCCAUUU
619

3457
UCUAUCUUUGACAAAAUCU
193
3457
UCUAUCUUUGACAAAAUCU
193
3475
AGAUUUUGUCAAAGAUAGA
620

3475
UACAGCACCAAGAGCGACG
194
3475
UACAGCACCAAGAGCGACG
194
3493
CGUCGCUCUUGGUGCUGUA
621

3493
GUGUGGUCUUACGGAGUAU
195
3493
GUGUGGUCUUACGGAGUAU
195
3511
AUACUCCGUAAGACCACAC
622

3511
UUGCUGUGGGAAAUCUUCU
196
3511
UUGCUGUGGGAAAUCUUCU
196
3529
AGAAGAUUUCCCACAGCAA
623

3529
UCCUUAGGUGGGUCUCCAU
197
3529
UCCUUAGGUGGGUCUCCAU
197
3547
AUGGAGACCCACCUAAGGA
624

3547
UACCCAGGAGUACAAAUGG
198
3547
UACCCAGGAGUACAAAUGG
198
3565
CCAUUUGUACUCCUGGGUA
625

3565
GAUGAGGACUUUUGCAGUC
199
3565
GAUGAGGACUUUUGCAGUC
199
3583
GACUGCAAAAGUCCUCAUC
626

3583
CGCCUGAGGGAAGGCAUGA
200
3583
CGCCUGAGGGAAGGCAUGA
200
3601
UCAUGCCUUCCCUCAGGCG
627

3601
AGGAUGAGAGCUCCUGAGU
201
3601
AGGAUGAGAGCUCCUGAGU
201
3619
ACUCAGGAGCUCUCAUCCU
628

3619
UACUCUACUCCUGAAAUCU
202
3619
UACUCUACUCCUGAAAUCU
202
3637
AGAUUUCAGGAGUAGAGUA
629

3637
UAUCAGAUCAUGCUGGACU
203
3637
UAUCAGAUCAUGCUGGACU
203
3655
AGUCCAGCAUGAUCUGAUA
630

3655
UGCUGGCACAGAGACCCAA
204
3655
UGCUGGCACAGAGACCCAA
204
3673
UUGGGUCUCUGUGCCAGCA
631

3673
AAAGAAAGGCCAAGAUUUG
205
3673
AAAGAAAGGCCAAGAUUUG
205
3691
CAAAUCUUGGCCUUUCUUU
632

3691
GCAGAACUUGUGGAAAAAC
206
3691
GCAGAACUUGUGGAAAAAC
206
3709
GUUUUUCCACAAGUUCUGC
633

3709
CUAGGUGAUUUGCUUCAAG
207
3709
CUAGGUGAUUUGCUUCAAG
207
3727
CUUGAAGCAAAUCACCUAG
634

3727
GCAAAUGUACAACAGGAUG
208
3727
GCAAAUGUACAACAGGAUG
208
3745
CAUCCUGUUGUACAUUUGC
635

3745
GGUAAAGACUACAUCCCAA
209
3745
GGUAAAGACUACAUCCCAA
209
3763
UUGGGAUGUAGUCUUUACC
636

3763
AUCAAUGCCAUACUGACAG
210
3763
AUCAAUGCCAUACUGACAG
210
3781
CUGUCAGUAUGGCAUUGAU
637

3781
GGAAAUAGUGGGUUUACAU
211
3781
GGAAAUAGUGGGUUUACAU
211
3799
AUGUAAACCCACUAUUUCC
638

3799
UACUCAACUCCUGCCUUCU
212
3799
UACUCAACUCCUGCCUUCU
212
3817
AGAAGGCAGGAGUUGAGUA
639

3817
UCUGAGGACUUCUUCAAGG
213
3817
UCUGAGGACUUCUUCAAGG
213
3835
CCUUGAAGAAGUCCUCAGA
640

3835
GAAAGUAUUUCAGCUCCGA
214
3835
GAAAGUAUUUCAGCUCCGA
214
3853
UCGGAGCUGAAAUACUUUC
641

3853
AAGUUUAAUUCAGGAAGCU
215
3853
AAGUUUAAUUCAGGAAGCU
215
3871
AGCUUCCUGAAUUAAACUU
642

3871
UCUGAUGAUGUCAGAUAUG
216
3871
UCUGAUGAUGUCAGAUAUG
216
3889
CAUAUCUGACAUCAUCAGA
643

3889
GUAAAUGCUUUCAAGUUCA
217
3889
GUAAAUGCUUUCAAGUUCA
217
3907
UGAACUUGAAAGCAUUUAC
644

3907
AUGAGCCUGGAAAGAAUCA
218
3907
AUGAGCCUGGAAAGAAUCA
218
3925
UGAUUCUUUCCAGGCUCAU
645

3925
AAAACCUUUGAAGAACUUU
219
3925
AAAACCUUUGAAGAACUUU
219
3943
AAAGUUCUUCAAAGGUUUU
646

3943
UUACCGAAUGCCACCUCCA
220
3943
UUACCGAAUGCCACCUCCA
220
3961
UGGAGGUGGCAUUCGGUAA
647

3961
AUGUUUGAUGACUACCAGG
221
3961
AUGUUUGAUGACUACCAGG
221
3979
CCUGGUAGUCAUCAAACAU
648

3979
GGCGACAGCAGCACUCUGU
222
3979
GGCGACAGCAGCACUCUGU
222
3997
ACAGAGUGCUGCUGUCGCC
649

3997
UUGGCCUCUCCCAUGCUGA
223
3997
UUGGCCUCUCCCAUGCUGA
223
4015
UCAGCAUGGGAGAGGCCAA
650

4015
AAGCGCUUCACCUGGACUG
224
4015
AAGCGCUUCACCUGGACUG
224
4033
CAGUCCAGGUGAAGCGCUU
651

4033
GACAGCAAACCCAAGGCCU
225
4033
GACAGCAAACCCAAGGCCU
225
4051
AGGCCUUGGGUUUGCUGUC
652

4051
UCGCUCAAGAUUGACUUGA
226
4051
UCGCUCAAGAUUGACUUGA
226
4069
UCAAGUCAAUCUUGAGCGA
653

4069
AGAGUAACCAGUAAAAGUA
227
4069
AGAGUAACCAGUAAAAGUA
227
4087
UACUUUUACUGGUUACUCU
654

4087
AAGGAGUCGGGGCUGUCUG
228
4087
AAGGAGUCGGGGCUGUCUG
228
4105
CAGACAGCCCCGACUCCUU
655

4105
GAUGUCAGCAGGCCCAGUU
229
4105
GAUGUCAGCAGGCCCAGUU
229
4123
AACUGGGCCUGCUGACAUC
656

4123
UUCUGCCAUUCCAGCUGUG
230
4123
UUCUGCCAUUCCAGCUGUG
230
4141
CACAGCUGGAAUGGCAGAA
657

4141
GGGCACGUCAGCGAAGGCA
231
4141
GGGCACGUCAGCGAAGGCA
231
4159
UGCCUUCGCUGACGUGCCC
658

4159
~AGCGCAGGUUCACCUACG
232
4159
AAGCGCAGGUUCACCUACG
232
4177
CGUAGGUGAACCUGCGCUU
659

4177
GACCACGCUGAGCUGGAAA
233
4177
GACCACGCUGAGCUGGAAA
233
4195
UUUCCAGCUCAGCGUGGUC
660

4195
AGGAAAAUCGCGUGCUGCU
234
4195
AGGAAAAUCGCGUGCUGCU
234
4213
AGCAGCACGCGAUUUUCCU
661

4213
UCCCCGCCCCCAGACUACA
235
4213
UCCCCGCCCCCAGACUACA
235
4231
UGUAGUCUGGGGGCGGGGA
662

4231
AACUCGGUGGUCCUGUACU
236
4231
AACUCGGUGGUCCUGUACU
236
4249
AGUACAGGACCACCGAGUU
663

4249
UCCACCCCACCCAUCUAGA
237
4249
UCCACCCCACCCAUCUAGA
237
4267
UCUAGAUGGGUGGGGUGGA
664

4267
AGUUUGACACGAAGCCUUA
238
4267
AGUUUGACACGAAGCCUUA
238
4285
UAAGGCUUCGUGUCAAACU
665

4285
AUUUCUAGAAGCACAUGUG
239
4285
AUUUCUAGAAGCACAUGUG
239
4303
CACAUGUGCUUCUAGAAAU
666

4303
GUAUUUAUACCCCCAGGAA
240
4303
GUAUUUAUACCCCCAGGAA
240
4321
UUCCUGGGGGUAUAAAUAC
667

4321
AACUAGCUUUUGCCAGUAU
241
4321
AACUAGCUUUUGCCAGUAU
241
4339
AUACUGGCAAAAGCUAGUU
668

4339
UUAUGCAUAUAUAAGUUUA
242
4339
UUAUGCAUAUAUAAGUUUA
242
4357
UAAACUUAUAUAUGCAUAA
669

4357
ACACCUUUAUCUUUCCAUG
243
4357
ACACCUUUAUCUUUCCAUG
243
4375
CAUGGAAAGAUAAAGGUGU
670

4375
GGGAGCCAGCUGCUUUUUG
244
4375
GGGAGCCAGCUGCUUUUUG
244
4393
CAAAAAGCAGCUGGCUCCC
671

4393
GUGAUUUUUUUAAUAGUGC
245
4393
GUGAUUUUUUUAAUAGUGC
245
4411
GCACUAUUAAAAAAAUCAC
672

4411
CUUUUUUUUUUUGACUAAC
246
4411
CUUUUUUUUUUUGACUAAC
246
4429
GUUAGUCAAAAAAAAAAAG
673

4429
CAAGAAUGUAACUCCAGAU
247
4429
CAAGAAUGUAACUCCAGAU
247
4447
AUCUGGAGUUACAUUCUUG
674

4447
UAGAGAAAUAGUGACAAGU
248
4447
UAGAGAAAUAGUGACAAGU
248
4465
ACUUGUCACUAUUUCUCUA
675

4465
UGAAGAACACUACUGCUAA
249
4465
UGAAGAACACUACUGCUAA
249
4483
UUAGCAGUAGUGUUCUUCA
676

4483
AAUCCUCAUGUUACUCAGU
250
4483
AAUCCUCAUGUUACUCAGU
250
4501
ACUGAGUAACAUGAGGAUU
677

4501
UGUUAGAGAAAUCCUUCCU
251
4501
UGUUAGAGAAAUCCUUCCU
251
4519
AGGAAGGAUUUCUCUAACA
678

4519
UAAACCCAAUGACUUCCCU
252
4519
UAAACCCAAUGACUUCCCU
252
4537
AGGGAAGUCAUUGGGUUUA
679

4537
UGCUCCAACCCCCGCCACC
253
4537
UGCUCCAACCCCCGCCACC
253
4555
GGUGGCGGGGGUUGGAGCA
680

4555
CUCAGGGCACGCAGGACCA
254
4555
CUCAGGGCACGCAGGACCA
254
4573
UGGUCCUGCGUGCCCUGAG
681

4573
AGUUUGAUUGAGGAGCUGC
255
4573
AGUUUGAUUGAGGAGCUGC
255
4591
GCAGCUCCUCAAUCAAACU
682

4591
CACUGAUCACCCAAUGCAU
256
4591
CACUGAUCACCCAAUGCAU
256
4609
AUGCAUUGGGUGAUCAGUG
683

4609
UCACGUACCCCACUGGGCC
257
4609
UCACGUACCCCACUGGGCC
257
4627
GGCCCAGUGGGGUACGUGA
684

4627
CAGCCCUGCAGCCCAAAAC
258
4627
CAGCCCUGCAGCCCAAAAC
258
4645
GUUUUGGGCUGCAGGGCUG
685

4645
CCCAGGGCAACAAGCCCGU
259
4645
CCCAGGGCAACAAGCCCGU
259
4663
ACGGGCUUGUUGCCCUGGG
686

4663
UUAGCCCCAGGGGAUCACU
260
4663
UUAGCCCCAGGGGAUCACU
260
4681
AGUGAUCCCCUGGGGCUAA
687

4681
UGGCUGGCCUGAGCAACAU
261
4681
UGGCUGGCCUGAGCAACAU
261
4699
AUGUUGCUCAGGCCAGCCA
688

4699
UCUCGGGAGUCCUCUAGCA
262
4699
UCUCGGGAGUCCUCUAGCA
262
4717
UGCUAGAGGACUCCCGAGA
689

4717
AGGCCUAAGACAUGUGAGG
263
4717
AGGCCUAAGACAUGUGAGG
263
4735
CCUCACAUGUCUUAGGCCU
690

4735
GAGGAAAAGGAAAAAAAGC
264
4735
GAGGAAAAGGAAAAAAAGC
264
4753
GCUUUUUUUCCUUUUCCUC
691

4753
CAAAAAGCAAGGGAGAAAA
265
4753
CAAAAAGCAAGGGAGAAAA
265
4771
UUUUCUCCCUUGCUUUUUG
692

4771
AGAGAAACCGGGAGAAGGC
266
4771
AGAGAAACCGGGAGAAGGC
266
4789
GCCUUCUCCCGGUUUCUCU
693

4789
CAUGAGAAAGAAUUUGAGA
267
4789
CAUGAGAAAGAAUUUGAGA
267
4807
UCUCAAAUUCUUUCUCAUG
694

4807
ACGCACCAUGUGGGCACGG
268
4807
ACGCACCAUGUGGGCACGG
268
4825
CCGUGCCCACAUGGUGCGU
695

4825
GAGGGGGACGGGGCUCAGC
269
4825
GAGGGGGACGGGGCUCAGC
269
4843
GCUGAGCCCCGUCCCCCUC
696

4843
CAAUGCCAUUUCAGUGGCU
270
4843
CAAUGCCAUUUCAGUGGCU
270
4861
AGCCACUGAAAUGGCAUUG
697

4861
UUCCCAGCUCUGACCCUUC
271
4861
UUCCCAGCUCUGACCCUUC
271
4879
GAAGGGUCAGAGCUGGGAA
698

4879
CUACAUUUGAGGGCCCAGC
272
4879
CUACAUUUGAGGGCCCAGC
272
4897
GCUGGGCCCUCAAAUGUAG
699

4897
CCAGGAGCAGAUGGACAGC
273
4897
CCAGGAGCAGAUGGACAGC
273
4915
GCUGUCCAUCUGCUCCUGG
700

4915
CGAUGAGGGGACAUUUUCU
274
4915
CGAUGAGGGGACAUUUUCU
274
4933
AGAAAAUGUCCCCUCAUCG
701

4933
UGGAUUCUGGGAGGCAAGA
275
4933
UGGAUUCUGGGAGGCAAGA
275
4951
UCUUGCCUCCCAGAAUCCA
702

4951
AAAAGGACAAAUAUCUUUU
276
4951
AAAAGGACAAAUAUCUUUU
276
4969
AAAAGAUAUUUGUCCUUUU
703

4969
UUUGGAACUAAAGCAAAUU
277
4969
UUUGGAACUAAAGCAAAUU
277
4987
AAUUUGCUUUAGUUCCAAA
704

4987
UUUAGACCUUUACCUAUGG
278
4987
UUUAGACCUUUACCUAUGG
278
5005
CCAUAGGUAAAGGUCUAAA
705

5005
GAAGUGGUUCUAUGUCCAU
279
5005
GAAGUGGUUCUAUGUCCAU
279
5023
AUGGACAUAGAACCACUUC
706

5023
UUCUCAUUCGUGGCAUGUU
280
5023
UUCUCAUUCGUGGCAUGUU
280
5041
AACAUGCCACGAAUGAGAA
707

5041
UUUGAUUUGUAGCACUGAG
281
5041
UUUGAUUUGUAGCACUGAG
281
5059
CUCAGUGCUACAAAUCAAA
708

5059
GGGUGGCACUCAACUCUGA
282
5059
GGGUGGCACUCAACUCUGA
282
5077
UCAGAGUUGAGUGCCACCC
709

5077
AGCCCAUACUUUUGGCUCC
283
5077
AGCCCAUACUUUUGGCUCC
283
5095
GGAGCCAAAAGUAUGGGCU
710

5095
CUCUAGUAAGAUGCACUGA
284
5095
CUCUAGUAAGAUGCACUGA
284
5113
UCAGUGCAUCUUACUAGAG
711

5113
AAAACUUAGCCAGAGUUAG
285
5113
AAAACUUAGCCAGAGUUAG
285
5131
CUAACUCUGGCUAAGUUUU
712

5131
GGUUGUCUCCAGGCCAUGA
286
5131
GGUUGUCUCCAGGCCAUGA
286
5149
UCAUGGCCUGGAGACAACC
713

5149
AUGGCCUUACACUGAAAAU
287
5149
AUGGCCUUACACUGAAAAU
287
5167
AUUUUCAGUGUPAGGCCAU
714

5167
UGUCACAUUCUAUUUUGGG
288
5167
UGUCACAUUCUAUUUUGGG
288
5185
CCCAAAAUAGPAUGUGACA
715

5185
GUAUUAAUAUAUAGUCCAG
289
5185
GUAUUAAUAUAUAGUCCAG
289
5203
CUGGACUAUAUAUUAAUAC
716

5203
GACACUUAACUCAAUUUCU
290
5203
GACACUUAACUCAAUUUCU
290
5221
AGAAAUUGAGUUAAGUGUC
717

5221
UUGGUAUUAUUCUGUUUUG
291
5221
UUGGUAUUAUUCUGUUUUG
291
5239
CAAAACAGAAUAAUACCAA
718

5239
GCACAGUUAGUUGUGAAAG
292
5239
GCACAGUUAGUUGUGAAAG
292
5257
CUUUCACAACUAACUGUGC
719

5257
GAAAGCUGAGAAGAAUGAA
293
5257
GAAAGCUGAGAAGAAUGAA
293
5275
UUCAUUCUUCUCAGCUUUC
720

5275
AAAUGCAGUCCUGAGGAGA
294
5275
AAAUGCAGUCCUGAGGAGA
294
5293
UCUCCUCAGGACUGCAUUU
721

5293
AGUUUUCUCCAUAUCAAAA
295
5293
AGUUUUCUCCAUAUCAAAA
295
5311
UUUUGAUAUGGAGAAAACU
722

5311
ACGAGGGCUGAUGGAGGAA
296
5311
ACGAGGGCUGAUGGAGGAA
296
5329
UUCCUCCAUCAGCCCUCGU
723

5329
AAAAGGUCAAUAAGGUCAA
297
5329
AAAAGGUCAAUAAGGUCAA
297
5347
UUGACCUUAUUGACCUUUU
724

5347
AGGGAAGACCCCGUCUCUA
298
5347
AGGGAAGACCCCGUCUCUA
298
5365
UAGAGACGGGGUCUUCCCU
725

5365
AUACCAACCAAACCAAUUC
299
5365
AUACCAACCAAACCAAUUC
299
5383
GAAUUGGUUUGGUUGGUAU
726

5383
CACCAACACAGUUGGGACC
300
5383
CACCAACACAGUUGGGACC
300
5401
GGUCCCAACUGUGUUGGUG
727

5401
CCAAAACACAGGAAGUCAG
301
5401
CCAAAACACAGGAAGUCAG
301
5419
CUGACUUCCUGUGUUUUGG
728

5419
GUCACGUUUCCUUUUCAUU
302
5419
GUCACGUUUCCUUUUCAUU
302
5437
AAUGAAAAGGAAACGUGAC
729

5437
UUAAUGGGGAUUCCACUAU
303
5437
UUAAUGGGGAUUCCACUAU
303
5455
AUAGUGGAAUCCCCAUUAA
730

5455
UCUCACACUAAUCUGAAAG
304
5455
UCUCACACUAAUCUGAAAG
304
5473
CUUUCAGAUUAGUGUGAGA
731

5473
GGAUGUGGAAGAGCAUUAG
305
5473
GGAUGUGGAAGAGCAUUAG
305
5491
CUAAUGCUCUUCCACAUCC
732

5491
GCUGGCGCAUAUUAAGCAC
306
5491
GCUGGCGCAUAUUAAGCAC
306
5509
GUGCUUAAUAUGCGCCAGC
733

5509
CUUUAAGCUCCUUGAGUAA
307
5509
CUUUAAGCUCCUUGAGUAA
307
5527
UUACUCAAGGAGCUUAAAG
734

5527
AAAAGGUGGUAUGUAAUUU
308
5527
AAAAGGUGGUAUGUAAUUU
308
5545
AAAUUACAUACCACCUUUU
735

5545
UAUGCAAGGUAUUUCUCCA
309
5545
UAUGCAAGGUAUUUCUCCA
309
5563
UGGAGAAAUACCUUGCAUA
736

5563
AGUUGGGACUCAGGAUAUU
310
5563
AGUUGGGACUCAGGAUAUU
310
5581
AAUAUCCUGAGUCCCAACU
737

5581
UAGUUAAUGAGCCAUCACU
311
5581
UAGUUAAUGAGCCAUCACU
311
5599
AGUGAUGGCUCAUUAACUA
738

5599
UAGAAGAAAAGCCCAUUUU
312
5599
UAGAAGAAAAGCCCAUUUU
312
5617
AAAAUGGGCUUUUCUUCUA
739

5617
UCAACUGCUUUGAAACUUG
313
5617
UCAACUGCUUUGAAACUUG
313
5635
CAAGUUUCAAAGCAGUUGA
740

5635
GCCUGGGGUCUGAGCAUGA
314
5635
GCCUGGGGUCUGAGCAUGA
314
5653
UCAUGCUCAGACCCCAGGC
741

5653
AUGGGAAUAGGGAGACAGG
315
5653
AUGGGAAUAGGGAGACAGG
315
5671
CCUGUCUCCCUAUUCCCAU
742

5671
GGUAGGAAAGGGCGCCUAC
316
5671
GGUAGGAAAGGGCGCCUAC
316
5689
GUAGGCGCCCUUUCCUACC
743

5689
CUCUUCAGGGUCUAAAGAU
317
5689
CUCUUCAGGGUCUAAAGAU
317
5707
AUCUUUAGACCCUGAAGAG
744

5707
UCAAGUGGGCCUUGGAUCG
318
5707
UCAAGUGGGCCUUGGAUCG
318
5725
CGAUCCAAGGCCCACUUGA
745

5725
GCUAAGCUGGCUCUGUUUG
319
5725
GCUAAGCUGGCUCUGUUUG
319
5743
CAAACAGAGCCAGCUUAGC
746

5743
GAUGCUAUUUAUGCAAGUU
320
5743
GAUGCUAUUUAUGCAAGUU
320
5761
AACUUGCAUAAAUAGCAUC
747

5761
UAGGGUCUAUGUAUUUAGG
321
5761
UAGGGUCUAUGUAUUUAGG
321
5779
CCUAAAUACAUAGACCCUA
748

5779
GAUGCGCCUACUCUUCAGG
322
5779
GAUGCGCCUACUCUUCAGG
322
5797
CCUGAAGAGUAGGCGCAUC
749

5797
GGUCUAAAGAUCAAGUGGG
323
5797
GGUCUAAAGAUCAAGUGGG
323
5815
CCCACUUGAUCUUUAGACC
750

5815
GCCUUGGAUCGCUAAGCUG
324
5815
GCCUUGGAUCGCUAAGCUG
324
5833
CAGCUUAGCGAUCCAAGGC
751

5833
GGCUCUGUUUGAUGCUAUU
325
5833
GGCUCUGUUUGAUGCUAUU
325
5851
AAUAGCAUCAAACAGAGCC
752

5851
UUAUGCAAGUUAGGGUCUA
326
5851
UUAUGCAAGUUAGGGUCUA
326
5869
UAGACCCUAACUUGCAUAA
753

5869
AUGUAUUUAGGAUGUCUGC
327
5869
AUGUAUUUAGGAUGUCUGC
327
5887
GCAGACAUCCUAAAUACAU
754

5887
CACCUUCUGCAGCCAGUCA
328
5887
CACCUUCUGCAGCCAGUCA
328
5905
UGACUGGCUGCAGAAGGUG
755

5905
AGAAGCUGGAGAGGCAACA
329
5905
AGAAGCUGGAGAGGCAACA
329
5923
UGUUGCCUCUCCAGCUUCU
756

5923
AGUGGAUUGCUGCUUCUUG
330
5923
AGUGGAUUGCUGCUUCUUG
330
5941
CAAGAAGCAGCAAUCCACU
757

5941
GGGGAGAAGAGUAUGCUUC
331
5941
GGGGAGAAGAGUAUGCUUC
331
5959
GAAGCAUACUCUUCUCCCC
758

5959
CCUUUUAUCCAUGUAAUUU
332
5959
CCUUUUAUCCAUGUAAUUU
332
5977
AAAUUACAUGGAUAAAAGG
759

5977
UAACUGUAGAACCUGAGCU
333
5977
UAACUGUAGAACCUGAGCU
333
5995
AGCUCAGGUUCUACAGUUA
760

5995
UCUAAGUAACCGAAGAAUG
334
5995
UCUAAGUAACCGAAGAAUG
334
6013
CAUUCUUCGGUUACUUAGA
761

6013
GUAUGCCUCUGUUCUUAUG
335
6013
GUAUGCCUCUGUUCUUAUG
335
6031
CAUAAGAACAGAGGCAUAC
762

6031
GUGCCACAUCCUUGUUUAA
336
6031
GUGCCACAUCCUUGUUUAA
336
6049
UUAAACAAGGAUGUGGCAC
763

6049
AAGGCUCUCUGUAUGAAGA
337
6049
AAGGCUCUCUGUAUGAAGA
337
6067
UCUUCAUACAGAGAGCCUU
764

6067
AGAUGGGACCGUCAUCAGC
338
6067
AGAUGGGACCGUCAUCAGC
338
6085
GCUGAUGACGGUCCCAUCU
765

6085
CACAUUCCCUAGUGAGCCU
339
6085
CACAUUCCCUAGUGAGCCU
339
6103
AGGCUCACUAGGGAAUGUG
766

6103
UACUGGCUCCUGGCAGCGG
340
6103
UACUGGCUCCUGGCAGCGG
340
6121
CCGCUGCCAGGAGCCAGUA
767

6121
GCUUUUGUGGAAGACUCAC
341
6121
GCUUUUGUGGAAGACUCAC
341
6139
GUGAGUCUUCCACAAAAGC
768

6139
CUAGCCAGAAGAGAGGAGU
342
6139
CUAGCCAGAAGAGAGGAGU
342
6157
ACUCCUCUCUUCUGGCUAG
769

6157
UGGGACAGUCCUCUCCACC
343
6157
UGGGACAGUCCUCUCCACC
343
6175
GGUGGAGAGGACUGUCCCA
770

6175
CAAGAUCUAAAUCCAAACA
344
6175
CAAGAUCUAAAUCCAAACA
344
6193
UGUUUGGAUUUAGAUCUUG
771

6193
AAAAGCAGGCUAGAGCCAG
345
6193
AAAAGCAGGCUAGAGCCAG
345
6211
CUGGCUCUAGCCUGCUUUU
772

6211
GAAGAGAGGACAAAUCUUU
346
6211
GAAGAGAGGACAAAUCUUU
346
6229
AAAGAUUUGUCCUCUCUUC
773

6229
UGUUGUUCCUCUUCUUUAC
347
6229
UGUUGUUCCUCUUCUUUAC
347
6247
GUAAAGAAGAGGAACAACA
774

6247
CACAUACGCAAACCACCUG
348
6247
CACAUACGCAAACCACCUG
348
6265
CAGGUGGUUUGCGUAUGUG
775

6265
GUGACAGCUGGCAAUUUUA
349
6265
GUGACAGCUGGCAAUUUUA
349
6283
UAAAAUUGCCAGCUGUCAC
776

6283
AUAAAUCAGGUAACUGGAA
350
6283
AUAAAUCAGGUAACUGGAA
350
6301
UUCCAGUUACCUGAUUUAU
777

6301
AGGAGGUUAAACUCAGAAA
351
6301
AGGAGGUUAAACUCAGAAA
351
6319
UUUCUGAGUUUAACCUCCU
778

6319
AAAAGAAGACCUCAGUCAA
352
6319
AAAAGAAGACCUCAGUCAA
352
6337
UUGACUGAGGUCUUCUUUU
779

6337
AUUCUCUACUUUUUUUUUU
353
6337
AUUCUCUACUUUUUUUUUU
353
6355
AAAAAAAAAAGUAGAGAAU
780

6355
UUUUUUUCCAAAUCAGAUA
354
6355
UUUUUUUCCAAAUCAGAUA
354
6373
UAUCUGAUUUGGAAAAAAA
781

6373
AAUAGCCCAGCAAAUAGUG
355
6373
AAUAGCCCAGCAAAUAGU
G 355
6391
CACUAUUUGCUGGGCUAUU
782

6391
GAUAACAAAUAAAACCUUA
356
6391
GAUAACAAAUAAAACCUUA
356
6409
UAAGGUUUUAUUUGUUAUC
783

6409
AGCUGUUCAUGUCUUGAUU
357
6409
AGCUGUUCAUGUCUUGAUU
357
6427
AAUCAAGACAUGAACAGCU
764

6427
UUCAAUAAUUAAUUCUUAA
358
6427
UUCAAUAAUUAAUUCUUAA
358
6445
UUAAGAAUUAAUUAUUGAA
785

6445
AUCAUUAAGAGACCAUAAU
359
6445
AUCAUUAAGAGACCAUAAU
359
6463
AUUAUGGUCUCUUAAUGAU
786

6463
UAAAUACUCCUUUUCAAGA
360
6463
UAAAUACUCCUUUUCAAGA
360
6481
UCUUGAAAAGGAGUAUUUA
787

6481
AGAAAAGCAAAACCAUUAG
361
6481
AGAAAAGCAAAACCAUUAG
361
6499
CUAAUGGUUUUGCUUUUCU
788

6499
GAAUUGUUACUCAGCUCCU
362
6499
GAAUUGUUACUCAGCUCCU
362
6517
AGGAGCUGAGUAACAAUUC
789

6517
UUCAAACUCAGGUUUGUAG
363
6517
UUCAAACUCAGGUUUGUAG
363
6535
CUACAAACCUGAGUUUGAA
790

6535
GCAUACAUGAGUCCAUCCA
364
6535
GCAUACAUGAGUCCAUCCA
364
6553
UGGAUGGACUCAUGUAUGC
791

6553
AUCAGUCAAAGAAUGGUUC
365
6553
AUCAGUCAAAGAAUGGUUC
365
6571
GAACCAUUCUUUGACUGAU
792

6571
CCAUCUGGAGUCUUAAUGU
366
6571
CCAUCUGGAGUCUUAAUGU
366
6589
ACAUUAAGACUCCAGAUGG
793

6589
UAGAAAGAAAAAUGGAGAC
367
6589
UAGAAAGAAAAAUGGAGAC
367
6607
GUCUCCAUUUUUCUUUCUA
794

6607
CUUGUAAUAAUGAGCUAGU
368
6607
CUUGUAAUAAUGAGCUAGU
368
6625
ACUAGCUCAUUAUUACAAG
795

6625
UUACAAAGUGCUUGUUCAU
369
6625
UUACAAAGUGCUUGUUCAU
369
6643
AUGAACAAGCACUUUGUAA
796

6643
UUAAAAUAGCACUGAAAAU
370
6643
UUAAAAUAGCACUGAAAAU
370
6661
AUUUUCAGUGCUAUUUUAA
797

6661
UUGAAACAUGAAUUAACUG
371
6661
UUGAAACAUGAAUUAACUG
371
6679
CAGUUAAUUCAUGUUUCAA
798

6679
GAUAAUAUUCCAAUCAUUU
372
6679
GAUAAUAUUCCAAUCAUUU
372
6697
AAAUGAUUGGAAUAUUAUC
799

6697
UGCCAUUUAUGACAAAAAU
373
6697
UGCCAUUUAUGACAAAAAU
373
6715
AUUUUUGUCAUAAAUGGCA
800

6715
UGGUUGGCACUAACAAAGA
374
6715
UGGUUGGCACUAACAAAGA
374
6733
UCUUUGUUAGUGCCAACCA
801

6733
AACGAGCACUUCCUUUCAG
375
6733
AACGAGCACUUCCUUUCAG
375
6751
CUGAAAGGAAGUGCUCGUU
802

6751
GAGUUUCUGAGAUAAUGUA
376
6751
GAGUUUCUGAGAUAAUGUA
376
6769
UACAUUAUCUCAGAAACUC
803

6769
ACGUGGAACAGUCUGGGUG
377
6769
ACGUGGAACAGUCUGGGUG
377
6787
CACCCAGACUGUUCCACGU
804

6787
GGAAUGGGGCUGAAACCAU
378
6787
GGAAUGGGGCUGAAACCAU
378
6805
AUGGUUUCAGCCCCAUUCC
805

6805
UGUGCAAGUCUGUGUCUUG
379
6805
UGUGCAAGUCUGUGUCUUG
379
6823
CAAGACACAGACUUGCACA
806

6823
GUCAGUCCAAGAAGUGACA
380
6823
GUCAGUCCAAGAAGUGACA
380
6841
UGUCACUUCUUGGACUGAC
807

6841
ACCGAGAUGUUAAUUUUAG
381
6841
ACCGAGAUGUUAAUUUUAG
381
6859
CUAAAAUUAACAUCUCGGU
808

6859
GGGACCCGUGCCUUGUUUC
382
6859
GGGACCCGUGCCUUGUUUC
382
6877
GAAACAAGGCACGGGUCCC
809

6877
CCUAGCCCACAAGAAUGCA
383
6877
CCUAGCCCACAAGAAUGCA
383
6895
UGCAUUCUUGUGGGCUAGG
810

6895
AAACAUCAAACAGAUACUC
384
6895
AAACAUCkAACAGAUACUC
384
6913
GAGUAUCUGUUUGAUGUUU
811

6913
CGCUAGCCUCAUUUAAAUU
385
6913
CGCUAGCCUCAUUUAAAUU
385
6931
AAUUUAAAUGAGGCUAGCG
812

6931
UGAUUAAAGGAGGAGUGCA
386
6931
UGAUUAAAGGAGGAGUGCA
386
6949
UGCACUCCUCCUUUAAUCA
813

6949
AUCUUUGGCCGACAGUGGU
387
6949
AUCUUUGGCCGACAGUGGU
387
6967
ACCACUGUCGGCCAAAGAU
814

6967
UGUAACUGUGUGUGUGUGU
388
6967
UGUAACUGUGUGUGUGUGU
388
6985
ACACACACACACAGUUACA
815

6985
UGUGUGUGUGUGUGUGUGU
389
6985
UGUGUGUGUGUGUGUGUGU
389
7003
ACACACACACACACACACA
816

7003
UGUGUGUGUGUGGGUGUGG
390
7003
UGUGUGUGUGUGGGUGUGG
390
7021
CCACACCCACACACACACA
817

7021
GGUGUAUGUGUGUUUUGUG
391
7021
GGUGUAUGUGUGUUUUGUG
391
7039
CACAAAACACACAUACACC
818

7039
GCAUAACUAUUUAAGGAAA
392
7039
GCAUAACUAUUUAAGGAAA
392
7057
UUUCCUUAAAUAGUUAUGC
819

7057
ACUGGAAUUUUAAAGUUAC
393
7057
ACUGGAAUUUUAAAGUUAC
393
7075
GUAACUUUAAAAUUCCAGU
820

7075
CUUUUAUACAAACCAAGAA
394
7075
CUUUUAUACAAACCAAGAA
394
7093
UUCUUGGUUUGUAUAAAAG
821

7093
AUAUAUGCUACAGAUAUAA
395
7093
AUAUAUGCUACAGAUAUAA
395
7111
UUAUAUCUGUAGCAUAUAU
822

7111
AGACAGACAUGGUUUGGUC
396
7111
AGACAGACAUGGUUUGGUC
396
7129
GACCAAACCAUGUCUGUCU
823

7129
CCUAUAUUUCUAGUCAUGA
397
7129
CCUAUAUUUCUAGUCAUGA
397
7147
UCAUGACUAGAAAUAUAGG
824

7147
AUGAAUGUAUUUUGUAUAC
398
7147
AUGAAUGUAUUUUGUAUAC
398
7165
GUAUACAAAAUACAUUCAU
825

7165
CCAUCUUCAUAUAAUAUAC
399
7165
CCAUCUUCAUAUAAUAUAC
399
7183
GUAUAUUAUAUGAAGAUGG
826

7183
CUUAAAAAUAUUUCUUAAU
400
7183
CUUAAAAAUAUUUCUUAAU
400
7201
AUUAAGAAAUAUUUUUAAG
827

7201
UUGGGAUUUGUAAUCGUAC
401
7201
UUGGGAUUUGUAAUCGUAC
401
7219
GUACGAUUACAAAUCCCAA
828

7219
CCAACUUAAUUGAUAAACU
402
7219
CCAACUUAAUUGAUAAACU
402
7237
AGUUUAUCAAUUAAGUUGG
829

7237
UUGGCAACUGCUUUUAUGU
403
7237
UUGGCAACUGCUUUUAUGU
403
7255
ACAUAAAAGCAGUUGCCAA
830

7255
UUCUGUCUCCUUCCAUAAA
404
7255
UUCUGUCUCCUUCCAUAAA
404
7273
UUUAUGGAAGGAGACAGAA
831

7273
AUUUUUCAAAAUACUAAUU
405
7273
AUUUUUCAAAAUACUAAUU
405
7291
AAUUAGUAUUUUGAAAAAU
832

7291
UCAACAAAGAAAAAGCUCU
406
7291
UCAACAAAGAAAAAGCUCU
406
7309
AGAGCUUUUUCUUUGUUGA
833

7309
UUUUUUUUCCUAAAAUAAA
407
7309
UUUUUUUUCCUAAAAUAAA
407
7327
UUUAUUUUAGGAAAAAAAA
834

7327
ACUCAAAUUUAUCCUUGUU
408
7327
ACUCAAAUUUAUCCUUGUU
408
7345
AACAAGGAUAAAUUUGAGU
835

7345
UUAGAGCAGAGAAAAAUUA
409
7345
UUAGAGCAGAGAAAAAUUA
409
7363
UAAUUUUUCUCUGCUCUAA
836

7363
AAGAAAAACUUUGAAAUGG
410
7363
AAGAAAAACUUUGAAAUGG
410
7381
CCAUUUCAAAGUUUUUCUU
837

7381
GUCUCAAAAAAUUGCUAAA
411
7381
GUCUCAAAAAAUUGCUAAA
411
7399
UUUAGCAAUUUUUUGAGAC
838

7399
AUAUUUUCAAUGGAAAACU
412
7399
AUAUUUUCAAUGGAAAACU
412
7417
AGUUUUCCAUUGAAAAUAU
839

7417
UAAAUGUUAGUUUAGCUGA
413
7417
UAAAUGUUAGUUUAGCUGA
413
7435
UCAGCUAAACUAACAUUUA
840

7435
AUUGUAUGGGGUUUUCGAA
414
7435
AUUGUAUGGGGUUUUCGAA
414
7453
UUCGAAAACCCCAUACAAU
841

7453
ACCUUUCACUUUUUGUUUG
415
7453
ACCUUUCACUUUUUGUUUG
415
7471
CAAACAAAAAGUGAAAGGU
842

7471
GUUUUACCUAUUUCACAAC
416
7471
GUUUUACCUAUUUCACAAC
416
7489
GUUGUGAAAUAGGUAAAAC
843

7489
CUGUGUAAAUUGCCAAUAA
417
7489
CUGUGUAAAUUGCCAAUAA
417
7507
UUAUUGGCAAUUUACACAG
844

7507
AUUCCUGUCCAUGAAAAUG
418
7507
AUUCCUGUCCAUGAAAAUG
418
7525
CAUUUUCAUGGACAGGAAU
845

7525
GCAAAUUAUCCAGUGUAGA
419
7525
GCAAAUUAUCCAGUGUAGA
419
7543
UCUACACUGGAUAAUUUGC
846

7543
AUAUAUUUGACCAUCACCC
420
7543
AUAUAUUUGACCAUCACCC
420
7561
GGGUGAUGGUCPAAUAUAU
847

7561
CUAUGGAUAUUGGCUAGUU
421
7561
CUAUGGAUAUUGGCUAGUU
421
7579
AACUAGCCAAUAUCCAUAG
848

7579
UUUGCCUUUAUUAAGCAAA
422
7579
UUUGCCUUUAUUAAGCAAA
422
7597
UUUGCUUAAUAAAGGCAAA
849

7597
AUUCAUUUCAGCCUGAAUG
423
7597
AUUCAUUUCAGCCUGAAUG
423
7615
CAUUCAGGCUGAAAUGAAU
850

7615
GUCUGCCUAUAUAUUCUCU
424
7615
GUCUGCCUAUAUAUUCUCU
424
7633
AGAGAAUAUAUAGGCAGAC
851

7633
UGCUCUUUGUAUUCUCCUU
425
7633
UGCUCUUUGUAUUCUCCUU
425
7651
AAGGAGAAUACAAAGAGCA
852

7651
UUGAACCCGUUAAAACAUC
426
7651
UUGAACCCGUUAAAACAUC
426
7669
GAUGUUUUAACGGGUUCAA
853

7662
AAAACAUCCUGUGGCACUC
427
7662
AAAACAUCCUGUGGCACUC
427
7680
GAGUGCCACAGGAUGUUUU
854

VEGFR2/KDR NM_002253.1

1
ACUGAGUCCCGGGACCCCG
855
1
ACUGAGUCCCGGGACCCCG
855
19
CGGGGUCCCGGGACUCAGU
1179

19
GGGAGAGCGGUCAGUGUGU
856
19
GGGAGAGCGGUCAGUGUGU
856
37
ACACACUGACCGCUCUCCC
1180

37
UGGUCGCUGCGUUUCCUCU
857
37
UGGUCGCUGCGUUUCCUCU
857
55
AGAGGAAACGCAGCGACCA
1181

55
UGCCUGCGCCGGGCAUCAC
858
55
UGCCUGCGCCGGGCAUCAC
858
73
GUGAUGCCCGGCGCAGGCA
1182

73
CUUGCGCGCCGCAGAAAGU
859
73
CUUGCGCGCCGCAGAAAGU
859
91
ACUUUCUGCGGCGCGCAAG
1183

91
UCCGUCUGGCAGCCUGGAU
860
91
UCCGUCUGGCAGCCUGGAU
860
109
AUCCAGGCUGCCAGACGGA
1184

109
UAUCCUCUCCUACCGGCAC
861
109
UAUCCUCUCCUACCGGCAC
861
127
GUGCCGGUAGGAGAGGAUA
1185

127
CCCGCAGACGCCCCUGCAG
862
127
CCCGCAGACGCCCCUGCAG
862
145
CUGCAGGGGCGUCUGCGGG
1186

145
GCCGCCGGUCGGCGCCCGG
863
145
GCCGCCGGUCGGCGCCCGG
863
163
CCGGGCGCCGACCGGCGGC
1187

163
GGCUCCCUAGCCCUGUGCG
864
163
GGCUCCCUAGCCCUGUGCG
864
181
CGCACAGGGCUAGGGAGCC
1188

181
GCUCAACUGUCCUGCGCUG
865
181
GCUCAACUGUCCUGCGCUG
865
199
CAGCGCAGGACAGUUGAGC
1189

199
GCGGGGUGCCGCGAGUUCC
866
199
GCGGGGUGCCGCGAGUUCC
866
217
GGAACUCGCGGCACCCCGC
1190

217
CACCUCCGCGCCUCCUUCU
867
217
CACCUCCGCGCCUCCUUCU
867
235
AGAAGGAGGCGCGGAGGUG
1191

235
UCUAGACAGGCGCUGGGAG
868
235
UCUAGACAGGCGCUGGGAG
868
253
CUCCCAGCGCCUGUCUAGA
1192

253
GAAAGAACCGGCUCCCGAG
869
253
GAAAGAACCGGCUCCCGAG
869
271
CUCGGGAGCCGGUUCUUUC
1193

271
GUUCUGGGCAUUUCGCCCG
870
271
GUUCUGGGCAUUUCGCCCG
870
289
CGGGCGAAAUGCCCAGAAC
1194

289
GGCUCGAGGUGCAGGAUGC
871
289
GGCUCGAGGUGCAGGAUGC
871
307
GCAUCCUGCACCUCGAGCC
1195

307
CAGAGCAAGGUGCUGCUGG
872
307
CAGAGCAAGGUGCUGCUGG
872
325
CCAGCAGCACCUUGCUCUG
1196

325
GCCGUCGCCCUGUGGCUCU
873
325
GCCGUCGCCCUGUGGCUCU
873
343
AGAGCCACAGGGCGACGGC
1197

343
UGCGUGGAGACCCGGGCCG
874
343
UGCGUGGAGACCCGGGCCG
874
361
CGGCCCGGGUCUCCACGCA
1198

361
GCCUCUGUGGGUUUGCCUA
875
361
GCCUCUGUGGGUUUGCCUA
875
379
UAGGCAAACCCACAGAGGC
1199

379
AGUGUUUCUCUUGAUCUGC
876
379
AGUGUUUCUCUUGAUCUGC
876
397
GCAGAUCAAGAGAAACACU
1200

397
CCCAGGCUCAGCAUACAAA
877
397
CCCAGGCUCAGCAUACAAA
877
415
UUUGUAUGCUGAGCCUGGG
1201

415
AAAGACAUACUUACAAUUA
878
415
AAAGACAUACUUACAAUUA
878
433
UAAUUGUAAGUAUGUCUUU
1202

433
AAGGCUAAUACAACUCUUC
879
433
AAGGCUAAUACAACUCUUC
879
451
GAAGAGUUGUAUUAGCCUU
1203

451
CAAAUUACUUGCAGGGGAC
880
451
CAAAUUACUUGCAGGGGAC
880
469
GUCCCCUGCAAGUAAUUUG
1204

469
CAGAGGGACUUGGACUGGC
881
469
CAGAGGGACUUGGACUGGC
881
487
GCCAGUCCAAGUCCCUCUG
1205

487
CUUUGGCCCAAUAAUCAGA
882
487
CUUUGGCCCAAUAAUCAGA
882
505
UCUGAUUAUUGGGCCAAAG
1206

505
AGUGGCAGUGAGCAAAGGG
883
505
AGUGGCAGUGAGCAAAGGG
883
523
CCCUUUGCUCACUGCCACU
1207

523
GUGGAGGUGACUGAGUGCA
884
523
GUGGAGGUGACUGAGUGCA
884
541
UGCACUCAGUCACCUCCAC
1208

541
AGCGAUGGCCUCUUCUGUA
885
541
AGCGAUGGCCUCUUCUGUA
885
559
UACAGAAGAGGCCAUCGCU
1209

559
AAGACACUCACAAUUCCAA
886
559
AAGACACUCACAAUUCCAA
886
577
UUGGAAUUGUGAGUGUCUU
1210

577
AAAGUGAUCGGAAAUGACA
887
577
AAAGUGAUCGGAAAUGACA
887
595
UGUCAUUUCCGAUCACUUU
1211

595
ACUGGAGCCUACAAGUGCU
888
595
ACUGGAGCCUACAAGUGCU
888
613
AGCACUUGUAGGCUCCAGU
1212

613
UUCUACCGGGAAACUGACU
889
613
UUCUACCGGGAAACUGACU
889
631
AGUCAGUUUCCCGGUAGAA
1213

631
UUGGCCUCGGUCAUUUAUG
890
631
UUGGCCUCGGUCAUUUAUG
890
649
CAUAAAUGACCGAGGCCAA
1214

649
GUCUAUGUUCAAGAUUACA
891
649
GUCUAUGUUCAAGAUUACA
891
667
UGUAAUCUUGAACAUAGAC
1215

667
AGAUCUCCAUUUAUUGCUU
892
667
AGAUCUCCAUUUAUUGCUU
892
685
AAGCAAUAAAUGGAGAUCU
1216

685
UCUGUUAGUGACCAACAUG
893
685
UCUGUUAGUGACCAACAUG
893
703
CAUGUUGGUCACUAACAGA
1217

703
GGAGUCGUGUACAUUACUG
894
703
GGAGUCGUGUACAUUACUG
894
721
CAGUAAUGUACACGACUCC
1218

721
GAGAACAAAAACAAAACUG
895
721
GAGAACAAAAACAAAACUG
895
739
CAGUUUUGUUUUUGUUCUC
1219

739
GUGGUGAUUCCAUGUCUCG
896
739
GUGGUGAUUCCAUGUCUCG
896
757
CGAGACAUGGAAUCACCAC
1220

757
GGGUCCAUUUCAAAUCUCA
897
757
GGGUCCAUUUCAAAUCUCA
897
775
UGAGAUUUGAAAUGGACCC
1221

775
AACGUGUCACUUUGUGCAA
898
775
AACGUGUCACUUUGUGCAA
898
793
UUGCACAAAGUGACACGUU
1222

793
AGAUACCCAGAAAAGAGAU
899
793
AGAUACCCAGAAAAGAGAU
899
811
AUCUCUUUUCUGGGUAUCU
1223

811
UUUGUUCCUGAUGGUAACA
900
811
UUUGUUCCUGAUGGUAACA
900
829
UGUUACCAUCAGGAACAAA
1224

829
AGAAUUUCCUGGGACAGCA
901
829
AGAAUUUCCUGGGACAGCA
901
847
UGCUGUCCCAGGAAAUUCU
1225

847
AAGAAGGGCUUUACUAUUC
902
847
AAGAAGGGCUUUACUAUUC
902
865
GAAUAGUAAAGCCCUUCUU
1226

865
CCCAGCUACAUGAUCAGCU
903
865
CCCAGCUACAUGAUCAGCU
903
883
AGCUGAUCAUGUAGCUGGG
1227

883
UAUGCUGGCAUGGUCUUCU
904
883
UAUGCUGGCAUGGUCUUCU
904
901
AGAAGACCAUGCCAGCAUA
1228

901
UGUGAAGCAAAAAUUAAUG
905
901
UGUGAAGCAAAAAUUAAUG
905
919
CAUUAAUUUUUGCUUCACA
1229

919
GAUGAAAGUUACCAGUCUA
906
919
GAUGAAAGUUACCAGUCUA
906
937
UAGACUGGUAACUUUCAUC
1230

937
AUUAUGUACAUAGUUGUCG
907
937
AUUAUGUACAUAGUUGUCG
907
955
CGACAACUAUGUACAUAAU
1231

955
GUUGUAGGGUAUAGGAUUU
908
955
GUUGUAGGGUAUAGGAUUU
908
973
AAAUCCUAUACCCUACAAC
1232

973
UAUGAUGUGGUUCUGAGUC
909
973
UAUGAUGUGGUUCUGAGUC
909
991
GACUCAGAACCACAUCAUA
1233

991
CCGUCUCAUGGAAUUGAAC
910
991
CCGUCUCAUGGAAUUGAAC
910
1009
GUUCAAUUCCAUGAGACGG
1234

1009
CUAUCUGUUGGAGAAAAGC
911
1009
CUAUCUGUUGGAGAAAAGC
911
1027
GCUUUUCUCCAACAGAUAG
1235

1027
CUUGUCUUAAAUUGUACAG
912
1027
CUUGUCUUAAAUUGUACAG
912
1045
CUGUACAAUUUAAGACAAG
1236

1045
GCAAGAACUGAACUAAAUG
913
1045
GCAAGAACUGAACUAAAUG
913
1063
CAUUUAGUUCAGUUCUUGC
1237

1063
GUGGGGAUUGACUUCAACU
914
1063
GUGGGGAUUGACUUCAACU
914
1081
AGUUGAAGUCAAUCCCCAC
1238

1081
UGGGAAUACCCUUCUUCGA
915
1081
UGGGAAUACCCUUCUUCGA
915
1099
UCGAAGAAGGGUAUUCCCA
1239

1099
AAGCAUCAGCAUAAGAAAC
916
1099
AAGCAUCAGCAUAAGAAAC
916
1117
GUUUCUUAUGCUGAUGCUU
1240

1117
CUUGUAAACCGAGACCUAA
917
1117
CUUGUAAACCGAGACCUAA
917
1135
UUAGGUCUCGGUUUACAAG
1241

1135
AAAACCCAGUCUGGGAGUG
918
1135
AAAACCCAGUCUGGGAGUG
918
1153
CACUCCCAGACUGGGUUUU
1242

1153
GAGAUGAAGAAAUUUUUGA
919
1153
GAGAUGAAGAAAUUUUUGA
919
1171
UCAAAAAUUUCUUCAUCUC
1243

1171
AGCACCUUAACUAUAGAUG
920
1171
AGCACCUUAACUAUAGAUG
920
1189
CAUCUAUAGUUAAGGUGCU
1244

1189
GGUGUAACCCGGAGUGACC
921
1189
GGUGUAACCCGGAGUGACC
921
1207
GGUCACUCCGGGUUACACC
1245

1207
CAAGGAUUGUACACCUGUG
922
1207
CAAGGAUUGUACACCUGUG
922
1225
CACAGGUGUACAAUCCUUG
1246

1225
GCAGCAUCCAGUGGGCUGA
923
1225
GCAGCAUCCAGUGGGCUGA
923
1243
UCAGCCCACUGGAUGCUGC
1247

1243
AUGACCAAGAAGAACAGCA
924
1243
AUGACCAAGAAGAACAGCA
924
1261
UGCUGUUCUUCUUGGUCAU
1248

1261
ACAUUUGUCAGGGUCCAUG
925
1261
ACAUUUGUCAGGGUCCAUG
925
1279
CAUGGACCCUGACAAAUGU
1249

1279
GAAAAACCUUUUGUUGCUU
926
1279
GAAAAACCUUUUGUUGCUU
926
1297
AAGCAACAAAAGGUUUUUC
1250

1297
UUUGGAAGUGGCAUGGAAU
927
1297
UUUGGAAGUGGCAUGGAAU
927
1315
AUUCCAUGCCACUUCCAAA
1251

1315
UCUCUGGUGGPAGCCACGG
928
1315
UCUCUGGUGGAAGCCACGG
928
1333
CCGUGGCUUCCACCAGAGA
1252

1333
GUGGGGGAGCGUGUCAGAA
929
1333
GUGGGGGAGCGUGUCAGAA
929
1351
UUCUGACACGCUCCCCCAC
1253

1351
AUCCCUGCGAAGUACCUUG
930
1351
AUCCCUGCGAAGUACCUUG
930
1369
CAAGGUACUUCGCAGGGAU
1254

1369
GGUUACCCACCCCCAGAAA
931
1369
GGUUACCCACCCCCAGAAA
931
1387
UUUCUGGGGGUGGGUAACC
1255

1387
AUAAAAUGGUAUAAAAAUG
932
1387
AUAAAAUGGUAUAAAAAUG
932
1405
CAUUUUUAUACCAUUUUAU
1256

1405
GGAAUACCCCUUGAGUCCA
933
1405
GGAAUACCCCUUGAGUCCA
933
1423
UGGACUCAAGGGGUAUUCC
1257

1423
AAUCACACAAUUAAAGCGG
934
1423
AAUCACACAAUUAAAGCGG
934
1441
CCGCUUUAAUUGUGUGAUU
1258

1441
GGGCAUGUACUGACGAUUA
935
1441
GGGCAUGUACUGACGAUUA
935
1459
UAAUCGUCAGUACAUGCCC
1259

1459
AUGGAAGUGAGUGAAAGAG
936
1459
AUGGAAGUGAGUGAAAGAG
936
1477
CUCUUUCACUCACUUCCAU
1260

1477
GACACAGGAAAUUACACUG
937
1477
GACACAGGAAAUUACACUG
937
1495
CAGUGUAAUUUCCUGUGUC
1261

1495
GUCAUCCUUACCAAUCCCA
938
1495
GUCAUCCUUACCAAUCCCA
938
1513
UGGGAUUGGUAAGGAUGAC
1262

1513
AUUUCAAAGGAGAAGCAGA
939
1513
AUUUCAAAGGAGAAGCAGA
939
1531
UCUGCUUCUCCUUUGAAAU
1263

1531
AGCCAUGUGGUCUCUCUGG
940
1531
AGCCAUGUGGUCUCUCUGG
940
1549
CCAGAGAGACCACAUGGCU
1264

1549
GUUGUGUAUGUCCCACCCC
941
1549
GUUGUGUAUGUCCCACCCC
941
1567
GGGGUGGGACAUACACAAC
1265

1567
CAGAUUGGUGAGAAAUCUC
942
1567
CAGAUUGGUGAGAAAUCUC
942
1585
GAGAUUUCUCACCAAUCUG
1266

1585
CUAAUCUCUCCUGUGGAUU
943
1585
CUAAUCUCUCCUGUGGAUU
943
1603
AAUCCACAGGAGAGAUUAG
1267

1603
UCCUACCAGUACGGCACCA
944
1603
UCCUACCAGUACGGCACCA
944
1621
UGGUGCCGUACUGGUAGGA
1268

1621
ACUCAAACGCUGACAUGUA
945
1621
ACUCAAACGCUGACAUGUA
945
1639
UACAUGUCAGCGUUUGAGU
1269

1639
ACGGUCUAUGCCAUUCCUC
946
1639
ACGGUCUAUGCCAUUCCUC
946
1657
GAGGAAUGGCAUAGACCGU
1270

1657
CCCCCGCAUCACAUCCACU
947
1657
CCCCCGCAUCACAUCCACU
947
1675
AGUGGAUGUGAUGCGGGGG
1271

1675
UGGUAUUGGCAGUUGGAGG
948
1675
UGGUAUUGGCAGUUGGAGG
948
1693
CCUCCAACUGCCAAUACCA
1272

1693
GAAGAGUGCGCCAACGAGC
949
1693
GAAGAGUGCGCCAACGAGC
949
1711
GCUCGUUGGCGCACUCUUC
1273

1711
CCCAGCCAAGCUGUCUCAG
950
1711
CCCAGCCAAGCUGUCUCAG
950
1729
CUGAGACAGCUUGGCUGGG
1274

1729
GUGACAAACCCAUACCCUU
951
1729
GUGACAAACCCAUACCCUU
951
1747
AAGGGUAUGGGUUUGUCAC
1275

1747
UGUGAAGAAUGGAGAAGUG
952
1747
UGUGPAGAAUGGAGAAGUG
952
1765
CACUUCUCCAUUCUUCACA
1276

1765
GUGGAGGACUUCCAGGGAG
953
1765
GUGGAGGACUUCCAGGGAG
953
1783
CUCCCUGGAAGUCCUCCAC
1277

1783
GGAAAUAAAAUUGAAGUUA
954
1783
GGAAAUAAAAUUGAAGUUA
954
1801
UAACUUCAAUUUUAUUUCC
1278

1801
AAUAAAAAUCAAUUUGCUC
955
1801
AAUAAAAAUCAAUUUGCUC
955
1819
GAGCAAAUUGAUUUUUAUU
1279

1819
CUAAUUGAAGGAAAAAACA
956
1819
CUAAUUGAAGGAAAAAACA
956
1837
UGUUUUUUCCUUCAAUUAG
1280

1837
AAAACUGUAAGUACCCUUG
957
1837
AAAACUGUAAGUACCCUUG
957
1855
CAAGGGUACUUACAGUUUU
1281

1855
GUUAUCCAAGCGGCAAAUG
958
1855
GUUAUCCAAGCGGCAAAUG
958
1873
CAUUUGCCGCUUGGAUAAC
1282

1873
GUGUCAGCUUUGUACAAAU
959
1873
GUGUCAGCUUUGUACAAAU
959
1891
AUUUGUACAAAGCUGACAC
1283

1891
UGUGAAGCGGUCAACAAAG
960
1891
UGUGAAGCGGUCAACAAAG
960
1909
CUUUGUUGACCGCUUCACA
1284

1909
GUCGGGAGAGGAGAGAGGG
961
1909
GUCGGGAGAGGAGAGAGGG
961
1927
CCCUCUCUCCUCUCCCGAC
1285

1927
GUGAUCUCCUUCCACGUGA
962
1927
GUGAUCUCCUUCCACGUGA
962
1945
UCACGUGGAAGGAGAUCAC
1286

1945
ACCAGGGGUCCUGAAAUUA
963
1945
ACCAGGGGUCCUGAAAUUA
963
1963
UAAUUUCAGGACCCCUGGU
1287

1963
ACUUUGCAACCUGACAUGC
964
1963
ACUUUGCAACCUGACAUGC
964
1981
GCAUGUCAGGUUGCAAAGU
1288

1981
CAGCCCACUGAGCAGGAGA
965
1981
CAGCCCACUGAGCAGGAGA
965
1999
UCUCCUGCUCAGUGGGCUG
1289

1999
AGCGUGUCUUUGUGGUGCA
966
1999
AGCGUGUCUUUGUGGUGCA
966
2017
UGCACCACAAAGACACGCU
1290

2017
ACUGCAGACAGAUCUACGU
967
2017
ACUGCAGACAGAUCUACGU
967
2035
ACGUAGAUCUGUCUGCAGU
1291

2035
UUUGAGAACCUCACAUGGU
968
2035
UUUGAGAACCUCACAUGGU
968
2053
ACCAUGUGAGGUUCUCAAA
1292

2053
UACAAGCUUGGCCCACAGC
969
2053
UACAAGCUUGGCCCACAGC
969
2071
GCUGUGGGCCAAGCUUGUA
1293

2071
CCUCUGCCAAUCCAUGUGG
970
2071
CCUCUGCCAAUCCAUGUGG
970
2089
CCACAUGGAUUGGCAGAGG
1294

2089
GGAGAGUUGCCCACACCUG
971
2089
GGAGAGUUGCCCACACCUG
971
2107
CAGGUGUGGGCAACUCUCC
1295

2107
GUUUGCAAGAACUUGGAUA
972
2107
GUUUGCAAGAACUUGGAUA
972
2125
UAUCCAAGUUCUUGCAAAC
1296

2125
ACUCUUUGGAAAUUGAAUG
973
2125
ACUCUUUGGAAAUUGAAUG
973
2143
CAUUCAAUUUCCAAAGAGU
1297

2143
GCCACCAUGUUCUCUAAUA
974
2143
GCCACCAUGUUCUCUAAUA
974
2161
UAUUAGAGAACAUGGUGGC
1298

2161
AGCACAAAUGACAUUUUGA
975
2161
AGCACAAAUGACAUUUUGA
975
2179
UCAAAAUGUCAUUUGUGCU
1299

2179
AUCAUGGAGCUUAAGAAUG
976
2179
AUCAUGGAGCUUAAGAAUG
976
2197
CAUUCUUAAGCUCCAUGAU
1300

2197
GCAUCCUUGCAGGACCAAG
977
2197
GCAUCCUUGCAGGACCAAG
977
2215
CUUGGUCCUGCAAGGAUGC
1301

2215
GGAGACUAUGUCUGCCUUG
978
2215
GGAGACUAUGUCUGCCUUG
978
2233
CAAGGCAGACAUAGUCUCC
1302

2233
GCUCAAGACAGGAAGACCA
979
2233
GCUCAAGACAGGAAGACCA
979
2251
UGGUCUUCCUGUCUUGAGC
1303

2251
AAGAAAAGACAUUGCGUGG
980
2251
AAGAAAAGACAUUGCGUGG
980
2269
CCACGCAAUGUCUUUUCUU
1304

2269
GUCAGGCAGCUCACAGUCC
981
2269
GUCAGGCAGCUCACAGUCC
981
2287
GGACUGUGAGCUGCCUGAC
1305

2287
CUAGAGCGUGUGGCACCCA
982
2287
CUAGAGCGUGUGGCACCCA
982
2305
UGGGUGCCACACGCUCUAG
1306

2305
ACGAUCACAGGAAACCUGG
983
2305
ACGAUCACAGGAAACCUGG
983
2323
CCAGGUUUCCUGUGAUCGU
1307

2323
GAGAAUCAGACGACAAGUA
984
2323
GAGAAUCAGACGACAAGUA
984
2341
UACUUGUCGUCUGAUUCUC
1308

2341
AUUGGGGAAAGCAUCGAAG
985
2341
AUUGGGGAAAGCAUCGAAG
985
2359
CUUCGAUGCUUUCCCCAAU
1309

2359
GUCUCAUGCACGGCAUCUG
986
2359
GUCUCAUGCACGGCAUCUG
986
2377
CAGAUGCCGUGCAUGAGAC
1310

2377
GGGAAUCCCCCUCCACAGA
987
2377
GGGAAUCCCCCUCCACAGA
987
2395
UCUGUGGAGGGGGAUUCCC
1311

2395
AUCAUGUGGUUUAAAGAUA
988
2395
AUCAUGUGGUUUAAAGAUA
988
2413
UAUCUUUAAACCACAUGAU
1312

2413
AAUGAGACCCUUGUAGAAG
989
2413
AAUGAGACCCUUGUAGAAG
989
2431
CUUCUACAAGGGUCUCAUU
1313

2431
GACUCAGGCAUUGUAUUGA
990
2431
GACUCAGGCAUUGUAUUGA
990
2449
UCAAUACAAUGCCUGAGUC
1314

2449
AAGGAUGGGAACCGGAACC
991
2449
AAGGAUGGGAACCGGAACC
991
2467
GGUUCCGGUUCCCAUCCUU
1315

2467
CUCACUAUCCGCAGAGUGA
992
2467
CUCACUAUCCGCAGAGUGA
992
2485
UCACUCUGCGGAUAGUGAG
1316

2485
AGGAAGGAGGACGAAGGCC
993
2485
AGGAAGGAGGACGAAGGCC
993
2503
GGCCUUCGUCCUCCUUCCU
1317

2503
CUCUACACCUGCCAGGCAU
994
2503
CUCUACACCUGCCAGGCAU
994
2521
AUGCCUGGCAGGUGUAGAG
1318

2521
UGCAGUGUUCUUGGCUGUG
995
2521
UGCAGUGUUCUUGGCUGUG
995
2539
CACAGCCAAGAACACUGCA
1319

2539
GCAAAAGUGGAGGCAUUUU
996
2539
GCAAAAGUGGAGGCAUUUU
996
2557
AAAAUGCCUCCACUUUUGC
1320

2557
UUCAUAAUAGAAGGUGCCC
997
2557
UUCAUAAUAGAAGGUGCCC
997
2575
GGGCACCUUCUAUUAUGAA
1321

2575
CAGGAAAAGACGAACUUGG
998
2575
CAGGAAAAGACGAACUUGG
998
2593
CCAAGUUCGUCUUUUCCUG
1322

2593
GAAAUCAUUAUUCUAGUAG
999
2593
GAAAUCAUUAUUCUAGUAG
999
2611
CUACUAGAAUAAUGAUUUC
1323

2611
GGCACGGCGGUGAUUGCCA
1000
2611
GGCACGGCGGUGAUUGCCA
1000
2629
UGGCAAUCACCGCCGUGCC
1324

2629
AUGUUCUUCUGGCUACUUC
1001
2629
AUGUUCUUCUGGCUACUUC
1001
2647
GAAGUAGCCAGAAGAACAU
1325

2647
CUUGUCAUCAUCCUACGGA
1002
2647
CUUGUCAUCAUCCUACGGA
1002
2665
UCCGUAGGAUGAUGACAAG
1326

2665
ACCGUUAAGCGGGCCAAUG
1003
2665
ACCGUUAAGCGGGCCAAUG
1003
2683
CAUUGGCCCGCUUAACGGU
1327

2683
GGAGGGGAACUGAAGACAG
1004
2683
GGAGGGGAACUGAAGACAG
1004
2701
CUGUCUUCAGUUCCCCUCC
1328

2701
GGCUACUUGUCCAUCGUCA
1005
2701
GGCUACUUGUCCAUCGUCA
1005
2719
UGACGAUGGACAAGUAGCC
1329

2719
AUGGAUCCAGAUGAACUCC
1006
2719
AUGGAUCCAGAUGAACUCC
1006
2737
GGAGUUCAUCUGGAUCCAU
1330

2737
CCAUUGGAUGAACAUUGUG
1007
2737
CCAUUGGAUGAACAUUGUG
1007
2755
CACAAUGUUCAUCCAAUGG
1331

2755
GAACGACUGCCUUAUGAUG
1008
2755
GAACGACUGCCUUAUGAUG
1008
2773
CAUCAUAAGGCAGUCGUUC
1332

2773
GCCAGCAAAUGGGAAUUCC
1009
2773
GCCAGCAAAUGGGAAUUCC
1009
2791
GGAAUUCCCAUUUGCUGGC
1333

2791
CCCAGAGACCGGCUGAAGC
1010
2791
CCCAGAGACCGGCUGAAGC
1010
2809
GCUUCAGCCGGUCUCUGGG
1334

2809
CUAGGUAAGCCUCUUGGCC
1011
2809
CUAGGUAAGCCUCUUGGCC
1011
2827
GGCCAAGAGGCUUACCUAG
1335

2827
CGUGGUGCCUUUGGCCAAG
1012
2827
CGUGGUGCCUUUGGCCAAG
1012
2845
CUUGGCCAAAGGCACCACG
1336

2845
GUGAUUGAAGCAGAUGCCU
1013
2845
GUGAUUGAAGCAGAUGCCU
1013
2863
AGGCAUCUGCUUCAAUCAC
1337

2863
UUUGGAAUUGACAAGACAG
1014
2863
UUUGGAAUUGACAAGACAG
1014
2881
CUGUCUUGUCAAUUCCAAA
1338

2881
GCAACUUGCAGGACAGUAG
1015
2881
GCAACUUGCAGGACAGUAG
1015
2899
CUACUGUCCUGCAAGUUGC
1339

2899
GCAGUCAAAAUGUUGAAAG
1016
2899
GCAGUCAAAAUGUUGAAAG
1016
2917
CUUUCAACAUUUUGACUGC
1340

2917
GAAGGAGCAACACACAGUG
1017
2917
GAAGGAGCAACACACAGUG
1017
2935
CACUGUGUGUUGCUCCUUC
1341

2935
GAGCAUCGAGCUCUCAUGU
1018
2935
GAGCAUCGAGCUCUCAUGU
1018
2953
ACAUGAGAGCUCGAUGCUC
1342

2953
UCUGAACUCAAGAUCCUCA
1019
2953
UCUGAACUCAAGAUCCUCA
1019
2971
UGAGGAUCUUGAGUUCAGA
1343

2971
AUUCAUAUUGGUCACCAUC
1020
2971
AUUCAUAUUGGUCACCAUC
1020
2989
GAUGGUGACCAAUAUGAAU
1344

2989
CUCAAUGUGGUCAACCUUC
1021
2989
CUCAAUGUGGUCAACCUUC
1021
3007
GAAGGUUGACCACAUUGAG
1345

3007
CUAGGUGCCUGUACCAAGC
1022
3007
CUAGGUGCCUGUACCAAGC
1022
3025
GCUUGGUACAGGCACCUAG
1346

3025
CCAGGAGGGCCACUCAUGG
1023
3025
CCAGGAGGGCCACUCAUGG
1023
3043
CCAUGAGUGGCCCUCCUGG
1347

3043
GUGAUUGUGGAAUUCUGCA
1024
3043
GUGAUUGUGGAAUUCUGCA
1024
3061
UGCAGAAUUCCACAAUCAC
1348

3061
AAAUUUGGAAACCUGUCCA
1025
3061
AAAUUUGGAAACCUGUCCA
1025
3079
UGGACAGGUUUCCAAAUUU
1349

3079
ACUUACCUGAGGAGCAAGA
1026
3079
ACUUACCUGAGGAGCAAGA
1026
3097
UCUUGCUCCUCAGGUAAGU
1350

3097
AGAAAUGAAUUUGUCCCCU
1027
3097
AGAAAUGAAUUUGUCCCCU
1027
3115
AGGGGACPAAUUCAUUUCU
1351

3115
UACAAGACCAAAGGGGCAC
1028
3115
UACAAGACCAAAGGGGCAC
1028
3133
GUGCCCCUUUGGUCUUGUA
1352

3133
CGAUUCCGUCAAGGGAAAG
1029
3133
CGAUUCCGUCAAGGGAAAG
1029
3151
CUUUCCCUUGACGGAAUCG
1353

3151
GACUACGUUGGAGCAAUCC
1030
3151
GACUACGUUGGAGCAAUCC
1030
3169
GGAUUGCUCCAACGUAGUC
1354

3169
CCUGUGGAUCUGAAACGGC
1031
3169
CCUGUGGAUCUGAAACGGC
1031
3187
GCCGUUUCAGAUCCACAGG
1355

3187
CGCUUGGACAGCAUCACCA
1032
3187
CGCUUGGACAGCAUCACCA
1032
3205
UGGUGAUGCUGUCCAAGCG
1356

3205
AGUAGCCAGAGCUCAGCCA
1033
3205
AGUAGCCAGAGCUCAGCCA
1033
3223
UGGCUGAGCUCUGGCUACU
1357

3223
AGCUCUGGAUUUGUGGAGG
1034
3223
AGCUCUGGAUUUGUGGAGG
1034
3241
CCUCCACAAAUCCAGAGCU
1358

3241
GAGAAGUCCCUCAGUGAUG
1035
3241
GAGAAGUCCCUCAGUGAUG
1035
3259
CAUCACUGAGGGACUUCUC
1359

3259
GUAGAAGAAGAGGAAGCUC
1036
3259
GUAGAAGAAGAGGAAGCUC
1036
3277
GAGCUUCCUCUUCUUCUAC
1360

3277
CCUGAAGAUCUGUAUAAGG
1037
3277
CCUGAAGAUCUGUAUAAGG
1037
3295
CCUUAUACAGAUCUUCAGG
1361

3295
GACUUCCUGACCUUGGAGC
1038
3295
GACUUCCUGACCUUGGAGC
1038
3313
GCUCCAAGGUCAGGAAGUC
1362

3313
CAUCUCAUCUGUUACAGCU
1039
3313
CAUCUCAUCUGUUACAGCU
1039
3331
AGCUGUAACAGAUGAGAUG
1363

3331
UUCCAAGUGGCUAAGGGCA
1040
3331
UUCCAAGUGGCUAAGGGCA
1040
3349
UGCCCUUAGCCACUUGGAA
1364

3349
AUGGAGUUCUUGGCAUCGC
1041
3349
AUGGAGUUCUUGGCAUCGC
1041
3367
GCGAUGCCAAGAACUCCAU
1365

3367
CGAAAGUGUAUCCACAGGG
1042
3367
CGAAAGUGUAUCCACAGGG
1042
3385
CCCUGUGGAUACACUUUCG
1366

3385
GACCUGGCGGCACGAAAUA
1043
3385
GACCUGGCGGCACGAAAUA
1043
3403
UAUUUCGUGCCGCCAGGUC
1367

3403
AUCCUCUUAUCGGAGAAGA
1044
3403
AUCCUCUUAUCGGAGAAGA
1044
3421
UCUUCUCCGAUPAGAGGAU
1368

3421
AACGUGGUUAAAAUCUGUG
1045
3421
AACGUGGUUAAAAUCUGUG
1045
3439
CACAGAUUUUPACCACGUU
1369

3439
GACUUUGGCUUGGCCCGGG
1046
3439
GACUUUGGCUUGGCCCGGG
1046
3457
CCCGGGCCAAGCCAAAGUC
1370

3457
GAUAUUUAUAAAGAUCCAG
1047
3457
GAUAUUUAUAAAGAUCCAG
1047
3475
CUGGAUCUUUAUAAAUAUC
1371

3475
GAUUAUGUCAGAAAAGGAG
1048
3475
GAUUAUGUCAGAAAAGGAG
1048
3493
CUCCUUUUCUGACAUAAUC
1372

3493
GAUGCUCGCCUCCCUUUGA
1049
3493
GAUGCUCGCCUCCCUUUGA
1049
3511
UCAAAGGGAGGCGAGCAUC
1373

3511
AAAUGGAUGGCCCCAGAAA
1050
3511
AAAUGGAUGGCCCCAGAAA
1050
3529
UUUCUGGGGCCAUCCAUUU
1374

3529
ACAAUUUUUGACAGAGUGU
1051
3529
ACAAUUUUUGACAGAGUGU
1051
3547
ACACUCUGUCAAAAAUUGU
1375

3547
UACACAAUCCAGAGUGACG
1052
3547
UACACAAUCCAGAGUGACG
1052
3565
CGUCACUCUGGAUUGUGUA
1376

3565
GUCUGGUCUUUUGGUGUUU
1053
3565
GUCUGGUCUUUUGGUGUUU
1053
3583
AAACACCAAAAGACCAGAC
1377

3583
UUGCUGUGGGAAAUAUUUU
1054
3583
UUGCUGUGGGAAAUAUUUU
1054
3601
AAAAUAUUUCCCACAGCAA
1378

3601
UCCUUAGGUGCUUCUCCAU
1055
3601
UCCUUAGGUGCUUCUCCAU
1055
3619
AUGGAGAAGCACCUAAGGA
1379

3619
UAUCCUGGGGUAAAGAUUG
1056
3619
UAUCCUGGGGUAAAGAUUG
1056
3637
CAAUCUUUACCCCAGGAUA
1380

3637
GAUGAAGAAUUUUGUAGGC
1057
3637
GAUGAAGAAUUUUGUAGGC
1057
3655
GCCUACAAAAUUCUUCAUC
1381

3655
CGAUUGAAAGAAGGAACUA
1058
3655
CGAUUGAAAGAAGGAACUA
1058
3673
UAGUUCCUUCUUUCAAUCG
1382

3673
AGAAUGAGGGCCCCUGAUU
1059
3673
AGAAUGAGGGCCCCUGAUU
1059
3691
AAUCAGGGGCCCUCAUUCU
1383

3691
UAUACUACACCAGAAAUGU
1060
3691
UAUACUACACCAGAAAUGU
1060
3709
ACAUUUCUGGUGUAGUAUA
1384

3709
UACCAGACCAUGCUGGACU
1061
3709
UACCAGACCAUGCUGGACU
1061
3727
AGUCCAGCAUGGUCUGGUA
1385

3727
UGCUGGCACGGGGAGCCCA
1062
3727
UGCUGGCACGGGGAGCCCA
1062
3745
UGGGCUCCCCGUGCCAGCA
1386

3745
AGUCAGAGACCCACGUUUU
1063
3745
AGUCAGAGACCCACGUUUU
1063
3763
AAAACGUGGGUCUCUGACU
1387

3763
UCAGAGUUGGUGGAACAUU
1064
3763
UCAGAGUUGGUGGAACAUU
1064
3781
AAUGUUCCACCAACUCUGA
1388

3781
UUGGGAAAUCUCUUGCAAG
1065
3781
UUGGGAAAUCUCUUGCAAG
1065
3799
CUUGCAAGAGAUUUCCCAA
1389

3799
GCUAAUGCUCAGCAGGAUG
1066
3799
GCUAAUGCUCAGCAGGAUG
1066
3817
CAUCCUGCUGAGCAUUAGC
1390

3817
GGCAAAGACUACAUUGUUC
1067
3817
GGCAAAGACUACAUUGUUC
1067
3835
GAACAAUGUAGUCUUUGCC
1391

3835
CUUCCGAUAUCAGAGACUU
1068
3835
CUUCCGAUAUCAGAGACUU
1068
3853
AAGUCUCUGAUAUCGGAAG
1392

3853
UUGAGCAUGGAAGAGGAUU
1069
3853
UUGAGCAUGGAAGAGGAUU
1069
3871
AAUCCUCUUCCAUGCUCAA
1393

3871
UCUGGACUCUCUCUGCCUA
1070
3871
UCUGGACUCUCUCUGCCUA
1070
3889
UAGGCAGAGAGAGUCCAGA
1394

3889
ACCUCACCUGUUUCCUGUA
1071
3889
ACCUCACCUGUUUCCUGUA
1071
3907
UACAGGAAACAGGUGAGGU
1395

3907
AUGGAGGAGGAGGAAGUAU
1072
3907
AUGGAGGAGGAGGAAGUAU
1072
3925
AUACUUCCUCCUCCUCCAU
1396

3925
UGUGACCCCAAAUUCCAUU
1073
3925
UGUGACCCCAAAUUCCAUU
1073
3943
AAUGGAAUUUGGGGUCACA
1397

3943
UAUGACAACACAGCAGGAA
1074
3943
UAUGACAACACAGCAGGAA
1074
3961
UUCCUGCUGUGUUGUCAUA
1398

3961
AUCAGUCAGUAUCUGCAGA
1075
3961
AUCAGUCAGUAUCUGCAGA
1075
3979
UCUGCAGAUACUGACUGAU
1399

3979
AACAGUAAGCGAAAGAGCC
1076
3979
AACAGUAAGCGAAAGAGCC
1076
3997
GGCUCUUUCGCUUACUGUU
1400

3997
CGGCCUGUGAGUGUAAAAA
1077
3997
CGGCCUGUGAGUGUAAAAA
1077
4015
UUUUUACACUCACAGGCCG
1401

4015
ACAUUUGAAGAUAUCCCGU
1078
4015
ACAUUUGAAGAUAUCCCGU
1078
4033
ACGGGAUAUCUUCAAAUGU
1402

4033
UUAGAAGAACCAGAAGUAA
1079
4033
UUAGAAGAACCAGAAGUAA
1079
4051
UUACUUCUGGUUCUUCUAA
1403

4051
AAAGUAAUCCCAGAUGACA
1080
4051
AAAGUAAUCCCAGAUGACA
1080
4069
UGUCAUCUGGGAUUACUUU
1404

4069
AACCAGACGGACAGUGGUA
1081
4069
AACCAGACGGACAGUGGUA
1081
4087
UACCACUGUCCGUCUGGUU
1405

4087
AUGGUUCUUGCCUCAGAAG
1082
4087
AUGGUUCUUGCCUCAGAAG
1082
4105
CUUCUGAGGCAAGAACCAU
1406

4105
GAGCUGAAAACUUUGGAAG
1083
4105
GAGCUGAAAACUUUGGAAG
1083
4123
CUUCCAAAGUUUUCAGCUC
1407

4123
GACAGAACCAAAUUAUCUC
1084
4123
GACAGAACCAAAUUAUCUC
1084
4141
GAGAUAAUUUGGUUCUGUC
1408

4141
CCAUCUUUUGGUGGAAUGG
1085
4141
CCAUCUUUUGGUGGAAUGG
1085
4159
CCAUUCCACCAAAAGAUGG
1409

4159
GUGCCCAGCAAAAGCAGGG
1086
4159
GUGCCCAGCAAAAGCAGGG
1086
4177
CCCUGCUUUUGCUGGGCAC
1410

4177
GAGUCUGUGGCAUCUGAAG
1087
4177
GAGUCUGUGGCAUCUGAAG
1087
4195
CUUCAGAUGCCACAGACUC
1411

4195
GGCUCAAACCAGACAAGCG
1088
4195
GGCUCAAACCAGACAAGCG
1088
4213
CGCUUGUCUGGUUUGAGCC
1412

4213
GGCUACCAGUCCGGAUAUC
1089
4213
GGCUACCAGUCCGGAUAUC
1089
4231
GAUAUCCGGACUGGUAGCC
1413

4231
CACUCCGAUGACACAGACA
1090
4231
CACUCCGAUGACACAGACA
1090
4249
UGUCUGUGUCAUCGGAGUG
1414

4249
ACCACCGUGUACUCCAGUG
1091
4249
ACCACCGUGUACUCCAGUG
1091
4267
CACUGGAGUACACGGUGGU
1415

4267
GAGGAAGCAGAACUUUUAA
1092
4267
GAGGAAGCAGAACUUUUAA
1092
4285
UUAAAAGUUCUGCUUCCUC
1416

4285
AAGCUGAUAGAGAUUGGAG
1093
4285
AAGCUGAUAGAGAUUGGAG
1093
4303
CUCCAAUCUCUAUCAGCUU
1417

4303
GUGCAAACCGGUAGCACAG
1094
4303
GUGCAAACCGGUAGCACAG
1094
4321
CUGUGCUACCGGUUUGCAC
1418

4321
GCCCAGAUUCUCCAGCCUG
1095
4321
GCCCAGAUUCUCCAGCCUG
1095
4339
CAGGCUGGAGAAUCUGGGC
1419

4339
GACUCGGGGACCACACUGA
1096
4339
GACUCGGGGACCACACUGA
1096
4357
UCAGUGUGGUCCCCGAGUC
1420

4357
AGCUCUCCUCCUGUUUAAA
1097
4357
AGCUCUCCUCCUGUUUAAA
1097
4375
UUUAAACAGGAGGAGAGCU
1421

4375
AAGGAAGCAUCCACACCCC
1098
4375
AAGGAAGCAUCCACACCCC
1098
4393
GGGGUGUGGAUGCUUCCUU
1422

4393
CAACUCCCGGACAUCACAU
1099
4393
CAACUCCCGGACAUCACAU
1099
4411
AUGUGAUGUCCGGGAGUUG
1423

4411
UGAGAGGUCUGCUCAGAUU
1100
4411
UGAGAGGUCUGCUCAGAUU
1100
4429
AAUCUGAGCAGACCUCUCA
1424

4429
UUUGAAGUGUUGUUCUUUC
1101
4429
UUUGAAGUGUUGUUCUUUC
1101
4447
GAAAGAACAACACUUCAAA
1425

4447
CCACCAGCAGGAAGUAGCC
1102
4447
CCACCAGCAGGAAGUAGCC
1102
4465
GGCUACUUCCUGCUGGUGG
1426

4465
CGCAUUUGAUUUUCAUUUC
1103
4465
CGCAUUUGAUUUUCAUUUC
1103
4483
GAAAUGAAAAUCAAAUGCG
1427

4483
CGACAACAGAAAAAGGACC
1104
4483
CGACAACAGAAAAAGGACC
1104
4501
GGUCCUUUUUCUGUUGUCG
1428

4501
CUCGGACUGCAGGGAGCCA
1105
4501
CUCGGACUGCAGGGAGCCA
1105
4519
UGGCUCCCUGCAGUCCGAG
1429

4519
AGUCUUCUAGGCAUAUCCU
1106
4519
AGUCUUCUAGGCAUAUCCU
1106
4537
AGGAUAUGCCUAGAAGACU
1430

4537
UGGAAGAGGCUUGUGACCC
1107
4537
UGGAAGAGGCUUGUGACCC
1107
4555
GGGUCACAAGCCUCUUCCA
1431

4555
CAAGAAUGUGUCUGUGUCU
1108
4555
CAAGAAUGUGUCUGUGUCU
1108
4573
AGACACAGACACAUUCUUG
1432

4573
UUCUCCCAGUGUUGACCUG
1109
4573
UUCUCCCAGUGUUGACCUG
1109
4591
CAGGUCAACACUGGGAGAA
1433

4591
GAUCCUCUUUUUUCAUUCA
1110
4591
GAUCCUCUUUUUUCAUUCA
1110
4609
UGAAUGAAAAAAGAGGAUC
1434

4609
AUUUAAAAAGCAUUAUCAU
1111
4609
AUUUAAAAAGCAUUAUCAU
1111
4627
AUGAUAAUGCUUUUUAAAU
1435

4627
UGCCCCUGCUGCGGGUCUC
1112
4627
UGCCCCUGCUGCGGGUCUC
1112
4645
GAGACCCGCAGCAGGGGCA
1436

4645
CACCAUGGGUUUAGAACAA
1113
4645
CACCAUGGGUUUAGAACAA
1113
4663
UUGUUCUAAACCCAUGGUG
1437

4663
AAGAGCUUCAAGCAAUGGC
1114
4663
AAGAGCUUCAAGCAAUGGC
1114
4681
GCCAUUGCUUGAAGCUCUU
1438

4681
CCCCAUCCUCAAAGAAGUA
1115
4681
CCCCAUCCUCAAAGAAGUA
1115
4699
UACUUCUUUGAGGAUGGGG
1439

4699
AGCAGUACCUGGGGAGCUG
1116
4699
AGCAGUACCUGGGGAGCUG
1116
4717
CAGCUCCCCAGGUACUGCU
1440

4717
GACACUUCUGUAAAACUAG
1117
4717
GACACUUCUGUAAAACUAG
1117
4735
CUAGUUUUACAGAAGUGUC
1441

4735
GAAGAUAAACCAGGCAACG
1118
4735
GAAGAUAAACCAGGCAACG
1118
4753
CGUUGCCUGGUUUAUCUUC
1442

4753
GUAAGUGUUCGAGGUGUUG
1119
4753
GUAAGUGUUCGAGGUGUUG
1119
4771
CAACACCUCGAACACUUAC
1443

4771
GAAGAUGGGAAGGAUUUGC
1120
4771
GAAGAUGGGAAGGAUUUGC
1120
4789
GCAAAUCCUUCCCAUCUUC
1444

4789
CAGGGCUGAGUCUAUCCAA
1121
4789
CAGGGCUGAGUCUAUCCAA
1121
4807
UUGGAUAGACUCAGCCCUG
1445

4807
AGAGGCUUUGUUUAGGACG
1122
4807
AGAGGCUUUGUUUAGGACG
1122
4825
CGUCCUAAACAAAGCCUCU
1446

4825
GUGGGUCCCAAGCCAAGCC
1123
4825
GUGGGUCCCAAGCCAAGCC
1123
4843
GGCUUGGCUUGGGACCCAC
1447

4843
CUUAAGUGUGGAAUUCGGA
1124
4843
CUUAAGUGUGGAAUUCGGA
1124
4861
UCCGAAUUCCACACUUAAG
1448

4861
AUUGAUAGAAAGGAAGACU
1125
4861
AUUGAUAGAAAGGAAGACU
1125
4879
AGUCUUCCUUUCUAUCAAU
1449

4879
UAACGUUACCUUGCUUUGG
1126
4879
UAACGUUACCUUGCUUUGG
1126
4897
CCAAAGCAAGGUAACGUUA
1450

4897
GAGAGUACUGGAGCCUGCA
1127
4897
GAGAGUACUGGAGCCUGCA
1127
4915
UGCAGGCUCCAGUACUCUC
1451

4915
AAAUGCAUUGUGUUUGCUC
1128
4915
AAAUGCAUUGUGUUUGCUC
1128
4933
GAGCAAACACAAUGCAUUU
1452

4933
CUGGUGGAGGUGGGCAUGG
1129
4933
CUGGUGGAGGUGGGCAUGG
1129
4951
CCAUGCCCACCUCCACCAG
1453

4951
GGGUCUGUUCUGAAAUGUA
1130
4951
GGGUCUGUUCUGAAAUGUA
1130
4969 UACAUUUCAGAACAGACCC
1454

4969
AAAGGGUUCAGACGGGGUU
1131
4969
AAAGGGUUCAGACGGGGUU
1131
4987
AACCCCGUCUGAACCCUUU
1455

4987
UUCUGGUUUUAGAAGGUUG
1132
4987
UUCUGGUUUUAGAAGGUUG
1132
5005
CAACCUUCUAAAACCAGAA
1456

5005
GCGUGUUCUUCGAGUUGGG
1133
5005
GCGUGUUCUUCGAGUUGGG
1133
5023
CCCAACUCGAAGAACACGC
1457

5023
GCUAAAGUAGAGUUCGUUG
1134
5023
GCUAAAGUAGAGUUCGUUG
1134
5041
CAACGAACUCUACUUUAGC
1458

5041
GUGCUGUUUCUGACUCCUA
1135
5041
GUGCUGUUUCUGACUCCUA
1135
5059
UAGGAGUCAGAAACAGCAC
1459

5059
AAUGAGAGUUCCUUCCAGA
1136
5059
AAUGAGAGUUCCUUCCAGA
1136
5077
UCUGGAAGGAACUCUCAUU
1460

5077
ACCGUUAGCUGUCUCCUUG
1137
5077
ACCGUUAGCUGUCUCCUUG
1137
5095
CAAGGAGACAGCUAACGGU
1461

5095
GCCAAGCCCCAGGAAGAAA
1138
5095
GCCAAGCCCCAGGAAGAAA
1138
5113
UUUCUUCCUGGGGCUUGGC
1462

5113
AAUGAUGCAGCUCUGGCUC
1139
5113
AAUGAUGCAGCUCUGGCUC
1139
5131
GAGCCAGAGCUGCAUCAUU
1463

5131
CCUUGUCUCCCAGGCUGAU
1140
5131
CCUUGUCUCCCAGGCUGAU
1140
5149
AUCAGCCUGGGAGACAAGG
1464

5149
UCCUUUAUUCAGAAUACCA
1141
5149
UCCUUUAUUCAGAAUACCA
1141
5167
UGGUAUUCUGAAUAAAGGA
1465

5167
ACAAAGAAAGGACAUUCAG
1142
5167
ACAAAGAAAGGACAUUCAG
1142
5185
CUGAAUGUCCUUUCUUUGU
1466

5185
GCUCAAGGCUCCCUGCCGU
1143
5185
GCUCAAGGCUCCCUGCCGU
1143
5203
ACGGCAGGGAGCCUUGAGC
1467

5203
UGUUGAAGAGUUCUGACUG
1144
5203
UGUUGAAGAGUUCUGACUG
1144
5221
CAGUCAGAACUCUUCAACA
1468

5221
GCACAAACCAGCUUCUGGU
1145
5221
GCACAAACCAGCUUCUGGU
1145
5239
ACCAGAAGCUGGUUUGUGC
1469

5239
UUUCUUCUGGAAUGAAUAC
1146
5239
UUUCUUCUGGAAUGAAUAC
1146
5257
GUAUUCAUUCCAGAAGAAA
1470

5257
CCCUCAUAUCUGUCCUGAU
1147
5257
CCCUCAUAUCUGUCCUGAU
1147
5275
AUCAGGACAGAUAUGAGGG
1471

5275
UGUGAUAUGUCUGAGACUG
1148
5275
UGUGAUAUGUCUGAGACUG
1148
5293
CAGUCUCAGACAUAUCACA
1472

5293
GAAUGCGGGAGGUUCAAUG
1149
5293
GAAUGCGGGAGGUUCAAUG
1149
5311
CAUUGAACCUCCCGCAUUC
1473

5311
GUGAAGCUGUGUGUGGUGU
1150
5311
GUGAAGCUGUGUGUGGUGU
1150
5329
ACACCACACACAGCUUCAC
1474

5329
UCAAAGUUUCAGGAAGGAU
1151
5329
UCAAAGUUUCAGGAAGGAU
1151
5347
AUCCUUCCUGAAACUUUGA
1475

5347
UUUUACCCUUUUGUUCUUC
1152
5347
UUUUACCCUUUUGUUCUUC
1152
5365
GAAGAACAAAAGGGUAAAA
1476

5365
CCCCCUGUCCCCAACCCAC
1153
5365
CCCCCUGUCCCCAACCCAC
1153
5383
GUGGGUUGGGGACAGGGGG
1477

5383
CUCUCACCCCGCAACCCAU
1154
5383
CUCUCACCCCGCAACCCAU
1154
5401
AUGGGUUGCGGGGUGAGAG
1478

5401
UCAGUAUUUUAGUUAUUUG
1155
5401
UCAGUAUUUUAGUUAUUUG
1155
5419
CAAAUAACUAAAAUACUGA
1479

5419
GGCCUCUACUCCAGUAAAC
1156
5419
GGCCUCUACUCCAGUAAAC
1156
5437
GUUUACUGGAGUAGAGGCC
1480

5437
CCUGAUUGGGUUUGUUCAC
1157
5437
CCUGAUUGGGUUUGUUCAC
1157
5455
GUGAACAAACCCAAUCAGG
1481

5455
CUCUCUGAAUGAUUAUUAG
1158
5455
CUCUCUGAAUGAUUAUUAG
1158
5473
CUAAUAAUCAUUCAGAGAG
1482

5473
GCCAGACUUCAAAAUUAUU
1159
5473
GCCAGACUUCAAAAUUAUU
1159
5491
AAUAAUUUUGAAGUCUGGC
1483

5491
UUUAUAGCCCAAAUUAUAA
1160
5491
UUUAUAGCCCAAAUUAUAA
1160
5509
UUAUAAUUUGGGCUAUAAA
1484

5509
ACAUCUAUUGUAUUAUUUA
1161
5509
ACAUCUAUUGUAUUAUUUA
1161
5527
UAAAUAAUACAAUAGAUGU
1485

5527
AGACUUUUAACAUAUAGAG
1162
5527
AGACUUUUAACAUAUAGAG
1162
5545
CUCUAUAUGUUAAAAGUCU
1486

5545
GCUAUUUCUACUGAUUUUU
1163
5545
GCUAUUUCUACUGAUUUUU
1163
5563
AAAAAUCAGUAGAAAUAGC
1487

5563
UGCCCUUGUUCUGUCCUUU
1164
5563
UGCCCUUGUUCUGUCCUUU
1164
5581
AAAGGACAGAACAAGGGCA
1488

5581
UUUUUCAAAAAAGAAAAUG
1165
5581
UUUUUCAAAAAAGAAAAUG
1165
5599
CAUUUUCUUUUUUGAAAAA
1489

5599
GUGUUUUUUGUUUGGUACC
1166
5599
GUGUUUUUUGUUUGGUACC
1166
5617
GGUACCAAACAAAAAACAC
1490

5617
CAUAGUGUGAAAUGCUGGG
1167
5617
CAUAGUGUGAAAUGCUGGG
1167
5635
CCCAGCAUUUCACACUAUG
1491

5635
GAACAAUGACUAUAAGACA
1168
5635
GAACAAUGACUAUAAGACA
1168
5653
UGUCUUAUAGUCAUUGUUC
1492

5653
AUGCUAUGGCACAUAUAUU
1169
5653
AUGCUAUGGCACAUAUAUU
1169
5671
AAUAUAUGUGCCAUAGCAU
1493

5671
UUAUAGUCUGUUUAUGUAG
1170
5671
UUAUAGUCUGUUUAUGUAG
1170
5689
CUACAUAAACAGACUAUAA
1494

5689
GAAACAAAUGUAAUAUAUU
1171
5689
GAAACAAAUGUAAUAUAUU
1171
5707
AAUAUAUUACAUUUGUUUC
1495

5707
UAAAGCCUUAUAUAUAAUG
1172
5707
UAAAGCCUUAUAUAUAAUG
1172
5725
CAUUAUAUAUPAGGCUUUA
1496

5725
GAACUUUGUACUAUUCACA
1173
5725
GAACUUUGUACUAUUCACA
1173
5743
UGUGAAUAGUACAkAGUUC
1497

5743
AUUUUGUAUCAGUAUUAUG
1174
5743
AUUUUGUAUCAGUAUUAUG
1174
5761
CAUAAUACUGAUACAAAAU
1498

5761
GUAGCAUAACAAAGGUCAU
1175
5761
GUAGCAUAACAAAGGUCAU
1175
5779
AUGACCUUUGUUAUGCUAC
1499

5779
UAAUGCUUUCAGCAAUUGA
1176
5779
UAAUGCUUUCAGCAAUUGA
1176
5797
UCAAUUGCUGAAAGCAUUA
1500

5797
AUGUCAUUUUAUUAAAGAA
1177
5797
AUGUCAUUUUAUUAAAGAA
1177
5815
UUCUUUAAUAAAAUGACAU
1501

5812
AGAACAUUGAAAAACUUGA
1178
5812
AGAACAUUGAAAAACUUGA
1178
5830
UCAAGUUUUUCAAUGUUCU
1502

VEGFR3/FILT4 NM_002020.1

1
ACCCACGCGCAGCGGCCGG
1503
1
ACCCACGCGCAGCGGCCGG
1503
19
CCGGCCGCUGCGCGUGGGU
1750

19
GAGAUGCAGCGGGGCGCCG
1504
19
GAGAUGCAGCGGGGCGCCG
1504
37
CGGCGCCCCGCUGCAUCUC
1751

37
GCGCUGUGCCUGCGACUGU
1505
37
GCGCUGUGCCUGCGACUGU
1505
55
ACAGUCGCAGGCACAGCGC
1752

55
UGGCUCUGCCUGGGACUCC
1506
55
UGGCUCUGCCUGGGACUCC
1506
73
GGAGUCCCAGGCAGAGCCA
1753

73
CUGGACGGCCUGGUGAGUG
1507
73
CUGGACGGCCUGGUGAGUG
1507
91
CACUCACCAGGCCGUCCAG
1754

91
GACUACUCCAUGACCCCCC
1508
91
GACUACUCCAUGACCCCCC
1508
109
GGGGGGUCAUGGAGUAGUC
1755

109
CCGACCUUGAACAUCACGG
1509
109
CCGACCUUGAACAUCACGG
1509
127
CCGUGAUGUUCAAGGUCGG
1756

127
GAGGAGUCACACGUCAUCG
1510
127
GAGGAGUCACACGUCAUCG
1510
145
CGAUGACGUGUGACUCCUC
1757

145
GACACCGGUGACAGCCUGU
1511
145
GACACCGGUGACAGCCUGU
1511
163
ACAGGCUGUCACCGGUGUC
1758

163
UCCAUCUCCUGCAGGGGAC
1512
163
UCCAUCUCCUGCAGGGGAC
1512
181
GUCCCCUGCAGGAGAUGGA
1759

181
CAGCACCCCCUCGAGUGGG
1513
181
CAGCACCCCCUCGAGUGGG
1513
199
CCCACUCGAGGGGGUGCUG
1760

199
GCUUGGCCAGGAGCUCAGG
1514
199
GCUUGGCCAGGAGCUCAGG
1514
217
CCUGAGCUCCUGGCCAAGC
1761

217
GAGGCGCCAGCCACCGGAG
1515
217
GAGGCGCCAGCCACCGGAG
1515
235
CUCCGGUGGCUGGCGCCUC
1762

235
GACAAGGACAGCGAGGACA
1516
235
GACAAGGACAGCGAGGACA
1516
253
UGUCCUCGCUGUCCUUGUC
1763

253
ACGGGGGUGGUGCGAGACU
1517
253
ACGGGGGUGGUGCGAGACU
1517
271
AGUCUCGCACCACCCCCGU
1764

271
UGCGAGGGCACAGACGCCA
1518
271
UGCGAGGGCACAGACGCCA
1518
289
UGGCGUCUGUGCCCUCGCA
1765

289
AGGCCCUACUGCAAGGUGU
1519
289
AGGCCCUACUGCAAGGUGU
1519
307
ACACCUUGCAGUAGGGCCU
1766

307
UUGCUGCUGCACGAGGUAC
1520
307
UUGCUGCUGCACGAGGUAC
1520
325
GUACCUCGUGCAGCAGCAA
1767

325
CAUGCCAACGACACAGGCA
1521
325
CAUGCCAACGACACAGGCA
1521
343
UGCCUGUGUCGUUGGCAUG
1768

343
AGCUACGUCUGCUACUACA
1522
343
AGCUACGUCUGCUACUACA
1522
361
UGUAGUAGCAGACGUAGCU
1769

361
AAGUACAUCAAGGCACGCA
1523
361
AAGUACAUCAAGGCACGCA
1523
379
UGCGUGCCUUGAUGUACUU
1770

379
AUCGAGGGCACCACGGCCG
1524
379
AUCGAGGGCACCACGGCCG
1524
397
CGGCCGUGGUGCCCUCGAU
1771

397
GCCAGCUCCUACGUGUUCG
1525
397
GCCAGCUCCUACGUGUUCG
1525
415
CGAACACGUAGGAGCUGGC
1772

415
GUGAGAGACUUUGAGCAGC
1526
415
GUGAGAGACUUUGAGCAGC
1526
433
GCUGCUCAAAGUCUCUCAC
1773

433
CCAUUCAUCAACAAGCCUG
1527
433
CCAUUCAUCAACAAGCCUG
1527
451
CAGGCUUGUUGAUGAAUGG
1774

451
GACACGCUCUUGGUCAACA
1528
451
GACACGCUCUUGGUCAACA
1528
469
UGUUGACCAAGAGCGUGUC
1775

469
AGGAAGGACGCCAUGUGGG
1529
469
AGGAAGGACGCCAUGUGGG
1529
487
CCCACAUGGCGUCCUUCCU
1776

487
GUGCCCUGUCUGGUGUCCA
1530
487
GUGCCCUGUCUGGUGUCCA
1530
505
UGGACACCAGACAGGGCAC
1777

505
AUCCCCGGCCUCAAUGUCA
1531
505
AUCCCCGGCCUCAAUGUCA
1531
523
UGACAUUGAGGCCGGGGAU
1778

523
ACGCUGCGCUCGCPAAGCU
1532
523
ACGCUGCGCUCGCAAAGCU
1532
541
AGCUUUGCGAGCGCAGCGU
1779

541
UCGGUGCUGUGGCCAGACG
1533
541
UCGGUGCUGUGGCCAGACG
1533
559
CGUCUGGCCACAGCACCGA
1780

559
GGGCAGGAGGUGGUGUGGG
1534
559
GGGCAGGAGGUGGUGUGGG
1534
577
CCCACACCACCUCCUGCCC
1781

577
GAUGACCGGCGGGGCAUGC
1535
577
GAUGACCGGCGGGGCAUGC
1535
595
GCAUGCCCCGCCGGUCAUC
1782

595
CUCGUGUCCACGCCACUGC
1536
595
CUCGUGUCCACGCCACUGC
1536
613
GCAGUGGCGUGGACACGAG
1783

613
CUGCACGAUGCCCUGUACC
1537
613
CUGCACGAUGCCCUGUACC
1537
631
GGUACAGGGCAUCGUGCAG
1784

631
CUGCAGUGCGAGACCACCU
1538
631
CUGCAGUGCGAGACCACCU
1538
649
AGGUGGUCUCGCACUGCAG
1785

649
UGGGGAGACCAGGACUUCC
1539
649
UGGGGAGACCAGGACUUCC
1539
667
GGAAGUCCUGGUCUCCCCA
1786

667
CUUUCCkACCCCUUCCUGG
1540
667
CUUUCCAACCCCUUCCUGG
1540
685
CCAGGAAGGGGUUGGAAAG
1787

685
GUGCACAUCACAGGCAACG
1541
685
GUGCACAUCACAGGCAACG
1541
703
CGUUGCCUGUGAUGUGCAC
1788

703
GAGCUCUAUGACAUCCAGC
1542
703
GAGCUCUAUGACAUCCAGC
1542
721
GCUGGAUGUCAUAGAGCUC
1789

721
CUGUUGCCCAGGAAGUCGC
1543
721
CUGUUGCCCAGGAAGUCGC
1543
739
GCGACUUCCUGGGCAACAG
1790

739
CUGGAGCUGCUGGUAGGGG
1544
739
CUGGAGCUGCUGGUAGGGG
1544
757
CCCCUACCAGCAGCUCCAG
1791

757
GAGAAGCUGGUCCUCAACU
1545
757
GAGAAGCUGGUCCUCAACU
1545
775
AGUUGAGGACCAGCUUCUC
1792

775
UGCACCGUGUGGGCUGAGU
1546
775
UGCACCGUGUGGGCUGAGU
1546
793
ACUCAGCCCACACGGUGCA
1793

793
UUUAACUCAGGUGUCACCU
1547
793
UUUAACUCAGGUGUCACCU
1547
811
AGGUGACACCUGAGUUAAA
1794

811
UUUGACUGGGACUACCCAG
1548
811
UUUGACUGGGACUACCCAG
1548
829
CUGGGUAGUCCCAGUCAAA
1795

829
GGGAAGCAGGCAGAGCGGG
1549
829
GGGAAGCAGGCAGAGCGGG
1549
847
CCCGCUCUGCCUGCUUCCC
1796

847
GGUAAGUGGGUGCCCGAGC
1550
847
GGUAAGUGGGUGCCCGAGC
1550
865
GCUCGGGCACCCACUUACC
1797

865
CGACGCUCCCAACAGACCC
1551
865
CGACGCUCCCAACAGACCC
1551
883
GGGUCUGUUGGGAGCGUCG
1798

883
CACACAGAACUCUCCAGCA
1552
883
CACACAGAACUCUCCAGCA
1552
901
UGCUGGAGAGUUCUGUGUG
1799

901
AUCCUGACCAUCCACAACG
1553
901
AUCCUGACCAUCCACAACG
1553
919
CGUUGUGGAUGGUCAGGAU
1800

919
GUCAGCCAGCACGACCUGG
1554
919
GUCAGCCAGCACGACCUGG
1554
937
CCAGGUCGUGCUGGCUGAC
1801

937
GGCUCGUAUGUGUGCAAGG
1555
937
GGCUCGUAUGUGUGCAAGG
1555
955
CCUUGCACACAUACGAGCC
1802

955
GCCAACAACGGCAUCCAGC
1556
955
GCCAACAACGGCAUCCAGC
1556
973
GCUGGAUGCCGUUGUUGGC
1803

973
CGAUUUCGGGAGAGCACCG
1557
973
CGAUUUCGGGAGAGCACCG
1557
991
CGGUGCUCUCCCGAAAUCG
1804

991
GAGGUCAUUGUGCAUGAAA
1558
991
GAGGUCAUUGUGCAUGAAA
1558
1009
UUUCAUGCACAAUGACCUC
1805

1009
AAUCCCUUCAUCAGCGUCG
1559
1009
AAUCCCUUCAUCAGCGUCG
1559
1027
CGACGCUGAUGAAGGGAUU
1806

1027
GAGUGGCUCAAAGGACCCA
1560
1027
GAGUGGCUCAAAGGACCCA
1560
1045
UGGGUCCUUUGAGCCACUC
1807

1045
AUCCUGGAGGCCACGGCAG
1561
1045
AUCCUGGAGGCCACGGCAG
1561
1063
CUGCCGUGGCCUCCAGGAU
1808

1063
GGAGACGAGCUGGUGAAGC
1562
1063
GGAGACGAGCUGGUGAAGC
1562
1081
GCUUCACCAGCUCGUCUCC
1809

1081
CUGCCCGUGAAGCUGGCAG
1563
1081
CUGCCCGUGAAGCUGGCAG
1563
1099
CUGCCAGCUUCACGGGCAG
1810

1099
GCGUACCCCCCGCCCGAGU
1564
1099
GCGUACCCCCCGCCCGAGU
1564
1117
ACUCGGGCGGGGGGUACGC
1811

1117
UUCCAGUGGUACAAGGAUG
1565
1117
UUCCAGUGGUACAAGGAUG
1565
1135
CAUCCUUGUACCACUGGAA
1812

1135
GGAAAGGCACUGUCCGGGC
1566
1135
GGAAAGGCACUGUCCGGGC
1566
1153
GCCCGGACAGUGCCUUUCC
1813

1153
CGCCACAGUCCACAUGCCC
1567
1153
CGCCACAGUCCACAUGCCC
1567
1171
GGGCAUGUGGACUGUGGCG
1814

1171
CUGGUGCUCAAGGAGGUGA
1568
1171
CUGGUGCUCAAGGAGGUGA
1568
1189
UCACCUCCUUGAGCACCAG
1815

1189
ACAGAGGCCAGCACAGGCA
1569
1189
ACAGAGGCCAGCACAGGCA
1569
1207
UGCCUGUGCUGGCCUCUGU
1816

1207
ACCUACACCCUCGCCCUGU
1570
1207
ACCUACACCCUCGCCCUGU
1570
1225
ACAGGGCGAGGGUGUAGGU
1817

1225
UGGAACUCCGCUGCUGGCC
1571
1225
UGGAACUCCGCUGCUGGCC
1571
1243
GGCCAGCAGCGGAGUUCCA
1818

1243
CUGAGGCGCAACAUCAGCC
1572
1243
CUGAGGCGCAACAUCAGCC
1572
1261
GGCUGAUGUUGCGCCUCAG
1819

1261
CUGGAGCUGGUGGUGAAUG
1573
1261
CUGGAGCUGGUGGUGAAUG
1573
1279
CAUUCACCACCAGCUCCAG
1820

1279
GUGCCCCCCCAGAUACAUG
1574
1279
GUGCCCCCCCAGAUACAUG
1574
1297
CAUGUAUCUGGGGGGGCAC
1821

1297
GAGAAGGAGGCCUCCUCCC
1575
1297
GAGAAGGAGGCCUCCUCCC
1575
1315
GGGAGGAGGCCUCCUUCUC
1822

1315
CCCAGCAUCUACUCGCGUC
1576
1315
CCCAGCAUCUACUCGCGUC
1576
1333
GACGCGAGUAGAUGCUGGG
1823

1333
CACAGCCGCCAGGCCCUCA
1577
1333
CACAGCCGCCAGGCCCUCA
1577
1351
UGAGGGCCUGGCGGCUGUG
1824

1351
ACCUGCACGGCCUACGGGG
1578
1351
ACCUGCACGGCCUACGGGG
1578
1369
CCCCGUAGGCCGUGCAGGU
1825

1369
GUGCCCCUGCCUCUCAGCA
1579
1369
GUGCCCCUGCCUCUCAGCA
1579
1387
UGCUGAGAGGCAGGGGCAC
1826

1387
AUCCAGUGGCACUGGCGGC
1580
1387
AUCCAGUGGCACUGGCGGC
1580
1405
GCCGCCAGUGCCACUGGAU
1827

1405
CCCUGGACACCCUGCAAGA
1581
1405
CCCUGGACACCCUGCAAGA
1581
1423
UCUUGCAGGGUGUCCAGGG
1828

1423
AUGUUUGCCCAGCGUAGUC
1582
1423
AUGUUUGCCCAGCGUAGUC
1582
1441
GACUACGCUGGGCAAACAU
1829

1441
CUCCGGCGGCGGCAGCAGC
1583
1441
CUCCGGCGGCGGCAGCAGC
1583
1459
GCUGCUGCCGCCGCCGGAG
1830

1459
CAAGACCUCAUGCCACAGU
1584
1459
CAAGACCUCAUGCCACAGU
1584
1477
ACUGUGGCAUGAGGUCUUG
1831

1477
UGCCGUGACUGGAGGGCGG
1585
1477
UGCCGUGACUGGAGGGCGG
1585
1495
CCGCCCUCCAGUCACGGCA
1832

1495
GUGACCACGCAGGAUGCCG
1586
1495
GUGACCACGCAGGAUGCCG
1586
1513
CGGCAUCCUGCGUGGUCAC
1833

1513
GUGAACCCCAUCGAGAGCC
1587
1513
GUGAACCCCAUCGAGAGCC
1587
1531
GGCUCUCGAUGGGGUUCAC
1834

1531
CUGGACACCUGGACCGAGU
1588
1531
CUGGACACCUGGACCGAGU
1588
1549
ACUCGGUCCAGGUGUCCAG
1835

1549
UUUGUGGAGGGAAAGAAUA
1589
1549
UUUGUGGAGGGAAAGAAUA
1589
1567
UAUUCUUUCCCUCCACAAA
1836

1567
AAGACUGUGAGCAAGCUGG
1590
1567
AAGACUGUGAGCAAGCUGG
1590
1585
CCAGCUUGCUCACAGUCUU
1837

1585
GUGAUCCAGAAUGCCAACG
1591
1585
GUGAUCCAGAAUGCCAACG
1591
1603
CGUUGGCAUUCUGGAUCAC
1838

1603
GUGUCUGCCAUGUACAAGU
1592
1603
GUGUCUGCCAUGUACAAGU
1592
1621
ACUUGUACAUGGCAGACAC
1839

1621
UGUGUGGUCUCCAACAAGG
1593
1621
UGUGUGGUCUCCAACAAGG
1593
1639
CCUUGUUGGAGACCACACA
1840

1639
GUGGGCCAGGAUGAGCGGC
1594
1639
GUGGGCCAGGAUGAGCGGC
1594
1657
GCCGCUCAUCCUGGCCCAC
1841

1657
CUCAUCUACUUCUAUGUGA
1595
1657
CUCAUCUACUUCUAUGUGA
1595
1675
UCACAUAGAAGUAGAUGAG
1842

1675
ACCACCAUCCCCGACGGCU
1596
1675
ACCACCAUCCCCGACGGCU
1596
1693
AGCCGUCGGGGAUGGUGGU
1843

1693
UUCACCAUCGAAUCCAAGC
1597
1693
UUCACCAUCGAAUCCAAGC
1597
1711
GCUUGGAUUCGAUGGUGAA
1844

1711
CCAUCCGAGGAGCUACUAG
1598
1711
CCAUCCGAGGAGCUACUAG
1598
1729
CUAGUAGCUCCUCGGAUGG
1845

1729
GAGGGCCAGCCGGUGCUCC
1599
1729
GAGGGCCAGCCGGUGCUCC
1599
1747
GGAGCACCGGCUGGCCCUC
1846

1747
CUGAGCUGCCAAGCCGACA
1600
1747
CUGAGCUGCCAAGCCGACA
1600
1765
UGUCGGCUUGGCAGCUCAG
1847

1765
AGCUACAAGUACGAGCAUC
1601
1765
AGCUACAAGUACGAGCAUC
1601
1783
GAUGCUCGUACUUGUAGCU
1848

1783
CUGCGCUGGUACCGCCUCA
1602
1783
CUGCGCUGGUACCGCCUCA
1602
1801
UGAGGCGGUACCAGCGCAG
1849

1801
AACCUGUCCACGCUGCACG
1603
1801
AACCUGUCCACGCUGCACG
1603
1819
CGUGCAGCGUGGACAGGUU
1850

1819
GAUGCGCACGGGAACCCGC
1604
1819
GAUGCGCACGGGAACCCGC
1604
1837
GCGGGUUCCCGUGCGCAUC
1851

1837
CUUCUGCUCGACUGCAAGA
1605
1837
CUUCUGCUCGACUGCAAGA
1605
1855
UCUUGCAGUCGAGCAGAAG
1852

1855
AACGUGCAUCUGUUCGCCA
1606
1855
AACGUGCAUCUGUUCGCCA
1606
1873
UGGCGAACAGAUGCACGUU
1853

1873
ACCCCUCUGGCCGCCAGCC
1607
1873
ACCCCUCUGGCCGCCAGCC
1607
1891
GGCUGGCGGCCAGAGGGGU
1854

1891
CUGGAGGAGGUGGCACCUG
1608
1891
CUGGAGGAGGUGGCACCUG
1608
1909
CAGGUGCCACCUCCUCCAG
1855

1909
GGGGCGCGCCACGCCACGC
1609
1909
GGGGCGCGCCACGCCACGC
1609
1927
GCGUGGCGUGGCGCGCCCC
1856

1927
CUCAGCCUGAGUAUCCCCC
1610
1927
CUCAGCCUGAGUAUCCCCC
1610
1945
GGGGGAUACUCAGGCUGAG
1857

1945
CGCGUCGCGCCCGAGCACG
1611
1945
CGCGUCGCGCCCGAGCACG
1611
1963
CGUGCUCGGGCGCGACGCG
1858

1963
GAGGGCCACUAUGUGUGCG
1612
1963
GAGGGCCACUAUGUGUGCG
1612
1981
CGCACACAUAGUGGCCCUC
1859

1981
GAAGUGCAAGACCGGCGCA
1613
1981
GAAGUGCAAGACCGGCGCA
1613
1999
UGCGCCGGUCUUGCACUUC
1860

1999
AGCCAUGACAAGCACUGCC
1614
1999
AGCCAUGACAAGCACUGCC
1614
2017
GGCAGUGCUUGUCAUGGCU
1861

2017
CACAAGAAGUACCUGUCGG
1615
2017
CACAAGAAGUACCUGUCGG
1615
2035
CCGACAGGUACUUCUUGUG
1862

2035
GUGCAGGCCCUGGAAGCCC
1616
2035
GUGCAGGCCCUGGAAGCCC
1616
2053
GGGCUUCCAGGGCCUGCAC
1863

2053
CCUCGGCUCACGCAGAACU
1617
2053
CCUCGGCUCACGCAGAACU
1617
2071
AGUUCUGCGUGAGCCGAGG
1864

2071
UUGACCGACCUCCUGGUGA
1618
2071
UUGACCGACCUCCUGGUGA
1618
2089
UCACCAGGAGGUCGGUCAA
1865

2089
AACGUGAGCGACUCGCUGG
1619
2089
AACGUGAGCGACUCGCUGG
1619
2107
CCAGCGAGUCGCUCACGUU
1866

2107
GAGAUGCAGUGCUUGGUGG
1620
2107
GAGAUGCAGUGCUUGGUGG
1620
2125
CCACCAAGCACUGCAUCUC
1867

2125
GCCGGAGCGCACGCGCCCA
1621
2125
GCCGGAGCGCACGCGCCCA
1621
2143
UGGGCGCGUGCGCUCCGGC
1868

2143
AGCAUCGUGUGGUACAAAG
1622
2143
AGCAUCGUGUGGUACAAAG
1622
2161
CUUUGUACCACACGAUGCU
1869

2161
GACGAGAGGCUGCUGGAGG
1623
2161
GACGAGAGGCUGCUGGAGG
1623
2179
CCUCCAGCAGCCUCUCGUC
1870

2179
GAAAAGUCUGGAGUCGACU
1624
2179
GAAAAGUCUGGAGUCGACU
1624
2197
AGUCGACUCCAGACUUUUC
1871

2197
UUGGCGGACUCCAACCAGA
1625
2197
UUGGCGGACUCCAACCAGA
1625
2215
UCUGGUUGGAGUCCGCCAA
1872

2215
AAGCUGAGCAUCCAGCGCG
1626
2215
AAGCUGAGCAUCCAGCGCG
1626
2233
CGCGCUGGAUGCUCAGCUU
1873

2233
GUGCGCGAGGAGGAUGCGG
1627
2233
GUGCGCGAGGAGGAUGCGG
1627
2251
CCGCAUCCUCCUCGCGCAC
1874

2251
GGACCGUAUCUGUGCAGCG
1628
2251
GGACCGUAUCUGUGCAGCG
1628
2269
CGCUGCACAGAUACGGUCC
1875

2269
GUGUGCAGACCCAAGGGCU
1629
2269
GUGUGCAGACCCAAGGGCU
1629
2287
AGCCCUUGGGUCUGCACAC
1876

2287
UGCGUCAACUCCUCCGCCA
1630
2287
UGCGUCAACUCCUCCGCCA
1630
2305
UGGCGGAGGAGUUGACGCA
1677

2305
AGCGUGGCCGUGGAAGGCU
1631
2305
AGCGUGGCCGUGGAAGGCU
1631
2323
AGCCUUCCACGGCCACGCU
1878

2323
UCCGAGGAUAAGGGCAGCA
1632
2323
UCCGAGGAUAAGGGCAGCA
1632
2341
UGCUGCCCUUAUCCUCGGA
1879

2341
AUGGAGAUCGUGAUCCUUG
1633
2341
AUGGAGAUCGUGAUCCUUG
1633
2359
CAAGGAUCACGAUCUCCAU
1880

2359
GUCGGUACCGGCGUCAUCG
1634
2359
GUCGGUACCGGCGUCAUCG
1634
2377
CGAUGACGCCGGUACCGAC
1881

2377
GCUGUCUUCUUCUGGGUCC
1635
2377
GCUGUCUUCUUCUGGGUCC
1635
2395
GGACCCAGAAGAAGACAGC
1882

2395
CUCCUCCUCCUCAUCUUCU
1636
2395
CUCCUCCUCCUCAUCUUCU
1636
2413
AGAAGAUGAGGAGGAGGAG
1883

2413
UGUAACAUGAGGAGGCCGG
1637
2413
UGUAACAUGAGGAGGCCGG
1637
2431
CCGGCCUCCUCAUGUUACA
1884

2431
GCCCACGCAGACAUCAAGA
1638
2431
GCCCACGCAGACAUCAAGA
1638
2449
UCUUGAUGUCUGCGUGGGC
1885

2449
ACGGGCUACCUGUCCAUCA
1639
2449
ACGGGCUACCUGUCCAUCA
1639
2467
UGAUGGACAGGUAGCCCGU
1886

2467
AUCAUGGACCCCGGGGAGG
1640
2467
AUCAUGGACCCCGGGGAGG
1640
2485
CCUCCCCGGGGUCCAUGAU
1887

2485
GUGCCUCUGGAGGAGCAAU
1641
2485
GUGCCUCUGGAGGAGCAAU
1641
2503
AUUGCUCCUCCAGAGGCAC
1888

2503
UGCGAAUACCUGUCCUACG
1642
2503
UGCGAAUACCUGUCCUACG
1642
2521
CGUAGGACAGGUAUUCGCA
1889

2521
GAUGCCAGCCAGUGGGAAU
1643
2521
GAUGCCAGCCAGUGGGAAU
1643
2539
AUUCCCACUGGCUGGCAUC
1890

2539
UUCCCCCGAGAGCGGCUGC
1644
2539
UUCCCCCGAGAGCGGCUGC
1644
2557
GCAGCCGCUCUCGGGGGAA
1891

2557
CACCUGGGGAGAGUGCUCG
1645
2557
CACCUGGGGAGAGUGCUCG
1645
2575
CGAGCACUCUCCCCAGGUG
1892

2575
GGCUACGGCGCCUUCGGGA
1646
2575
GGCUACGGCGCCUUCGGGA
1646
2593
UCCCGAAGGCGCCGUAGCC
1893

2593
AAGGUGGUGGAAGCCUCCG
1647
2593
AAGGUGGUGGAAGCCUCCG
1647
2611
CGGAGGCUUCCACCACCUU
1894

2611
GCUUUCGGCAUCCACAAGG
1648
2611
GCUUUCGGCAUCCACAAGG
1648
2629
CCUUGUGGAUGCCGAAAGC
1895

2629
GGCAGCAGCUGUGACACCG
1649
2629
GGCAGCAGCUGUGACACCG
1649
2647
CGGUGUCACAGCUGCUGCC
1896

2647
GUGGCCGUGAAAAUGCUGA
1650
2647
GUGGCCGUGAAAAUGCUGA
1650
2665
UCAGCAUUUUCACGGCCAC
1897

2665
AAAGAGGGCGCCACGGCCA
1651
2665
AAAGAGGGCGCCACGGCCA
1651
2683
UGGCCGUGGCGCCCUCUUU
1898

2683
AGCGAGCAGCGCGCGCUGA
1652
2683
AGCGAGCAGCGCGCGCUGA
1652
2701
UCAGCGCGCGCUGCUCGCU
1899

2701
AUGUCGGAGCUCAAGAUCC
1653
2701
AUGUCGGAGCUCAAGAUCC
1653
2719
GGAUCUUGAGCUCCGACAU
1900

2719
CUCAUUCACAUCGGCAACC
1654
2719
CUCAUUCACAUCGGCAACC
1654
2737
GGUUGCCGAUGUGAAUGAG
1901

2737
CACCUCAACGUGGUCAACC
1655
2737
CACCUCAACGUGGUCAACC
1655
2755
GGUUGACCACGUUGAGGUG
1902

2755
CUCCUCGGGGCGUGCACCA
1656
2755
CUCCUCGGGGCGUGCACCA
1656
2773
UGGUGCACGCCCCGAGGAG
1903

2773
AAGCCGCAGGGCCCCCUCA
1657
2773
AAGCCGCAGGGCCCCCUCA
1657
2791
UGAGGGGGCCCUGCGGCUU
1904

2791
AUGGUGAUCGUGGAGUUCU
1658
2791
AUGGUGAUCGUGGAGUUCU
1658
2809
AGAACUCCACGAUCACCAU
1905

2809
UGCAAGUACGGCAACCUCU
1659
2809
UGCAAGUACGGCAACCUCU
1659
2827
AGAGGUUGCCGUACUUGCA
1906

2827
UCCAACUUCCUGCGCGCCA
1660
2827
UCCAACUUCCUGCGCGCCA
1660
2845
UGGCGCGCAGGAAGUUGGA
1907

2845
AAGCGGGACGCCUUCAGCC
1661
2845
AAGCGGGACGCCUUCAGCC
1661
2863
GGCUGAAGGCGUCCCGCUU
1908

2863
CCCUGCGCGGAGAAGUCUC
1662
2863
CCCUGCGCGGAGAAGUCUC
1662
2881
GAGACUUCUCCGCGCAGGG
1909

2881
CCCGAGCAGCGCGGACGCU
1663
2881
CCCGAGCAGCGCGGACGCU
1663
2899
AGCGUCCGCGCUGCUCGGG
1910

2899
UUCCGCGCCAUGGUGGAGC
1664
2899
UUCCGCGCCAUGGUGGAGC
1664
2917
GCUCCACCAUGGCGCGGAA
1911

2917
CUCGCCAGGCUGGAUCGGA
1665
2917
CUCGCCAGGCUGGAUCGGA
1665
2935
UCCGAUCCAGCCUGGCGAG
1912

2935
AGGCGGCCGGGGAGCAGCG
1666
2935
AGGCGGCCGGGGAGCAGCG
1666
2953
CGCUGCUCCCCGGCCGCCU
1913

2953
GACAGGGUCCUCUUCGCGC
1667
2953
GACAGGGUCCUCUUCGCGC
1667
2971
GCGCGAAGAGGACCCUGUC
1914

2971
CGGUUCUCGAAGACCGAGG
1668
2971
CGGUUCUCGAAGACCGAGG
1668
2989
CCUCGGUCUUCGAGAACCG
1915

2989
GGCGGAGCGAGGCGGGCUU
1669
2989
GGCGGAGCGAGGCGGGCUU
1669
3007
AAGCCCGCCUCGCUCCGCC
1916

3007
UCUCCAGACCAAGAAGCUG
1670
3007
UCUCCAGACCAAGAAGCUG
1670
3025
CAGCUUCUUGGUCUGGAGA
1917

3025
GAGGACCUGUGGCUGAGCC
1671
3025
GAGGACCUGUGGCUGAGCC
1671
3043
GGCUCAGCCACAGGUCCUC
1918

3043
CCGCUGACCAUGGAAGAUC
1672
3043
CCGCUGACCAUGGAAGAUC
1672
3061
GAUCUUCCAUGGUCAGCGG
1919

3061
CUUGUCUGCUACAGCUUCC
1673
3061
CUUGUCUGCUACAGCUUCC
1673
3079
GGAAGCUGUAGCAGACAAG
1920

3079
CAGGUGGCCAGAGGGAUGG
1674
3079
CAGGUGGCCAGAGGGAUGG
1674
3097
CCAUCCCUCUGGCCACCUG
1921

3097
GAGUUCCUGGCUUCCCGAA
1675
3097
GAGUUCCUGGCUUCCCGAA
1675
3115
UUCGGGAAGCCAGGAACUC
1922

3115
AAGUGCAUCCACAGAGACC
1676
3115
AAGUGCAUCCACAGAGACC
1676
3133
GGUCUCUGUGGAUGCACUU
1923

3133
CUGGCUGCUCGGAACAUUC
1677
3133
CUGGCUGCUCGGAACAUUC
1677
3151
GAAUGUUCCGAGCAGCCAG
1924

3151
CUGCUGUCGGAAAGCGACG
1678
3151
CUGCUGUCGGAAAGCGACG
1678
3169
CGUCGCUUUCCGACAGCAG
1925

3169
GUGGUGAAGAUCUGUGACU
1679
3169
GUGGUGAAGAUCUGUGACU
1679
3187
AGUCACAGAUCUUCACCAC
1926

3187
UUUGGCCUUGCCCGGGACA
1680
3187
UUUGGCCUUGCCCGGGACA
1680
3205
UGUCCCGGGCAAGGCCAAA
1927

3205
AUCUACAAAGACCCCGACU
1681
3205
AUCUACAAAGACCCCGACU
1681
3223
AGUCGGGGUCUUUGUAGAU
1928

3223
UACGUCCGCAAGGGCAGUG
1682
3223
UACGUCCGCAAGGGCAGUG
1682
3241
CACUGCCCUUGCGGACGUA
1929

3241
GCCCGGCUGCCCCUGAAGU
1683
3241
GCCCGGCUGCCCCUGAAGU
1683
3259
ACUUCAGGGGCAGCCGGGC
1930

3259
UGGAUGGCCCCUGAAAGCA
1684
3259
UGGAUGGCCCCUGAAAGCA
1684
3277
UGCUUUCAGGGGCCAUCCA
1931

3277
AUCUUCGACAAGGUGUACA
1685
3277
AUCUUCGACAAGGUGUACA
1685
3295
UGUACACCUUGUCGAAGAU
1932

3295
ACCACGCAGAGUGACGUGU
1686
3295
ACCACGCAGAGUGACGUGU
1686
3313
ACACGUCACUCUGCGUGGU
1933

3313
UGGUCCUUUGGGGUGCUUC
1687
3313
UGGUCCUUUGGGGUGCUUC
1687
3331
GAAGCACCCCAAAGGACCA
1934

3331
CUCUGGGAGAUCUUCUCUC
1688
3331
CUCUGGGAGAUCUUCUCUC
1688
3349
GAGAGAAGAUCUCCCAGAG
1935

3349
CUGGGGGCCUCCCCGUACC
1689
3349
CUGGGGGCCUCCCCGUACC
1689
3367
GGUACGGGGAGGCCCCCAG
1936

3367
CCUGGGGUGCAGAUCAAUG
1690
3367
CCUGGGGUGCAGAUCAAUG
1690
3385
CAUUGAUCUGCACCCCAGG
1937

3385
GAGGAGUUCUGCCAGCGCG
1691
3385
GAGGAGUUCUGCCAGCGCG
1691
3403
CGCGCUGGCAGAACUCCUC
1938

3403
GUGAGAGACGGCACAAGGA
1692
3403
GUGAGAGACGGCACAAGGA
1692
3421
UCCUUGUGCCGUCUCUCAC
1939

3421
AUGAGGGCCCCGGAGCUGG
1693
3421
AUGAGGGCCCCGGAGCUGG
1693
3439
CCAGCUCCGGGGCCCUCAU
1940

3439
GCCACUCCCGCCAUACGCC
1694
3439
GCCACUCCCGCCAUACGCC
1694
3457
GGCGUAUGGCGGGAGUGGC
1941

3457
CACAUCAUGCUGAACUGCU
1695
3457
CACAUCAUGCUGAACUGCU
1695
3475
AGCAGUUCAGCAUGAUGUG
1942

3475
UGGUCCGGAGACCCCAAGG
1696
3475
UGGUCCGGAGACCCCAAGG
1696
3493
CCUUGGGGUCUCCGGACCA
1943

3493
GCGAGACCUGCAUUCUCGG
1697
3493
GCGAGACCUGCAUUCUCGG
1697
3511
CCGAGAAUGCAGGUCUCGC
1944

3511
GACCUGGUGGAGAUCCUGG
1698
3511
GACCUGGUGGAGAUCCUGG
1698
3529
CCAGGAUCUCCACCAGGUC
1945

3529
GGGGACCUGCUCCAGGGCA
1699
3529
GGGGACCUGCUCCAGGGCA
1699
3547
UGCCCUGGAGCAGGUCCCC
1946

3547
AGGGGCCUGCAAGAGGAAG
1700
3547
AGGGGCCUGCAAGAGGAAG
1700
3565
CUUCCUCUUGCAGGCCCCU
1947

3565
GAGGAGGUCUGCAUGGCCC
1701
3565
GAGGAGGUCUGCAUGGCCC
1701
3583
GGGCCAUGCAGACCUCCUC
1948

3583
CCGCGCAGCUCUCAGAGCU
1702
3583
CCGCGCAGCUCUCAGAGCU
1702
3601
AGCUCUGAGAGCUGCGCGG
1949

3601
UCAGAAGAGGGCAGCUUCU
1703
3601
UCAGAAGAGGGCAGCUUCU
1703
3619
AGAAGCUGCCCUCUUCUGA
1950

3619
UCGCAGGUGUCCACCAUGG
1704
3619
UCGCAGGUGUCCACCAUGG
1704
3637
CCAUGGUGGACACCUGCGA
1951

3637
GCCCUACACAUCGCCCAGG
1705
3637
GCCCUACACAUCGCCCAGG
1705
3655
CCUGGGCGAUGUGUAGGGC
1952

3655
GCUGACGCUGAGGACAGCC
1706
3655
GCUGACGCUGAGGACAGCC
1706
3673
GGCUGUCCUCAGCGUCAGC
1953

3673
CCGCCAAGCCUGCAGCGCC
1707
3673
CCGCCAAGCCUGCAGCGCC
1707
3691
GGCGCUGCAGGCUUGGCGG
1954

3691
CACAGCCUGGCCGCCAGGU
1708
3691
CACAGCCUGGCCGCCAGGU
1708
3709
ACCUGGCGGCCAGGCUGUG
1955

3709
UAUUACAACUGGGUGUCCU
1709
3709
UAUUACAACUGGGUGUCCU
1709
3727
AGGACACCCAGUUGUAAUA
1956

3727
UUUCCCGGGUGCCUGGCCA
1710
3727
UUUCCCGGGUGCCUGGCCA
1710
3745
UGGCCAGGCACCCGGGAAA
1957

3745
AGAGGGGCUGAGACCCGUG
1711
3745
AGAGGGGCUGAGACCCGUG
1711
3763
CACGGGUCUCAGCCCCUCU
1958

3763
GGUUCCUCCAGGAUGAAGA
1712
3763
GGUUCCUCCAGGAUGAAGA
1712
3781
UCUUCAUCCUGGAGGAACC
1959

3781
ACAUUUGAGGAAUUCCCCA
1713
3781
ACAUUUGAGGAAUUCCCCA
1713
3799
UGGGGAAUUCCUCAAAUGU
1960

3799
AUGACCCCAACGACCUACA
1714
3799
AUGACCCCAACGACCUACA
1714
3817
UGUAGGUCGUUGGGGUCAU
1961

3817
AAAGGCUCUGUGGACAACC
1715
3817
AAAGGCUCUGUGGACAACC
1715
3835
GGUUGUCCACAGAGCCUUU
1962

3835
CAGACAGACAGUGGGAUGG
1716
3835
CAGACAGACAGUGGGAUGG
1716
3853
CCAUCCCACUGUCUGUCUG
1963

3853
GUGCUGGCCUCGGAGGAGU
1717
3853
GUGCUGGCCUCGGAGGAGU
1717
3871
ACUCCUCCGAGGCCAGCAC
1964

3871
UUUGAGCAGAUAGAGAGCA
1718
3871
UUUGAGCAGAUAGAGAGCA
1718
3889
UGCUCUCUAUCUGCUCAAA
1965

3889
AGGCAUAGACAAGAAAGCG
1719
3889
AGGCAUAGACAAGAAAGCG
1719
3907
CGCUUUCUUGUCUAUGCCU
1966

3907
GGCUUCAGGUAGCUGAAGC
1720
3907
GGCUUCAGGUAGCUGAAGC
1720
3925
GCUUCAGCUACCUGAAGCC
1967

3925
CAGAGAGAGAGAAGGCAGC
1721
3925
CAGAGAGAGAGAAGGCAGC
1721
3943
GCUGCCUUCUCUCUCUCUG
1968

3943
CAUACGUCAGCAUUUUCUU
1722
3943
CAUACGUCAGCAUUUUCUU
1722
3961
AAGAAAAUGCUGACGUAUG
1969

3961
UCUCUGCACUUAUAAGAAA
1723
3961
UCUCUGCACUUAUAAGAAA
1723
3979
UUUCUUAUAAGUGCAGAGA
1970

3979
AGAUCAAAGACUUUAAGAC
1724
3979
AGAUCAAAGACUUUAAGAC
1724
3997
GUCUUAAAGUCUUUGAUCU
1971

3997
CUUUCGCUAUUUCUUCUAC
1725
3997
CUUUCGCUAUUUCUUCUAC
1725
4015
GUAGAAGAAAUAGCGAAAG
1972

4015
CUGCUAUCUACUACAAACU
1726
4015
CUGCUAUCUACUACAAACU
1726
4033
AGUUUGUAGUAGAUAGCAG
1973

4033
UUCAAAGAGGAACCAGGAG
1727
4033
UUCAAAGAGGAACCAGGAG
1727
4051
CUCCUGGUUCCUCUUUGAA
1974

4051
GGACAAGAGGAGCAUGAAA
1728
4051
GGACAAGAGGAGCAUGAAA
1728
4069
UUUCAUGCUCCUCUUGUCC
1975

4069
AGUGGACAAGGAGUGUGAC
1729
4069
AGUGGACAAGGAGUGUGAC
1729
4087
GUCACACUCCUUGUCCACU
1976

4087
CCACUGAAGCACCACAGGG
1730
4087
CCACUGAAGCACCACAGGG
1730
4105
CCCUGUGGUGCUUCAGUGG
1977

4105
GAGGGGUUAGGCCUCCGGA
1731
4105
GAGGGGUUAGGCCUCCGGA
1731
4123
UCCGGAGGCCUAACCCCUC
1978

4123
AUGACUGCGGGCAGGCCUG
1732
4123
AUGACUGCGGGCAGGCCUG
1732
4141
CAGGCCUGCCCGCAGUCAU
1979

4141
GGAUAAUAUCCAGCCUCCC
1733
4141
GGAUAAUAUCCAGCCUCCC
1733
4159
GGGAGGCUGGAUAUUAUCC
1980

4159
CACAAGAAGCUGGUGGAGC
1734
4159
CACAAGAAGCUGGUGGAGC
1734
4177
GCUCCACCAGCUUCUUGUG
1981

4177
CAGAGUGUUCCCUGACUCC
1735
4177
CAGAGUGUUCCCUGACUCC
1735
4195
GGAGUCAGGGAACACUCUG
1982

4195
CUCCAAGGAAAGGGAGACG
1736
4195
CUCCAAGGAAAGGGAGACG
1736
4213
CGUCUCCCUUUCCUUGGAG
1983

4213
GCCCUUUCAUGGUCUGCUG
1737
4213
GCCCUUUCAUGGUCUGCUG
1737
4231
CAGCAGACCAUGAAAGGGC
1984

4231
GAGUAACAGGUGCCUUCCC
1738
4231
GAGUAACAGGUGCCUUCCC
1738
4249
GGGAAGGCACCUGUUACUC
1985

4249
CAGACACUGGCGUUACUGC
1739
4249
CAGACACUGGCGUUACUGC
1739
4267
GCAGUAACGCCAGUGUCUG
1986

4267
CUUGACCAAAGAGCCCUCA
1740
4267
CUUGACCAAAGAGCCCUCA
1740
4285
UGAGGGCUCUUUGGUCAAG
1987

4285
AAGCGGCCCUUAUGCCAGC
1741
4285
AAGCGGCCCUUAUGCCAGC
1741
4303
GCUGGCAUAAGGGCCGCUU
1988

4303
CGUGACAGAGGGCUCACCU
1742
4303
CGUGACAGAGGGCUCACCU
1742
4321
AGGUGAGCCCUCUGUCACG
1989

4321
UCUUGCCUUCUAGGUCACU
1743
4321
UCUUGCCUUCUAGGUCACU
1743
4339
AGUGACCUAGAAGGCAAGA
1990

4339
UUCUCACAAUGUCCCUUCA
1744
4339
UUCUCACAAUGUCCCUUCA
1744
4357
UGAAGGGACAUUGUGAGAA
1991

4357
AGCACCUGACCCUGUGCCC
1745
4357
AGCACCUGACCCUGUGCCC
1745
4375
GGGCACAGGGUCAGGUGCU
1992

4375
CGCCGAUUAUUCCUUGGUA
1746
4375
CGCCGAUUAUUCCUUGGUA
1746
4393
UACCAAGGAAUAAUCGGCG
1993

4393
AAUAUGAGUAAUACAUCAA
1747
4393
AAUAUGAGUAAUACAUCAA
1747
4411
UUGAUGUAUUACUCAUAUU
1994

4411
AAGAGUAGUAUUAAAAGCU
1748
4411
AAGAGUAGUAUUAAAAGCU
1748
4429
AGCUUUUAAUACUACUCUU
1995

4429
UAAUUAAUCAUGUUUAUAA
1749
4429
UAAUUAAUCAUGUUUAUAA
1749
4447
UUAUAAACAUGAUUAAUUA
1996

VEGF NM_003376.3

3
GCGGAGGCUUGGGGCAGCC
1997
3
GCGGAGGCUUGGGGCAGCC
1997
21
GGCUGCCCCAAGCCUCCGC
2093

21
CGGGUAGCUCGGAGGUCGU
1998
21
CGGGUAGCUCGGAGGUCGU
1998
39
ACGACCUCCGAGCUACCCG
2094

39
UGGCGCUGGGGGCUAGCAC
1999
39
UGGCGCUGGGGGCUAGCAC
1999
57
GUGCUAGCCCCCAGCGCCA
2095

57
CCAGCGCUCUGUCGGGAGG
2000
57
CCAGCGCUCUGUCGGGAGG
2000
75
CCUCCCGACAGAGCGCUGG
2096

75
GCGCAGCGGUUAGGUGGAC
2001
75
GCGCAGCGGUUAGGUGGAC
2001
93
GUCCACCUAACCGCUGCGC
2097

93
CCGGUCAGCGGACUCACCG
2002
93
CCGGUCAGCGGACUCACCG
2002
111
CGGUGAGUCCGCUGACCGG
2098

111
GGCCAGGGCGCUCGGUGCU
2003
111
GGCCAGGGCGCUCGGUGCU
2003
129
AGCACCGAGCGCCCUGGCC
2099

129
UGGAAUUUGAUAUUCAUUG
2004
129
UGGAAUUUGAUAUUCAUUG
2004
147
CAAUGAAUAUCAAAUUCCA
2100

147
GAUCCGGGUUUUAUCCCUC
2005
147
GAUCCGGGUUUUAUCCCUC
2005
165
GAGGGAUAAAACCCGGAUC
2101

165
CUUCUUUUUUCUUAAACAU
2006
165
CUUCUUUUUUCUUAAACAU
2006
183
AUGUUUAAGAAAAAAGAAG
2102

183
UUUUUUUUUAAAACUGUAU
2007
183
UUUUUUUUUAAAACUGUAU
2007
201
AUACAGUUUUAAAAAAAAA
2103

201
UUGUUUCUCGUUUUAAUUU
2008
201
UUGUUUCUCGUUUUAAUUU
2008
219
AAAUUAAAACGAGAAACAA
2104

219
UAUUUUUGCUUGCCAUUCC
2009
219
UAUUUUUGCUUGCCAUUCC
2009
237
GGAAUGGCAAGCAAAAAUA
2105

237
CCCACUUGAAUCGGGCCGA
2010
237
CCCACUUGAAUCGGGCCGA
2010
255
UCGGCCCGAUUCAAGUGGG
2106

255
ACGGCUUGGGGAGAUUGCU
2011
255
ACGGCUUGGGGAGAUUGCU
2011
273
AGCAAUCUCCCCAAGCCGU
2107

273
UCUACUUCCCCAAAUCACU
2012
273
UCUACUUCCCCAAAUCACU
2012
291
AGUGAUUUGGGGAAGUAGA
2108

291
UGUGGAUUUUGGAAACCAG
2013
291
UGUGGAUUUUGGAAACCAG
2013
309
CUGGUUUCCAAAAUCCACA
2109

309
GCAGAAAGAGGAAAGAGGU
2014
309
GCAGAAAGAGGAAAGAGGU
2014
327
ACCUCUUUCCUCUUUCUGC
2110

327
UAGCAAGAGCUCCAGAGAG
2015
327
UAGCAAGAGCUCCAGAGAG
2015
345
CUCUCUGGAGCUCUUGCUA
2111

345
GAAGUCGAGGAAGAGAGAG
2016
345
GAAGUCGAGGAAGAGAGAG
2016
363
CUCUCUCUUCCUCGACUUC
2112

363
GACGGGGUCAGAGAGAGCG
2017
363
GACGGGGUCAGAGAGAGCG
2017
381
CGCUCUCUCUGACCCCGUC
2113

381
GCGCGGGCGUGCGAGCAGC
2018
381
GCGCGGGCGUGCGAGCAGC
2018
399
GCUGCUCGCACGCCCGCGC
2114

399
CGAAAGCGACAGGGGCAAA
2019
399
CGAAAGCGACAGGGGCAAA
2019
417
UUUGCCCCUGUCGCUUUCG
2115

417
AGUGAGUGACCUGCUUUUG
2020
417
AGUGAGUGACCUGCUUUUG
2020
435
CAAAAGCAGGUCACUCACU
2116

435
GGGGGUGACCGCCGGAGCG
2021
435
GGGGGUGACCGCCGGAGCG
2021
453
CGCUCCGGCGGUCACCCCC
2117

453
GCGGCGUGAGCCCUCCCCC
2022
453
GCGGCGUGAGCCCUCCCCC
2022
471
GGGGGAGGGCUCACGCCGC
2118

471
CUUGGGAUCCCGCAGCUGA
2023
471
CUUGGGAUCCCGCAGCUGA
2023
489
UCAGCUGCGGGAUCCCAAG
2119

489
ACCAGUCGCGCUGACGGAC
2024
489
ACCAGUCGCGCUGACGGAC
2024
507
GUCCGUCAGCGCGACUGGU
2120

507
CAGACAGACAGACACCGCC
2025
507
CAGACAGACAGACACCGCC
2025
525
GGCGGUGUCUGUCUGUCUG
2121

525
CCCCAGCCCCAGCUACCAC
2026
525
CCCCAGCCCCAGCUACCAC
2026
543
GUGGUAGCUGGGGCUGGGG
2122

543
CCUCCUCCCCGGCCGGCGG
2027
543
CCUCCUCCCCGGCCGGCGG
2027
561
CCGCCGGCCGGGGAGGAGG
2123

561
GCGGACAGUGGACGCGGCG
2028
561
GCGGACAGUGGACGCGGCG
2028
579
CGCCGCGUCCACUGUCCGC
2124

579
GGCGAGCCGCGGGCAGGGG
2029
579
GGCGAGCCGCGGGCAGGGG
2029
597
CCCCUGCCCGCGGCUCGCC
2125

597
GCCGGAGCCCGCGCCCGGA
2030
597
GCCGGAGCCCGCGCCCGGA
2030
615
UCCGGGCGCGGGCUCCGGC
2126

615
AGGCGGGGUGGAGGGGGUC
2031
615
AGGCGGGGUGGAGGGGGUC
2031
633
GACCCCCUCCACCCCGCCU
2127

633
CGGGGCUCGCGGCGUCGCA
2032
633
CGGGGCUCGCGGCGUCGCA
2032
651
UGCGACGCCGCGAGCCCCG
2128

651
ACUGAAACUUUUCGUCCAA
2033
651
ACUGAAACUUUUCGUCCAA
2033
669
UUGGACGAAAAGUUUCAGU
2129

669
ACUUCUGGGCUGUUCUCGC
2034
669
ACUUCUGGGCUGUUCUCGC
2034
667
GCGAGAACAGCCCAGAAGU
2130

687
CUUCGGAGGAGCCGUGGUC
2035
687
CUUCGGAGGAGCCGUGGUC
2035
705
GACCACGGCUCCUCCGAAG
2131

705
CCGCGCGGGGGAAGCCGAG
2036
705
CCGCGCGGGGGAAGCCGAG
2036
723
CUCGGCUUCCCCCGCGCGG
2132

723
GCCGAGCGGAGCCGCGAGA
2037
723
GCCGAGCGGAGCCGCGAGA
2037
741
UCUCGCGGCUCCGCUCGGC
2133

741
AAGUGCUAGCUCGGGCCGG
2038
741
AAGUGCUAGCUCGGGCCGG
2038
759
CCGGCCCGAGCUAGCACUU
2134

759
GGAGGAGCCGCAGCCGGAG
2039
759
GGAGGAGCCGCAGCCGGAG
2039
777
CUCCGGCUGCGGCUCCUCC
2135

777
GGAGGGGGAGGAGGAAGAA
2040
777
GGAGGGGGAGGAGGAAGAA
2040
795
UUCUUCCUCCUCCCCCUCC
2136

795
AGAGAAGGAAGAGGAGAGG
2041
795
AGAGAAGGAAGAGGAGAGG
2041
813
CCUCUCCUCUUCCUUCUCU
2137

813
GGGGCCGCAGUGGCGACUC
2042
813
GGGGCCGCAGUGGCGACUC
2042
831
GAGUCGCCACUGCGGCCCC
2138

831
CGGCGCUCGGAAGCCGGGC
2043
831
CGGCGCUCGGAAGCCGGGC
2043
849
GCCCGGCUUCCGAGCGCCG
2139

849
CUCAUGGACGGGUGAGGCG
2044
849
CUCAUGGACGGGUGAGGCG
2044
867
CGCCUCACCCGUCCAUGAG
2140

867
GGCGGUGUGCGCAGACAGU
2045
867
GGCGGUGUGCGCAGACAGU
2045
885
ACUGUCUGCGCACACCGCC
2141

885
UGCUCCAGCCGCGCGCGCU
2046
885
UGCUCCAGCCGCGCGCGCU
2046
903
AGCGCGCGCGGCUGGAGCA
2142

903
UCCCCAGGCCCUGGCCCGG
2047
903
UCCCCAGGCCCUGGCCCGG
2047
921
CCGGGCCAGGGCCUGGGGA
2143

921
GGCCUCGGGCCGGGGAGGA
2048
921
GGCCUCGGGCCGGGGAGGA
2048
939
UCCUCCCCGGCCCGAGGCC
2144

939
AAGAGUAGCUCGCCGAGGC
2049
939
AAGAGUAGCUCGCCGAGGC
2049
957
GCCUCGGCGAGCUACUCUU
2145

957
CGCCGAGGAGAGCGGGCCG
2050
957
CGCCGAGGAGAGCGGGCCG
2050
975
CGGCCCGCUCUCCUCGGCG
2146

975
GCCCCACAGCCCGAGCCGG
2051
975
GCCCCACAGCCCGAGCCGG
2051
993
CCGGCUCGGGCUGUGGGGC
2147

993
GAGAGGGAGCGCGAGCCGC
2052
993
GAGAGGGAGCGCGAGCCGC
2052
1011
GCGGCUCGCGCUCCCUCUC
2148

1011
CGCCGGCCCCGGUCGGGCC
2053
1011
CGCCGGCCCCGGUCGGGCC
2053
1029
GGCCCGACCGGGGCCGGCG
2149

1029
CUCCGAAACCAUGAACUUU
2054
1029
CUCCGAAACCAUGAACUUU
2054
1047
AAAGUUCAUGGUUUCGGAG
2150

1047
UCUGCUGUCUUGGGUGCAU
2055
1047
UCUGCUGUCUUGGGUGCAU
2055
1065
AUGCACCCAAGACAGCAGA
2151

1065
UUGGAGCCUUGCCUUGCUG
2056
1065
UUGGAGCCUUGCCUUGCUG
2056
1083
CAGCAAGGCAAGGCUCCAA
2152

1083
GCUCUACCUCCACCAUGCC
2057
1083
GCUCUACCUCCACCAUGCC
2057
1101
GGCAUGGUGGAGGUAGAGC
2153

1101
CAAGUGGUCCCAGGCUGCA
2058
1101
CAAGUGGUCCCAGGCUGCA
2058
1119
UGCAGCCUGGGACCACUUG
2154

1119
ACCCAUGGCAGAAGGAGGA
2059
1119
ACCCAUGGCAGPAGGAGGA
2059
1137
UCCUCCUUCUGCCAUGGGU
2155

1137
AGGGCAGAAUCAUCACGAA
2060
1137
AGGGCAGAAUCAUCACGAA
2060
1155
UUCGUGAUGAUUCUGCCCU
2156

1155
AGUGGUGAAGUUCAUGGAU
2061
1155
AGUGGUGAAGUUCAUGGAU
2061
1173
AUCCAUGAACUUCACCACU
2157

1173
UGUCUAUCAGCGCAGCUAC
2062
1173
UGUCUAUCAGCGCAGCUAC
2062
1191
GUAGCUGCGCUGAUAGACA
2158

1191
CUGCCAUCCAAUCGAGACC
2063
1191
CUGCCAUCCAAUCGAGACC
2063
1209
GGUCUCGAUUGGAUGGCAG
2159

1209
CCUGGUGGACAUCUUCCAG
2064
1209
CCUGGUGGACAUCUUCCAG
2064
1227
CUGGAAGAUGUCCACCAGG
2160

1227
GGAGUACCCUGAUGAGAUC
2065
1227
GGAGUACCCUGAUGAGAUC
2065
1245
GAUCUCAUCAGGGUACUCC
2161

1245
CGAGUACAUCUUCAAGCCA
2066
1245
CGAGUACAUCUUCAAGCCA
2066
1263
UGGCUUGAAGAUGUACUCG
2162

1263
AUCCUGUGUGCCCCUGAUG
2067
1263
AUCCUGUGUGCCCCUGAUG
2067
1281
CAUCAGGGGCACACAGGAU
2163

1281
GCGAUGCGGGGGCUGCUGC
2068
1281
GCGAUGCGGGGGCUGCUGC
2068
1299
GCAGCAGCCCCCGCAUCGC
2164

1299
CAAUGACGAGGGCCUGGAG
2069
1299
CAAUGACGAGGGCCUGGAG
2069
1317
CUCCAGGCCCUCGUCAUUG
2165

1317
GUGUGUGCCCACUGAGGAG
2070
1317
GUGUGUGCCCACUGAGGAG
2070
1335
CUCCUCAGUGGGCACACAC
2166

1335
GUCCAACAUCACCAUGCAG
2071
1335
GUCCAACAUCACCAUGCAG
2071
1353
CUGCAUGGUGAUGUUGGAC
2167

1353
GAUUAUGCGGAUCAAACCU
2072
1353
GAUUAUGCGGAUCAAACCU
2072
1371
AGGUUUGAUCCGCAUAAUC
2168

1371
UCACCAAGGCCAGCACAUA
2073
1371
UCACCAAGGCCAGCACAUA
2073
1389
UAUGUGCUGGCCUUGGUGA
2169

1389
AGGAGAGAUGAGCUUCCUA
2074
1389
AGGAGAGAUGAGCUUCCUA
2074
1407
UAGGAAGCUCAUCUCUCCU
2170

1407
ACAGCACAACAAAUGUGPA
2075
1407
ACAGCACAACAAAUGUGAA
2075
1425
UUCACAUUUGUUGUGCUGU
2171

1425
AUGCAGACCAAAGAAAGAU
2076
1425
AUGCAGACCAAAGAAAGAU
2076
1443
AUCUUUCUUUGGUCUGCAU
2172

1443
UAGAGCAAGACAAGAAAAA
2077
1443
UAGAGCAAGACAAGAAAAA
2077
1461
UUUUUCUUGUCUUGCUCUA
2173

1461
AAAAUCAGUUCGAGGAAAG
2078
1461
AAAAUCAGUUCGAGGAAAG
2078
1479
CUUUCCUCGAACUGAUUUU
2174

1479
GGGAAAGGGGCAAAAACGA
2079
1479
GGGAAAGGGGCAAAAACGA
2079
1497
UCGUUUUUGCCCCUUUCCC
2175

1497
AAAGCGCAAGAAAUCCCGG
2080
1497
AAAGCGCAAGAAAUCCCGG
2080
1515
CCGGGAUUUCUUGCGCUUU
2176

1515
GUAUAAGUCCUGGAGCGUU
2081
1515
GUAUAAGUCCUGGAGCGUU
2081
1533
AACGCUCCAGGACUUAUAC
2177

1533
UCCCUGUGGGCCUUGCUCA
2082
1533
UCCCUGUGGGCCUUGCUCA
2082
1551
UGAGCAAGGCCCACAGGGA
2178

1551
AGAGCGGAGAAAGCAUUUG
2083
1551
AGAGCGGAGAAAGCAUUUG
2083
1569
CAAAUGCUUUCUCCGCUCU
2179

1569
GUUUGUACAAGAUCCGCAG
2084
1569
GUUUGUACAAGAUCCGCAG
2084
1587
CUGCGGAUCUUGUACAAAC
2180

1587
GACGUGUAAAUGUUCCUGC
2085
1587
GACGUGUAAAUGUUCCUGC
2085
1605
GCAGGAACAUUUACACGUC
2181

1605
CAAAAACACAGACUCGCGU
2086
1605
CAAAAACACAGACUCGCGU
2086
1623
ACGCGAGUCUGUGUUUUUG
2182

1623
UUGCAAGGCGAGGCAGCUU
2087
1623
UUGCAAGGCGAGGCAGCUU
2087
1641
AAGCUGCCUCGCCUUGCAA
2183

1641
UGAGUUAAACGAACGUACU
2088
1641
UGAGUUAAACGAACGUACU
2088
1659
AGUACGUUCGUUUAACUCA
2184

1659
UUGCAGAUGUGACAAGCCG
2089
1659
UUGCAGAUGUGACAAGCCG
2089
1677
CGGCUUGUCACAUCUGCAA
2185

1677
GAGGCGGUGAGCCGGGCAG
2090
1677
GAGGCGGUGAGCCGGGCAG
2090
1695
CUGCCCGGCUCACCGCCUC
2186

1695
GGAGGAAGGAGCCUCCCUC
2091
1695
GGAGGAAGGAGCCUCCCUC
2091
1713
GAGGGAGGCUCCUUCCUCC
2187

1703
GAGCCUCCCUCAGGGUUUC
2092
1703
GAGCCUCCCUCAGGGUUUC
2092
1721
GAAACCCUGAGGGAGGCUC
2188

TABLE III

VEGF and/or VEGFR Synthetic Modified siNA Constructs

Target

Seq
Cmpd

Seq

Pos
Target
ID
#
Aliases
Sequence
ID

VEGFR1

298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:298U21 sense siNA
UGUCUGCUUCUCACAGGAUTT
2709

1956
GAAGGAGAGGACCUGAAACUGUC
2286

FLT1:1956U21 sense siNA
AGGAGAGGACCUGAAACUGTT
2710

1957
AAGGAGAGGACCUGAAACUGUCU
2287

FLT1:1957U21 sense siNA
GGAGAGGACCUGAAACUGUTT
2711

2787
GCAUUUGGCAUUAAGAAAUCACC
2288

FLT1:2787U21 sense siNA
AUUUGGCAUUAAGAAAUCATT
2712

298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:316L21 antisense siNA
AUCCUGUGAGAAGCAGACATT
2713

(298C)

1956
GAAGGAGAGGACCUGAAACUGUC
2286

FLT1:1974L21 antisense siNA
CAGUUUCAGGUCCUCUCCUTT
2714

(1956C)

1957
AAGGAGAGGACCUGAAACUGUCU
2287

FLT1:1975L21 antisense siNA
ACAGUUUCAGGUCCUCUCCTT
2715

(1957C)

2787
GCAUUUGGCAUUAAGAAAUCACC
2288

FLT1:2805L21 antisense siNA
UGAUUUCUUAAUGCCAAAUTT
2716

(2787C)

298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:298U21 sense siNA stab04
B uGucuGcuucucAcAGGAuTT B
2717

1956
GAAGGAGAGGACCUGAAACUGUC
2286

FLT1:1956U21 sense siNA
B AGGAGAGGAccuGAAAcuGTT B
2718

stab04

1957
AAGGAGAGGACCUGAAACUGUCU
2287

FLT1:1957U21 sense siNA
B GGAGAGGAccuGAAAcuGuTT B
2719

stab04

2787
GCAUUUGGCAUUAAGAAAUCACC
2288

FLT1:2787U21 sense siNA
B AuuuGGcAuuAAGAAAucATT B
2720

stab04

298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:316L21 antisense siNA
AuccuGuGAGAAGcAGAcATsT
2721

(298C) stab05

1956
GAAGGAGAGGACCUGAAACUGUC
2286

FLT1:1974121 antisense siNA
cAGuuucAGGuccucuccuTsT
2722

(1956C) stab05

1957
AAGGAGAGGACCUGAAACUGUCU
2287

FLT1:1975L21 antisense siNA
AcAGuuucAGGuccucuccTsT
2723

(1957C) stab05

2787
GCAUUUGGCAUUAAGAAAUCACC
2288

FLT1:2805121 antisense siNA
uGAuuucuuAAuGccAAAuTsT
2724

(2787C) stab05

298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:298U21 sense siNA stab07
B uGucuGcuucucAcAGGAuTT B
2725

1956
GAAGGAGAGGACCUGAAACUGUC
2286
37387
FLT1:1956U21 sense siNA
B AGGAGAGGAccuGAAAcuGTT B
2726

stab07

1957
AAGGAGAGGACCUGAAACUGUCU
2287
37388
FLT1:1957U21 sense siNA
B GGAGAGGACcuGAAAcuGuTT B
2727

stab07

2787
GCAUUUGGCAUUAAGAAAUCACC
2288
37404
FLT1:2787U21 sense siNA
B AuuuGGcAuuAAGAAAucATT B
2728

stab07

298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:316L21 antisense siNA

AuccuGuGAGAAGcAGAcATsT
2729

(298C) stab11

1956
GAAGGAGAGGACCUGAAACUGUC
2286

FLT1:1974L21 antisense siNA
cAGuuucAGGuccucuccuTsT
2730

(1956C) stab11

1957
AAGGAGAGGACCUGAAACUGUCU
2287

FLT1:1975121 antisense siNA

AcAGuuucAGGuccucuccTsT
2731

(1957C) stab11

2787
GCAUUUGGCAUUAAGAAAUCACC
2288

FLT1:2805121 antisense siNA
uGAuuucuuAAuGccAAAuTsT
2732

(2787C) stab11

349
AACUGAGUUUAAAAGGCACCCAG
2289
31209
FLT1:367L21 antisense siNA
GAcucAAAuuuuccGuGGGTsT
2733

(349C) stab05 inv

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31210
FLT1:2967L21 antisense siNA
cGuuccucccGGAGAcuAcTsT
2734

(2949C) stab05 inv

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31211
FLT1:3930L21 antisense siNA
GGAccuuucuuAGuuuuGGTsT
2735

(3912C) stab05 inv

349
AACUGAGUUUAAAAGGCACCCAG
2289
31212
FLT1:349U21 sense siNA stab07
B cccAcGGAAAAuuuGAGucTT B
2736

inv

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31213
FLT1:2949U21 sense siNA stab07
B GuAGucucCGGGAGGAAcGTT B
2737

inv

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31214
FLT1:3912U21 sense siNA stab07
B ccAAAAcuAAGAAAGGuCcTT B
2738

inv

349
AACUGAGUUUAAAAGGCACCCAG
2289
31215
FLT1:367L21 antisense siNA

GAcucAAAuuuuccGuGGGTsT
2739

(349C) stab08 inv

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31216
FLT1:2967121 antisense siNA
cGuuccucccGGAGAcuAcTsT
2740

(2949C) stab08 inv

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31217
FLT1:3930121 antisense siNA

GGAccuuucuuAGuuuuGGTsT
2741

(3912C) stab08 inv

349
AACUGAGUUUAAAAGGCACCCAG
2289
31270
FLT1:349U21 sense siNA stab09
B CUGAGUUUAAAAGGCACCCTT B
2742

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31271
FLT1:2949U21 sense siNA
B GCAAGGAGGGCCUCUGAUGTT B
2743

stab09

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31272
FLT1:3912U21 sense siNA stab09
B CCUGGAAAGAAUCAAAACCTT B
2744

349
AACUGAGUUUAAAAGGCACCCAG
2289
31273
FLT1:367L21 antisense siNA (349C)
GGGUGCCUUUUAAACUCAGTsT
2745

stab10

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31274
FLT1:2967L21 antisense siNA
CAUCAGAGGCCCUCCUUGCTsT
2746

(2949C) stab10

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31275
FLT1:3930L21 antisense siNA
GGUUUUGAUUCUUUCCAGGTsT
2747

(3912C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
31276
FLT1:349U21 sense siNA stab09
B CCCACGGAAAAUUUGAGUCTT B
2748

inv

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31277
FLT1:2949U21 sense siNA
B GUAGUCUCCGGGAGGAACGTT B
2749

stab09 inv

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31278
FLT1:3912U21 sense siNA
B CCAAAACUAAGAAAGGUCCTT B
2750

stab09 inv

349
AACUGAGUUUAAAAGGCACCCAG
2289
31279
FLT1:367L21 antisense siNA
GACUCAAAUUUUCCGUGGGTsT
2751

(349C) stab10 inv

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31280
FLT1:2967121 antisense siNA
CGUUCCUCCCGGAGACUACTsT
2752

(2949C) stab10 inv

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31281
FLT1:3930L21 antisense siNA
GGACCUUUCUUAGUUUUGGTsT
2753

(3912C) stab10 inv

2340
AACAACCACAAAAUACAACAAGA
2292
31424
FLT1:2358121 antisense siNA
uuGuuGuAuuuuGuGGuuGXsX
2754

(2340C) stab11 3′-BrdU

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31425
FLT1:2967L21 antisense siNA
cAucAGAGGcccuccuuGcXsX
2755

(2949C) stab11 3′-BrdU

2340
AACAACCACAAAAUACAACAAGA
2292
31442
FLT1:2358L21 antisense siNA
uuGuuGuAuuuuGuGGuuGXsT
2756

(2340C) stab11 3′-BrdU

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31443
FLT1:2967L21 antisense siNA
cAucAGAGGcccuccuuGcXsT
2757

(2949C) stab11 3′-BrdU

2340
AACAACCACAAAAUACAACAAGA
2292
31449
FLT1:2340U21 sense siNA
B CAACCACAAAAUACAACAATT B
2758

stab09

2340
AACAACCACAAAAUACAACAAGA
2292
31450
FLT1:2340U21 sense siNA
B AACAACAUAAAACACCAACTT B
2759

inv stab09

2340
AACAACCACAAAAUACAACAAGA
2292
31451
FLT1:2358121 antisense
UUGUUGUAUUUUGUGGUUGTsT
2760

siNA (2340C) stab10

2340
AACAACCACAAAAUACAACAAGA
2292
31452
FLT1:2358121 antisense siNA
GUUGGUGUUUUAUGUUGUUTsT
2761

(2340C) inv stab10

2340
AACAACCACAAAAUACAACAAGA
2292
31509
FLT1:2358L21 antisense siNA
uuGuuGuAuuuuGuGGuuGTsT
2762

(2340C)

349
AACUGAGUUUAAAAGGCACCCAG
2289
31794
2x cholesterol + R31194
(H)2 ZTa
2763

FLT1:349U21 sense siNA stab07
B cuGAGuuuAAAAGGcAcccTT B

349
AACUGAGUUUAAAAGGCACCCAG
2289
31795
2x cholesterol + R31212
(H)2 ZTa
2764

FLT1:349U21 sense siNA stab07
B cccACGGAAAAuuuGAGucTT B

inv

349
AACUGAGUUUAAAAGGCACCCAG
2289
31796
2x cholesterol + R31270
(H)2 ZTA
2765

FLT1:349U21 sense siNA stab09
B CUGAGUUUAAAAGGCACCCTT B

349
AACUGAGUUUAAAAGGCACCCAG
2289
31797
2x cholesterol + R31276
(H)2 ZTA
2766

FLT1:349U21 sense siNA stab09
B CCCACGGAAAAUUUGAGUCTT B

inv

349
AACUGAGUUUAAAAGGCACCCAG
2289
31798
2x C18 phospholipid + R31194
(L)2 ZTa
2767

FLT1:349U21 sense siNA stab07
B cuGAGuuuAAAAGGCAcccTT B

349
AACUGAGUUUAAAAGGCACCCAG
2289
31799
2x C18 phospholipid + R31212
(L)2 ZTa B
2768

FLT1:349U21 sense siNA stab07
cccAcGGAAAAuuuGAGucTT B

inv

349
AACUGAGUUUAAAAGGCACCCAG
2289
31800
2x C18 phospholipid + R31270
(L)2 ZTA B
2769

FLT1:349U21 sense siNA stab09
CUGAGUUUAAAAGGCACCCTT B

349
AACUGAGUUUAAAAGGCACCCAG
2289
31801
2x C18 phospholipid + R31276
(L)2 ZTA B
2770

FLT1:349U21 sense siNA stab09
CCCACGGAAAAUUUGAGUCTT B

inv

3645
CAUGCUGGACUGCUGGCAC
2293
32235
FLT1:3645U21 sense siNA
CAUGCUGGACUGCUGGCACTT
2771

3646
AUGCUGGACUGCUGGCACA
2294
32236
FLT1:3646U21 sense siNA
AUGCUGGACUGCUGGCACATT
2772

3647
UGCUGGACUGCUGGCACAG
2295
32237
FLT1:3647U21 sense siNA
UGCUGGACUGCUGGCACAGTT
2773

3645
CAUGCUGGACUGCUGGCAC
2293
32250
FLT1:3663L21 antisense siNA
GUGCCAGCAGUCCAGCAUGTT
2774

(3645C)

3646
AUGCUGGACUGCUGGCACA
2294
32251
FLT1:3664L21 antisense siNA
UGUGCCAGCAGUCCAGCAUTT
2775

(3646C)

3647
UGCUGGACUGCUGGCACAG
2295
32252
FLT1:3665121 antisense siNA
CUGUGCCAGCAGUCCAGCAU
2776

(3647C)

349
AACUGAGUUUAAAAGGCACCCAG
2289
32278
FLT1:349U21 sense siNA stab16
B CUGAGUUUAAAAGGCACCCTT B
2777

349
AACUGAGUUUAAAAGGCACCCAG
2289
32279
FLT1:349U21 sense siNA stab18
B cuGAGuuuAAAAGGcAcccTT B
2778

349
AACUGAGUUUAAAAGGCACCCAG
2289
32280
FLT1:349U21 sense siNA inv
B CCCACGGAAAAUUUGAGUCTT B
2779

stab16

349
AACUGAGUUUAAAAGGCACCCAG
2289
32281
FLT1:349U21 sense siNA inv
B cccAcGGAAAAuuuGAGucTT B
2780

stab18

346
CUGAACUGAGUUUAAAAGGCACC
2296
32282
FLT1:346U21 sense siNA stab09
B GAACUGAGUUUAAAAGGCATT B
2781

347
UGAACUGAGUUUAAAAGGCACCC
2297
32283
FLT1:347U21 sense siNA stab09
B AACUGAGUUUAAAAGGCACTT B
2782

348
GAACUGAGUUUAAAAGGCACCCA
2298
32284
FLT1:348U21 sense siNA stab09
B ACUGAGUUUAAAAGGCACCTT B
2783

350
ACUGAGUUUAAAAGGCACCCAGC
2299
32285
FLT1:350U21 sense siNA stab09
B UGAGUUUAAAAGGCACCCATT B
2784

351
CUGAGUUUAAAAGGCACCCAGCA
2300
32286
FLT1:351U21 sense siNA stab09
B GAGUUUAAAAGGCACCCAGTT B
2785

352
UGAGUUUAAAAGGCACCCAGCAC
2301
32287
FLT1:352U21 sense siNA stab09
B AGUUUAAAAGGCACCCAGCTT B
2786

353
GAGUUUAAAAGGCACCCAGCACA
2302
32288
FLT1:353U21 sense siNA stab09
B GUUUAAAAGGCACCCAGCATT B
2787

346
CUGAACUGAGUUUAAAAGGCACC
2296
32289
FLT1:364L21 antisense siNA
UGCCUUUUAAACUCAGUUCTsT
2788

(346C) stab10

347
UGAACUGAGUUUAAAAGGCACCC
2297
32290
FLT1:365L21 antisense siNA
GUGCCUUUUAAACUCAGUUTsT
2789

(347C) stab10

348
GAACUGAGUUUAAAAGGCACCCA
2298
32291
FLT1:366L21 antisense siNA
GGUGCCUUUUAAACUCAGUTsT
2790

(348C) stab10

350
ACUGAGUUUAAAAGGCACCCAGC
2299
32292
FLT1:368121 antisense siNA
UGGGUGCCUUUUAAACUCATsT
2791

(350C) stab10

351
CUGAGUUUAAAAGGCACCCAGCA
2300
32293
FLT1:369121 antisense siNA
CUGGGUGCCUUUUAAAACUCTsT
2792

(351C) stab10

352
UGAGUUUAAAAGGCACCCAGCAC
2301
32294
FLT1:370121 antisense siNA
GCUGGGUGCCUUUUAAACUTsT
2793

(3520) stab10

353
GAGUUUAAAAGGCACCCAGCACA
2302
32295
FLT1:371121 antisense siNA
UGCUGGGUGCCUUUUAAACTsT
2794

(353C) stab10

346
CUGAACUGAGUUUAAAAGGCACC
2296
32296
FLT1:346U21 sense siNA inv
B ACGGAAAAUUUGAGUCAAGTT B
2795

stab09

347
UGAACUGAGUUUAAAAGGCACCC
2297
32297
FLT1:347U21 sense siNA inv
B CACGGAAAAUUUGAGUCAATT B
2796

stab09

348
GAACUGAGUUUAAAAGGCACCCA
2298
32298
FLT1:348U21 sense siNA inv
B CCACGGAAAAUUUGAGUCATT B
2797

stab09

350
ACUGAGUUUAAAAGGCACCCAGC
2299
32299
FLT1:350U21 sense siNA inv
B ACCCACGGAAAAUUUGAGUTT B
2798

stab09

351
CUGAGUUUAAAAGGCACCCAGCA
2300
32300
FLT1:351U21 sense siNA inv
B GACCCACGGAAAAUUUGAGTT B
2799

stab09

352
UGAGUUUAAAAGGCACCCAGCAC
2301
32301
FLT1:352U21 sense siNA inv
B CGACCCACGGAAAAUUUGATT B
2800

stab09

353
GAGUUUAAAAGGCACCCAGCACA
2302
32302
FLT1:353U21 sense siNA inv
B ACGACCCACGGAAAAUUUGTT B
2801

stab09

346
CUGAACUGAGUUUAAAAGGCACC
2296
32303
FLT1:364L21 antisense siNA
CUUGACUCAAAUUUUCCGUTsT
2802

(346C) inv stab10

347
UGAACUGAGUUUAAAAGGCACCC
2297
32304
FLT1:365L21 antisense siNA
UUGACUCAAAUUUUCCGUGTsT
2803

(347C) inv stab10

348
GAACUGAGUUUAAAAGGCACCCA
2298
32305
FLT1:366121 antisense siNA

(348C) inv stab10
UGACUCAAAUUUUCCGUGGTsT
2804

350
ACUGAGUUUAAAAGGCACCCAGC
2299
32306
FLT1:368L21 antisense siNA
ACUCAAAUUUUCCGUGGGUTsT
2805

(350C) inv stab10

351
CUGAGUUUAAAAGGCACCCAGCA
2300
32307
FLT1:369L21 antisense siNA
UCAAAUUUUCCGUGGGUCTST
2806

(351C) inv stab10

352
UGAGUUUAAAAGGCACCCAGCAC
2301
32308
FLT1:370L21 antisense siNA
UCAAAUUUUCCGUGGGUCGTsT
2807

(352C) inv stab10

353
GAGUUUAAAAGGCACCCAGCACA
2302
32309
FLT1:371121 antisense siNA
CAAAUUUUCCGUGGGUCGUTST
2808

(353C) inv stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
32338
FLT1:367L21 antisense siNA
UGGGUGCCUUUUAAACUCAGXST
2809

(349C) stab10 3′-Brd

349
AACUGAGUUUAAAAGGCACCCAG
2289
32718
pGGGUGCCUUUUAAACUC
GAGUUUAAAAG B
2810

FLT1:367121 antisense siNA

(349C) v1 5′p

349
AACUGAGUUUAAAAGGCACCCAG
2289
32719
pGGGUGCCUUUUAAACUCAG
GAGUUUAAAAG B
2811

FLT1:367L21 antisense siNA

(349C) v2 5′p

2967
AAGCAAGGAGGGCCUCUGAUGGU
2290
32720
FLT1:2967L21 antisense
pCAUCAGAGGCCCUCCUUGC
2812

siNA (2949C) v1 5′p
AAGGAGGGCCUCU B

2967
AAGCAAGGAGGGCCUCUGAUGGU
2290
32721
FLT1:2967121 antisense siNA
pCAUCAGAGGCCCUCCUU
2813

(2949C) v2 5′p
AAGGAGGGCCUCUG B

2967
AAGCAAGGAGGGCCUCUGAUGGU
2290
32722
FLT1:2967121 antisense siNA
pCAUCAGAGGCCCUCCU
2814

(2949C) v3 5′p
AGGAGGGCCUCUG B

346
CUGAACUGAGUUUAAAAGGCACC
2296
32748
FLT1:346U21 sense siNA
B GAAcuGAGuuuAAAAGGcATT B
2815

stab07

347
UGAACUGAGUUUAAAAGGCACCC
2297
32749
FLT1:347U21 sense siNA stab07
B AAcuGAGuuuAAAAGGcAcTT B
2816

348
GAACUGAGUUUAAAAGGCACCCA
2298
32750
FLT1:348U21 sense siNA stab07
B AcuGAGuuuAAAAGGcAccTT B
2817

350
ACUGAGUUUAAAAGGCACCCAGC
2299
32751
FLT1:350U21 sense siNA stab07
B uGAGuuuAAAAGGcAcccATT B
2818

351
CUGAGUUUAAAAGGCACCCAGCA
2300
32752
FLT1:351U21 sense siNA stab07
B GAGuuuAAAAGGcAcccAGTT B
2819

352
UGAGUUUAAAAGGCACCCAGCAC
2301
32753
FLT1:352U21 sense siNA stab07
B AGuuuAAAAGGcAcccAGcTT B
2820

353
GAGUUUAAAAGGCACCCAGCACA
2302
32754
FLT1:353U21 sense siNA stab07
B GuuuAAAAGGcAcccAGcATT B
2821

346
CUGAACUGAGUUUAAAAGGCACC
2296
32755
FLT1:364121 antisense siNA
uGccuuuuAAAcucAGuucTsT
2822

(346C) stab08

347
UGAACUGAGUUUAAAAGGCACCC
2297
32756
FLT1:365121 antisense siNA

GuGccuuuuAAAcucAGuuTsT
2823

(347C) stab08

348
GAACUGAGUUUAAAAGGCACCCA
2298
32757
FLT1:366121 antisense siNA

GGuGccuuuuAAAcucAGuTsT
2824

(348C) stab08

350
ACUGAGUUUAAAAGGCACCCAGC
2299
32758
FLT1:368121 antisense siNA
uGGGuGccuuuuAAAcucATsT
2825

(350C) stab08

351
CUGAGUUUAAAAGGCACCCAGCA
2300
32759
FLT1:369L21 antisense siNA
cuGGGuGccuuuuAAAcucTsT
2826

(351C) stab08

352
UGAGUUUAAAAGGCACCCAGCAC
2301
32760
FLT1:370L21 antisense siNA

GcuGGGuGccuuuuAAAcuTsT
2827

(352C) stab08

353
GAGUUUAAAAGGCACCCAGCACA
2302
32761
FLT1:371L21 antisense siNA
uGcuGGGuGccuuuuAAAcTsT
2828

(353C) stab08

346
CUGAACUGAGUUUAAAAGGCACC
2296
32772
FLT1:346U21 sense siNA inv
B AcGGAAAAuuuGAGucAAGTT B
2829

stab07

347
UGAACUGAGUUUAAAAGGCACCC
2297
32773
FLT1:347U21 sense siNA inv
B cAcGGAAAAuuuGAGucAATT B
2830

stab07

348
GAACUGAGUUUAAAAGGCACCCA
2298
32774
FLT1:348U21 sense siNA inv
B ccAcGGAAAAuuuGAGucATT B
2831

stab07

350
ACUGAGUUUAAAAGGCACCCAGC
2299
32775
FLT1:350U21 sense siNA inv
B AcccAcGGAAAAuuuGAGuTT B
2832

stab07

351
CUGAGUUUAAAAGGCACCCAGCA
2300
32776
FLT1:351U21 sense siNA inv
B GAcccAcGGAAAAuuuGAGTT B
2833

stab07

352
UGAGUUUAAAAGGCACCCAGCAC
2301
32777
FLT1:352U21 sense siNA inv
B cGAcccAcGGAAAAuuuGATT B
2834

stab07

353
GAGUUUAAAAGGCACCCAGCACA
2302
32778
FLT1:353U21 sense siNA inv
B AcGAcccAcGGAAAAuuuGTT B
2835

stab07

346
CUGAACUGAGUUUAAAAGGCACC
2296
32779
FLT1:364121 antisense siNA
cuuGAcucAAAuuuuccGuTsT
2836

(346C) inv stab08

347
UGAACUGAGUUUAAAAGGCACCC
2297
32780
FLT1:365121 antisense siNA
uuGAcucAAAuuuuccGuGTsT
2837

(347C) inv stab08

348
GAACUGAGUUUAAAAGGCACCCA
2298
32781
FLT1:366L21 antisense siNA
uGAcucAAAuuuuccGuGGTsT
2838

(348C) inv stab08

350
ACUGAGUUUAAAAGGCACCCAGC
2299
32782
FLT1:368L21 antisense siNA

AcucAAAuuuuccGuGGGuTsT
2839

(350C) inv stab08

351
CUGAGUUUAAAAGGCACCCAGCA
2300
32783
FLT1:369121 antisense siNA
cucAAAuuuuccGuGGGucTsT
2840

(351C) inv stab08

352
UGAGUUUAAAAGGCACCCAGCAC
2301
32784
FLT1:370L21 antisense siNA
ucAAAuuuuccGuGGGucGTsT
2841

(352C) inv stab08

353
GAGUUUAAAAGGCACCCAGCACA
2302
32785
FLT1:371121 antisense siNA
cAAAuuuuccGuGGGucGuTsT
2842

(353C) inv stab08

349
AACUGAGUUUAAAAGGCACCCAG
2289
33121
FLT1:349U21 sense siNA stab22
CUGAGUUUAAAAGGCAO0CTTB
2843

349
AACUGAGUUUAAAAGGCACCCAG
2289
33321
FLT1:367L21 antisense siNA
pGGGuGccuuuuAAAcucAGTsT
2844

(349C) stab08 + 5′ P

349
AACUGAGUUUAAAAGGCACCCAG
2289
33338
FLT1:367L21 antisense siNA
L GGGuGccuuuuAAAcucAGTsT
2845

(349C) stab08 + 5′ aminoL

349
AACUGAGUUUAAAAGGCACCCAG
2289
33553
FLT1:367L21 antisense siNA
L GGGuGccuuuuAAAcucAGTsT
2846

(349C) stab08 + 5′ aminoL

349
AACUGAGUUUAAAAGGCACCCAG
2289
33571
FLT1:367L21 antisense siNA
GGUGCCUUUUAAACUCAGTT
2847

(349C) stab10 + 5′I

3645
AUCAUGCUGGACUGCUGGCACAG
2189
33725
FLT1:3645U21 sense siNA
B cAuGcuGGAcuGcuGGcAcTT B
2848

stab07

3646
UCAUGCUGGACUGCUGGCACAGA
2195
33726
FLT1:3646U21 sense siNA
B AuGcuGGAcuGcuGGcAcATT B
2849

stab07

3645
AUCAUGCUGGACUGCUGGCACAG
2189
33731
FLT1:3663L21 antisense siNA

GuGccAGcAGuccAGcAuGTsT
2850

(3645C) stab08

3646
UCAUGCUGGACUGCUGGCACAGA
2195
33732
FLT1:3664L21 antisense siNA
uGuGccAGcAGuccAGcAuTsT
2851

(3646C) stab08

3645
AUCAUGCUGGACUGCUGGCACAG
2189
33737
FLT1:3645U21 sense siNA
B CAUGCUGGACUGCUGGCACTT B
2852

stab09

3646
UCAUGCUGGACUGCUGGCACAGA
2195
33738
FLT1:3646U21 sense siNA
B AUGCUGGACUGCUGGCACATT B
2853

stab09

3645
AUCAUGCUGGACUGCUGGCACAG
2189
33743
FLT1:3663L21 antisense siNA
GUGCCAGCAGUCCAGCAUGTsT
2854

(3645C) stab10

3646
UCAUGCUGGACUGCUGGCACAGA
2195
33744
FLT1:3664L21 antisense siNA
UGUGCCAGCAGUCCAGCAUTsT
2855

(3646C) stab10

3645
AUCAUGCUGGACUGCUGGCACAG
2189
33749
FLT1:3645U21 sense siNA inv
B cAcGGucGuCAGGucGuAcTT B
2856

stab07

3646
UCAUGCUGGACUGCUGGCACAGA
2195
33750
FLT1:3646U21 sense siNA inv
B AcAcGGucGuCAGGucGuATT B
2857

stab07

3645
AUCAUGCUGGACUGCUGGCACAG
2189
33755
FLT1:3663L21 antisense siNA

GuAcGAccuGAcGAccGUGTsT
2858

(3645C) inv stab08

3646
UCAUGCUGGACUGCUGGCACAGA
2195
33756
FLT1:3664L21 antisense siNA
uAcGAccuGAcGAccGuGuTsT
2859

(3646C) inv stab08

3645
AUCAUGCUGGACUGCUGGCACAG
2189
33761
FLT1:3645U21 sense siNA inv
B CACGGUCGUCAGGUCGUACTT B
2860

stab09

3646
UCAUGCUGGACUGCUGGCACAGA
2195
33762
FLT1:3646U21 sense siNA inv
B ACACGGUCGUCAGGUCGUATT B
2861

stab09

3645
AUCAUGCUGGACUGCUGGCACAG
2189
33767
FLT1:3663L21 antisense siNA
GUACGACCUGACGACCGUGTsT
2862

(3645C) inv stab10

3646
UCAUGCUGGACUGCUGGCACAGA
2195
33768
FLT1:3664L21 antisense siNA
UACGACCUGACGACCGUGUTsT
2863

(3646C) inv stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34487
FLT1:349U21 sense siNA stab09
B CsUsGAGUUUsAsAsAsAsGGCA
2864

w/block PS
CCsCsTsT B

349
AACUGAGUUUAAAAGGCACCCAG
2289
34488
FLT1:367L21 antisense siNA
GGGsUsGsCsCsUUUUAAsAsCsUs
2865

(349C) stab10 w/block PS
CsAGTsT

349
AACUGAGUUUAAAAGGCACCCAG
2289
34489
FLT1:349U21 sense siNA stab09
B CsCsCACGGAsAsAsAsUsUUGAG

inv w/block PS
UsCsT5TB
2866

349
AACUGAGUUUAAAAGGCACCCAG
2289
34490
FLT1:367L21 antisense siNA
GACsUsCsAsAsAUUUUCsCsGsUs

(349C) stab10 inv w/block PS
GsGGTsT
2867

349
AACUGAGUUUAAAAGGCACCCAG
2289
29694
FLT1:349U21 sense siNA stab01
CsUsGsAsGsUUUAAAAGGCACCC
2868

TsT

2340
AACAACCACAAAAUACAACAAGA
2292
29695
FLT1:2340U21 sense siNA
CsAsAsCsCsACAAAAUACAACAA
2869

stab01
TsT

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
29696
FLT1:3912U21 sense siNA
CsCsUsGsGsAAAGAAUCAAAACC
2870

stab01
TsT

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
29697
FLT1:2949U21 sense siNA
GsCsAsAsGsGAGGGCCUCUGATT
2871

stab01

349
AACUGAGUUUAAAAGGCACCCAG
2289
29698
FLT1:367L21 antisense siNA
GsGsGsUsGsCCUUUUAAACUCA
2872

(349C) stab01
GTsT

2340
AACAACCACAAAAUACAACAAGA
2292
29699
FLT1:2358L21 antisense siNA
UsUsGsUsUsGUAUUUUGUGGUU
2873

(2340C) stab01
GTsT

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
29700
FLT1:3930L21 antisense siNA
GsGsUsUsUsUGAUUCUUUCCAG
2874

(3912C) stab01
GTsT

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
29701
FLT1:2967L21 antisense siNA
CsAsUsCsAsGAGGCCCUCCUUG
2875

(2949C) stab01
CTsT

349
AACUGAGUUUAAAAGGCACCCAG
2289
29702
FLT1:349U21 sense siNA stab03
csusGsAsGuuuAAAAGGcAcscsc
2876

sTsT

2340
AACAACCACAAAAUACAACAAGA
2292
29703
FLT1:2340U21 sense siNA stab03
csAsAscscAcAAAAuAcAAcsAsA
2877

sTsT

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
29704
FLT1:3912U21 sense siNA stab03
cscsusGsGAAAGAAucAAAAscsc
2878

sTsT

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
29705
FLT1:2949U21 sense siNA stab03
GscsAsAsGGAGGGccucuGAsusG
2879

sTsT

349
AACUGAGUUUAAAAGGCACCCAG
2289
29706
FLT1:367121 antisense siNA
GsGsGsUsGsCsCsUsUsUsUsAsA
2880

(349C) stab02
sAsCsUsCsAsGsTsT

340
AACAACCACAAAAUACAACAAGA
2292
29707
FLT1:2358121 antisense siNA
UsUsGsUsUsGsUsAsUsUsUsUsG
2881

(2340C) stab02
sUsGsGsUsUsGsTsT

912
AGCCUGGAAAGAAUCAAAACCUU
2291
29708
FLT1:3930L21 antisense siNA
GsGsUsUsUsUsGsAsUsUsCsUsU
2882

(3912C) stab02
sUsCsCsAsGsGsTsT

949
AAGCAAGGAGGGCCUCUGAUGGU
2290
29709
FLT1:2967L21 antisense siNA
CsAsUsCsAsGsAsGsGsCsCsCsU
2883

(2949C) stab02
sCsCsUsUsGsCsTsT

2340
AACAACCACAAAAUACAACAAGA
2292
29981
FLT1:2340U21 sense siNA
CAACCACAAAAUACAACAAGA
2884

Native

2340
AACAACCACAAAAUACAACAAGA
2292
29982
FLT1:2358L21 antisense siNA
UUGUUGUAUUUUGUGGUUGUU
2885

(2340C) Native

2340
AACAACCACAAAAUACAACAAGA
2292
29983
FLT1:2340U21 sense siNA
AsAsCsAsAsCAUAAAACACCAAC
2886

stab01 inv
TsT

2340
AACAACCACAAAAUACAACAAGA
2292
29984
FLT1:2358121 antisense siNA
GsUsUsGsGsUGUUUUAUGUUGU
2887

(2340C) stab01 inv
UTsT

2340
AACAACCACAAAAUACAACAAGA
2292
29985
FLT1:2340U21 sense siNA
AsAscsAsAcAuAAAAcAccAsA
2888

stab03 inv
scsTsT

2340
AACAACCACAAAAUACAACAAGA
2292
29986
FLT1:2358L21 antisense siNA
GsUsUsGsGsUsGsUsUsUsUsAsU
2889

(2340C) stab02 inv
sGsUsUsGsUsUsTsT

2340
AACAACCACAAAAUACAACAAGA
2292
29987
FLT1:2340U21 sense siNA inv
AGAACAACAUAAAACACCAAC
2890

Native

2340
AACAACCACAAAAUACAACAAGA
2292
29988
FLT1:2358121 antisense siNA
UUGUUGGUGUUUUAUGUUGUU
2891

(2340C) inv Native

2340
AACAACCACAAAAUACAACAAGA
2292
30075
FLT1:2340U21 sense siNA
CAACCACAAAAUACAACAATT
2892

2340
AACAACCACAAAAUACAACAAGA
2292
30076
FLT1:2358L21 antisense siNA
UUGUUGUAUUUUGUGGUUGTT
2893

(2340C)

2342
AACAACCACAAAAUACAACAAGA
2292
30077
FLT1:2342U21 sense siNA inv
AGAACAACAUAAAACACCATT
2894

2340
AACAACCACAAAAUACAACAAGA
2292
30078
FLT1:2358L21 antisense siNA
UUGUUGGUGUUUUAUGUUGTT
2895

(2340C) inv

2340
AACAACCACAAAAUACAACAAGA
2292
30187
FLT1:2358L21 antisense siNA
uuGuuGuAuuuuGuGGuuGTT
2896

(2340C) 2′-F U,C

2340
AACAACCACAAAAUACAACAAGA
2292
30190
FLT1:2358121 antisense siNA
uuGuuGuAuuuuGuGGuuGXX
2897

(2340C) nitroindole

2340
AACAACCACAAAAUACAACAAGA
2292
30193
FLT1:2358L21 antisense siNA
uuGuuGuAuuuuGuGGuuGZZ
2898

(2340C) nitropyrole

2340
AACAACCACAAAAUACAACAAGA
2292
30196
FLT1:2340U21 sense siNA
B CAACCAcAAAAuAcAACAATT B
2899

stab04

2340
AACAACCACAAAAUACAACAAGA
2292
30199
FLT1:2340U21 sense siNA
cAAccAcAAAAuAcAACAATT
2900

sense iB caps

2340
AACAACCACAAAAUACAACAAGA
2292
30340
FLT1:2358L21 antisense
uuGuuGuAuuuuGuGGuuGTX
2901

siNA (2340C) 3′dT

2340
AACAACCACAAAAUACAACAAGA
2292
30341
FLT1:2358L21 antisense siNA
uuGuuGuAuuuuGuGGuuGTGly
2902

(2340C) glyceryl

2340
AACAACCACAAAAUACAACAAGA
2292
30342
FLT1:2358L21 antisense siNA
uuGuuGuAuuuuGuGGuuGTU
2903

(2340C) 3′OMeU

2340
AACAACCACAAAAUACAACAAGA
2292
30343
FLT1:2358L21 antisense siNA
uuGuuGuAuuuuGuGGuuGTt
2904

(2340C) L-dT

2340
AACAACCACAAAAUACAACAAGA
2292
30344
FLT1:2358L21 antisense siNA
uuGuuGuAuuuuGuGGuuGTu
2905

(2340C) L-rU

2340
AACAACCACAAAAUACAACAAGA
2292
30345
FLT1:2358121 antisense siNA
uuGuuGuAuuuuGuGGuuGTD
2906

(2340C) idT

2340
AACAACCACAAAAUACAACAAGA
2292
30346
FLT1:2358121 antisense siNA
uuGuuGuAuuuuGuGGuuGXT
2907

(2340C) 3′dT

2340
AACAACCACAAAAUACAACAAGA
2292
30416
FLT1:2358121 antisense siNA
uuGuuGuAuuuuGuGGuuGTsT
2908

(2340C) stab05

1184
UCGUGUAAGGAGUGGACCAUCAU
2303
30777
FLT1:1184U21 sense siNA
B GuGuAAGGAGuGGAccAucTT B
2909

stab04

3503
UUACGGAGUAUUGCUGUGGGAAA
2304
30778
FLT1:3503U21 sense siNA
B AcGGAGuAuuGcuGuGGGATT B
2910

stab04

4715
UAGCAGGCCUAAGACAUGUGAGG
2305
30779
FLT1:4715U21 sense siNA
B GcAGGccuAAGAcAuGuGATT B
2911

stab04

4753
AGCAAAAAGCAAGGGAGAAAAGA
2306
30780
FLT1:4753U21 sense siNA
B cAAAAAGcAAGGGAGAAAATT B
2912

stab04

1184
UCGUGUAAGGAGUGGACCAUCAU
2303
30781
FLT1:1202L21 antisense siNA
GAuGGuccAcuccuuAcAcTsT
2913

(1184C) stab05

3503
UUACGGAGUAUUGCUGUGGGAAA
2304
30782
FLT1:3521121 antisense siNA
ucccAcAGcAAuAcuccGuTsT
2914

(3503C) stab05

4715
UAGCAGGCCUAAGACAUGUGAGG
2305
30783
FLT1:4733L21 antisense siNA
ucAcAuGucuuAGGccuGcTsT
2915

(4715C) stab05

4753
AGCAAAAAGCAAGGGAGAAAAGA
2306
30784
FLT1:4771L21 antisense siNA
uuuucucccuuGcuuuuuGTsT
2916

(4753C) stab05

2340
AACAACCACAAAAUACAACAAGA
2292
30955
FLT1:2340U21 sense siNA
B cAAccAcAAAAuAcAAcAATT B
2917

stab07

2340
AACAACCACAAAAUACAACAAGA
2292
30956
FLT1:2358121 antisense siNA
uuGuuGuAuuuuGuGGuuGTsT
2918

(2340C) stab08

2340
AACAACCACAAAAUACAACAAGA
2292
30963
FLT1:2340U21 sense siNA inv
AACAACAUAAAACACCAACTT
2919

2340
AACAACCACAAAAUACAACAAGA
2292
30964
FLT1:2358L21 antisense siNA
GUUGGUGUUUUAUGUUGUUTT
2920

(2340C) inv

2340
AACAACCACAAAAUACAACAAGA
2292
30965
FLT1:2340U21 sense siNA
B AACAAcAuAAAAcAcCAACTT B
2921

stab04 inv

2340
AACAACCACAAAAUACAACAAGA
2292
30966
FLT1:2358L21 antisense siNA
GuuGGuGuuuuAuGuuGuuTsT
2922

(2340C) stab05 inv

2340
AACAACCACAAAAUACAACAAGA
2292
30967
FLT1:2340U21 sense siNA
B AAcAAcAuAAAAcAccAAcTT B
2923

stab07 inv

2340
AACAACCACAAAAUACAACAAGA
2292
30968
FLT1:2358L21 antisense siNA

GuuGGuGuuuuAuGuuGuuTsT
2924

(2340C) stab08 inv

349
AACUGAGUUUAAAAGGCACCCAG
2289
31182
FLT1:349U21 sense siNA stab00
CUGAGUUUAAAAGGCACCCTT
2925

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31183
FLT1:2949U21 sense siNA TT
GCAAGGAGGGCCUCUGAUGTT
2926

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31184
FLT1:3912U21 sense siNA TT
CCUGGAAAGAAUCAAAACCTT
2927

349
AACUGAGUUUAAAAGGCACCCAG
2289
31185
FLT1:367L21 antisense siNA
GGGUGCCUUUUAAACUCAGTT
2928

(349C) stab00

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31186
FLT1:2967L21 antisense siNA
TTCAUCAGAGGCCCUCCUUGCTT
2929

(2949C)

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31187
FLT1:3930L21 antisense siNA
TTGGUUUUGAUUCUUUCCAGGTT
2930

(3912C)

349
AACUGAGUUUAAAAGGCACCCAG
2289
31188
FLT1:349U21 sense siNA stab04
B cuGAGuuuAAAAGGcAcccTT B
2931

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31189
FLT1:2949U21 sense siNA
B GcAAGGAGGGccucuGAuGTT B
2932

stab04

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31190
FLT1:3912U21 sense siNA
B ccuGGAAAGAAucAAAAccTT B
2933

stab04

349
AACUGAGUUUAAAAGGCACCCAG
2289
31191
FLT1:367L21 antisense siNA
GGGuGccuuuuAAAcucAGTsT
2934

(349C) stab05

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31192
FLT1:2967L21 antisense siNA
cAucAGAGGcccuccuuGcTsT
2935

(2949C) stab05

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31193
FLT1:3930L21 antisense siNA
GGuuuuGAuucuuuccAGGTsT
2936

(3912C) stab05

349
AACUGAGUUUAAAAGGCACCCAG
2289
31194
FLT1:349U21 sense siNA stab07
B cuGAGuuuAAAAGGcAcccTT B
2937

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31195
FLT1:2949U21 sense siNA
B GcAAGGAGGGccucuGAuGTT B
2938

stab07

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31196
FLT1:3912U21 sense siNA
B ccuGGAAAGAAucAAAAccTT B
2939

stab07

349
AACUGAGUUUAAAAGGCACCCAG
2289
31197
FLT1:367L21 antisense siNA

GGGuGccuuuuAAAcucAGTsT
2940

(349C) stab08

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31198
FLT1:2967L21 antisense siNA
cAucAGAGGcccuccuuGcTsT
2941

(2949C) stab08

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31199
FLT1:3930121 antisense siNA

GGuuuuGAuucuuuccAGGTsT
2942

(3912C) stab08

349
AACUGAGUUUAAAAGGCACCCAG
2289
31200
FLT1:349U21 sense siNA inv TT
CCCACGGAAAAUUUGAGUCTT
2943

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31201
FLT1:2949U21 sense siNA inv
GUAGUCUCCGGGAGGAACGTT
2944

TT

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31202
FLT1:3912U21 sense siNA inv
CCAAAACUAAGAAAGGUCCTT
2945

TT

349
AACUGAGUUUAAAAGGCACCCAG
2289
31203
FLT1:367121 antisense siNA
GACUCAAAUUUUCCGUGGGTT
2946

(349C) inv TT

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31204
FLT1:2967L21 antisense siNA
CGUUCCUCCCGGAGACUACTT
2947

(2949C) inv TT

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31205
FLT1:3930L21 antisense siNA
GGACCUUUCUUAGUUUUGGTT
2948

(3912C) inv TT

349
AACUGAGUUUAAAAGGCACCCAG
2289
31206
FLT1:349U21 sense siNA
B CccAcGGAAAAuuuGAGucTT B
2949

stab04 inv

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31207
FLT1:2949U21 sense siNA
B GuAGucuccGGGAGGAAcGTT B
2950

stab04 inv

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31208
FLT1:3912U21 sense siNA
B ccAAAAcuAAGAAAGGuccTT B
2951

stab04 inv

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31510
FLT1:2967L21 antisense siNA
B cAucAGAGGcccuccuuGcTsT
2952

(2949C) stab11

349
AACUGAGUUUAAAAGGCACCCAG
2289
31511
FLT1:367121 antisense siNA

GGGuGccuuuuAAAcucAGTsT
2953

(349C) stab11

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31512
FLT1:3930L21 antisense siNA

GGuuuuGAuucuuuccAGGTsT
2954

(3912C) stab11

2340
AACAACCACAAAAUACAACAAGA
2292
31513
FLT1:2358L21 antisense siNA

GuuGGuGuuuuAuGuuGuuTsT
2955

(2340C) inv stab11

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31514
FLT1:2967L21 antisense siNA
cGuuccucccGGAGAcuAcTsT
2956

(2949C) inv stab11

349
AACUGAGUUUAAAAGGCACCCAG
2289
31515
FLT1:367121 antisense siNA

GAcucAAAuuuuccGuGGGTsT
2957

(349C) inv stab11

3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31516
FLT1:3930L21 antisense siNA

GGAccuuucuuAGuuuuGGTsT
2958

(3912C) inv stab11

349
AACUGAGUUUAAAAGGCACCCAG
2289
34426
5′n-1 C31270 FLT1:349U21
CUGAGUUUAAAAGGCACCCTT B
2843

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34427
5′n-2 C31270 FLT1:349U21
UGAGUUUAAAAGGCACCCTT B
2959

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34428
5′n-3 C31270 FLT1:349U21
GAGUUUAAAAGGCACCCTT B
2960

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34429
5′n-4 C31270 FLT1:349U21
AGUUUAAAAGGCACCCTT B
2961

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34430
5′n-5 C31270 FLT1:349U21
GUUUAAAAGGCACCCTT B
2962

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34431
5′n-7 C31270 FLT1:349U21
UUAAAAGGCACCCTT B
2963

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34432
5′n-9 C31270 FLT1:349U21
AAAAGGCACCCTT B
2964

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34433
3′n-1 C31270 FLT1:349U21
B CUGAGUUUAAAAGGCACCCTT
296S

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34434
3′n-2 C31270 FLT1:349U21
B CUGAGUUUAAAAGGCACCCT
2966

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34435
3′n-3 C31270 FLT1:349U21
B CUGAGUUUAAAAGGCACCC
2967

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34436
3′n-4 C31270 FLT1:349U21
B CUGAGUUUAAAAGGCACC
2968

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34437
3′n-5 C31270 FLT1:349U21
B CUGAGUUUAAAAGGCAC
2969

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34438
3′n-7 C31270 FLT1:349U21
B CUGAGUUUAAAAGGC
2970

sense siNA stab09

349
AACUGAGUUUAAAAGGCACCCAG
2289
34439
5′n-1 C31273 FLT1:367L21
GGUGCCUUUUAAACUCAGTsT
2971

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34440
5′n-2 C31273 FLT1:367L21
GUGCCUUUUAAACUCAGTsT
2972

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34441
5′n-3 C31273 FLT1:367L21
UGCCUUUUAAACUCAGTsT
2973

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34442
5′n-4 C31273 FLT1:367L21
GCCUUUUAAACUCAGTsT
2974

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34443
5′n-5 C31273 FLT1:367L21
CCUUUUAAACUCAGTsT
2975

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34444
3′n-1 C31273 FLT1:367L21
GGGUGCCUUUUAAACUCAGT
2976

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34445
3′n-2 C31273 FLT1:367121
GGGUGCCUUUUAAACUCAG
2977

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34446
3′n-3 C31273 FLT1:367L21
GGGUGCCUUUUAAACUCA
2978

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34447
3′n-4 C31273 FLT1:367121
GGGUGCCUUUUAAACUC
2979

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34448
3′n-5 C31273 FLT1:367121
GGGUGCCUUUUAAACU
2980

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34449
3′n-7 C31273 FLT1:367L21
GGGUGCCUUUUAAA
2981

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34450
3′n-9 C31273 FLT1:367L21
GGGUGCCUUUUA
2982

antisense siNA (349C) stab10

349
AACUGAGUUUAAAAGGCACCCAG
2289
34452
FLT1:367L21 antisense siNA

CUACCAGCGAGUUUGUAGUUUA

2983

(349C) scram1 + A15 all 2′OMe

AAAAAAAAAAAAACA

349
AACUGAGUUUAAAAGGCACCCAG
2289
34453
FLT1:367121 antisense siNA

CUACCAGCGAGUUUGUAGUUUA

2984

(349C) scram1 + A20 all 2′OMe

AAAAAAAAAAAAAAAAAsA

349
AACUGAGUUUAAAAGGCACCCAG
2289
34454
FLT1:367121 antisense siNA

CUACCAGCGAGUUUGUAGUUUA

2985

(349C) scram1 + A25 all 2′OMe

AAAAAAAAAAAAAAAAAAAAAA

AsA

349
AACUGAGUUUAAAAGGCACCCAG
2289
34455
FLT1:367L21 antisense siNA

CUACCAGCGAGUUUGUAGUUUA

2986

(349C) scram1 + A30 all 2′OMe

AAAAAAAAAAAAAAAAAAAAAA

AAAAAAsA

1501
ACCUCACUGCCACUCUAAUUGUC
2307
34676
FLT1:1501U21 sense siNA
CUCACUGCCACUCUAAUUGTT
2987

stab00

1502
CCUCACUGCCACUCUAAUUGUCA
2308
34677
FLT1:1502U21 sense siNA
UCACUGCCACUCUAAUUGUTT
2988

stab00

1503
CUCACUGCCACUCUAAUUGUCAA
2309
34678
FLT1:1503U21 sense siNA
CACUGCCACUCUAAUUGUCTT
2989

stab00

5353
AAGACCCCGUCUCUAUACCAACC
2310
34679
FLT1:5353U21 sense siNA
GACCCCGUCUCUAUACCAATT
2990

stab00

1501
ACCUCACUGCCACUCUAAUUGUC
2307
34684
FLT1:1519L21 (1501C) siRNA
CAAUUAGAGUGGCAGUGAGTT
2991

stab00

1502
CCUCACUGCCACUCUAAUUGUCA
2308
34685
FLT1:1520L21 (1502C) siRNA
ACAAUUAGAGUGGCAGUGATT
2992

stab00

1503
CUCACUGCCACUCUAAUUGUCAA
2309
34686
FLT1:1521L21 (1503C) siRNA
GACAAUUAGAGUGGCAGUGTT
2993

stab00

5353
AAGACCCCGUCUCUAUACCAACC
2310
34687
FLT1:5371L21 (5353C) siRNA
UUGGUAUAGAGACGGGGUCTT
2994

stab00

349
AACUGAGUUUAAAAGGCACCCAG
2289
35117
FLT1:349U21 sense siNA stab07
B cuGAGuuuAAAAGGCACCCTT B
2995

N1

349
AACUGAGUUUAAAAGGCACCCAG
2289
35118
FLT1:367L21 antisense siNA

GGGUGCcuuuuAAAcucAGTsT
2996

(349C) stab08 N1

349
AACUGAGUUUAAAAGGCACCCAG
2289
35119
FLT1:367L21 antisense siNA

GGGUGccuuuuAAAcucAGTsT
2997

(349C) stab08 N2

349
AACUGAGUUUAAAAGGCACCCAG
2289
35120
FLT1:367121 antisense siNA

GGGUGccuuuuAAAcucAGTsT
2998

(349C) stab08 N3

349
AACUGAGUUUAAAAGGCACCCAG
2289
35121
FLT1:367L21 antisense siNA

GGGuGccuuuuAAAcucAGTsT
2999

(349C) stab25

349
AACUGAGUUUAAAAGGCACCCAG
2289
35122
FLT1:367L21 antisense siNA

GGGuGccuuuuAAAcucAGTsT
3000

(349C) stab08 N5

349
AACUGAGUUUAAAAGGCACCCAG
2289
35123
FLT1:367L21 antisense siNA
GGGuGccuuuuAAAcucAGTsT
3001

(349C) stab24

346
CUGAACUGAGUUUAAAAGGCACC
2296
35814
FLT1:346U21 sense siNA
B GAAcuGAGuuuAAAAGGcATT B
3002

stab23

346
CUGAACUGAGUUUAAAAGGCACC
2296
35815
FLT1:346U21 sense siNA stab07
B GAAcuGAGuuuAAAAGGCATT B
3003

N2

346
CUGAACUGAGUUUAAAAGGCACC
2296
35816
FLT1:364L21 antisense siNA
UGccuuuuAAAcucAGuucTsT
3004

(346C) stab24

346
CUGAACUGAGUUUAAAAGGCACC
2296
35817
FLT1:364L21 antisense siNA

UGccuuuuAAAcucAGuucTsT
3005

(346C) stab08 N2

346
CUGAACUGAGUUUAAAAGGCACC
2296
35818
FLT1:364121 antisense siNA
UGCcuuuuAAAcucAGuucTsT
3006

(346C) sdtab24

346
CUGAACUGAGUUUAAAAGGCACC
2296
35909
FLT1:346U21 sense siNA stab07

GAAcuGAGuUuAAAAGGcATT
3007

J1

346
CUGAACUGAGUUUAAAAGGCACC
2296
35910
FLT1:364L21 antisense siNA

UGccuuuUAAAcucAGUucTsT
3008

(346C) stab08 J1

47
GAGCGGGCUCCGGGGCUCGGGUG
2311
36152
FLT1:47U21 sense siNA stab00
GCGGGCUCCGGGGCUCGGGTT
3009

121
CUGGCUGGAGCCGCGAGACGGGC
2312
36153
FLT1:121U21 sense siNAstab00
GGCUGGAGCCGCGAGACGGTT
3010

122
UGGCUGGAGCCGCGAGACGGGCG
2313
36154
FLT1:122U21 sense siNA stab00
GCUGGAGCCGCGAGACGGGTT
3011

251
CAUGGUCAGCUACUGGGACACCG
2314
36155
FLT1:251U21 sense siNA stab00
UGGUCAGCUACUGGGACACTT
3012

252
AUGGUCAGCUACUGGGACACCGG
2315
36156
FLT1:252U21 sense siNA stab00
GGUCAGCUACUGGGACACCTT
3013

354
AGUUUAAAAGGCACCCAGCACAU
2316
36157
FLT1:354U21 sense siNA stab00
UUUAAAAGGCACCCAGCACTT
3014

419
AGCAGCCCAUAAAUGGUCUUUGC
2317
36158
FLT1:419U21 sense siNA stab00
CAGCCCAUAAAUGGUCUUUTT
3015

594
UCAAAGAAGAAGGAAACAGAAUC
2318
36159
FLT1:594U21 sense siNA stab00
AAAGAAGAAGGAAACAGAATT
3016

595
CAAAGAAGAAGGAAACAGAAUCU
2319
36160
FLT1:595U21 sense siNA stab00
AAGAAGAAGGAAACAGAAUTT
3017

709
AGCUCGUCAUUCCCUGCCGGGUU
2320
36161
FLT1:709U21 sense siNA stab00
CUCGUCAUUCCCUGCCGGGTT
3018

710
GCUCGUCAUUCCCUGCCGGGUUA
2321
36162
FLT1:710U21 sense siNA stab00
UCGUCAUUCCCUGCCGGGUTT
3019

758
AAAAAAGUUUCCACUUGACACUU
2322
36163
FLT1:758U21 sense siNA stab00
AAAAGUUUCCACUUGACACTT
3020

759
AAAAAGUUUCCACUUGACACUUU
2323
36164
FLT1:759U21 sense siNA stab00
AAAGUUUCCACUUGACACUTT
3021

796
AACGCAUAAUCUGGGACAGUAGA
2324
36165
FLT1:796U21 sense siNA stab00
CGCAUAAUCUGGGACAGUATT
3022

797
ACGCAUAAUCUGGGACAGUAGAA
2325
36166
FLT1:797U21 sense siNA stab00
GCAUAAUCUGGGACAGUAGTT
3023

798
CGCAUAAUCUGGGACAGUAGAAA
2326
36167
FLT1:798U21 sense siNA stab00
CAUAAUCUGGGACAGUAGATT
3024

799
GCAUAAUCUGGGACAGUAGAAAG
2327
36168
FLT1:799U21 sense siNA stab00
AUAAUCUGGGACAGUAGAATT
3025

1220
CACCUCAGUGCAUAUAUAUGAUA
2328
36169
FLT1:1220U21 sense siNA
CCUCAGUGCAUAUAUAUGATT
3026

stab00

1438
CUGAAGAGGAUGCAGGGAAUUAU
2329
36170
FLT1:1438U21 sense siNA
GAAGAGGAUGCAGGGAAUUTT
3027

stab00

1541
UUACGAAAAGGCCGUGUCAUCGU
2330
36171
FLT1:1541U21 sense siNA
ACGAAAAGGCCGUGUCAUCTT
3028

stab00

1640
AAUCAAGUGGUUCUGGCACCCCU
2331
36172
FLT1:1640U21 sense siNA
UCAAGUGGUUCUGGCACCCTT
3029

stab00

1666
ACCAUAAUCAUUCCGAAGCAAGG
2332
36173
FLT1:1666U21 sense siNA
CAUAAUCAUUCCGAAGCAATT
3030

stab00

1877
GACUGUGGGAAGAAACAUAAGCU
2333
36174
FLT1:1877U21 sense siNA
CUGUGGGAAGAAACAUAAGTT
3031

stab00

2247
AACCUCAGUGAUCACACAGUGGC
2334
36175
FLT1:2247U21 sense siNA
CCUCAGUGAUCACACAGUGTT
3032

stab00

2248
ACCUCAGUGAUCACACAGUGGCC
2335
36176
FLT1:2248U21 sense siNA
CUCAGUGAUCACACAGUGGTT
3033

stab00

2360
AGAGCCUGGAAUUAUUUUAGGAC
2336
36177
FLT1:2360U21 sense siNA
AGCCUGGAAUUAUUUUAGGTT
3034

stab00

2415
ACAGAAGAGGAUGAAGGUGUCUA
2337
36178
FLT1:2415U21 sense siNA
AGAAGAGGAUGAAGGUGUCTT
3035

stab00

2514
UCUAAUCUGGAGCUGAUCACUCU
2338
36179
FLT1:2514U21 sense siNA
UAAUCUGGAGCUGAUCACUTT
3036

stab00

2518
AUCUGGAGCUGAUCACUCUAACA
2339
36180
FLT1:2518U21 sense siNA
CUGGAGCUGAUCACUCUAATT
3037

stab00

2703
AGCAAGUGGGAGUUUGCCCGGGA
2340
36181
FLT1:2703U21 sense siNA
CAAGUGGGAGUUUGCCCGGTT
3038

stab00

2795
CAUUAAGAAAUCACCUACGUGCC
2341
36182
FLT1:2795U21 sense siNA
UUAAGAAAUCACCUACGUGTT
3039

stab00

2965
UGAUGGUGAUUGUUGAAUACUGC
2342
36183
FLT1:2965U21 sense siNA
AUGGUGAUUGUUGAAUACUTT
3040

stab00

3074
GAAAGAAAAAAUGGAGCCAGGCC
2343
36184
FLT1:3074U21 sense siNA
AAGAAAAAAUGGAGCCAGGTT
3041

stab00

3100
AACAAGGCAAGAAACCAAGACUA
2344
36185
FLT1:3100U21 sense siNA
CAAGGCAAGAAACCAAGACTT
3042

stab00

3101
ACAAGGCAAGAAACCAAGACUAG
2345
36186
FLT1:3101U21 sense siNA
AAGGCAAGAAACCAAGACUTT
3043

stab00

3182
GAGUGAUGUUGAGGAAGAGGAGG
2346
36187
FLT1:3182U21 sense siNA
GUGAUGUUGAGGAAGAGGATT
3044

stab00

3183
AGUGAUGUUGAGGAAGAGGAGGA
2347
36188
FLT1:3183U21 sense siNA
UGAUGUUGAGGAAGAGGAGTT
3045

stab00

3253
CUUACAGUUUUCAAGUGGCCAGA
2348
36189
FLT1:3253U21 sense siNA
UACAGUUUUCAAGUGGCCATT
3046

stab00

3254
UUACAGUUUUCAAGUGGCCAGAG
2349
36190
FLT1:3254U21 sense siNA
ACAGUUUUCAAGUGGCCAGTT
3047

stab00

3260
UUUUCAAGUGGCCAGAGGCAUGG
2350
36191
FLT1:3260U21 sense siNA
UUCAAGUGGCCAGAGGCAUTT
3048

stab00

3261
UUUCAAGUGGCCAGAGGCAUGGA
2351
36192
FLT1:3261U21 sense siNA
UCAAGUGGCCAGAGGCAUGTT
3049

stab00

3294
UCCAGAAAGUGCAUUCAUCGGGA
2352
36193
FLT1:3294U21 sense siNA
CAGAAAGUGCAUUCAUCGGTT
3050

stab00

3323
AGCGAGAAACAUUCUUUUAUCUG
2353
36194
FLT1:3323U21 sense siNA
CGAGAAACAUUCUUUUAUCTT
3051

stab00

3324
GCGAGAAACAUUCUUUUAUCUGA
2354
36195
FLT1:3324U21 sense siNA
GAGAAACAUUCUUUUAUCUTT
3052

stab00

3325
CGAGAAACAUUCUUUUAUCUGAG
2355
36196
FLT1:3325U21 sense siNA
AGAAACAUUCUUUUAUCUGTT
3053

stab00

3513
UUGCUGUGGGAAAUCUUCUCCUU
2356
36197
FLT1:3513U21 sense siNA
GCUGUGGGAAAUCUUCUCCTT
3054

stab00

3812
UGCCUUCUCUGAGGACUUCUUCA
2357
36198
FLT1:3812U21 sense siNA
CCUUCUCUGAGGACUUCUUTT
3055

stab00

3864
UCAGGAAGCUCUGAUGAUGUCAG
2358
36199
FLT1:3864U21 sense siNA
AGGAAGCUCUGAUGAUGUCTT
3056

stab00

3865
CAGGAAGCUCUGAUGAUGUCAGA
2359
36200
FLT1:3865U21 sense siNA
GGAAGCUCUGAUGAUGUCATT
3057

stab00

3901
UCAAGUUCAUGAGCCUGGAAAGA
2360
36201
FLT1:3901U21 sense siNA
AAGUUCAUGAGCCUGGAAATT
3058

stab00

3902
CAAGUUCAUGAGCCUGGAAAGAA
2361
36202
FLT1:3902U21 sense siNA
AGUUCAUGAGCCUGGAAAGTT
3059

stab00

3910
UGAGCCUGGAAAGAAUCAAAACC
2362
36203
FLT1:3910U21 sense siNA
AGCCUGGAAAGAAUCAAAATT
3060

stab00

4136
CAGCUGUGGGCACGUCAGCGAAG
2363
36204
FLT1:4136U21 sense siNA
GCUGUGGGCACGUCAGCGATT
3061

stab00

4154
CGAAGGCAAGCGCAGGUUCACCU
2364
36205
FLT1:4154U21 sense siNA
AAGGCAAGCGCAGGUUCACTT
3062

stab00

4635
UGCAGCCCAAAACCCAGGGCAAC
2365
36206
FLT1:4635U21 sense siNA
CAGCCCAAAACCCAGGGCATT
3063

stab00

4945
GAGGCAAGAAAAGGACAAAUAUC
2366
36207
FLT1:4945U21 sense siNA
GGCAAGAAAAGGACAAAUATT
3064

stab00

5090
UUGGCUCCUCUAGUAAGAUGCAC
2367
36208
FLT1:5090U21 sense siNA
GGCUCCUCUAGUAAGAUGCTT
3065

stab00

5137
GUCUCCAGGCCAUGAUGGCCUUA
2368
36209
FLT1:5137U21 sense siNA
CUCCAGGCCAUGAUGGCCUTT
3066

stab00

5138
UCUCCAGGCCAUGAUGGCCUUAC
2369
36210
FLT1:5138U21 sense siNA
UCCAGGCCAUGAUGGCCUUTT
3067

stab00

5354
AGACCCCGUCUCUAUACCAACCA
2370
36211
FLT1:5354U21 sense siNA
ACCCCGUCUCUAUACCAACTT
3068

stab00

5356
ACCCCGUCUCUAUACCAACCAAA
2371
36212
FLT1:5356U21 sense siNA
CCCGUCUCUAUACCAACCATT
3069

stab00

5357
CCCCGUCUCUAUACCAACCAAAC
2372
36213
FLT1:5357U21 sense siNA
CCGUCUCUAUACCAACCAATT
3070

stab00

5707
GAUCAAGUGGGCCUUGGAUCGCU
2373
36214
FLT1:5707U21 sense siNA
UCAAGUGGGCCUUGGAUCGTT
3071

stab00

5708
AUCAAGUGGGCCUUGGAUCGCUA
2374
36215
FLT1:5708U21 sense siNA
CAAGUGGGCCUUGGAUCGCTT
3072

stab00

47
GAGCGGGCUCCGGGGCUCGGGUG
2311
36216
FLT1:65L21 antisense siNA
CCCGAGCCCCGGAGCCCGCTT
3073

(47C) stab00

121
CUGGCUGGAGCCGCGAGACGGGC
2312
36217
FLT1:139L21 antisense siNA
CCGUCUCGCGGCUCCAGCCTT
3074

(121C) stab00

122
UGGCUGGAGCCGCGAGACGGGCG
2313
36218
FLT1:140L21 antisense siNA
CCCGUCUCGCGGCUCCAGCTT
3075

(122C) stab00

251
CAUGGUCAGCUACUGGGACACCG
2314
36219
FLT1:269121 antisense siNA
GUGUCCCAGUAGCUGACCATT
3076

(251C) stab00

252
AUGGUCAGCUACUGGGACACCGG
2315
36220
FLT1:270121 antisense siNA
GGUGUCCCAGUAGCUGACCTT
3077

(252C) stab00

354
AGUUUAAAAGGCACCCAGCACAU
2316
36221
FLT1:372121 antisense siNA
GUGCUGGGUGCCUUUUAAATT
3078

(354C)

419
AGCAGCCCAUAAAUGGUCUUUGC
2317
36222
FLT1:437L21 antisense siNA
AAAGACCAUUUAUGGGCUGTT
3079

(419C) stab00

594
UCAAAGAAGAAGGAAACAGAAUC
2318
36223
FLT1:612L21 antisense siNA
UUCUGUUUCCUUCUUCUUUTT
3080

(594C) stab00

595
CAAAGAAGAAGGAAACAGAAUCU
2319
36224
FLT1:613121 antisense siNA
AUUCUGUUUCCUUCUUCUUTT
3081

(595C) stab00

709
AGCUCGUCAUUCCCUGCCGGGUU
2320
36225
FLT1:727L21 antisense siNA
CCCGGCAGGGAAUGACGAGTT
3082

(709C) stab00

710
GCUCGUCAUUCCCUGCCGGGUUA
2321
36226
FLT1:728121 antisense siNA
ACCCGGCAGGGAAUGACGATT
3083

(710C) stab00

758
AAAAAAGUUUCCACUUGACACUU
2322
36227
FLT1:776121 antisense siNA
GUGUCAAGUGGAAACUUUUTT
3084

(758C) stab00

759
AAAAAGUUUCCACUUGACACUUU
2323
36228
FLT1:777121 antisense siNA
AGUGUCAAGUGGAAACUUUTT
3085

(759C) stab00

796
AACGCAUAAUCUGGGACAGUAGA
2324
36229
FLT1:814L21 antisense siNA
UACUGUCCCAGAUUAUGCGTT
3086

(796C) stab00

797
ACGCAUAAUCUGGGACAGUAGAA
2325
36230
FLT1:815L21 antisense siNA
CUACUGUCCCAGAUUAUGCTT
3087

(797C) stab00

798
CGCAUAAUCUGGGACAGUAGAAA
2326
36231
FLT1:816L21 antisense siNA
UCUACUGUCCCAGAUUAUGTT
3088

(798C) stab00

799
GCAUAAUCUGGGACAGUAGAAAG
2327
36232
FLT1:817L21 antisense siNA
UUCUACUGUCCCAGAUUAUTT
3089

(799C) stab00

1220
CACCUCAGUGCAUAUAUAUGAUA
2328
36233
FLT1:1238L21 antisense siNA
UCAUAUAUAUGCACUGAGGTT
3090

(1220C) stab00

1438
CUGAAGAGGAUGCAGGGAAUUAU
2329
36234
FLT1:1456L21 antisense siNA
AAUUCCCUGCAUCCUCUUCTT
3091

(1438C) stab00

1541
UUACGAAAAGGCCGUGUCAUCGU
2330
36235
FLT1:1559L21 antisense siNA
GAUGACACGGCCUUUUCGUTT
3092

(1541C) stab00

1640
AAUCAAGUGGUUCUGGCACCCCU
2331
36236
FLT1:1658L21 antisense siNA
GGGUGCCAGAACCACUUGATT
3093

(1640C) stab00

1666
ACCAUAAUCAUUCCGAAGCAAGG
2332
36237
FLT1:1684L21 antisense siNA
UUGCUUCGGAAUGAUUAUGTT
3094

(1666C) stab00

1877
GACUGUGGGAAGAAACAUAAGCU
2333
36238
FLT1:1895L21 antisense siNA
CUUAUGUUUCUUCCCACAGTT
3095

(1877C) stab00

2247
AACCUCAGUGAUCACACAGUGGC
2334
36239
FLT1:2265L21 antisense siNA
CACUGUGUGAUOACUGAGGTT
3096

(2247C) stab00

2248
ACCUCAGUGAUCACACAGUGGOC
2335
36240
FLT1:2266L21 antisense siNA
COACUGUGUGAUCACUGAGTT
3097

(2248C) stab00

2360
AGAGCCUGGAAUUAUUUUAGGAC
2336
36241
FLT1:2378L21 antisense siNA
CCUAAAAUAAUUCCAGGCUTT
3098

(2360C) stab00

2415
ACAGAAGAGGAUGAAGGUGUCUA
2337
36242
FLT1:2433L21 antisense siNA
GACACCUUCAUCCUCUUCUTT
3099

(2415C) stab00

2514
UCUAAUCUGGAGCUGAUCACUCU
2338
36243
FLT1:2532121 antisense siNA
AGUGAUCAGCUCCAGAUUATT
3100

(2514C) stab00

2518
AUCUGGAGCUGAUCACUCUAACA
2339
36244
FLT1:2536L21 antisense siNA
UUAGAGUGAUCAGCUCCAGTT
3101

(2518C) stab00

2703
AGCAAGUGGGAGUUUGCCCGGGA
2340
36245
FLT1:2721L21 antisense siNA
CCGGGCAAACUCCCACUUGTT
3102

(2703C) stab00

2795
CAUUAAGAAAUCACCUACGUGCC
2341
36246
FLT1:2813L21 antisense siNA
CACGUAGGUGAUUUCUUAATT
3103

(2795C) stab00

2965
UGAUGGUGAUUGUUGAAUACUGC
2342
36247
FLT1:2983121 antisense siNA
AGUAUUCAACAAUCACCAUTT
3104

(2965C) stab00

3074
GAAAGAAAAAAUGGAGCCAGGCC
2343
36248
FLT1:3092121 antisense siNA
CCUGGCUCCAUUUUUUCUUTT
3105

(3074C) stab00

3100
AACAAGGCAAGAAACCAAGACUA
2344
36249
FLT1:3118L21 antisense siNA
GUCUUGGUUUCUUGCCUUGTT
3106

(3100C) stab00

3101
ACAAGGCAAGAAACCAAGACUAG
2345
36250
FLT1:3119L21 antisense siNA
AGUCUUGGUUUCUUGCCUUTT
3107

(3101C) stab00

3182
GAGUGAUGUUGAGGAAGAGGAGG
2346
36251
FLT1:3200L21 antisense siNA
UCCUCUUCCUCAACAUCACTT
3108

(3182C) stab00

3183
AGUGAUGUUGAGGAAGAGGAGGA
2347
36252
FLT1:3201L21 antisense siNA
CUCCUCUUCCUCAACAUCATT
3109

(3183C) stab00

3253
CUUACAGUUUUCAAGUGGCCAGA
2348
36253
FLT1:3271L21 antisense siNA
UGGCCACUUGAAAACUGUATT
3110

(3253C) stab00

3254
UUACAGUUUUCAAGUGGCCAGAG
2349
36254
FLT1:3272L21 antisense siNA
CUGGCCACUUGAAAACUGUTT
3111

(3254C) stab00

3260
UUUUCAAGUGGCCAGAGGCAUGG
2350
36255
FLT1:3278121 antisense siNA
AUGCCUCUGGCCACUUGAATT
3112

(3260C) stab00

3261
UUUCAAGUGGCCAGAGGCAUGGA
2351
36256
FLT1:3279121 antisense siNA
CAUGCCUCUGGCCACUUGATT
3113

(3261C) stab00

3294
UCCAGAAAGUGCAUUCAUCGGGA
2352
36257
FLT1:3312121 antisense siNA
CCGAUGAAUGCACUUUCUGTT
3114

(3294C) stab00

3323
AGCGAGAAACAUUCUUUUAUCUG
2353
36258
FLT1:3341121 antisense siNA
GAUAAAAGAAUGUUUCUCGTT
3115

(3323C) stab00

3324
GCGAGAAACAUUCUUUUAUCUGA
2354
36259
FLT1:3342L21 antisense siNA
AGAUAAAAGAAUGUUUCUCTT
3116

(3324C) stab00

3325
CGAGAAACAUUCUUUUAUCUGAG
2355
36260
FLT1:3343L21 antisense siNA
CAGAUAAAAGAAUGUUUCUTT
3117

(3325C) stab00

3513
UUGCUGUGGGAAAUCUUCUCCUU
2356
36261
FLT1:3531L21 antisense siNA
GGAGAAGAUUUCCCACAGCTT
3118

(3513C) stab00

3812
UGCCUUCUCUGAGGACUUCUUCA
2357
36262
FLT1:3830L21 antisense siNA
AAGAAGUCCUCAGAGAAGGTT
3119

(3812C) stab00

3864
UCAGGAAGCUCUGAUGAUGUCAG
2358
36263
FLT1:3882L21 antisense siNA
GACAUCAUCAGAGCUUCCUTT
3120

(3864C) stab00

3865
CAGGAAGCUCUGAUGAUGUCAGA
2359
36264
FLT1:3883L21 antisense siNA
UGACAUCAUCAGAGCUUCCTT
3121

(3865C) stab00

3901
UCAAGUUCAUGAGCCUGGAAAGA
2360
36265
FLT1:3919121 antisense siNA
UUUCCAGGCUCAUGAACUUTT
3122

(3901C) stab00

3902
CAAGUUCAUGAGCCUGGAAAGAA
2361
36266
FLT1:3920L21 antisense siNA
CUUUCCAGGCUCAUGAACUTT
3123

(3902C) stab00

3910
UGAGCCUGGAAAGAAUCAAAACC
2362
36267
FLT1:3928L21 antisense siNA
UUUUGAUUCUUUCCAGGCUTT
3124

(3910C) stab00

4136
CAGCUGUGGGCACGUCAGCGAAG
2363
36268
FLT1:4154L21 antisense siNA
UCGCUGACGUGCCCACAGCTT
3125

(4136C) stab00

4154
CGAAGGCAAGCGCAGGUUCACCU
2364
36269
FLT1:4172L21 antisense siNA
GUGAACCUGCGCUUGOOUUTT
3126

(4154C) stab00

4635
UGCAGCCCAAAACCCAGGGCAAC
2365
36270
FLT1:4653121 antisense siNA
UGCCCUGGGUUUUGGGCUGTT
3127

(4635C) stab00

4945
GAGGCAAGAAAAGGACAAAUAUC
2366
36271
FLT1:4963121 antisense siNA
UAUUUGUCOUUUUCUUGCCTT
3128

(49450) stab00

5090
UUGGCUCOUCUAGUAAGAUGCAC
2367
36272
FLT1:5108121 antisense siNA
GCAUCUUACUAGAGGAGCCTT
3129

(5090C) stab00

5137
GUCUCCAGGCCAUGAUGGCCUUA
2368
36273
FLT1:5155121 antisense siNA
AGGOCAUCAUGGOCUGGAGTT
3130

(5137C) stab00

5138
UCUCCAGGCCAUGAUGGCCUUAC
2369
36274
FLT1:5156L21 antisense siNA
AAGGCCAUCAUGGCCUGGATT
3131

(5138C) stab00

5354
AGACCCCGUCUCUAUACCAACCA
2370
36275
FLT1:5372121 antisense siNA
GUUGGUAUAGAGACGGGGUTT
3132

(5354C) stab00

5356
ACCCCGUCUCUAUACCAACCAAA
2371
36276
FLT1:5374L21 antisense siNA
UGGUUGGUAUAGAGACGGGTT
3133

(5356C) stab00

5357
CCCCGUCUCUAUACCAACCAAAC
2372
36277
FLT1:5375121 antisense siNA
UUGGUUGGUAUAGAGACGGTT
3134

(5357C) stab00

5707
GAUCAAGUGGGCCUUGGAUCGCU
2373
36278
FLT1:5725121 antisense siNA
CGAUCCAAGGCCOACUUGATT
3135

(5707C) stab00

5708
AUCAAGUGGGCCUUGGAUCGCUA
2374
36279
FLT1:5726121 antisense siNA
GCGAUCCAAGGCCCACUUGTT
3136

(5708C) stab00

346
CUGAACUGAGUUUAAAAGGCACC
2296
36431
FLT1:346U21 sense siNA stab00
GAACUGAGUUUAAAAGGCATT
3137

346
CUGAACUGAGUUUAAAAGGCACC
2296
36439
FLT1:364121 antisense siNA
UGCCUUUUAAACUCAGUUCTT
3138

(346C) stab00

349
AACUGAGUUUAAAAGGCACCCAG
2289
36457
FLT1:349U19 sense siNA
CUGAGUUUAAAAGGCACCC
3139

stab00-3′TT

349
AACUGAGUUUAAAAGGCACCCAG
2289
36458
FLT1:367121 antisense siNA
B GGGUGCCUUUUAAACUCAGTsT B
3140

(349C) stab10 +5′ & 3′ iB

349
AACUGAGUUUAAAAGGCACCCAG
2289
36459
FLT1:367L19 siRNA (349C)
B GGGUGCCUUUUAAACUCAG
3141

stab00 +5′ iB −3 TT

349
AACUGAGUUUAAAAGGCACCCAG
2289
36460
FLT1:349U21 sense siNA
cuGAGuuuAAAAGGcAccc1T
3142

stab07 −5′ & 3′ iB

349
AACUGAGUUUAAAAGGCACCCAG
2289
36461
FLT1:349U21 sense siNA
cuGAGuuuAAAAGGcAccc
3143

stab07 −5′ iB −3 TTB

349
AACUGAGUUUAAAAGGCACCCAG
2289
36462
FLT1:367L19 siRNA (349C)

GGGuGccuuuuAAAcucAG
3144

stab08 −3′ TTB

2338
AAAACAACCACAAAAUACAACAA
2375
37389
FLT1:2338U21 sense siNA
B AAcAAccAcAAAAuAcAAcTT B
3145

stab07

2342
CAACCACAAAAUACAACAAGAGC
2376
37390
FLT1:2342U21 sense siNA
B AccAcAAAAuAcAAcAAGATT B
3146

stab07

2365
CUGGAAUUAUUUUAGGACCAGGA
2377
37391
FLT1:2365U21 sense siNA
B GGAAuuAuuuuAGGAccAGTT B
3147

stab07

2391
AGCACGCUGUUUAUUGAAAGAGU
2378
37392
FLT1:2391U21 sense siNA
B cAcGcuGuuuAuuGAAAGATT B
3148

stab07

2392
GCACGCUGUUUAUUGAAAGAGUC
2379
37393
FLT1:2392U21 sense siNA
B AcGcuGuuuAuuGAAAGAGTT B
3149

stab07

2393
CACGCUGUUUAUUGAAAGAGUCA
2380
37394
FLT1:2393U21 sense siNA
B cGcuGuuuAuuGAAAGAGuTT B
3150

stab07

2394
ACGCUGUUUAUUGAAAGAGUCAC
2381
37395
FLT1:2394U21 sense siNA
B GcuGuuuAuuGAAAGAGucTT B
3151

stab07

2395
CGCUGUUUAUUGAAAGAGUCACA
2382
37396
FLT1:2395U21 sense siNA
B cuGuuuAuuGAAAGAGucATT B
3152

stab07

2396
GCUGUUUAUUGAAAGAGUCACAG
2383
37397
FLT1:2396U21 sense siNA
B uGuuuAuuGAAAGAGuCAcTT B
3153

stab07

2397
CUGUUUAUUGAAAGAGUCACAGA
2384
37398
FLT1:2397U21 sense siNA
B GuuuAuuGAAAGAGucAcATT B
3154

stab07

2398
UGUUUAUUGAAAGAGUCACAGAA
2385
37399
FLT1:2398U21 sense siNA
B uuuAuuGAAAGAGucAcAGTT B
3155

stab07

2697
GAUGCCAGCAAGUGGGAGUUUGC
2386
37400
FLT1:2697U21 sense siNA
B uGccAGcAAGuGGGAGuuuTT B
3156

stab07

2699
UGCCAGCAAGUGGGAGUUUGCCC
2387
37401
FLT1:2699U21 sense siNA
B ccAGcAAGuGGGAGuuuGcTT B
3157

stab07

2785
CAGCAUUUGGCAUUAAGAAAUCA
2388
37402
FLT1:2785U21 sense siNA
B GcAuuuGGcAuuAAGAAAuTT B
3158

stab07

2786
AGCAUUUGGCAUUAAGAAAUCAC
2389
37403
FLT1:2786U21 sense siNA
B cAuuuGGcAuuAAGAAAucTT B
3159

stab07

2788
CAUUUGGCAUUAAGAAAUCACCU
2390
37405
FLT1:2788U21 sense siNA
B uuuGGcAuuAAGAAAucAcTT B
3160

stab07

2789
AUUUGGCAUUAAGAAAUCACCUA
2391
37406
FLT1:2789U21 sense siNA
B uuGGcAuuAAGAAAucAccTT B
3161

stab07

2812
CGUGCCGGACUGUGGCUGUGAAA
2392
37407
FLT1:2812U21 sense siNA
B uGccGGAcuGuGGcuGuGATT B
3162

stab07

2860
GCGAGUACAAAGCUCUGAUGACU
2393
37408
FLT1:2860U21 sense siNA
B GAGuAcAAAGcucuGAuGATT B
3163

stab07

2861
CGAGUACAAAGCUCUGAUGACUG
2394
37409
FLT1:2861U21 sense siNA
B AGuAcAAAGCucuGAuGAcTT B
3164

stab07

2947
CCAAGCAAGGAGGGCCUCUGAUG
2395
37410
FLT1:2947U21 sense siNA
B AAGcAAGGAGGGccucuGATT B
3165

stab07

2950
AGCAAGGAGGGCCUCUGAUGGUG
2396
37411
FLT1:2950U21 sense siNA
B cAAGGAGGGccucuGAuGGTT B
3166

stab07

2952
CAAGGAGGGCCUCUGAUGGUGAU
2397
37412
FLT1:2952U21 sense siNA
B AGGAGGGccucuGAuGGuGTT B
3167

stab07

2953
AAGGAGGGCCUCUGAUGGUGAUU
2398
37413
FLT1:2953U21 sense siNA
B GGAGGGcCucuGAuGGuGATT B
3168

stab07

2954
AGGAGGGCCUCUGAUGGUGAUUG
2399
37414
FLT1:2954U21 sense siNA
B GAGGGccucuGAuGGuGAuTT B
3169

stab07

3262
UUCAAGUGGCCAGAGGCAUGGAG
2400
37415
FLT1:3262U21 sense siNA
B cAAGuGGccAGAGGcAuGGTT B
3170

stab07

3263
UCAAGUGGCCAGAGGCAUGGAGU
2401
37416
FLT1:3263U21 sense siNA
B AAGuGGccAGAGGCAuGGATT B
3171

stab07

3266
AGUGGCCAGAGGCAUGGAGUUCC
2402
37417
FLT1:3266U21 sense siNA
B uGGccAGAGGcAuGGAGuuTT B
3172

stab07

3911
GAGCCUGGAAAGAAUCAAAACCU
2403
37418
FLT1:3911U21 sense siNA
B GccuGGAAAGAAucAAAAcTT B
3173

stab07

4419
UUUUUUGACUAACAAGAAUGUAA
2404
37419
FLT1:4419U21 sense siNA
B uuuuGAcuAAcAAGAAuGuTT B
3174

stab07

346
CUGAACUGAGUUUAAAAGGCACC
2296
37420
FLT1:364L21 antisense siNA
UGCcuuuuAAAcucAGuuCTT
3175

(346C) stab26

347
UGAACUGAGUUUAAAAGGCACCC
2297
37421
FLT1:365L21 antisense siNA
GUGccuuuuAAAcucAGuuTT
3176

(347C) stab26

349
AACUGAGUUUAAAAGGCACCCAG
2289
37422
FLT1:367L21 antisense siNA
GGGUGCCUUUUAAAcucAGTT
3177

(349C) stab26

351
CUGAGUUUAAAAGGCACCCAGCA
2300
37423
FLT1:369L21 antisense siNA
CUGGGuGccuuuuAAAcucTT
3178

(351C) stab26

353
GAGUUUAAAAGGCACCCAGCACA
2302
37424
FLT1:371121 antisense siNA
UGCuGGGuGccuuuuAAAcTT
3179

(353C) stab26

1956
GAAGGAGAGGACCUGAAACUGUC
2286
37425
FLT1:1974L21 antisense siNA
CAGuuucAGGuccucuccuTT
3180

(1956C) stab26

1957
AAGGAGAGGACCUGAAACUGUCU
2287
37426
FLT1:1975121 antisense siNA
ACAGuuucAGGuccucuccTT
3181

(1957C) stab26

2338
AAAACAACCACAAAAUACAACAA
2375
37427
FLT1:2356L21 antisense siNA
GUUGuAuuuuGuGGuuGuuTT
3182

(2338C) stab26

2340
AACAACCACAAAAUACAACAAGA
2292
37428
FLT1:2358L21 antisense siNA
UUGuuGuAuuuuGuGGuuGTT
3183

(2340C) stab26

2342
CAACCACAAAAUACAACAAGAGC
2376
37429
FLT1:2360121 antisense siNA
UCUuGuuGuAuuuuGuGGuTT
3184

(2342C) stab26

2365
CUGGAAUUAUUUUAGGACCAGGA
2377
37430
FLT1:2383L21 antisense siNA
CUGGuccuAAAAuAAuuccTT
3185

(2365C) stab26

2391
AGCACGCUGUUUAUUGAAAGAGU
2378
37431
FLT1:2409L21 antisense siNA
UCUuuCAAuAAAcAGcGuGTT
3186

(2391C) stab26

2392
GCACGCUGUUUAUUGAAAGAGUC
2379
37432
FLT1:2410121 antisense siNA
CUCuuucAAuAAAcAGcGuTT
3187

(2392C) stab26

2393
CACGCUGUUUAUUGAAAGAGUCA
2380
37433
FLT1:2411L21 antisense siNA
ACUcuuucAAuAAAcAGcGTT
3188

(2393C) stab26

2394
ACGCUGUUUAUUGAAAGAGUCAC
2381
37434
FLT1:2412L21 antisense siNA
GACucuuucAAuAAAcAGcTT
3189

stab26

2395
CGCUGUUUAUUGAAAGAGUCACA
2382
37435
FLT1:2413121 antisense siNA
UGAcucuuucAAuAAAcAGTT
3190

(2395C) stab26

2396
GCUGUUUAUUGAAAGAGUCACAG
2383
37436
FLT1:2414L21 antisense siNA
GUGAcucuuucAAuAAAcATT
3191

(2396C) stab26

2397
CUGUUUAUUGAAAGAGUCACAGA
2384
37437
FLT1:2415121 antisense siNA
UGUGAcucuuucAAuAAAcTT
3192

(2397C) stab26

2398
UGUUUAUUGAAAGAGUCACAGAA
2385
37438
FLT1:2416121 antisense siNA
CUGuGAcucuuucAAuAAATT
3193

(2398C) stab26

2697
GAUGCCAGCAAGUGGGAGUUUGC
2386
37439
FLT1:2715L21 antisense siNA
AAAcucccAcuuGcuGGcATT
3194

(2697C) stab26

2699
UGCCAGCAAGUGGGAGUUUGCCC
2387
37440
FLT1:2717121 antisense siNA
GCAAAcucccAcuuGcuGGTT
3195

(2699C) stab26

2785
CAGCAUUUGGCAUUAAGAAAUCA
2388
37441
FLT1 :2803L21 antisense siNA
AUUucuuAAuGccAAAuGcTT
3196

(2785C) stab26

2786
AGCAUUUGGCAUUAAGAAAUCAC
2389
37442
FLT1 :2804121 antisense siNA
GAUuucuuAAuGccAAAuGTT
3197

(2786C) stab26

2787
GCAUUUGGCAUUAAGAAAUCACC
2288
37443
FLT1 :2805L21 antisense siNA
UGAuuucuuAAuGccAAAuTT
3198

(2787C) stab26

2788
CAUUUGGCAUUAAGAAAUCACCU
2390
37444
FLT1:2806L21 antisense siNA
GUGAuuucuuAAuGccAAATT
3199

(2788C) stab26

2789
AUUUGGCAUUAAGAAAUCACCUA
2391
37445
FLT1:2807L21 antisense siNA
GGUGAuuucuuAAuGccAATT
3200

(2789C) stab26

2812
CGUGCCGGACUGUGGCUGUGAAA
2392
37446
FLT1:2830L21 antisense siNA
UCAcAGccAcAGuccGGcATT
3201

(2812C) stab26

2860
GCGAGUACAAAGCUCUGAUGACU
2393
37447
FLT1:2878L21 antisense siNA
UCAucAGAGcuuuGuAcucTT
3202

(2860C) stab26

2861
CGAGUACAAAGCUCUGAUGACUG
2394
37448
FLT1:2879L21 antisense siNA
GUCAucAGAGcuuuGuAcuTT
3203

(2861C) stab26

2947
CCAAGCAAGGAGGGCCUCUGAUG
2395
37449
FLT1:2965L21 antisense siNA
UCAGAGGcccuccuuGcuuTT
3204

(2947C) stab26

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
37450
FLT1:2967L21 antisense siNA
CAUcAGAGGcccuccuuGcTT
3205

(2949C) stab26

2950
AGCAAGGAGGGCCUCUGAUGGUG
2396
37451
FLT1:2968L21 antisense siNA
CCAucAGAGGcccuccuuGTT
3206

(2950C) stab26

2952
CAAGGAGGGCCUCUGAUGGUGAU
2397
37452
FLT1:2970L21 antisense siNA
CACcAucAGAGGcccuccuTT
3207

(2952C) stab26

2953
AAGGAGGGCCUCUGAUGGUGAUU
2398
37453
FLT1:2971L21 antisense siNA
UCAccAucAGAGGcccuccTT
3208

(2953C) stab26

2954
AGGAGGGCCUCUGAUGGUGAUUG
2399
37454
FLT1:2972L21 antisense siNA
AUCAccAucAGAGGcccucTT
3209

(2954C) stab26

3262
UUCAAGUGGCCAGAGGCAUGGAG
2400
37455
FLT1:3280L21 antisense siNA
CCAuGccucuGGccAcuuGTT
3210

(3262C) stab26

3263
UCAAGUGGCCAGAGGCAUGGAGU
2401
37456
FLT1:3281L21 antisense siNA
UCCAuGccucuGGccAcuuTT
3211

(3263C) stab26

3266
AGUGGCCAGAGGCAUGGAGUUCC
2402
37457
FLT1:3284121 antisense siNA
AACuccAuGccucuGGccATT
3212

(3266C) stab26

3911
GAGCCUGGAAAGAAUCAAAACCU
2403
37458
FLT1:3929121 antisense siNA
GUUuuGAuucuuuccAGGcTT
3213

(3911C) stab26

4419
UUUUUUGACUAACAAGAAUGUAA
2404
37459
FLT1:4437L21 antisense siNA
ACAuucuuGuuAGucAAAATT
3214

(4419C) stab26

3646
UCAUGCUGGACUGCUGGCACAGA
2195
37576
FLT1:3664121 antisense siNA
UGUGccAGcAGuccAGcAuTT
3215

(3646C) stab26

349
AACUGAGUUUAAAAGGCACCCAG
2289
38285
5′CB 31270 FLT1:349U21 sense
CBUGAGUUUAAAAGGCACCCTT B
3216

siNA stab09

VEGFR2

3304
UGACCUUGGAGCAUCUCAUCUGU
2405

KDR:3304U21 sense siNA stab04
B AccuuGGAGcAucucAucuTT B
3217

3894
UCACCUGUUUCCUGUAUGGAGGA
2406

KDR:3894U21 sense siNA stab04
B AccuGuuuccuGuAuGGAGTT B
3218

3304
UGACCUUGGAGCAUCUCAUCUGU
2405

KDR:3322L21 antisense siNA
AGAuGAGAuGcucoAAGGuTsT
3219

(3304C) stab05

3894
UCACCUGUUUCCUGUAUGGAGGA
2406

KDR:3912L21 antisense siNA
cuccAuAcAGGAAAcAGGuTsT
3220

(3894C) stab05

3304
UGACCUUGGAGCAUCUCAUCUGU
2405

KDR:3304U21 sense siNA stab07
B AccuuGGAGcAucucAucuTT B
3221

3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32766
KDR:3894U21 sense siNA stab07
B AccuGuuuccuGuAuGGAGTT B
3222

3304
UGACCUUGGAGCAUCUCAUCUGU
2405

KDR:3322L21 antisense siNA

AGAuGAGAuGcuccAAGGuTsT
3223

(3304C) stab11

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407

KDR:3872L21 antisense siNA

GAAuccucuuccAuGcucATsT
3224

(3854C) stab11

3894
UCACCUGUUUCCUGUAUGGAGGA
2406

KDR:3912L21 antisense siNA
cuccAuAcAGGAAAcAGGuTsT
3225

(3894C) stab11

3948
GACAACACAGCAGGAAUCAGUCA
2408

KDR:3966L21 antisense siNA

AcuGAuuccuGcuGuGuuGTsT
3226

(3948C) stab11

3076
UGUCCACUUACCUGAGGAGCAAG
2409
30785
KDR:3076U21 sense siNA stab04
B uccACuuAcCuGAGGAGCATT B
3227

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
30786
KDR:3854U21 sense siNA stab04
B uGAGcAuGGAAGAGGAuucTT B
3228

4089
AUGGUUCUUGCCUCAGAAGAGCU
2410
30787
KDR:4089U21 sense siNA stab04
B GGuucuuGcCuCAGAAGAGTT B
3229

4191
UCUGAAGGCUCAAACCAGACAAG
2411
30788
KDR:4191U21 sense siNA stab04
B uGAAGGCucAAAccAGAcATT B
3230

3076
UGUCCACUUACCUGAGGAGCAAG
2409
30789
KDR:3094L21 antisense siNA
uGcuccucAGGuAAGuGGATsT
3231

(3076C) stab05

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
30790
KDR:3872L21 antisense siNA
GAAuccucuuccAuGcucATsT
3232

(3854C) stab05

4089
AUGGUUCUUGCCUCAGAAGAGCU
2410
30791
KDR:4107L21 antisense siNA
cucuucuGAGGcAAGAAccTsT
3233

(4089C) stab05

4191
UCUGAAGGCUCAAACCAGACAAG
2411
30792
KDR:4209L21 antisense siNA
uGucuGGuuuGAGccuucATsT
3234

(4191C) stab05

3076
UGUCCACUUACCUGAGGAGCAAG
2409
31426
KDR:3076U21 sense siNA
UCCACUUACCUGAGGAGCATT
3235

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31435
KDR:3854U21 sense siNA
UGAGCAUGGAAGAGGAUUCTT
3236

4089
AUGGUUCUUGCCUCAGAAGAGCU
2410
31428
KDR:4089U21 sense siNA
GGUUCUUGCCUCAGAAGAGTT
3237

4191
UCUGAAGGCUCAAACCAGACAAG
2411
31429
KDR:4191U21 sense siNA
UGAAGGCUCAAACCAGACATT
3238

3076
UGUCCACUUACCUGAGGAGCAAG
2409
31430
KDR:3094L21 antisense siNA
UGCUCCUCAGGUAAGUGGATT
3239

(3076C)

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31439
KDR:3872L21 antisense siNA
GAAUCCUCUUCCAUGCUCATT
3240

(3854C)

4089
AUGGUUCUUGCCUCAGAAGAGCU
2410
31432
KDR:4107L21 antisense siNA
CUCUUCUGAGGCAAGAACCTT
3241

(4089C)

4191
UCUGAAGGCUCAAACCAGACAAG
2411
31433
KDR:4209L21 antisense siNA
UGUCUGGUUUGAGCCUUCATT
3242

(4191C)

3304
UGACCUUGGAGCAUCUCAUCUGU
2405
31434
KDR:3304U21 sense siNA
ACCUUGGAGCAUCUCAUCUTT
3243

3894
UCACCUGUUUCCUGUAUGGAGGA
2406
31436
KDR:3894U21 sense siNA
ACCUGUUUCCUGUAUGGAGTT
3244

3948
GACAACACAGCAGGAAUCAGUCA
2408
31437
KDR:3948U21 sense siNA
CAACACAGCAGGAAUCAGUTT
3245

3304
UGACCUUGGAGCAUCUCAUCUGU
2405
31438
KDR:3322L21 antisense siNA
AGAUGAGAUGCUCCAAGGUTT
3246

(3304C)

3894
UCACCUGUUUCCUGUAUGGAGGA
2406
31440
KDR:3912L21 antisense siNA
CUCCAUACAGGAAACAGGUTT
3247

(3894C)

3948
GACAACACAGCAGGAAUCAGUCA
2408
31441
KDR:3966L21 antisense siNA
ACUGAUUCCUGCUGUGUUGTT
3248

(3948C)

3948
GACAACACAGCAGGAAUCAGUCA
2408
31856
KDR:3948U21 sense siNA stab04
B cAAcAcAGcAGGAAucAGuTT B
3249

3948
GACAACACAGCAGGAAUCAGUCA
2408
31857
KDR:3966L21 antisense siNA
AcuGAuuccuGcuGuGuuGTsT
3250

(3948C) stab05

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31858
KDR:3854U21 sense siNA stab07
B uGAGcAuGGAAGAGGAuucTT B
3251

3948
GACAACACAGCAGGAAUCAGUCA
2408
31859
KDR:3948U21 sense siNA stab07
B cAAAGcAGGAAucAGuTT B
3252

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31860
KDR:3872L21 antisense siNA

GAAuccucuuccAuGcucATsT
3253

(3854C) stab08

3948
GACAACACAGCAGGAAUCAGUCA
2408
31861
KDR:3966L21 antisense siNA

AcuGAuuccuGcuGuGuuGTsT
3254

(3948C) stab08

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31862
KDR:3854U21 sense siNA stab09
B UGAGCAUGGAAGAGGAUUCTT B
3255

3948
GACAACACAGCAGGAAUCAGUCA
2408
31863
KDR:3948U21 sense siNA stab09
B CAACACAGCAGGAAUCAGUTT B
3256

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31864
KDR:3872L21 antisense siNA
GAAUCCUCUUCCAUGCUCATsT
3257

(3854C) stab10

3948
GACAACACAGCAGGAAUCAGUCA
2408
31865
KDR:3966L21 antisense siNA
ACUGAUUCCUGCUGUGUUGTsT
3258

(3948C) stab10

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31878
KDR:3854U21 sense siNA inv
B cuuAGGAGAAGGuAcGAGuTT B
3259

stab04

3948
GACAACACAGCAGGAAUCAGUCA
2408
31879
KDR:3948U21 sense siNA inv
B uGAcuAAGGAcGAcAcAAcTT B
3260

stab04

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31880
KDR:3872L21 antisense siNA
AcucGuAccuucuccuAAGTsT
3261

(3854C) inv stab05

3948
GACAACACAGCAGGAAUCAGUCA
2408
31881
KDR:3966L21 antisense siNA
GuuGuGucGuccuuAGucATsT
3262

(3948C) inv stab05

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31882
KDR:3854U21 sense siNA inv
B cuuAGGAGAAGGuAcGAGuTT B
3263

stab07

3948
GACAACACAGCAGGAAUCAGUCA
2408
31883
KDR:3948U21 sense siNA inv
B uGAcuAAGGAcGAcAcAAcTT B
3264

stab07

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31884
KDR:3872L21 antisense siNA

AcucGuAccuucuccuAAGTsT
3265

(3854C) inv stab08

3948
GACAACACAGCAGGAAUCAGUCA
2408
31885
KDR:3966L21 antisense siNA

GuuGuGucGuccuuAGucATsT
3266

(3948C) inv stab08

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31886
KDR:3854U21 sense siNA inv
B CUUAGGAGAAGGUACGAGUTT B
3267

stab09

3948
GACAACACAGCAGGAAUCAGUCA
2408
31887
KDR:3948U21 sense siNA inv
B UGACUAAGGACGACACAACTT B
3268

stab09

3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31888
KDR:3872L21 antisense siNA

(3854C) inv stab10
ACUCGUACCUUCUCCUAAGTsT
3269

3948
GACAACACAGCAGGAAUCAGUCA
2408
31889
KDR:3966L21 antisense siNA
GUUGUGUCGUCCUUAGUCATsT
3270

(3948C) inv stab10

2764
CCUUAUGAUGCCAGCAAAU
2412
32238
KDR:2764U21 sense siNA
CCUUAUGAUGCCAGCAAAUTT
3271

2765
CUUAUGAUGCCAGCAAAUG
2413
32239
KDR:2765U21 sense siNA
CUUAUGAUGCCAGCAAAUGTT
3272

2766
UUAUGAUGCCAGCAAAUGG
2414
32240
KDR:2766U21 sense siNA
UUAUGAUGCCAGCAAAUGGTT
3273

2767
UAUGAUGCCAGCAAAUGGG
2415
32241
KDR:2767U21 sense siNA
UAUGAUGCCAGCAAAUGGGTT
3274

2768
AUGAUGCCAGCAAAUGGGA
2416
32242
KDR:2768U21 sense siNA
AUGAUGCCAGCAAAUGGGATT
3275

3712
CAGACCAUGCUGGACUGCU
2417
32243
KDR:3712U21 sense siNA
CAGACCAUGCUGGACUGCUTT
3276

3713
AGACCAUGCUGGACUGCUG
2418
32244
KDR:3713U21 sense siNA
AGACCAUGCUGGACUGCUGTT
3277

3714
GACCAUGCUGGACUGCUGG
2419
32245
KDR:3714U21 sense siNA
GACCAUGCUGGACUGCUGGTT
3278

3715
ACCAUGCUGGACUGCUGGC
2420
32246
KDR:3715U21 sense siNA
ACCAUGCUGGACUGCUGGCTT
3279

3716
CCAUGCUGGACUGCUGGCA
2421
32247
KDR:3716U21 sense siNA
CCAUGCUGGACUGCUGGCATT
3280

3811
CAGGAUGGCAAAGACUACA
2422
32248
KDR:3811U21 sense siNA
CAGGAUGGCAAAGACUACATT
3281

3812
AGGAUGGCAAAGACUACAU
2423
32249
KDR:3812U21 sense siNA
AGGAUGGCAAAGACUACAUTT
3282

2764
CCUUAUGAUGCCAGCAAAU
2412
32253
KDR:2782L21 antisense siNA
AUUUGCUGGCAUCAUAAGGTT
3283

(2764C)

2765
CUUAUGAUGCCAGCAAAUG
2413
32254
KDR:2783L21 antisense siNA
CAUUUGCUGGCAUCAUAAGTT
3284

(2765C)

2766
UUAUGAUGCCAGCAAAUGG
2414
32255
KDR:2784L21 antisense siNA
CCAUUUGCUGGCAUCAUAATT
3285

(2766C)

2767
UAUGAUGCCAGCAAAUGGG
2415
32256
KDR:2785L21 antisense siNA
CCCAUUUGCUGGCAUCAUATT
3286

(2767C)

2768
AUGAUGCCAGCAAAUGGGA
2416
32257
KDR:2786L21 antisense siNA
UCCCAUUUGCUGGCAUCAUTT
3287

(2768C)

3712
CAGACCAUGCUGGACUGCU
2417
32258
KDR:3730L21 antisense siNA
AGCAGUCCAGCAUGGUCUGTT
3288

(3712C)

3713
AGACCAUGCUGGACUGCUG
2418
32259
KDR:3731L21 antisense siNA
CAGCAGUCCAGCAUGGUCUTT
3289

(3713C)

3714
GACCAUGCUGGACUGCUGG
2419
32260
KDR:3732L21 antisense siNA
CCAGCAGUCCAGCAUGGUCTT
3290

(3714C)

3715
ACCAUGCUGGACUGCUGGC
2420
32261
KDR:3733L21 antisense siNA
GCCAGCAGUCCAGCAUGGUTT
3291

(3715C)

3716
CCAUGCUGGACUGCUGGCA
2421
32262
KDR:3734L21 antisense siNA
UGCCAGCAGUCCAGCAUGGTT
3292

(3716C)

3811
CAGGAUGGCAAAGACUACA
2422
32263
KDR:3829L21 antisense siNA
UGUAGUCUUUGCCAUCCUGTT
3293

(3811C)

3812
AGGAUGGCAAAGACUACAU
2423
32264
KDR:3830L21 antisense siNA
AUGUAGUCUUUGCCAUCCUTT
3294

(3812C)

3304
UGACCUUGGAGCAUCUCAUCUGU
2405
32310
KDR:3304U21 sense siNA
B ACCUUGGAGCAUCUCAUCUTT B
3295

stab09

3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32311
KDR:3894U21 sense siNA
B ACCUGUUUCCUGUAUGGAGTT B
3296

stab09

3304
UGACCUUGGAGCAUCUCAUCUGU
2405
32312
KDR:3322L21 antisense
AGAUGAGAUGCUCCAAGGUTST
3297

siNA (3304C) stab10

3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32313
KDR:3912L21 antisense siNA
CUCCAUACAGGAAACAGGUTsT
3298

(3894C) stab10

3304
UGACCUUGGAGCAUCUCAUCUGU
2405
32314
KDR:3304U21 sense siNA inv
B UCUACUCUACGAGGUUCCATT B
3299

stab09

3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32315
KDR:3894U21 sense siNA inv
B GAGGUAUGUCCUUUGUCCATT B
3300

stab09

3304
UGACCUUGGAGCAUCUCAUCUGU
2405
32316
KDR:3322L21 antisense siNA
UGGAACCUCGUAGAGUAGATsT
3301

(3304C) inv stab10

3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32317
KDR:3912L21 antisense siNA
UGGACAAAGGACAUACCUCTsT
3302

(3894C) inv stab10

828
AACAGAAUUUCCUGGGACAGCAA
2424
32762
KDR:828U21 sense siNA stab07
B cAGAAuuuccuGGGAcAGcTT B
3303

3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32763
KDR:3310U21 sense siNA stab07
B GAGcAucucAucuGuuAcATT B
3304

3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32764
KDR:3758U21 sense siNA stab07
B cGuuuucAGAGuuGGuGGATT B
3305

3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32765
KDR:3893U21 sense siNA stab07
B cAccuGuuuccuGuAuGGATT B
3306

828
AACAGAAUUUCCUGGGACAGCAA
2424
32767
KDR:846L21 antisense siNA

GcuGucccAGGAAAuucuGTsT
3307

(828C) stab08

3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32768
KDR:3328L21 antisense siNA
uGuAAcAGAuGAGAuGcucTsT
3308

(3310C) stab08

3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32769
KDR:3776L21 antisense siNA
uccAccAAcucuGAAAAcGTsT
3309

(3758C) stab08

3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32770
KDR:3911L21 antisense siNA
uccAuAcAGGAAAcAGGuGTsT
3310

(3893C) stab08

3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32771
KDR:3912L21 antisense siNA
cuccAuAcAGGAAAcAGGuTsT
3311

(3894C) stab08

828
AACAGAAUUUCCUGGGACAGCAA
2424
32786
KDR:828U21 sense siNA inv
B cGAcAGGGuccuuuAAGAcTT B
3312

stab07

3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32787
KDR:3310U21 sense siNA inv
B AcAuuGucuAcucuAcGAGTT B
3313

stab07

3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32788
KDR:3758U21 sense siNA inv
B AGGuGGuuGAGAcuuuuGcTT B
3314

stab07

3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32789
KDR:3893U21 sense siNA inv
B AGGuAuGuccuuuGuccAcTT B
3315

stab07

3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32790
KDR:3894U21 sense siNA inv
B GAGGuAuGuccuuuGuccATT B
3316

stab07

828
AACAGAAUUUCCUGGGACAGCAA
2424
32791
KDR:846L21 antisense siNA

GucuuAAAGGAcccuGucGTsT
3317

(828C) inv stab08

3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32792
KDR:3328L21 antisense siNA
cucGuAGAGuAGAcAAuGuTsT
3318

(3310C) inv stab08

3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32793
KDR:3776L21 antisense siNA

GcAAAAGucucAAccAccuTsT
3319

(3758C) inv stab08

3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32794
KDR:3911L21 antisense siNA

GuGGAcAAAGGAcAuAccuTsT
3320

(3893C) inv stab08

3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32795
KDR:3912L21 antisense siNA
uGGAcAAAGGAcAuAccucTsT
3321

(3894C) inv stab08

828
AACAGAAUUUCCUGGGACAGCAA
2424
32958
KDR:828U21 sense siNA stab09
B CAGAAUUUCCUGGGACAGCTT B
3322

3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32959
KDR:3310U21 sense siNA stab09
B GAGCAUCUCAUCUGUUACATT B
3323

3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32960
KDR:3758U21 sense siNA stab09
B CGUUUUCAGAGUUGGUGGATT B
3324

3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32961
KDR:3893U21 sense siNA stab09
B CACCUGUUUCCUGUAUGGATT B
3325

828
AACAGAAUUUCCUGGGACAGCAA
2424
32963
KDR:846L21 antisense siNA
GCUGUCCCAGGAAAUUCUGTsT
3326

(828C) stab10

3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32964
KDR:3328L21 antisense siNA
UGUAACAGAUGAGAUGCUCTsT
3327

(3310C) stab10

3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32965
KDR:3776L21 antisense siNA
UCCACCAACUCUGAAAACGTsT
3328

(3758C) stab10

3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32966
KDR:3911L21 antisense siNA
UCCAUACAGGAAACAGGUGTsT
3329

(3893C) stab10

828
AACAGAAUUUCCUGGGACAGCAA
2424
32988
KDR:828U21 sense siNA inv
B CGACAGGGUCCUUUAAGACTT B
3330

stab09

3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32989
KDR:3310U21 sense siNA inv
B ACAUUGUCUACUCUACGAGTT B
3331

stab09

3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32990
KDR:3758U21 sense siNA inv
B AGGUGGUUGAGACUUUUGCTT B
3332

stab09

3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32991
KDR:3893U21 sense siNA inv
B AGGUAUGUCCUUUGUCCACTT B
3333

stab09

828
AACAGAAUUUCCUGGGACAGCAA
2424
32993
KDR:846L21 antisense siNA
GUCUUAAAGGACCCUGUCGTsT
3334

(828C) inv stab10

3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32994
KDR:3328L21 antisense siNA
CUCGUAGAGUAGACAAUGUTsT
3335

(3310C) inv stab10

3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32995
KDR:3776L21 antisense siNA
GCAAAAGUCUCAACCACCUTsT
3336

(3758C) inv stab10

3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32996
KDR:3911L21 antisense siNA
GUGGACAAAGGACAUACCUTsT
3337

(3893C) inv stab10

2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33727
KDR:2767U21 sense siNA stab07
B uAuGAuGccAGcAAAuGGGTT B
3338

2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33728
KDR:2768U21 sense siNA stab07
B AuGAuGccAGcAAAuGGGATT B
3339

3715
AGACCAUGCUGGACUGCUGGCAC
2241
33729
KDR:3715U21 sense siNA stab07
B AccAuGcuGGAcuGcuGGcTT B
3340

3716
GACCAUGCUGGACUGCUGGCACG
2247
33730
KDR:3716U21 sense siNA stab07
B ccAuGcuGGAcuGcuGGcATT B
3341

2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33733
KDR:2785L21 antisense siNA
cccAuuuGcuGGcAucAuATsT
3342

(2767C) stab08

2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33734
KDR:2786L21 antisense siNA
ucccAuuuGcuGGcAucAuTsT
3343

(2768C) stab08

3715
AGACCAUGCUGGACUGCUGGCAC
2241
33735
KDR3733L21 antisense siNA

GccAGcAGuccAGcAuGGuTsT
3344

(3715C) stab08

3716
GACCAUGCUGGACUGCUGGCACG
2247
33736
KDR:3734L21 antisense siNA
uGccAGcAGuccAGcAuGGTsT
3345

(3716C) stab08

2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33739
KDR:2767U21 sense siNA
B UAUGAUGCCAGCAAAUGGGTT B
3346

stab09

2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33740
KDR:2768U21 sense siNA
B AUGAUGCCAGCAAAUGGGATT B
3347

stab09

3715
AGACCAUGCUGGACUGCUGGCAC
2241
33741
KDR:3715U21 sense siNA
B ACCAUGCUGGACUGCUGGCTT B
3348

stab09

3716
GACCAUGCUGGACUGCUGGCACG
2247
33742
KDR:3716U21 sense siNA
B CCAUGCUGGACUGCUGGCATT B
3349

stab09

2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33745
KDR:2785L21 antisense siNA
CCCAUUUGCUGGCAUCAUATsT
3350

(2767C) stab10

2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33746
KDR:2786L21 antisense siNA
UCCCAUUUGCUGGCAUCAUTsT
3351

(2768C) stab10

3715
AGACCAUGCUGGACUGCUGGCAC
2241
33747
KDR:3733L21 antisense siNA
GCCAGCAGUCCAGCAUGGUTsT
3352

(3715C) stab10

3716
GACCAUGCUGGACUGCUGGCACG
2247
33748
KDR:3734L21 antisense siNA
UGCCAGCAGUCCAGCAUGGTsT
3353

(3716C) stab10

2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33751
KDR:2767U21 sense siNA inv
B GGGuAAAcGAccGuAGuAuTT B
3354

stab07

2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33752
KDR:2768U21 sense siNA inv
B AGGGuAAAcGAccGuAGuATT B
3355

stab07

3715
AGACCAUGCUGGACUGCUGGCAC
2241
33753
KDR:3715U21 sense siNA inv
B cGGucGucAGGucGuAccATT B
3356

stab07

3716
GACCAUGCUGGACUGCUGGCACG
2247
33754
KDR:3716U21 sense siNA inv
B AcGGucGucAGGucGuAccTT B
3357

stab07

2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33757
KDR:2785L21 antisense siNA

AuAcuAcGGucGuuuAcccTsT
3358

(2767C) inv stab08

2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33758
KDR:2786L21 antisense siNA
uAcuAcGGucGuuuAcccuTsT
3359

(2768C) inv stab08

3715
AGACCAUGCUGGACUGCUGGCAC
2241
33759
KDR:3733L21 antisense siNA
uGGuAcGAccuGAcGAccGTsT
3360

(3715C) inv stab08

3716
GACCAUGCUGGACUGCUGGCACG
2247
33760
KDR:3734L21 antisense siNA

GGuAcGAccuGAcGAccGuTsT
3361

(3716C) inv stab08

2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33763
KDR:2767U21 sense siNA inv
B GGGUAAACGACCGUAGUAUTT B
3362

stab09

2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33764
KDR:2768U21 sense siNA inv
B AGGGUAAACGACCGUAGUATT B
3363

stab09

3715
AGACCAUGCUGGACUGCUGGCAC
2241
33765
KDR:3715U21 sense siNA inv
B CGGUCGUCAGGUCGUACCATT B
3364

stab09

3716
GACCAUGCUGGACUGCUGGCACG
2247
33766
KDR:3716U21 sense siNA inv
B ACGGUCGUCAGGUCGUACCTT B
3365

stab09

2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33769
KDR:2785L21 antisense siNA
AUACUACGGUCGUUUACCCTsT
3366

(2767C) inv stab10

2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33770
KDR:2786L21 antisense siNA
UACUACGGUCGUUUACCCUTsT
3367

(2768C) inv stab10

3715
AGACCAUGCUGGACUGCUGGCAC
2241
33771
KDR3733L21 antisense siNA
UGGUACGACCUGACGACCGTsT
3368

(3715C) inv stab10

3716
GACCAUGCUGGACUGCUGGCACG
2247
33772
KDR:3734L21 antisense siNA
GGUACGACCUGACGACCGUTsT
3369

(3716C) inv stab10

3715
AGACCAUGCUGGACUGCUGGCAC
2241
34502
KDR:3733L21 antisense siNA

GccAGcAGuccAGcAuGGuTT B
3370

(3715C) stab19

3715
AGACCAUGCUGGACUGCUGGCAC
2241
34503
KDR:3733L21 antisense siNA

GccAGcAGuccAGcAuGGTT
3371

(3715C) stab08 Blunt

3715
AGACCAUGCUGGACUGCUGGCAC
2241
34504
KDR:3733L21 antisense siNA
uGGuAcGAccuGAcGAccGTT B
3372

(3715C) inv stab19

3715
AGACCAUGCUGGACUGCUGGCAC
2241
34505
KDR:3733L21 antisense siNA
uGGuAcGAccuGAcGAccG
3373

(3715C) inv stab08 Blunt

503
UCAGAGUGGCAGUGAGCAAAGGG
2428
34680
KDR:503U21 sense siNA stab00
AGAGUGGCAGUGAGCAAAGTT
3374

503
UCAGAGUGGCAGUGAGCAAAGGG
2428
34688
KDR:521L21 (503C) siRNA
CUUUGCUCACUGCCACUCUTT
3375

stab00

3715
AGACCAUGCUGGACUGCUGGCAC
2241
35124
KDR:3715U21 sense siNA stab04
B AccAuGcuGGAcuGcuGGcTT B
3376

3715
AGACCAUGCUGGACUGCUGGCAC
2241
35125
KDR:3715U21 sense siNA stab07
B AccAuGcuGGAcuGCUGGCTT B
3377

N1

3715
AGACCAUGCUGGACUGCUGGCAC
2241
35126
KDR:3733L21 antisense siNA

GCCAGCAGuccAGcAuGGuTsT
3378

(3715C) stab08 N1

3715
AGACCAUGCUGGACUGCUGGCAC
2241
35127
KDR:3733L21 antisense siNA

GCCAGcAGuccAGcAuGGuTsT
3379

(3715C) stab08 N2

3715
AGACCAUGCUGGACUGCUGGCAC
2241
35128
KDR:3733L21 antisense siNA

GCCAGcAGuccAGcAuGGuTsT
3380

(3715C) stab08 N3

3715
AGACCAUGCUGGACUGCUGGCAC
2241
35129
KDR:3733L21 antisense siNA

GCCAGcAGuccAGcAuGGuTsT
3381

(3715C) stab25

3715
AGACCAUGCUGGACUGCUGGCAC
2241
35130
KDR:3733L21 antisense siNA

GCcAGcAGuccAGcAuGGuTsT
3382

(3715C) stab08 N5

3715
AGACCAUGCUGGACUGCUGGCAC
2241
35131
KDR:3733L21 antisense siNA
GccAGcAGuccAGcAuGGuTsT
3383

(3715C) stab24

83
CCGCAGAAAGUCCGUCUGGCAGC
2429
36280
KDR:83U21 sense siNA stab00
GCAGAAAGUCCGUCUGGCATT
3384

84
CGCAGAAAGUCCGUCUGGCAGCC
2430
36281
KDR:84U21 sense siNA stab00
CAGAAAGUCCGUCUGGCAGTT
3385

85
GCAGAAAGUCCGUCUGGCAGCCU
2431
36282
KDR:85U21 sense siNA stab00
AGAAAGUCCGUCUGGCAGCTT
3386

99
UGGCAGCCUGGAUAUCCUCUCCU
2432
36283
KDR:99U21 sense siNA stab00
GCAGCCUGGAUAUCCUCUCTT
3387

100
GGCAGCCUGGAUAUCCUCUCCUA
2433
36284
KDR:100U21 sense siNA stab00
CAGCCUGGAUAUCCUCUCCTT
3388

161
CCCGGGCUCCCUAGCCCUGUGCG
2434
36285
KDR:161U21 sense siNA stab00
CGGGCUCCCUAGCCCUGUGTT
3389

162
CCGGGCUCCCUAGCCCUGUGCGC
2435
36286
KDR:162U21 sense siNA stab00
GGGCUCCCUAGCCCUGUGCTT
3390

229
CCUCCUUCUCUAGACAGGCGCUG
2436
36287
KDR:229U21 sense siNA stab00
UCCUUCUCUAGACAGGCGCTT
3391

230
CUCCUUCUCUAGACAGGCGCUGG
2437
36288
KDR:230U21 sense siNA stab00
CCUUCUCUAGACAGGCGCUTT
3392

231
UCCUUCUCUAGACAGGCGCUGGG
2438
36289
KDR:231U21 sense siNA stab00
CUUCUCUAGACAGGCGCUGTT
3393

522
AGGGUGGAGGUGACUGAGUGCAG
2439
36290
KDR:522U21 sense siNA stab00
GGUGGAGGUGACUGAGUGCTT
3394

523
GGGUGGAGGUGACUGAGUGCAGC
2440
36291
KDR:523U21 sense siNA stab00
GUGGAGGUGACUGAGUGCATT
3395

888
GCUGGCAUGGUCUUCUGUGAAGC
2441
36292
KDR:888U21 sense siNA stab00
UGGCAUGGUCUUCUGUGAATT
3396

889
CUGGCAUGGUCUUCUGUGAAGCA
2442
36293
KDR:889U21 sense siNA stab00
GGCAUGGUCUUCUGUGAAGTT
3397

905
UGAAGCAAAAAUUAAUGAUGAAA
2443
36294
KDR:905U21 sense siNA stab00
AAGCAAAAAUUAAUGAUGATT
3398

906
GAAGCAAAAAUUAAUGAUGAAAG
2444
36295
KDR:906U21 sense siNA stab00
AGCAAAAAUUAAUGAUGAATT
3399

1249
CCAAGAAGAACAGCACAUUUGUC
2445
36296
KDR:1249U21 sense siNA stab00
AAGAAGAACAGCACAUUUGTT
3400

1260
AGCACAUUUGUCAGGGUCCAUGA
2446
36297
KDR:1260U21 sense siNA stab00
CACAUUUGUCAGGGUCCAUTT
3401

1305
AGUGGCAUGGAAUCUCUGGUGGA
2447
36298
KDR:1305U21 sense siNA stab00
UGGCAUGGAAUCUCUGGUGTT
3402

1315
AAUCUCUGGUGGAAGCCACGGUG
2448
36299
KDR:1315U21 sense siNA stab00
UCUCUGGUGGAAGCCACGGTT
3403

1541
GGUCUCUCUGGUUGUGUAUGUCC
2449
36300
KDR:1541U21 sense siNA stab00
UCUCUCUGGUUGUGUAUGLTT
3404

1542
GUCUCUCUGGUUGUGUAUGUCCC
2450
36301
KDR:1542U21 sense siNA stab00
CUCUCUGGUUGUGUAUGUCTT
3405

1588
UAAUCUCUCCUGUGGAUUCCUAC
2451
36302
KDR:1588U21 sense siNA stab00
AUCUCUCCUGUGGAUUCCUTT
3406

1589
AAUCUCUCCUGUGGAUUCCUACC
2452
36303
KDR:1589U21 sense siNA stab00
UCUCUCCUGUGGAUUCCUATT
3407

1875
GUGUCAGCUUUGUACAAAUGUGA
2453
36304
KDR:1875U21 sense siNA stab00
GUCAGCUUUGUACAAAUGUTT
3408

2874
GACAAGACAGCAACUUGCAGGAC
2454
36305
KDR:2874U21 sense siNA stab00
CAAGACAGCAACUUGCAGGTT
3409

2875
ACAAGACAGCAACUUGCAGGACA
2455
36306
KDR:2875U21 sense siNA stab00
AAGACAGCAACUUGCAGGATT
3410

2876
CAAGACAGCAACUUGCAGGACAG
2456
36307
KDR:2876U21 sense siNA stab00
AGACAGCAACUUGCAGGACTT
3411

3039
CUCAUGGUGAUUGUGGAAUUCUG
2457
36308
KDR:3039U21 sense siNA stab00
CAUGGUGAUUGUGGAAUUCTT
3412

3040
UCAUGGUGAUUGUGGAAUUCUGC
2458
36309
KDR:3040U21 sense siNA stab00
AUGGUGAUUGUGGAAUUCUTT
3413

3249
UCCCUCAGUGAUGUAGAAGAAGA
2459
36310
KDR:3249U21 sense siNA stab00
CCUCAGUGAUGUAGAAGAATT
3414

3263
AGAAGAAGAGGAAGCUCCUGAAG
2460
36311
KDR:3263U21 sense siNA stab00
AAGAAGAGGAAGCUCCUGATT
3415

3264
GAAGAAGAGGAAGCUCCUGAAGA
2461
36312
KDR:3264U21 sense siNA stab00
AGAAGAGGAAGCUCCUGAATT
3416

3269
AGAGGAAGCUCCUGAAGAUCUGU
2462
36313
KDR:3269U21 sense siNA stab00
AGGAAGCUCCUGAAGAUCUTT
3417

3270
GAGGAAGCUCCUGAAGAUCUGUA
2463
36314
KDR:3270U21 sense siNA stab00
GGAAGCUCCUGAAGAUCUGTT
3418

3346
AGGGCAUGGAGUUCUUGGCAUCG
2464
36315
KDR:3346U21 sense siNA stab00
GGCAUGGAGUUCUUGGCAUTT
3419

3585
UUGCUGUGGGAAAUAUUUUCCUU
2465
36316
KDR:3585U21 sense siNA stab00
GCUGUGGGAAAUAUUUUCCTT
3420

3586
UGCUGUGGGAAAUAUUUUCCUUA
2466
36317
KDR:3586U21 sense siNA stab00
CUGUGGGAAAUAUUUUCCUTT
3421

3860
CAUGGAAGAGGAUUCUGGACUCU
2467
36318
KDR:3860U21 sense siNA stab00
UGGAAGAGGAUUCUGGACUTT
3422

3877
GACUCUCUCUGCCUACCUCACCU
2468
36319
KDR:3877U21 sense siNA stab00
CUCUCUCUGCCUACCUCACTT
3423

3878
ACUCUCUCUGCCUACCUCACCUG
2469
36320
KDR:3878U21 sense siNA stab00
UCUCUCUGCCUACCUCACCTT
3424

4287
AAGCUGAUAGAGAUUGGAGUGCA
2470
36321
KDR:4287U21 sense siNA stab00
GCUGAUAGAGAUUGGAGUGTT
3425

4288
AGCUGAUAGAGAUUGGAGUGCAA
2471
36322
KDR:4288U21 sense siNA stab00
CUGAUAGAGAUUGGAGUGCTT
3426

4318
GCACAGCCCAGAUUCUCCAGCCU
2472
36323
KDR:4318U21 sense siNA stab00
ACAGCCCAGAUUCUCCAGCTT
3427

4319
CACAGCCCAGAUUCUCCAGCCUG
2473
36324
KDR:4319U21 sense siNA stab00
CAGCCCAGAUUCUCCAGCCTT
3428

4320
ACAGCCCAGAUUCUCCAGCCUGA
2474
36325
KDR:4320U21 sense siNA stab00
AGCCCAGAUUCUCCAGCCUTT
3429

4321
CAGCCCAGAUUCUCCAGCCUGAC
2475
36326
KDR:4321U21 sense siNA stab00
GCCCAGAUUCUCCAGCCUGTT
3430

4359
AGCUCUCCUCCUGUUUAAAAGGA
2476
36327
KDR:4359U21 sense siNA stab00
CUCUCCUCCUGUUUAAAAGTT
3431

4534
UAUCCUGGAAGAGGCUUGUGACC
2477
36328
KDR:4534U21 sense siNA stab00
UCCUGGAAGAGGCUUGUGATT
3432

4535
AUCCUGGAAGAGGCUUGUGACCC
2478
36329
KDR:4535U21 sense siNA stab00
CCUGGAAGAGGCUUGUGACTT
3433

4536
UCCUGGAAGAGGCUUGUGACCCA
2479
36330
KDR:4536U21 sense siNA stab00
CUGGAAGAGGCUUGUGACCTT
3434

4539
UGGAAGAGGCUUGUGACCCAAGA
2480
36331
KDR:4539U21 sense siNA stab00
GAAGAGGCUUGUGACCCAATT
3435

4769
UGUUGAAGAUGGGAAGGAUUUGC
2481
36332
KDR:4769U21 sense siNA stab00
UUGAAGAUGGGAAGGAUUUTT
3436

4934
UCUGGUGGAGGUGGGCAUGGGGU
2482
36333
KDR:4934U21 sense siNA stab00
UGGUGGAGGUGGGCAUGGGTT
3437

5038
UCGUUGUGCUGUUUCUGACUCCU
2483
36334
KDR:5038U21 sense siNA stab00
GUUGUGCUGUUUCUGACUCTT
3438

5039
CGUUGUGCUGUUUCUGACUCCUA
2484
36335
KDR:5039U21 sense siNA stab00
UUGUGCUGUUUCUGACUCCTT
3439

5040
GUUGUGCUGUUUCUGACUCCUAA
2485
36336
KDR:5040U21 sense siNA stabOG
UGUGCUGUUUCUGACUCCUTT
3440

5331
UCAAAGUUUCAGGAAGGAUUUUA
2486
36337
KDR:5331U21 sense siNA stab00
AAAGUUUCAGGAAGGAUUUTT
3441

5332
CAAAGUUUCAGGAAGGAUUUUAC
2487
36338
KDR:5332U21 sense siNA stab00
AAGUUUCAGGAAGGAUUUUTT
3442

5333
AAAGUUUCAGGAAGGAUUUUACC
2488
36339
KDR:5333U21 sense siNA stab00
AGUUUCAGGAAGGAUUUUATT
3443

5587
UCAAAAAAGAAAAUGUGUUUUUU
2489
36340
KDR:5587U21 sense siNA stab00
AAAAAAGAAAAUGUGUUUUTT
3444

5737
CUAUUCACAUUUUGUAUCAGUAU
2490
36341
KDR:5737U21 sense siNA stab00
AUUCACAUUUUGUAUCAGUTT
3445

5738
UAUUCACAUUUUGUAUCAGUAUU
2491
36342
KDR:5738U21 sense siNA stab00
UUCACAUUUUGUAUCAGUATT
3446

5739
AUUCACAUUUUGUAUCAGUAUUA
2492
36343
KDR:5739U21 sense siNA stab00
UCACAUUUUGUAUCAGUAUTT
3447

83
CCGCAGAAAGUCCGUCUGGCAGC
2429
36344
KDR:101L21 antisense siNA
UGCCAGACGGACUUUCUGCTT
3448

(83C) stab00

84
CGCAGAAAGUCCGUCUGGCAGCC
2430
36345
KDR:102L21 antisense siNA
CUGCCAGACGGACUUUCUGTT
3449

(84C) stab00

85
GCAGAAAGUCCGUCUGGCAGCCU
2431
36346
KDR:103L21 antisense siNA
GCUGCCAGACGGACUUUCUTT
3450

(85C) stab00

99
UGGCAGCCUGGAUAUCCUCUCCU
2432
36347
KDR:117L21 antisense siNA
GAGAGGAUAUCCAGGCUGCTT
3451

(99C) stab00

100
GGCAGCCUGGAUAUCCUCUCCUA
2433
36348
KDR:118L21 antisense siNA
GGAGAGGAUAUCCAGGCUGTT
3452

(100C) stab00

161
CCCGGGCUCCCUAGCCCUGUGCG
2434
36349
KDR:179L21 antisense siNA
CACAGGGCUAGGGAGCCCGTT
3453

(161C) stab00

162
CCGGGCUCCCUAGCCCUGUGCGC
2435
36350
KDR:180L21 antisense siNA
GCACAGGGCUAGGGAGCCCTT
3454

(162C) stab00

229
CCUCCUUCUCUAGACAGGCGCUG
2436
36351
KDR:247L21 antisense siNA
GCGCCUGUCUAGAGAAGGATT
3455

(229C) stab00

230
CUCCUUCUCUAGACAGGCGCUGG
2437
36352
KDR:248L21 antisense siNA
AGCGCCUGUCUAGAGAAGGTT
3456

(230C) stab00

231
UCCUUCUCUAGACAGGCGCUGGG
2438
36353
KDR:249L21 antisense siNA
CAGCGCCUGUCUAGAGAAGTT
3457

(231C) stab00

522
AGGGUGGAGGUGACUGAGUGCAG
2439
36354
KDR:540L21 antisense siNA
GCACUCAGUCACCUCCACCTT
3458

(522C) stab00

523
GGGUGGAGGUGACUGAGUGCAGC
2440
36355
KDR:541L21 antisense siNA
UGCACUCAGUCACCUCCACTT
3459

(523C) stab00

888
GCUGGCAUGGUCUUCUGUGAAGC
2441
36356
KDR:906L21 antisense siNA
UUCACAGAAGACCAUGCCATT
3460

(888C) stab00

889
CUGGCAUGGUCUUCUGUGAAGCA
2442
36357
KDR:907L21 antisense siNA
CUUCACAGAAGACCAUGCCTT
3461

(889C) stab00

905
UGAAGCAAAAAUUAAUGAUGAAA
2443
36358
KDR:923L21 antisense siNA
UCAUCAUUAAUUUUUGCUUTT
3462

(905C) stab00

906
GAAGCAAAAAUUAAUGAUGAAAG
2444
36359
KDR:924L21 antisense siNA
UUCAUCAUUAAUUUUUGCUTT
3463

(906C) stab00

1249
CCAAGAAGAACAGCACAUUUGUC
2445
36360
KDR:1267121 antisense siNA
CAAAUGUGCUGUUCUUCUUTT
3464

(1249C) stab00

1260
AGCACAUUUGUCAGGGUCCAUGA
2446
36361
KDR:1278L21 antisense siNA
AUGGACCCUGACAAAUGUGTT
3465

(1260C) stab00

1305
AGUGGCAUGGAAUCUCUGGUGGA
2447
36362
KDR:1323L21 antisense siNA
CACCAGAGAUUCCAUGCCATT
3466

(1305C) stab00

1315
AAUCUCUGGUGGAAGCCACGGUG
2448
36363
KDR:1333L21 antisense siNA
CCGUGGCUUCCACCAGAGATT
3467

(1315C) stab00

1541
GGUCUCUCUGGUUGUGUAUGUCC
2449
36364
KDR:1559L21 antisense siNA
ACAUACACAACCAGAGAGATT
3468

(1541C) stab00

1542
GUCUCUCUGGUUGUGUAUGUCCC
2450
36365
KDR:1560L21 antisense siNA
GACAUACACAACCAGAGAGTT
3469

(1542C) stab00

1588
UAAUCUCUCCUGUGGAUUCCUAC
2451
36366
KDR:1606L21 antisense siNA
AGGAAUCCACAGGAGAGAUTT
3470

(1588C) stab00

1589
AAUCUCUCCUGUGGAUUCCUACC
2452
36367
KDR:1607L21 antisense siNA
UAGGAAUCCACAGGAGAGATT
3471

(1589C) stab00

1875
GUGUCAGCUUUGUACAAAUGUGA
2453
36368
KDR:1893L21 antisense siNA
ACAUUUGUACAAAGCUGACTT
3472

(1875C) stab00

2874
GACAAGACAGCAACUUGCAGGAC
2454
36369
KDR:2892L21 antisense siNA
CCUGCAAGUUGCUGUCUUGTT
3473

(2874C) stab00

2875
ACAAGACAGCAACUUGCAGGACA
2455
36370
KDR:2893L21 antisense siNA
UCCUGCAAGUUGCUGUCUUTT
3474

(2875C) stab00

2876
CAAGACAGCAACUUGCAGGACAG
2456
36371
KDR:2894L21 antisense siNA
GUCCUGCAAGUUGCUGUCUTT
3475

(2876C) stab00

3039
CUCAUGGUGAUUGUGGAAUUCUG
2457
36372
KDR:3057L21 antisense siNA
GAAUUCCACAAUCAOCAUGTT
3476

(3039C) stab00

3040
UCAUGGUGAUUGUGGAAUUCUGC
2458
36373
KDR:3058L21 antisense siNA
AGAAUUCCACAAUCACCAUTT
3477

(3040C) stab00

3249
UCCCUCAGUGAUGUAGAAGAAGA
2459
36374
KDR:3267L21 antisense siNA
UUCUUCUACAUCACUGAGGTT
3478

(3249C) stab00

3263
AGAAGAAGAGGAAGCUCCUGAAG
2460
36375
KDR:3281L21 antisense siNA
UCAGGAGCUUCCUCUUCUUTT
3479

(3263C) stab00

3264
GAAGAAGAGGAAGCUCCUGAAGA
2461
36376
KDR:3282L21 antisense siNA
UUCAGGAGCUUCCUCUUCUTT
3480

(3264C) stab00

3269
AGAGGAAGCUCCUGAAGAUCUGU
2462
36377
KDR:3287L21 antisense siNA
AGAUCUUCAGGAGCUUCCUTT
3481

(3269C) stab00

3270
GAGGAAGCUCCUGAAGAUCUGUA
2463
36378
KDR:3288L21 antisense siNA
CAGAUCUUCAGGAGOUUCCTT
3482

(3270C) stab00

3346
AGGGCAUGGAGUUCUUGGCAUCG
2464
36379
KDR:3364121 antisense siNA
AUGCCAAGAACUCCAUGCCTT
3483

(3346C) stab00

3585
UUGCUGUGGGAAAUAUUUUCCUU
2465
36380
KDR:3603L21 antisense siNA
GGAPAAUAUUUCCCACAGCTT
3484

(3585C) stab00

3586
UG0UGUGGGAAAUAUUUUCCUUA
2466
36381
KDR3604L21 antisense siNA
AGGAAAAUAUUUCCCACAGTT
3485

(3586C) stab00

3860
CAUGGAAGAGGAUUCUGGACUCU
2467
36382
KDR:3878L21 antisense siNA
AGUCCAGAAUCCUCUUCCATT
3486

(3860C) stab00

3877
GACUCUCUCUGCCUACCUCACCU
2468
36383
KDR3895121 antisense siNA
GUGAGGUAGGCAGAGAGAGTT
3487

(3877C) stab00

3878
ACUCUCUCUGCCUACCUCACCUG
2469
36384
KDR:3896L21 antisense siNA
GGUGAGGUAGGCAGAGAGATT
3488

(3878C) stab00

4287
AAGCUGAUAGAGAUUGGAGUGCA
2470
36385
KDR:4305L21 antisense siNA
CACUCCAAUCUCUAU0AGCTT
3489

(4287C) stab00

4288
AGCUGAUAGAGAUUGGAGUGCAA
2471
36386
KDR:4306L21 antisense siNA
GCACUCCAAUCUCUAUCAGTT
3490

(4288C) stab00

4318
GCACAGCCCAGAUUCUCCAGCCU
2472
36387
KDR:4336L21 antisense siNA
GCUGGAGAAUCUGGGCUGTT
3491

(4318C) stab00

4319
CACAGCCCAGAUUCUCCAGCCUG
2473
36388
KDR4337L21 antisense siNA
GGCUGGAGAAUCUGGGCUGTT
3492

(4319C) stab00

4320
ACAGCCCAGAUUCUCCAGCCUGA
2474
36389
KDR:4338L21 antisense siNA
AGGCUGGAGAAUCUGGGCUTT
3493

(4320C) stab00

4321
CAGCCCAGAUUCUCCAGCCUGAC
2475
36390
KDR:4339L21 antisense siNA
CAGGCUGGAGAAUCUGGGCTT
3494

(4321C) stab00

4359
AGCUCUCCUCCUGUUUAAAAGGA
2476
36391
KDR:4377L21 antisense siNA
CUUUUPAACAGGAGGAGAGTT
3495

(4359C) stab00

4534
UAUCCUGGAAGAGGCUUGUGACC
2477
36392
KDR:4552L21 antisense siNA
UCACAAGCCUCUUCCAGGATT
3496

(4534C) stab00

4535
AUCCUGGAAGAGGCUUGUGACCC
2478
36393
KDR:4553L21 antisense siNA
GUCACAAGCCUCUUCCAGGTT
3497

(4535C) stab00

4536
UCCUGGAAGAGGCUUGUGACCCA
2479
36394
KDR:4554L21 antisense siNA
GGUCACAAGCCUCUUCCAGTT
3498

(4536C) stab00

4539
UGGAAGAGGCUUGUGACCCAAGA
2480
36395
KDR:4557L21 antisense siNA
UUGGGUCACAAGCCUCUUCTT
3499

(4539C) stab00

4769
UGUUGAAGAUGGGAAGGAUUUGC
2481
36396
KDR:4787L21 antisense siNA
AAAUCCUUCCCAUCUUCAATT
3500

(4769C) stab00

4934
UCUGGUGGAGGUGGGCAUGGGGU
2482
36397
KDR:4952L21 antisense siNA
CCCAUGCCCACCUCCACCATT
3501

(4934C) stab00

5038
UCGUUGUGCUGUUUCUGACUCCU
2483
36398
KDR:5056L21 antisense siNA
GAGUCAGAAACAGCACAACTT
3502

(5038C) stab00

5039
CGUUGUGCUGUUUCUGACUCCUA
2484
36399
KDR:5057L21 antisense siNA
GGAGUCAGAAACAGCACAATT
3503

(5039C) stab00

5040
GUUGUGCUGUUUCUGACUCCUAA
2485
36400
KDR:5058L21 antisense siNA
AGGAGUCAG4AAACAGCACATT
3504

(5040C) stab00

5331
UCAAAGUUUCAGGAAGGAUUUUA
2486
36401
KDR:5349L21 antisense siNA
AAAUCCUUCCUGAAACUUUTT
3505

(5331C) stab00

5332
CAAAGUUUCAGGAAGGAUUUUAC
2487
36402
KDR:5350L21 antisense siNA
AAAAUCCUUCCUGAAACUUTT
3506

(5332C) stab00

5333
AAAGUUUCAGGAAGGAUUUUACC
2488
36403
KDR:5351L21 antisense siNA
UAAAAUCCUUCCUGAAACUTT
3507

(5333C) stab00

5587
UCAAAAAAGAAAAUGUGUUUUUU
2489
36404
KDR:5605L21 antisense siNA
AAAACACAUUUUCUUUUUUTT
3508

(5587C) stab00

5737
CUAUUCACAUUUUGUAUCAGUAU
2490
36405
KDR:5755L21 antisense siNA
ACUGAUACAAAAUGUGAAUTT
3509

(5737C) stab00

5738
UAUUCACAUUUUGUAUCAGUAUU
2491
36406
KDR:5756L21 antisense siNA
UACUGAUACAAAAUGUGAATT
3510

(5738C) stab00

5379
AUUCACAUUUUGUAUCAGUAUUA
2492
36407
KDR:5757L21 antisense siNA
AUACUGAUACAAAAUGUGATT
3511

(5739C) stab00

359
GGCCGCCUCUGUGGGUUUGCCUA
2493
37460
KDR:359U21 sense siNA stab07
B ccGccucuGuGGGuuuGccTT B
3512

360
GCCGCCUCUGUGGGUUUGCCUAG
2494
37461
KDR:360U21 sense siNA stab07
B cGccucuGuGGGuuuGccuTT B
3513

799
ACCCAGAAAAGAGAUUUGUUCCU
2495
37462
KDR:799U21 sense siNA stab07
B ccAGAAAAGAGAuuuGuucTT B
3514

826
GUAACAGAAUUUCCUGGGACAGC
2496
37463
KDR:826U21 sense siNA stab07
B AAcAGAAuuuccuGGGAcATT B
3515

1027
AGCUUGUCUUAAAUUGUACAGCA
2497
37464
KDR:1027U21 sense siNA stab07
B cuuGucuuAAAuuGuAcAGTT B
3516

1827
GAAGGAAAAAACAAAACUGUAAG
2498
37465
KDR:1827U21 sense siNA stab07
B AGGAAAAAAcAAAAcuGuATT B
3517

1828
AAGGAAAAAACAAAACUGUAAGU
2499
37466
KDR:1828U21 sense siNA stab07
B GGAAAAAAcAAAAcuGuAATT B
3518

1947
ACCAGGGGUCCUGAAAUUACUUU
2500
37467
KDR:1947U21 sense siNA stab07
B cAGGGGuccuGAAAuuAcuTT B
3519

2247
AAGACCAAGAAAAGACAUUGCGU
2501
37468
KDR:2247U21 sense siNA stab07
B GAccAAGAAAAGAcAuuGcTT B
3520

2501
AGGCCUCUACACCUGCCAGGCAU
2502
37469
KDR:2501U21 sense siNA stab07
B GccucuAcAccuGccAGGcTT B
3521

2624
GAUUGCCAUGUUCUUCUGGCUAC
2503
37470
KDR:2624U21 sense siNA stab07
B uuGccAuGuucuucuGGcuTT B
3522

2685
GGAGGGGAACUGAAGACAGGCUA
2504
37471
KDR:2685U21 sense siNA stab07
B AGGGGAAcuGAAGAcAGGcTT B
3523

2688
GGGGAACUGAAGACAGGCUACUU
2505
37472
KDR:2688U21 sense siNA stab07
B GGAAcuGAAGAcAGGcuAcTT B
3524

2689
GGGAACUGAAGACAGGCUACUUG
2506
37473
KDR:2689U21 sense siNA stab07
B GAAcuGAAGAcAGGcuAcuTT B
3525

2690
GGAACUGAAGACAGGCUACUUGU
2507
37474
KDR:2690U21 sense siNA stab07
B AAcuGAAGAcAGGcuAcuuTT B
3526

2692
AACUGAAGACAGGCUACUUGUCC
2508
37475
KDR:2692U21 sense siNA stab07
B cuGAAGAcAGGcuAcuuGuTT B
3527

2762
ACUGCCUUAUGAUGCCAGCAAAU
2509
37476
KDR:2762U21 sense siNA stab07
B uGccuuAuGAuGccAGcAATT B
3528

3187
GGCGCUUGGACAGCAUCACCAGU
2510
37477
KDR:3187U21 sense siNA stab07
B cGcuuGGAcAGcAucAccATT B
3529

3293
UAAGGACUUCCUGACCUUGGAGC
2511
37478
KDR:3293U21 sense siNA stab07
B AGGAcuuccuGAccuuGGATT B
3530

3306
ACCUUGGAGCAUCUCAUCUGUUA
2512
37479
KDR:3306U21 sense siNA stab07
B cuuGGAGcAucucAucuGuTT B
3531

3308
CUUGGAGCAUCUCAUCUGUUACA
2513
37480
KDR:3308U21 sense siNA stab07
B uGGAGcAucucAucuGuuATT B
3532

3309
UUGGAGCAUCUCAUCUGUUACAG
2514
37481
KDR:3309U21 sense siNA stab07
B GGAGcAucucAucuGuuAcTT B
3533

3312
GAGCAUCUCAUCUGUUACAGCUU
2515
37482
KDR:3312U21 sense siNA stab07
B GcAucucAucuGuuAcAGcTT B
3534

3320
CAUCUGUUACAGCUUCCAAGUGG
2516
37483
KDR:3320U21 sense siNA stab07
B ucuGuuAcAGcuuccAAGuTT B
3535

3324
UGUUACAGCUUCCAAGUGGCUAA
2517
37484
KDR:3324U21 sense siNA stab07
B uuAcAGcuuccAAGuGGcuTT B
3536

3334
UCCAAGUGGCUAAGGGCAUGGAG
2518
37485
KDR:3334U21 sense siNA stab07
B cAAGuGGcuAAGGGCAuGGTT B
3537

3346
AGGGCAUGGAGUUCUUGGCAUCG
2464
37486
KDR:3346U21 sense siNA stab07
B GGcAuGGAGuucuuGGcAuTT B
3538

3347
GGGCAUGGAGUUCUUGGCAUCGC
2519
37487
KDR:3347U21 sense siNA stab07
B GcAuGGAGuucuuGGcAucTT B
3539

3857
GAGCAUGGAAGAGGAUUCUGGAC
2520
37488
KDR:3857U21 sense siNA stab07
B GcAuGGAAGAGGAuucuGGTT B
3540

3858
AGCAUGGAAGAGGAUUCUGGACU
2521
37489
KDR:3858U21 sense siNA stab07
B cAuGGAAGAGGAuucuGGATT B
3541

3860
CAUGGAAGAGGAUUCUGGACUCU
2467
37490
KDR:3860U21 sense siNA stab07
B uGGAAGAGGAuucuGGAcuTT B
3542

3883
CUCUGCCUACCUCACCUGUUUCC
2522
37491
KDR:3883U21 sense siNA stab07
B cuGccuAccucAccuGuuuTT B
3543

3884
UCUGCCUACCUCACCUGUUUCCU
2523
37492
KDR:3884U21 sense siNA stab07
B uGccuAccucAccuGuuucTT B
3544

3885
CUGCCUACCUCACCUGUUUCCUG
2524
37493
KDR:3885U21 sense siNA stab07
B GccuAccucAccuGuuuccTT B
3545

3892
CCUCACCUGUUUCCUGUAUGGAG
2525
37494
KDR:3892U21 sense siNA stab07
B ucAccuGuuuccuGuAuGGTT B
3546

3936
AAAUUCCAUUAUGACAACACAGC
2526
37495
KDR:3936U21 sense siNA stab07
B AuuccAuuAuGAcAAcAcATT B
3547

3940
UCCAUUAUGACAACACAGCAGGA
2527
37496
KDR:3940U21 sense siNA stab07
B cAuuAuGAcAAcAcAGcAGTT B
3548

359
GGCCGCCUCUGUGGGUUUGCCUA
2493
37497
KDR:377L21 antisense siNA
GGCAAAcccAcAGAGGcGG1T
3549

(359C) stab26

360
GCCGCCUCUGUGGGUUUGCCUAG
2494
37498
KDR:378L21 antisense siNA
AGGcAAAcccAcAGAGGcGTT
3550

(360C) stab26

799
ACCCAGAAAAGAGAUUUGUUCCU
2495
37499
KDR:817L21 antisense siNA
GAAcAAAucucuuuucuGGTT
3551

(799C) stab26

826
GUAACAGAAUUUCCUGGGACAGC
2496
37500
KDR:844L21 antisense siNA
UGUcccAGGAAAuucuGuuTT
3552

(826C) stab26

1027
AGCUUGUCUUAAAUUGUACAGCA
2497
37501
KDR:1045L21 antisense siNA
CUGuAcAAuuuAAGAcAAGTT
3553

(1027C) stab26

1827
GAAGGAAAAAACAAAACUGUAAG
2498
37502
KDR:1845L21 antisense siNA
UACAGuuuuGuuuuuuccuTT
3554

(1827C) stab26

1828
AAGGAAAAAACAAAACUGUAAGU
2499
37503
KDR:1846L21 antisense siNA
UUAcAGuuuuGuuuuuuccTT
3555

(1828C) stab26

1947
ACCAGGGGUCCUGAAAUUACUUU
2500
37504
KDR:1965L21 antisense siNA
AGUAAuuucAGGAccccuGTT
3556

(1947c) stab26

2247
AAGACCAAGAAAAGACAUUGCGU
2501
37505
KDR:2265L21 antisense siNA
GCAAuGucuuuucuuGGucTT
3557

(2247C) stab26

2501
AGGCCUCUACACCUGCCAGGCAU
2502
37506
KDR:2519L21 antisense siNA
GCCuGGcAGGuGuAGAGGcTT
3558

(2501C) stab26

2624
GAUUGCCAUGUUCUUCUGGCUAC
2503
37507
KDR:2642L21 antisense siNA
AGCcAGAAGAAcAuGGcAATT
3559

(2624C) stab26

2685
GGAGGGGAACUGAAGACAGGCUA
2504
37508
KDR:2703L21 antisense siNA
GCCuGucuucAGuuccccuTT
3560

(2685C) stab26

2688
GGGGAACUGAAGACAGGCUACUU
2505
37509
KDR:2706L21 antisense siNA
GUAGccuGucuucAGuuccTT
3561

(2688C) stab26

2689
GGGAACUGAAGACAGGCUACUUG
2506
37510
KDR:2707L21 antisense siNA
AGUAGccuGucuucAGuucTT
3562

(2689C) stab26

2690
GGAACUGAAGACAGGCUACUUGU
2507
37511
KDR:2708L21 antisense siNA
AAGuAGccuGucuucAGuuTT
3563

(2690C) stab26

2692
AACUGAAGACAGGCUACUUGUCC
2508
37512
KDR:2710L21 antisense siNA
ACAAGuAGccuGucuucAGTT
3564

(2692C) stab26

2762
ACUGCCUUAUGAUGCCAGCAAAU
2509
37513
KDR:2780L21 antisense siNA
UUGcuGGcAucAuAAGGcATT
3565

(2762C) stab26

3187
GGCGCUUGGACAGCAUCACCAGU
2510
37514
KDR:3205L21 antisense siNA
UGGuGAuGcuGuccAAGcGTT
3566

(3187C) stab26

3293
UAAGGACUUCCUGACCUUGGAGC
2511
37515
KDR:3311L21 antisense siNA
UCCAAGGucAGGAAGuccuTT
3567

(3293C) stab26

3306
ACCUUGGAGCAUCUCAUCUGUUA
2512
37516
KDR:3324L21 antisense siNA
ACAGAuGAGAuGcuccAAGTT
3568

(3306C) stab26

3308
CUUGGAGCAUCUCAUCUGUUACA
2513
37517
KDR:3326L21 antisense siNA
UAAcAGAuGAGAuGcuccATT
3569

(3308C) stab26

3309
UUGGAGCAUCUCAUCUGUUACAG
2514
37518
KDR3327L21 antisense siNA
GUAAcAGAuGAGAuGcuccTT
3570

(3309C) stab26

3312
GAGCAUCUCAUCUGUUACAGCUU
2515
37519
KDR:3330L21 antisense siNA
GCUGuAAcAGAuGAGAuGcTT
3571

(3312C) stab26

3320
CAUCUGUUACAGCUUCCAAGUGG
2516
37520
KDR:3338L21 antisense siNA
ACUuGGAAGcuGuAAcAGATT
3572

(3320C) stab26

3324
UGUUACAGCUUCCAAGUGGCUAA
2517
37521
KDR:3342L21 antisense siNA
AGCcAcuuGGAAGcuGuAATT
3573

(3324C) stab26

3334
UCCAAGUGGCUAAGGGCAUGGAG
2518
37522
KDR.3352L21 antisense siNA
CCAuGcccuuAGccAcuuGTT
3574

(3334C) stab26

3346
AGGGCAUGGAGUUCUUGGCAUCG
2464
37523
KDR:3364L21 antisense siNA
AUGcCAAGAAcuccAuGccTT
3575

(3346C) stab26

3347
GGGCAUGGAGUUCUUGGCAUCGC
2519
37524
KDR:3365L21 antisense siNA
GAUGccAAGAAcuccAuGcTT
3576

(3347C) stab26

3758
CACGUUUUCAGAGUUGGUGGAAC
2426
37525
KDR:3776L21 antisense siNA
UCCAccAAcucuGAAAAcGTT
3577

(3758C) stab26

3857
GAGCAUGGAAGAGGAUUCUGGAC
2520
37526
KDR:3875L21 antisense siNA
CCAGAAuccucuuccAuGcTT
3578

(3857C) stab26

3858
AGCAUGGAAGAGGAUUCUGGACU
2521
37527
KDR:3876L21 antisense siNA
UCCAGAAuccucuuccAuGTT
3579

(3858C) stab26

3860
CAUGGAAGAGGAUUCUGGACUCU
2467
37528
KDR:3878L21 antisense siNA
AGUccAGAAuccucuuccATT
3580

(3860C) stab26

3883
CUCUGCCUACCUCACCUGUUUCC
2522
37529
KDR:3901L21 antisense siNA
AAAcAGGuGAGGuAGGcAGTT
3581

(3883C) stab26

3884
UCUGCCUACCUCACCUGUUUCCU
2523
37530
KDR:3902L21 antisense siNA
GAAAcAGGuGAGGuAGGcATT
3582

(3884C) stab26

3885
CUGCCUACCUCACCUGUUUCCUG
2524
37531
KDR:3903L21 antisense siNA
GGAAAcAGGuGAGGuAGGcTT
3583

(3885C) stab26

3892
CCUCACCUGUUUCCUGUAUGGAG
2525
37532
KDR:391 0121 antisense siNA
CCAuAcAGGAAAcAGGUGATT
3584

(3892C) stab26

3893
CUCACCUGUUUCCUGUAUGGAGG
2427
37533
KDR:391 1121 antisense siNA
UCCAuAcAGGAAAcAGGuGTT
3585

(3893C) stab26

3936
AAAUUCCAUUAUGACAACACAGC
2526
37534
KDR:3954L21 antisense siNA
UGUGuuGucAuAAuGGAAuTT
3586

(3936C) stab26

3940
UCCAUUAUGACAACACAGCAGGA
2527
37535
KDR:3958L21 antisense siNA
CUGcuGuGuuGucAuAAuGTT
3587

(3940C) stab26

3948
GACAACACAGCAGGAAUCAGUCA
2408
37536
KDR:3966L21 antisense siNA
ACUGAuuccuGcuGuGuuGTT
3588

(3948C) stab26

VEGFR3

2011
AGCACUGCCACAAGAAGUACCUG
2528
31904
FLT4:2011U21 sense siNA
CACUGCCACAAGAAGUACCTT
3589

3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3921U21 sense siNA
GAAG0AGAGAGAGAGAAGGTT
3590

4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4038U21 sense siNA
AGAGGAACCAGGAGGACAATT
3591

4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4054U21 sense siNA
CAAGAGGAGCAUGAAAGUGTT
3592

2011
AGCACUGCCACAAGAAGUACCUG
2528
31908
FLT4:2029L21 antisense
GGUACUUCUUGUGGCAGUGTT
3593

siNA (2011C)

3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3939L21 antisense siNA
CCUUCUCUCUCUCUGCUUCTT
3594

(3921C)

4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4056L21 antisense siNA
UUGUCCUCCUGGUUCCUCUTT
3595

(4038C)

4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4072L21 antisense siNA
CACUUUCAUGCUCCUCUUGTT
3596

(4054C)

2011
AGCACUGCCACAAGAAGUACCUG
2528

FLT4:2011U21 sense siNA
B cAcuGccAcAAGAAGuAccTT B
3597

stab04

3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3921U21 sense siNA
B GAAGcAGAGAGAGAGAAGGTT B
3598

stab04

4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4038U21 sense siNA
B AGAGGAAccAGGAGGAcAATT B
3599

stab04

4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4054U21 sense siNA stab04
B cAAGAGGAGCAuGAAAGuGTT B
3600

2011
AGCACUGCCACAAGAAGUACCUG
2528

FLT4:2029L21 antisense siNA
GGuAcuucuuGuGGcAGuGTsT
3601

(2011C) stab05

3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3939L21 antisense siNA
ccuucucucucucuGcuucTsT
3602

(3921C) stab05

4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4056L21 antisense siNA
uuGuccuccuGGuuccucuTsT
3603

(4038C) stab05

4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4072L21 antisense siNA
cAcuuucAuGcuccucuuGTsT
3604

(4054C) stab05.

2011
AGCACUGCCACAAGAAGUACCUG
2528

FLT4:2011U21 sense siNA stab07
B cAcuGccACAAGAAGuAccTT B
3605

3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3921U21 sense siNA stab07
B GAAGcAGAGAGAGAGAAGGTT B
3606

4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4038U21 sense siNA stab07
B AGAGGAAccAGGAGGAcAATT B
3607

4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4054U21 sense siNA stab07
B cAAGAGGAGcAuGAAAGuGTT B
3608

2011
AGCACUGCCACAAGAAGUACCUG
2528

FLT4:2029L21 antisense siNA

GGuAcuucuuGuGGcAGuGTsT
3609

(2011C) stab11

3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3939L21 antisense siNA
ccuucucucucucuGcuucTsT
3610

(3921C) stab11

4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4056L21 antisense siNA
uuGuccuccuGGuuccucuTsT
3611

(4038C) stab11

4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4072L21 antisense siNA
cAcuuucAuGcuccucuuGTsT
3612

(4054C) stab11

1666
ACUUCUAUGUGACCACCAUCCCC
2532
31902
FLT4:1666U21 sense siNA
UUCUAUGUGACCACCAUCCTT
3613

2009
CAAGCACUGCCACAAGAAGUACC
2533
31903
FLT4:2009U21 sense siNA
AGCACUGCCACAAGAAGUATT
3614

2815
AGUACGGCAACCUCUCCAACUUC
2534
31905
FLT4:2815U21 sense siNA
UACGGCAACCUCUCCAACUTT
3615

1666
ACUUCUAUGUGACCACCAUCCCC
2532
31906
FLT4:1684L21 antisense siNA
GGAUGGUGGUCACAUAGAATT
3616

(1666C)

2009
CAAGCACUGCCACAAGAAGUACC
2533
31907
FLT4:2027L21 antisense siNA
ACUUCUUGUGGCAGUGCUTT
3617

(2009C)

2815
AGUACGGCAACCUCUCCAACUUC
2534
31909
FLT4:2833L21 antisense siNA
AGUUGGAGAGGUUGCCGUATT
3618

(2815C)

1609
CUGCCAUGUACAAGUGUGUGGUC
2535
34383
FLT4:1609U21 sense siNA stab09
B GCCAUGUACAAGUGUGUGGTT B
3619

1666
ACUUCUAUGUGACCACCAUCCCC
2532
34384
FLT4:1666U21 sense siNA stab09
B UUCUAUGUGACCACCAUCCTT B
3620

2009
CAAGCACUGCCACAAGAAGUACC
2533
34385
FLT4:2009U21 sense siNA stab09
B AGCACUGCCACAAGAAGUATT B
3621

2011
AGCACUGCCACAAGAAGUACCUG
2528
34386
FLT4:2011U21 sense siNA stab09
B CACUGCCACAAGAAGUACCTT B
3622

2014
ACUGCCACAAGAAGUACCUGUCG
2536
34387
FLT4:2014U21 sense siNA stab09
B UGCCACAAGAAGUACCUGUTT B
3623

2815
AGUACGGCAACCUCUCCAACUUC
2534
34388
FLT4:2815U21 sense siNA stab09
B UACGGCAACCUCUCCAACUTT B
3624

3172
UGGUGAAGAUCUGUGACUUUGGC
2537
34389
FLT4:3172U21 sense siNA stab09
B GUGAAGAUCUGUGACUUUGTT B
3625

3176
GAAGAUCUGUGACUUUGGCCUUG
2538
34390
FLT4:3176U21 sense siNA stab09
B AGAUCUGUGACUUUGGCCUTT B
3626

1609
CUGCCAUGUACAAGUGUGUGGUC
2535
34391
FLT4:1627L21 antisense siNA
CCACACACUUGUACAUGGCTsT
3627

(1609C) stab10

1666
ACUUCUAUGUGACCACCAUCCCC
2532
34392
FLT4:1684L21 antisense siNA
GGAUGGUGGUCACAUAGAATsT
3628

(1666C) stab10

2009
CAAGCACUGCCACAAGAAGUACC
2533
34393
FLT4:2027L21 antisense siNA
UACUUCUUGUGGCAGUGCUTsT
3629

(2009C) stab10

2011
AGCACUGCCACAAGAAGUACCUG
2528
34394
FLT4:2029L21 antisense siNA
GGUACUUCUUGUGGCAGUGTsT
3630

(2011C) stab10

2014
ACUGCCACAAGAAGUACCUGUCG
2536
34395
FLT4:2032L21 antisense siNA
ACAGGUACUUCUUGUGGCATsT
3631

(2014C) stab10

2815
AGUACGGCAACCUCUCCAACUUC
2534
34396
FLT4:2833L21 antisense siNA
AGUUGGAGAGGUUGCCGUATsT
3632

(2815C) stab10

3172
UGGUGAAGAUCUGUGACUUUGGC
2537
34397
FLT4:3190L21 antisense siNA
CAAAGUCACAGAUCUUCACTsT
3633

(3172C) stab10

3176
GAAGAUCUGUGACUUUGGCCUUG
2538
34398
FLT4:3194L21 antisense siNA
AGGCCAAAGUCACAGAUCUTsT
3634

(3176C) stab10

1609
CUGCCAUGUACAAGUGUGUGGUC
2535
34399
FLT4:1627L21 antisense siNA
ccAcAcAcuuGuAcAuGGcTsT
3635

(1609C) stab08

1666
ACUUCUAUGUGACCACCAUCCCC
2532
34400
FLT4:1684L21 antisense siNA

GGAuGGuGGucAcAuAGAATsT
3636

(1666C) stab08

2009
CAAGCACUGCCACAAGAAGUACC
2533
34401
FLT4:2027L21 antisense siNA
uAcuucuuGuGGcAGuGcuTsT
3637

(2009C) stab08

2011
AGCACUGCCACAAGAAGUACCUG
2528
34402
FLT4:2029L21 antisense siNA

GGuAcuucuuGuGGcAGuGTsT
3638

(2011C) stab08

2014
ACUGCCACAAGAAGUACCUGUCG
2536
34403
FLT4:2032L21 antisense siNA

AcAGGuAcuucuuGuGGcATsT
3639

(2014C) stab08

2815
AGUACGGCAACCUCUCCAACUUC
2534
34404
FLT4:2833L21 antisense siNA

AGuuGGAGAGGuuGccGuATsT
3640

(2815C) stab08

3172
UGGUGAAGAUCUGUGACUUUGGC
2537
34405
FLT4:3190L21 antisense siNA
cAAAGucAcAGAucuucAcTsT
3641

(3172C) stab08

3176
GAAGAUCUGUGACUUUGGCCUUG
2538
34406
FLT4:3194L21 antisense siNA

AGGccAAAGucAcAGAucuTsT
3642

(3176C) stab08

VEGF

329
AGCAAGAGCUCCAGAGAGAAGUCG
2539
32166
VEGF:331U21 sense siNA
AAGAGCUCCAGAGAGAAGUTT
3643

414
CAAAGUGAGUGACCUGCUUUUGG
2540
32167
VEGF:416U21 sense siNA
AAGUGAGUGACCUGCUUUUTT
3644

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541
32168
VEGF:1153U21 sense siNA
GAAGUGGUGAAGUUCAUGGTT
3645

1212
GGUGGACAUCUUCCAGGAGUACC
2542
32525
VEGF:1214U21 sense siNA
UGGACAUCUUCCAGGAGUATT
3646

1213
GUGGACAUCUUCCAGGAGUACCC
2543
32526
VEGF:1215U21 sense siNA
GGACAUCUUCCAGGAGUACTT
3647

1215
GGACAUCUUCCAGGAGUACCCUG
2544
32527
VEGF:1217U21 sense siNA
ACAUCUUCCAGGAGUACCCTT
3648

1334
AGUCCAACAUCACCAUGCAGAUU
2545
32169
VEGF:1336U21 sense siNA
UCCAACAUCACCAUGCAGATT
3649

1650
CGAACGUACUUGCAGAUGUGACA
2546
32540
VEGF:1652U21 sense siNA
AACGUACUUGCAGAUGUGATT
3650

329
GCAAGAGCUCCAGAGAGAAGUCG
2539
32170
VEGF:349L21 antisense siNA
ACUUCUCUCUGGAGCUCUUTT
3651

(331C)

414
CAAAGUGAGUGACCUGCUUUUGG
2540
32171
VEGF:434L21 antisense siNA
AAAAGCAGGUCACUCACUUTT
3652

(416C)

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541
32172
VEGF:1171L21 antisense siNA
CCAUGAACUUCACCACUUCTT
3653

(1153C)

1212
GGUGGACAUCUUCCAGGAGUACC
2542
32543
VEGF:1232L21 antisense siNA
UACUCCUGGAAGAUGUCCATT
3654

(1214C)

1213
GUGGACAUCUUCCAGGAGUACCC
2543
32544
VEGF:1233L21 antisense siNA
GUACUCCUGGAAGAUGUCCTT
3655

(1215C)

1215
GGACAUCUUCCAGGAGUACCCUG
2544
32545
VEGF:1235L21 antisense siNA
GGGUACUCCUGGAAGAUGUTT
3656

(1217C)

1334
AGUCCAACAUCACCAUGCAGAUU
2545
32173
VEGF:1354L21 antisense siNA
UCUGCAUGGUGAUGUUGGATT
3657

(1336C)

1650
CGAACGUACUUGCAGAUGUGACA
2546
32558
VEGF:1670L21 antisense siNA
UCACAUCUGCAAGUACGUUTT
3658

(1652C)

329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:331U21 sense siNA stab04
B AAGAGcuccAGAGAGAAGuTT B
3659

414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:416U21 sense siNA stab04
B AAGuGAGuGAccuGcuuuuTT B
3660

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1153U21 sense siNA
B GAAGUGGuGAAGuucAuGGTT B
3661

stab04

1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1214U21 sense siNA
B uGGAcAucuuccAGGAGuATT B
3662

stab04

1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1215U21 sense siNA
B GGAcAucuuccAGGAGuAcTT B
3663

stab04

1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1217U21 sense siNA
B AcAucuuccAGGAGuAcccTT B
3664

stab04

1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1336U21 sense siNA
B uccAAcAucAccAuGcAGATT B
3665

stab04

1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1652U21 sense siNA
B AAcGuAcuuGcAGAuGuGATT B
3666

stab04

329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA
AcuucucucuGGAGcucuuTsT
3667

(331C) stab05

414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA
AAAAGcAGGucAcucAcuuTsT
3668

(416C) stab05

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA
ccAuGA.AcuucAccAcuucTsT
3669

(1153C) stab05

1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1232L21 antisense siNA
uAcuccuGGAAGAuGuccATsT
3670

(1214C) stab05

1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1233L21 antisense siNA
GuAcuccuGGAAGAuGuccTsT
3671

(1215C) stab05

1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA
GGGuAcuccuGGAAGAuGuTsT
3672

(1217C) stab05

1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA
ucuGcAuGGuGAuGuuGGATsT
3673

(1336C) stab05

1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA
ucAcAucuGcAAGuAcGuuTsT
3674

(1652C) stab05

329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:331U21 sense siNA stab07
B AAGAGcuccAGAGAGAAGuTT B
3675

414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:416U21 sense siNA stab07
B AAGuGAGuGAccuGcuuuuTT B
3676

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1153U21 sense siNA
B GAAGuGGuGAAGuucAuGGTT B
3677

stab07

1212
GGUGGACAUCUUCCAGGAGUACC
2542
33977
VEGF:1214U21 sense siNA
B uGGAcAucuuccAGGAGuATT B
3678

stab07

1213
GUGGACAUCUUCCAGGAGUACCC
2543
33978
VEGF:1215U21 sense siNA
B GGAcAucuuccAGGAGuAcTT B
3679

stab07

1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1217U21 sense siNA
B AcAucuuccAGGAGuAcccTT B
3680

stab07

1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1336U21 sense siNA
B uccAAcAucAccAuGcAGATT B
3681

stab07

1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1652U21 sense siNA
B AAcGuAcuuGcAGAuGuGATT B
3682

stab07

329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA

AcuucucucuGGAGcucuuTsT
3683

(331C) stab11

414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA

AAAAGcAGGucAcucAcuuTsT
3684

(416C) stab11

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA
ccAuGAAcuucAccAcuucTsT
3685

(1153C) stab11

1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1232L21 antisense siNA
uAcuccuGGAAGAuGuccATsT
3686

(1214C) stab11

1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1233L21 antisense siNA

GuAcuccuGGAAGAuGuccTsT
3687

(1215C) stab11

1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA

GGGuAcuccuGGAAGAuGuTsT
3688

(1217C) stab11

1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA
ucuGcAuGGuGAuGuuGGATsT
3689

(1336C) stab11

1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA
ucAcAucuGcAAGuAcGuuTsT
3690

(1652C) stab11

329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:331U21 sense siNA stab18
B AAGAGcuccAGAGAGAAGuTT B
3691

414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:416U21 sense siNA stab18
B AAGuGAGuGAccuGcuuuuTT B
3692

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1153U21 sense siNA
B GAAGuGGuGAAGuucAuGGTT B
3693

stab18

1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1214U21 sense siNA
B uGGAcAucuuccAGGAGuATT B
3694

stab18

1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1215U21 sense siNA
B GGAcAucuuccAGGAGuAcTT B
3695

stab18

1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1217U21 sense siNA
B AcAucuuccAGGAGuAcccTT B
3696

stab18

1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1336U21 sense siNA
B uccAAcAucAccAuGcAGATT B
3697

stab18

1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1652U21 sense siNA
B AAcGuAcuuGcAGAuGuGATT B
3698

stab18

329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA

AcuucucucuGGAGcucuuTsT
3699

(331C) stab08

414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA

AAAAGcAGGucAcucAcuuTsT
3700

(416C) stab08

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171121 antisense siNA
ccAuGAAcuucAccAcuucTsT
3701

(1153C) stab08

1212
GGUGGACAUCUUCCAGGAGUACC
2542
33983
VEGF:1232L21 antisense siNA
uAcuccuGGAAGAuGuccATsT
3702

(1214C) stab08

1213
GUGGACAUCUUCCAGGAGUACCC
2543
33984
VEGF:1233L21 antisense siNA

GuAcuccuGGAAGAuGuccTsT
3703

(1215C) stab08

1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA

GGGuAcuccuGGAAGAuGuTsT
3704

(1217C) stab08

1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354121 antisense siNA
ucuGcAuGGuGAuGuuGGATsT
3705

(1336C) stab08

1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA
ucAcAucuGcAAGuAcGuuTsT
3706

(1652C) stab08

329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:331U21 sense siNA stab09
B AAGAGCUCCAGAGAGAAGUTT B
3707

414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:416U21 sense siNA stab09
B AAGUGAGUGACCUGCUUUUTT B
3708

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1153U21 sense siNA
B GAAGUGGUGAAGUUCAUGGTT B
3709

stab09

1212
GGUGGACAUCUUCCAGGAGUACC
2542
33965
VEGF:1214U21 sense siNA
B UGGACAUCUUCCAGGAGUATT B
3710

stab09

1213
GUGGACAUCUUCCAGGAGUACCC
2543
33966
VEGF:1215U21 sense siNA
B GGACAUCUUCCAGGAGUACTT B
3711

stab09

1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1217U21 sense siNA
B ACAUCUUCCAGGAGUACCCTT B
3712

stab09

1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1336U21 sense siNA
B UCCAACAUCACCAUGCAGATT B
3713

stab09

1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1652U21 sense siNA
B AACGUACUUGCAGAUGUGATT B
3714

stab09

329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA
ACUUCUCUCUGGAGCUCUUTsT
3715

(331C) stab10

414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA
AAAAGCAGGUCACUCACUUTsT
3716

(416C) stab10

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA
CCAUGAACUUCACCACUUCTsT
3717

(1153C) stab10

1212
GGUGGACAUCUUCCAGGAGUACC
2542
33971
VEGF:1232L21 antisense siNA
UACUCCUGGAAGAUGUCCATsT
3718

(1214C) stab10

1213
GUGGACAUCUUCCAGGAGUACCC
2543
33972
VEGF:1233L21 antisense siNA
GUACUCCUGGAAGAUGUCCTsT
3719

(1215C) stab10

1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA
GGGUACUCCUGGAAGAUGUTsT
3720

(1217C) stab10

1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA
UCUGCAUGGUGAUGUUGGATsT
3721

(1336C) stab10

1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA
UCACAUCUGCAAGUACGUUTsT
3722

(1652C) stab10

329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA
AcuucucucuGGAGcucuuTT B
3723

(331C) stab19

414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:43AL21 antisense siNA
AAAAGcAGGucAcucAcuuTT B
3724

(416C) stab19

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA
ccAuGAAcuucAccAcuucTT B
3725

(1153C) stab19

1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1232L21 antisense siNA
uAcuccuGGAAGAuGuccATT B
3726

(1214C) stab19

1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1233L21 antisense siNA

GuAcuccuGGAAGAuGuccTT B
3727

(1215C) stab19

1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA

GGGuAcuccuGGAAGAuGuTT B
3728

(1217C) stab19

1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA
ucuGcAuGGuGAuGuuGGATT B
3729

(1336C) stab19

1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA
ucAcAucuGcAAGuAcGuuTT B
3730

(1652C) stab19

329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA
ACUUCUCUCUGGAGCUCUUTT B
3731

(331C) stab22

414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA
AAAAGCAGGUCACUCACUUTT B
3732

(416C) stab22

1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA
CCAUGAACUUCACCACUUCTT B
3733

(1153C) stab22

1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1232L21 antisense siNA
UACUCCUGGAAGAUGUCCATT B
3734

(1214C) stab22

1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1233L21 antisense siNA
GUACUCCUGGAAGAUGUCCTT B
3735

(1215C) stab22

1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA
GGGUACUCCUGGAAGAUGUTT B
3736

(1217C) stab22

1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA
UCUGCAUGGUGAUGUUGGATT B
3737

(1336C) stab22

1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA
UCACAUCUGCAAGUACGUUTT B
3738

(1652C) stab22

1207
AGACCCUGGUGGACAUCUUCCAG
2547
32524
VEGF:1207U21 sense siNA
ACCCUGGUGGACAUCUUCCTT
3739

stab00

1358
UAUGCGGAUCAAACCUCACCAAG
2548
32528
VEGF:1358U21 sense siNA
UGCGGAUCAAACCUCACCATT
3740

stab00

1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32529
VEGF:1419U21 sense siNA
AUGUGAAUGCAGACCAAAGTT
3741

stab00

1420
AAUGUGAAUGCAGACCAAAGAAA
2550
32530
VEGF:1420U21 sense siNA
UGUGAAUGCAGACCAAAGATT
3742

stab00

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32531
VEGF:1421U21 sense siNA
GUGAAUGCAGACCAAAGAATT
3743

stab00

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
32532
VEGF:1423U21 sense siNA
GAAUGCAGACCAAAGAAAGTT
3744

stab00

1587
CAGACGUGUAAAUGUUCCUGCAA
2553
32533
VEGF:1587U21 sense siNA
GACGUGUAAAUGUUCCUGCTT
3745

stab00

1591
CGUGUAAAUGUUCCUGCAAAAAC
2554
32534
VEGF:1591U21 sense siNA
UGUAAAUGUUCCUGCAAAATT
3746

stab00

1592
GUGUAAAUGUUCCUGCAAAAACA
2555
32535
VEGF:1592U21 sense siNA
GUAAAUGUUCCUGCAAAAATT
3747

stab00

1593
UGUAAAUGUUCCUGCAAAAACAC
2556
32536
VEGF:1593U21 sense siNA
UAAAUGUUCCUGCAAAAACTT
3748

stab00

1594
GUAAAUGUUCCUGCAAAAACACA
2557
32537
VEGF:1594U21 sense siNA
AAAUGUUCCUGCAAAAAAATT
3749

stab00

1604
CUGCAAAAACACAGACUCGCGUU
2558
32538
VEGF:1604U21 sense siNA
GCAAAAACACAGACUCGCGTT
3750

stab00

1637
GCAGCUUGAGUUAAACGAACGUA
2559
32539
VEGF:1637U21 sense siNA
AGCUUGAGUUAAACGAACGTT
3751

stab00

1656
CGUACUUGCAGAUGUGACAAGCC
2560
32541
VEGF:1656U21 sense siNA
UACUUGCAGAUGUGACAAGTT
3752

stab00

1207
AGACCCUGGUGGACAUCUUCCAG
2547
32542
VEGF:1225L21 antisense siNA
GGAAGAUGUCCACCAGGGUTT
3753

(1207C) stab00

1358
UAUGCGGAUCAAACCUCACCAAG
2548
32546
VEGF:1376L21 antisense siNA
UGGUGAGGUUUGAUCCGCATT
3754

(1358C) stab00

1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32547
VEGF:1437L21 antisense siNA
CUUUGGUCUGCAUUCACAUTT
3755

(1419C) stab00

1420
AAUGUGAAUGCAGACCAAAGAAA
2550
32548
VEGF:1438L21 antisense siNA
UCUUUGGUCUGCAUUCACATT
3756

(1420C) stab00

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32549
VEGF:1439L21 antisense siNA
UUCUUUGGUCUGCAUUCACTT
3757

(1421C) stab00

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
32550
VEGF:1441L21 antisense siNA
CUUUCUUUGGUCUGCAUUCTT
3758

(1423C) stab00

1587
CAGACGUGUAAAUGUUCCUGCAA
2553
32551
VEGF:1605L21 antisense siNA
GCAGGAACAUUUACACGUCTT
3759

(1587C) stab00

1591
CGUGUAAAUGUUCCUGCAAAAAC
2554
32552
VEGF:1609L21 antisense siNA
UUUUGCAGGAACAUUUACATT
3760

(1591C) stab00

1592
GUGUAAAUGUUCCUGCAAAAACA
2555
32553
VEGF:1610L21 antisense siNA
UUUUUGCAGGAACAUUUACTT
3761

(1592C) stab00

1593
UGUAAAUGUUCCUGCAAAAACAC
2556
32554
VEGF:1611L21 antisense siNA
GUUUUUGCAGGAACAUUUATT
3762

(1593C) stab00

1594
GUAAAUGUUCCUGCAAAAACACA
2557
32555
VEGF:1612L21 antisense siNA
UGUUUUUGCAGGAACAUUUTT
3763

(1594C) stab00

1604
CUGCAAAAACACAGACUCGCGUU
2558
32556
VEGF:1622L21 antisense siNA
CGCGAGUCUGUGUUUUUGCTT
3764

(1604C) stab00

1637
GCAGCUUGAGUUAAA0GAACGUA
2559
32557
VEGF:1655L21 antisense siNA
CGUUOGUUUAACUCAAGCUTT
3765

(1637C) stab00

1656
CGUACUUGCAGAUGUGACAAGCC
2560
32559
VEGF:1674L21 antisense siNA
CUUGUCACAUCUGCAAGUATT
3766

(1656C) stab00

1206
GAGACCCUGGUGGACAUCUUCCA
2561
32560
VEGF:1206U21 sense siNA
GACCCUGGUGGACAUCUUCTT
3767

stab00

1208
GACCCUGGUGGACAUCUUCCAGG
2562
32561
VEGF:1208U21 sense siNA
CCCUGGUGGACAUCUUCCATT
3768

stab00

1551
UCAGAGCGGAGAAAGCAUUUGUU
2563
32562
VEGF:1551U21 sense siNA
AGAGCGGAGAAAGCAUUUGTT
3769

stab00

1582
AU0CGCAGACGUGUAAAUGUUCC
2564
32563
VEGF:1582U21 sense siNA
CCGCAGACGUGUAAAUGUUTT
3770

stab00

1584
CCGCAGACGUGUAAAUGUUCCUG
2565
32564
VEGF:1584U21 sense siNA
GCAGACGUGUAAAUGUUCCTT
3771

stab00

1585
CGCAGACGUGUAAAUGUUCCUGC
2566
32565
VEGF:1585U21 sense siNA
CAGACGUGUAAAUGUUCCUTT
3772

stab00

1589
GACGUGUAAAUGUUCCUGCAAAA
2567
32566
VEGF:1589U21 sense siNA
CGUGUAAAUGUUCCUGCAATT
3773

stab00

1595
UAAAUGUUCCUGCAAAAACACAG
2568
32567
VEGF:1595U21 sense siNA
AAUGUUCCUGCAAAAACACTT
3774

stab00

1596
AAAUGUUCCUGCAAAAACACAGA
2569
32568
VEGF:1596U21 sense siNA
AUGUUCCUGCAAAAACACATT
3775

stab00

1602
UCCUGCAAAAACACAGACUCGCG
2570
32569
VEGF:1602U21 sense siNA
CUGCAAAAACACAGACUCGTT
3776

stab00

1603
CCUGCAAAAACACAGACUCGCGU
2571
32570
VEGF:1603U21 sense siNA
UGCAAAAACACAGACUCGCTT
3777

stab00

1630
AGGCGAGGCAGCUUGAGUUAAAC
2572
32571
VEGF:1630U21 sense siNA
GCGAGGCAGCUUGAGUUAATT
3778

stab00

1633
CGAGGCAGCUUGAGUUAAACGAA
2573
32572
VEGF:1633U21 sense siNA
AGGCAGCUUGAGUUAAACGTT
3779

stab00

1634
GAGGCAGCUUGAGUUAAACGAAC
2574
32573
VEGF:1634U21 sense siNA
GGCAGCUUGAGUUAAACGATT
3780

stab00

1635
AGGCAGCUUGAGUUAAACGAACG
2575
32574
VEGF:1635U21 sense siNA
GCAGCUUGAGUUAAACGAATT
3781

stab00

1636
GGCAGCUUGAGUUAAACGAACGU
2576
32575
VEGF:1636U21 sense siNA
CAGCUUGAGUUAAACGAACTT
3782

stab00

1648
UAAACGAACGUACUUGCAGAUGU
2577
32576
VEGF:1648U21 sense siNA
AACGAACGUACUUGCAGAUTT
3783

stab00

1649
AAACGAACGUACUUGCAGAUGUG
2578
32577
VEGF:1649U21 sense siNA
ACGAACGUACUUGCAGAUGTT
3784

stab00

1206
GAGACCCUGGUGGACAUCUUCCA
2561
32578
VEGF:1224L21 antisense siNA
GAAGAUGUCCACCAGGGUCTT
3785

(1206C) stab00

1208
GACCCUGGUGGACAUCUUCCAGG
2562
32579
VEGF:1226L21 antisense siNA
GGAAGAAUGUCCACCAGGGTT
3786

(1208C) stab00

1551
UCAGAGCGGAGAAAGCAUUUGUU
2563
32580
VEGF:1569L21 antisense siNA
CAAAUGCUUUCUCCGCUCUTT
3787

(1551C) stab00

1582
AUCCGCAGACGUGUAAAUGUUCC
2564
32581
VEGF:1600L21 antisense siNA
AACAUUUACACGUCUGCGGTT
3788

(1582C) stab00

1584
CCGCAGACGUGUAAAUGUUCCUG
2565
32582
VEGF:1602L21 antisense siNA
GGAACAUUUACACGUCUGCTT
3789

(1584C) stab00

1585
CGCAGACGUGUAAAUGUUCCUGC
2566
32583
VEGF:1603L21 antisense siNA
AGGAACAUUUACACGUCUGTT
3790

(1585C) stab00

1589
GACGUGUAAAUGUUCCUGCAAAA
2567
32584
VEGF:1607L21 antisense siNA
UUGCAGGAACAUUUACACGTT
3791

(1589C) stab00

1595
UAAAUGUUCCUGCAAAAACACAG
2568
32585
VEGF:1613L21 antisense siNA
GUGUUUUUGCAGGAACAUUTT
3792

(1595C) stab00

1596
AAAUGUUCCUGCAAAAACACAGA
2569
32586
VEGF:1614L21 antisense siNA
UGUGUUUUUGCAGGAACAUTT
3793

(1596C) stab00

1602
UCCUGCAAAAACACAGACUCGCG
2570
32587
VEGF:1620L21 antisense siNA
CGAGUCUGUGUUUUUGCAGTT
3794

(1602C) stab00

1603
CCUGCAAAAACACAGACUCGCGU
2571
32588
VEGF:1621L21 antisense siNA
GCGAGUCUGUGUUUUUGCATT
3795

(1603C) stab00

1630
AGGCGAGGCAGCUUGAGUUAAAC
2572
32589
VEGF:1648L21 antisense siNA
UUAACUCAAGCUGCCUCGCTT
3796

(1630C) stab00

1633
CGAGGCAGCUUGAGUUAAACGAA
2573
32590
VEGF:1651L21 antisense siNA
CGUUUAACUCAAGCUGCCUTT
3797

(1633C) stab00

1634
GAGGCAGCUUGAGUUAAACGAAC
2574
32591
VEGF:1652L21 antisense siNA
UCGUUUAACUCAAGCUGCCTT
3798

(1634C) stab00

1635
AGGCAGCUUGAGUUAAACGAACG
2575
32592
VEGF:1653L21 antisense siNA
UUCGUUUAACUCAAGCUGCTT
3799

(1635C) stab00

1636
GGCAGCUUGAGUUAAACGAACGU
2576
32593
VEGF:1654L21 antisense siNA
GUUCGUUUAACUCAAGCUGTT
3800

(1636C) stab00

1648
UAAACGAACGUACUUGCAGAUGU
2577
32594
VEGF:1666L21 antisense siNA
AUCUGCAAGUACGUUCGUUTT
3801

(1648C) stab00

1649
AAACGAACGUACUUGCAGAUGUG
2578
32595
VEGF:1667L21 antisense siNA
CAUCUGCAAGUACGUUCGUTT
3802

(1649C) stab00

1358
UAUGCGGAUCAAACCUCACCAAG
2548
32968
VEGF:1358U21 sense siNA
B uGcGGAucAAAccucAccATT B
3803

stab07

1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32969
VEGF:1419U21 sense siNA
B AuGuGAAuGcAGAccAAAGTT B
3804

stab07

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32970
VEGF:1421U21 sense siNA
B GuGAAuGcAGAccAAAGAATT B
3805

stab07

1596
AAAUGUUCCUGCAAAAACACAGA
2569
32971
VEGF:1596U21 sense siNA
B AuGuuccuGcAAAAAcAcATT B
3806

stab07

1636
GGCAGCUUGAGUUAAACGAACGU
2576
32972
VEGF:1636U21 sense siNA
B cAGcuuGAGuuAAAcGAAcTT B
3807

stab07

1358
UAUGCGGAUCAAACCUCACCAAG
2548
32973
VEGF:1376L21 antisense siNA
uGGuGAGGuuuGAuccGcATsT
3808

(1358C) stab08

1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32974
VEGF:1437L21 antisense siNA
cuuuGGucuGcAuucAcAuTsT
3809

(1419C) stab08

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32975
VEGF:1439L21 antisense siNA
uucuuuGGucuGcAuucAcTsT
3810

(1421C) stab08

1596
AAAUGUUCCUGCAAAAACACAGA
2569
32976
VEGF:1614L21 antisense siNA
uGuGuuuuuGcAGGAAcAuTsT
3811

(1596C) stab08

1636
GGCAGCUUGAGUUAAACGAACGU
2576
32977
VEGF:1654L21 antisense siNA

GuucGuuuAAcucAAGcuGTsT
3812

(1636C) stab08

1358
UAUGCGGAUCAAACCUCACCAAG
2548
32978
VEGF:1358U21 sense siNA
B UGCGGAUCAAACCUCACCATT B
3813

stab09

1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32979
VEGF:1419U21 sense siNA
B AUGUGAAUGCAGACCAAAGTT B
3814

stab09

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32980
VEGF:1421U21 sense siNA
B GUGAAUGCAGACCAAAGAATT B
3815

stab09

1596
AAAUGUUCCUGCAAAAACACAGA
2569
32981
VEGF:1596U21 sense siNA
B AUGUUCCUGCAAAAACACATT B
3816

stab09

1636
GGCAGCUUGAGUUAAACGAACGU
2576
32982
VEGF:1636U21 sense siNA
B CAGCUUGAGUUAAACGAACTT B
3817

stab09

1358
UAUGCGGAUCAAACCUCACCAAG
2548
32983
VEGF:1376L21 antisense siNA
UGGUGAGGUUUGAUCCGCATsT
3818

(1358C) stab10

1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32984
VEGF:1437L21 antisense siNA
CUUUGGUCUGCAUUCACAUTsT
3819

(1419C) stab10

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32985
VEGF:1439L21 antisense siNA
UUCUUUGGUCUGCAUUCACTsT
3820

(1421C) stab10

1596
AAAUGUUCCUGCAAAAACACAGA
2569
32986
VEGF:1614L21 antisense siNA
UGUGUUUUUGCAGGAACAUTsT
3821

(1596C) stab10

1636
GGCAGCUUGAGUUAAACGAACGU
2576
32987
VEGF:1654L21 antisense siNA
GUUCGUUUAACUCAAGCUGTsT
3822

(1636C) stab10

1358
UAUGCGGAUCAAACCUCACCAAG
2548
32998
VEGF:1358U21 sense siNA inv
B AccAcuccAAAcuAGGcGuTT B
3823

stab07

1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32999
VEGF:1419U21 sense siNA inv
B GAAAccAGAcGuAAGuGuATT B
3824

stab07

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
33000
VEGF:1421U21 sense siNA inv
B AAGAAAccAGAcGuAAGuGTT B
3825

stab07

1596
AAAUGUUCCUGCAAAAACACAGA
2569
33001
VEGF:1596U21 sense siNA inv
B AcAcAAAAAcGuccuuGuATT B
3826

stab07

1636
GGCAGCUUGAGUUAAACGAACGU
2576
33002
VEGF:1636U21 sense siNA inv
B cAAGcAAAuuGAGuucGAcTT B
3827

stab07

1358
UAUGCGGAUCAAACCUCACCAAG
2548
33003
VEGF:1376L21 antisense siNA

AcGccuAGuuuGGAGuGGuTsT
3828

(1358C) inv stab08

1419
AAAUGUGAAUGCAGACCAAAGAA
2549
33004
VEGF:1437L21 antisense siNA
uAcAcuuAcGucuGGuuucTsT
3829

(1419C) inv stab08

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
33005
VEGF:1439L21 antisense siNA
cAcuuAcGucuGGuuucuuTsT
3830

(1421C) inv stab08

1596
AAAUGUUCCUGCAAAAACACAGA
2569
33006
VEGF:1614L21 antisense siNA
uAcAAGGAcGuuuuuGuGuTsT
3831

(1596C) inv stab08

1636
GGCAGCUUGAGUUAAACGAACGU
2576
33007
VEGF:1654L21 antisense siNA

GucGAAcucAAuuuGcuuGTsT
3832

(1636C) inv stab08

1358
UAUGCGGAUCAAACCUCACCAAG
2548
33008
VEGF:1358U21 sense siNA inv
B ACCACUCCAAACUAGGCGUTT B
3833

stab09

1419
AAAUGUGAAUGCAGACCAAAGAA
2549
33009
VEGF:1419U21 sense siNA inv
B GAAACCAGACGUAAGUGUATT B
3834

stab09

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
33010
VEGF:1421U21 sense siNA inv
B AAGAAACCAGACGUAAGUGTT B
3835

stab09

1596
AAAUGUUCCUGCAAAAACACAGA
2569
33011
VEGF:1596U21 sense siNA inv
B ACACAAAAACGUCCUUGUATT B
3836

stab09

1636
GGCAGCUUGAGUUAAACGAACGU
2576
33012
VEGF:1636U21 sense siNA inv
B CAAGCAAAUUGAGUUCGACTT B
3837

stab09

1358
UAUGCGGAUCAAACCUCACCAAG
2548
33013
VEGF:1376L21 antisense siNA
ACGCCUAGUUUGGAGUGGUTsT
3838

(1358C) inv stab10

1419
AAAUGUGAAUGCAGACCAAAGAA
2549
33014
VEGF:1437L21 antisense siNA
UACACUUACGUCUGGUUUCTsT
3839

(1419C) inv stab10

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
33015
VEGF:1439L21 antisense siNA
CACUUACGUCUGGUUUCUUTsT
3840

(1421C) inv stab10

1596
AAAUGUUCCUGCAAAAACACAGA
2569
33016
VEGF:1614L21 antisense siNA
UACAAGGACGUUUUUGUGUTsT
3841

(1596C) inv stab10

1636
GGCAGCUUGAGUUAAACGAACGU
2576
33017
VEGF:1654L21 antisense siNA
GUCGAACUCAAUUUGCUUGTsT
3842

(1636C) inv stab10

1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33968
VEGF:1420U21 sense siNA
B UGUGAAUGCAGACCAAAGATT B
3843

stab09

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
33970
VEGF:1423U21 sense siNA
B GAAUGCAGACCAAAGAAAGTT B
3844

stab09

1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33974
VEGF:1438L21 antisense
UCUUUGGUCUGCAUUCACATsT
3845

siNA (1420C) stab10

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
33976
VEGF:1441L21 antisense
CUUUCUUUGGUCUGCAUUCTST
3846

siNA (1423C) stab10

1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33980
VEGF:1420U21 sense siNA
B uGuGAAuGcAGAccAAAGATT B
3847

stab07

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
33982
VEGF:1423U21 sense siNA
B GAAuGcAGAccAAAGAAAGTT B
3848

stab07

1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33986
VEGF:1438L21 antisense siNA
ucuuuGGucuGcAuucAcATsT
3849

(1420C) stab08

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
33988
VEGF:1441L21 antisense siNA
cuuucuuuGGucuGcAuucTsT
3850

(1423C) stab08

1214
GGUGGACAUCUUCCAGGAGUACC
2542
33989
VEGF:1214U21 sense siNA inv
B AUGAGGACCUUCUACAGGUTT B
3851

stab09

1215
GUGGACAUCUUCCAGGAGUACCC
2543
33990
VEGF:1215U21 sense siNA inv
B CAUGAGGACCUUCUACAGGTT B
3852

stab09

1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33992
VEGF:1420U21 sense siNA inv
B AGAAACCAGACGUAAGUGUTT B
3853

stab09

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
33994
VEGF:1423U21 sense siNA inv
B GAAAGAAACCAGACGUAAGTT B
3854

stab09

1214
GGUGGACAUCUUCCAGGAGUACC
2542
33995
VEGF:1232L21 antisense siNA
ACCUGUAGAAGGUCCUCAUTsT
3855

(1214C) inv stab10

1215
GUGGACAUCUUCCAGGAGUACCC
2543
33996
VEGF:1233L21 antisense siNA
CCUGUAGAAGGUCCUCAUGTsT
3856

(1215C) inv stab10

1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33998
VEGF:1438L21 antisense siNA
ACACUUACGUCUGGUUUCUTsT
3857

(1420C) inv stab10

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
34000
VEGF:1441L21 antisense siNA
CUUACGUCUGGUUUCUUUCTsT
3858

(1423C) inv stab10

1214
GGUGGACAUCUUCCAGGAGUACC
2542
34001
VEGF:1214U21 sense siNA inv
B AuGAGGAccuucuAcAGGuTT B
3859

stab07

1215
GUGGACAUCUUCCAGGAGUACCC
2543
34002
VEGF:1215U21 sense siNA inv
B cAuGAGGAccuucuAcAGGTT B
3860

stab07

1420
AAUGUGAAUGCAGACCAAAGAAA
2550
34004
VEGF:1420U21 sense siNA inv
B AGAAAccAGAcGuAAGuGuTT B
3861

stab07

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
34006
VEGF:1423U21 sense siNA inv
B GAAAGAAAccAGAcGuAAGTT B
3862

stab07

1214
GGUGGACAUCUUCCAGGAGUACC
2542
34007
VEGF:1232L21 antisense siNA

AccuGuAGAAGGuccucAuTsT
3863

(1214C) inv stab08

1215
GUGGACAUCUUCCAGGAGUACCC
2543
34008
VEGF:1233L21 antisense siNA
ccuGuAGAAGGuccucAuGTsT
3864

(1215C) inv stab08

1420
AAUGUGAAUGCAGACCAAAGAAA
2550
34010
VEGF:1438L21 antisense siNA

AcAcuuAcGucuGGuuucuTsT
3865

(1420C) inv stab08

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
34012
VEGF:1441L21 antisense siNA
cuuAcGucuGGuuucuuucTsT
3866

(1423C) inv stab08

1366
AAACCUCACCAAGGCCAGCACAU
2579
34062
VEGF:1366U21 sense siNA
ACCUCACCAAGGCCAGCACTT
3867

stab00 (HVEGF5)

1366
AAACCUCACCAAGGCCAGCACAU
2579
34084
VEGF:1384L21 antisense
GUGCUGGCCUUGGUGAGGUTT
3868

siNA (1366C) stab00

(HVEGF5)

1366
AAACCUCACCAAGGCCAGCACAU
2579
34066
VEGF:1366U21 sense siNA
B AccucACcAAGGCCAGCAcTT B
3869

stab07 (HVEGF5)

1366
AAACCUCACCAAGGCCAGCACAU
2579
34068
VEGF:1384L21 antisense siNA
GuGcuGGccuuGGuGAGGuTsT
3870

(1366C) stab08 (HVEGF5)

1366
AAACCUCACCAAGGCCAGCACAU
2579
34070
VEGF:1366U21 sense siNA
B ACCUCACCAAGGCCAGCACTT B
3871

stab09 (HVEGF5)

1366
AAACCUCACCAAGGCCAGCACAU
2579
34072
VEGF:1384L21 antisense siNA
GUGCUGGCCUUGGUGAGGUTsT
3872

(1366C) stab10 (HVEGF5)

1366
AAACCUCACCAAGGCCAGCACAU
2579
34074
VEGF:1366U21 sense siNA inv
CACGACCGGAACCACUCCATT
3873

stab00 (HVEGF5)

1366
AAACCUCACCAAGGCCAGCACAU
2579
34076
VEGF:1384L21 antisense siNA

(1366C) inv stab00 (HVEGF5)
UGGAGUGGUUCCGGUCGUGTT
3874

1366
AAACCUCACCAAGGCCAGCACAU
2579
34078
VEGF:1366U21 sense siNA inv
B cAcGAccGGAAccAcuccATT B
3875

stab07 (HVEGF5)

1366
AAACCUCACCAAGGCCAGCACAU
2579
34080
VEGF:1384L21 antisense siNA
uGGAGuGGuuccGGucGuGTsT
3876

(1366C) inv stab08 (HVEGF5)

1366
AAACCUCACCAAGGCCAGCACAU
2579
34082
VEGF:1366U21 sense siNA inv
B CACGACCGGAACCACUCCATT B
3877

stab09 (HVEGF5)

1366
AAACCUCACCAAGGCCAGCACAU
2579
34084
VEGF:1384L21 antisense siNA
UGGAGUGGUUCCGGUCGUGTsT
3878

(1366C) inv stab10 (HVEGF5)

360
AGAGAGACGGGGUCAGAGAGAGC
2580
34681
VEGF:360U21 sense siNA
AGAGACGGGGUCAGAGAGATT
3879

stab00

1562
AAAGCAUUUGUUUGUACAAGAUC
2581
34682
VEGF:1562U21 sense siNA
AGCAUUUGUUUGUACAAGATT
3880

stab00

360
AGAGAGACGGGGUCAGAGAGAGC
2580
34689
VEGF:378L21 (360C) siRNA
UCUCUCUGACCCCGUCUCUTT
3881

stab00

1562
AAAGCAUUUGUUUGUACAAGAUC
2581
34690
VEGF:1580L21 (1562C) siRNA
UCUUGUACAAACAAAUGCUTT
3882

stab00

162
UCCCUCUUCUUUUUUCUUAAACA
2582
36002
VEGF:162U21 sense siNA
CCUCUUCUUUUUUCUUAAATT
3883

stab00

163
CCCUCUUCUUUUUUCUUAAACAU
2583
36003
VEGF:163U21 sense siNA
CUCUUCUUUUUUCUUAAACTT
3884

stab00

164
CCUCUUCUUUUUUCUUAAACAUU
2584
36004
VEGF:164U21 sense siNA
UCUUCUUUUUUCUUAAACATT
3885

stab00

166
UCUUCUUUUUUCUUAAACAUUUU
2585
36005
VEGF:166U21 sense siNA
UUCUUUUUUCUUAAACAUUTT
3886

stab00

169
UCUUUUUUCUUAAACAUUUUUUU
2586
36006
VEGF:169U21 sense siNA
UUUUUUCUUAAACAUUUUUTT
3887

stab00

171
UUUUUUCUUAAACAUUUUUUUUU
2587
36007
VEGF:171U21 sense siNA
UUUUCUUAAACAUUUUUUUTT
3888

stab00

172
UUUUUCUUAAACAUUUUUUUUUA
2588
36008
VEGF:172U21 sense siNA
UUUCUUAAACAUUUUUUUUTT
3889

stab00

181
AACAUUUUUUUUUAAAACUGUAU
2589
36009
VEGF:181U21 sense siNA
CAUUUUUUUUUAAAACUGUTT
3890

stab00

187
UUUUUUUAAAACUGUAUUGUUUC
2590
36010
VEGF:187U21 sense siNA
UUUUUAAAACUGUAUUGUUTT
3891

stab00

188
UUUUUUAAAACUGUAUUGUUUCU
2591
36011
VEGF:188U21 sense siNA
UUUUAAAACUGUAUUGUUUTT
3892

stab00

192
UUAAAACUGUAUUGUUUCUCGUU
2592
36012
VEGF:192U21 sense siNA
AAAACUGUAUUGUUUCUCGTT
3893

stab00

202
AUUGUUUCUCGUUUUAAUUUAUU
2593
36013
VEGF:202U21 sense siNA
UGUUUCUCGUUUUAAUUUATT
3894

stab00

220
UUAUUUUUGCUUGCCAUUCCCCA
2594
36014
VEGF:220U21 sense siNA
AUUUUUGCUUGCCAUUCCCTT
3895

stab00

237
UCCCCACUUGAAUCGGGCCGACG
2595
36015
VEGF:237U21 sense siNA
CCCACUUGAAUCGGGCCGATT
3896

stab00

238
CCCCACUUGAAUCGGGCCGACGG
2596
36016
VEGF:238U21 sense siNA
CCACUUGAAUCGGGCCGACTT
3897

stab00

338
CUCCAGAGAGAAGUCGAGGAAGA
2597
36017
VEGF:338U21 sense siNA
CCAGAGAGAAGUCGAGGAATT
3898

stab00

339
UCCAGAGAGAAGUCGAGGAAGAG
2598
36018
VEGF:339U21 sense siNA
CAGAGAGAAGUCGAGGAAGTT
3899

stab00

371
GUCAGAGAGAGCGCGCGGGCGUG
2599
36019
VEGF:371U21 sense siNA
CAGAGAGAGCGCGCGGGCGTT
3900

stab00

484
GCAGCUGACCAGUCGCGCUGACG
2600
36020
VEGF:484U21 sense siNA
AGCUGACCAGUCGCGCUGATT
3901

stab00

598
GGCCGGAGCCCGCGCCCGGAGGC
2601
36021
VEGF:598U21 sense siNA
CCGGAGCCCGCGCCCGGAGTT
3902

stab00

599
GCCGGAGCCCGCGCCCGGAGGCG
2602
36022
VEGF:599U21 sense siNA stab00
CGGAGCCCGCGCCCGGAGGTT
3903

600
CCGGAGCCCGCGCCCGGAGGCGG
2603
36023
VEGF:600U21 sense siNA stab00
GGAGCCCGCGCCCGGAGGCTT
3904

652
CACUGAAACUUUUCGUCCAACUU
2604
36024
VEGF:652U21 sense siNA stab00
CUGAAACUUUUCGUCCAACTT
3905

653
ACUGAAACUUUUCGUCCAACUUC
2605
36025
VEGF:653U21 sense siNA stab00
UGAAACUUUUCGUCCAACUTT
3906

654
CUGAAACUUUUCGUCCAACUUCU
2606
36026
VEGF:654U21 sense siNA stab00
GAAACUUUUCGUCCAACUUTT
3907

658
AACUUUUCGUCCAACUUCUGGGC
2607
36027
VEGF:658U21 sense siNA stab00
CUUUUCGUCCAACUUCUGGTT
3908

672
CUUCUGGGCUGUUCUCGCUUCGG
2608
36028
VEGF:672U21 sense siNA stab00
UCUGGGCUGUUCUCGCUUCTT
3909

674
UCUGGGCUGUUCUCGCUUCGGAG
2609
36029
VEGF:674U21 sense siNA stab00
UGGGCUGUUCUCGCUUCGGTT
3910

691
UCGGAGGAGCCGUGGUCCGCGCG
2610
36030
VEGF:691U21 sense siNA stab00
GGAGGAGCCGUGGUCCGCGTT
3911

692
CGGAGGAGCCGUGGUCCGCGCGG
2611
36031
VEGF:692U21 sense siNA stab00
GAGGAGCCGUGGUCCGCGCTT
3912

758
CCGGGAGGAGCCGCAGCCGGAGG
2612
36032
VEGF:758U21 sense siNA stab00
GGGAGGAGCCGCAGCCGGATT
3913

759
CGGGAGGAGCCGCAGCCGGAGGA
2613
36033
VEGF:759U21 sense siNA stab00
GGAGGAGCCGCAGCCGGAGTT
3914

760
GGGAGGAGCCGCAGCCGGAGGAG
2614
36034
VEGF:760U21 sense siNA stab00
GAGGAGCCGCAGCCGGAGGTT
3915

795
GAAGAGAAGGAAGAGGAGAGGGG
2615
36035
VEGF:795U21 sense siNA stab00
AGAGAAGGAAGAGGAGAGGTT
3916

886
GUGCUCCAGCCGCGCGCGCUCCC
2616
36036
VEGF:886U21 sense siNA stab00
GCUCCAGCCGCGCGCGCUCTT
3917

977
GCCCCACAGCCCGAGCCGGAGAG
2617
36037
VEGF:977U21 sense siNA stab00
CCCACAGCCCGAGCCGGAGTT
3918

978
CCCCACAGCCCGAGCCGGAGAGG
2618
36038
VEGF:978U21 sense siNA stab00
CCACAGCCCGAGCCGGAGATT
3919

1038
ACCAUGAACUUCUGCUGUUCUUG
2619
36039
VEGF:1038U21 sense siNA
CAUGAACUUUCUGCUGUCUTT
3920

stab00

1043
GAACUUUCUGCUGUCUUGGGUGC
2620
36040
VEGF:1043U21 sense siNA
ACUUUCUGCUGUCUUGGGUTT
3921

stab00

1049
UCUGCUGUCUUGGGUGCAUUGGA
2621
36041
VEGF:1049U21 sense siNA
UGCUGUCUUGGGUGCAUUGTT
3922

stab00

1061
GGUGCAUUGGAGCCUUGCCUUGC
2622
36042
VEGF:1061U21 sense siNA
UGCAUUGGAGCCUUGCCUUTT
3923

stab00

1072
GCCUUGCCUUGCUGCUCUACCUC
2623
36043
VEGF:1072U21 sense siNA
CUUGCCUUGCUGCUCUACCTT
3924

stab00

1088
UCACCUCCACCAUGCCAAGUGGU
2624
36044
VEGF:1088U21 sense siNA
ACCUCCACCAUGCCAAGUGTT
3925

stab00

1089
CUCCUCCACCAUGCCAAGUGGUC
2625
36045
VEGF:1089U21 sense siNA
CCUCCACCAUGCCAAGUGGTT
3926

stab00

1095
CACCAUGCCAAGUGGUCCCAGGC
2626
36046
VEGF:1095U21 sense siNA
CCAUGCCAAGUGGUCCCAGTT
3927

stab00

1110
UCCCAGGCUGCACCCAUGGCAGA
2627
36047
VEGF:1110U21 sense siNA
CCAGGCUGCACCCAUGGCATT
3928

stab00

1175
AUUCUAUCAGCGCAGCUACUGCC
2628
36048
VEGF:1175U21 sense siNA
UCUAUCAGCGCAGCUACUGTT
3929

stab00

1220
CAUCUUCCAGGAGUACCCUGAUG
2629
36049
VEGF:1220U21 sense siNA
UCUUCCAGGAGUACCCUGATT
3930

stab00

1253
CAUCUUCAAGCCAUCCUGUGUGC
2630
36050
VEGF:1253U21 sense siNA
UCUUCAAGCCAUCCUGUGUTT
3931

stab00

1300
CUAAUGACGAGGGCCUGGAGUGU
2631
36051
VEGF:1300U21 sense siNA
AAUGACGAGGGCCUGGAGUTT
3932

stab00

1309
CGGGCCUGGAGUGUGUGCCCACU
2632
36052
VEGF:1309U21 sense siNA
GGCCUGGAGUGUGUGCCCATT
3933

stab00

1326
CCCACUGAGGAGUCCAACAUCAC
2633
36053
VEGF:1326U21 sense siNA
CACUGAGGAGUCCAACAUCTT
3934

stab00

1338
UCCAACAUCACCAUGCAGAUUAU
2634
36054
VEGF:1338U21 sense siNA
CAACAUCACCAUGCAGAUUTT
3935

stab00

1342
ACAUCACCAUGCAGAUUAUGCGG
2635
36055
VEGF:1342U21 sense siNA
AUCACCAUGCAGAUUAUGCTT
3936

stab00

1351
UGCAGAUUAUGCGGAUCAAACCU
2636
36056
VEGF:1351U21 sense siNA
CAGAUUAUGCGGAUCAAACTT
3937

stab00

1352
GCAGAUUAUGCGGAUCAAACCUC
2637
36057
VEGF:1352U21 sense siNA
AGAUUAUGCGGAUCAAACCTT
3938

stab00

1353
CAGAUUAUGCGGAUCAAACCUCA
2638
36058
VEGF:1353U21 sense siNA
GAUUAUGCGGAUCAAACCUTT
3939

stab00

1389
AUAGGAGAGAUGAGCUUCCUACA
2639
36059
VEGF:1389U21 sense siNA
AGGAGAGAUGAGCUUCCUATT
3940

stab00

1398
GAGAGCUUCCUACAGCACAACAA
2640
36060
VEGF:1398U21 sense siNA
GAGCUUCCUACAGCACAACTT
3941

stab00

1401
AGCUUCCUACAGCACAACAAAUG
2641
36061
VEGF:1401U21 sense siNA
CUUCCUACAGCACAACAAATT
3942

stab00

1407
CCACAGCACAACAAAUGUGAAUG
2642
36062
VEGF:1407U21 sense siNA
ACAGCACAACAAAUGUGAATT
3943

stab00

1408
UACAGCACAACAAAUGUGAAUGC
2643
36063
VEGF:1408U21 sense siNA
CAGCACAACAAAUGUGAAUTT
3944

stab00

1417
ACAAAUGUGAAUGCAGACCAAAG
2644
36064
VEGF:1417U21 sense siNA
AAAUGUGAAUGCAGACCAATT
3945

stab00

162
UCCCUCUUCUUUUUUCUUAAACA
2582
36065
VEGF:180L21 antisense siNA
UUUAAGAAAAAAGAAGAGGTT
3946

(162C) stab00

163
CCCUCUUCUUUUUUCUUAAACAU
2583
36066
VEGF:181L21 antisense siNA
GUUUAAGAAAAAAGAAGAGTT
3947

(163C) stab00

164
CCUCUUCUUUUUUCUUAAACAUU
2584
36067
VEGF:182L21 antisense siNA
UGUUUAAGAAAAAAGAAGATT
3948

(164C) stab00

166
UCUUCUUUUUUCUUAAACAUUUU
2585
36068
VEGF:184L21 antisense siNA
AAUGUUUAAGAAAAAAGAATT
3949

(166C) stab00

169
UCUUUUUUCUUAAACAUUUUUUU
2586
36069
VEGF:187L21 antisense siNA
AAAAAUGUUUAAGAAAAAATT
3950

(169C) stab00

171
UUUUUUCUUAAACAUUUUUUUUU
2587
36070
VEGF:189L21 antisense siNA
AAAAAAAUGUUUAAGAAAATT
3951

(171C) stab00

172
UUUUUCUUAAACAUUUUUUUUUA
2588
36071
VEGF:190L21 antisense siNA
AAAAAAAAUGUUUAAGAAATT
3952

(172C) stab00

181
AACAUUUUUUUUUAAAACUGUAU
2589
36072
VEGF:199L21 antisense siNA
ACAGUUUUAAAAAAAAAUGTT
3953

(181C) stab00

187
UUUUUUUAAAACUGUAUUGUUUC
2590
36073
VEGF:205L21 antisense siNA
AACAAUACAGUUUUAAAAATT
3954

(187C) stab00

188
UUUUUUAAAACUGUAUUGUUUCU
2591
36074
VEGF:206L21 antisense siNA
AAACAAUACAGUUUUAAAATT
3955

(188C) stab00

192
UUAAAACUGUAUUGUUUCUCGUU
2592
36075
VEGF:210L21 antisense siNA
CGAGAAACAAUACAGUUUUTT
3956

(192C) stab00

202
AUUGUUUCUCGUUUUAAUUUAUU
2593
36076
VEGF:220L21 antisense siNA
UAAAUUAAAACGAGAAACATT
3957

(202C) stab00

220
UUAUUUUUGCUUGCCAUUCCCCA
2594
36077
VEGF:238L21 antisense siNA
GGGAAUGGCAAGCAAAAAUTT
3958

(220C) stab00

237
UCCCCACUUGAAUCGGGCCGACG
2595
36078
VEGF:255L21 antisense siNA
UCGGCCCGAUUCAAGUGGGTT
3959

(237C) stab00

238
CCCCACUUGAAUCGGGCCGACGG
2596
36079
VEGF:258L21 antisense siNA
GUCGGCCCGAUUCAAGUGGTT
3960

(238C) stab00

338
CUCCAGAGAGAAGUCGAGGAAGA
2597
36080
VEGF:356L21 antisense siNA
UUCCUCGACUUCUCUCUGGTT
3961

(338C) stab00

339
UCCAGAGAGAAGUCGAGGAAGAG
2598
36081
VEGF:357L21 antisense siNA
CUUCCUCGACUUCUCUCUGTT
3962

(339C) stab00

371
GUCAGAGAGAGCGCGCGGGCGUG
2599
36082
VEGF:389L21 antisense siNA
CGCCCGCGCGCUCUCUCUGTT
3963

(371C) stab00

484
GCAGCUGACCAGUCGCGCUGACG
2600
36083
VEGF:502L21 antisense siNA
UCAGCGCGACUGGUCAGCUTT
3964

(484C) stab00

598
GGCCGGAGCCCGCGCCCGGAGGC
2601
36084
VEGF:616L21 antisense siNA
CUCCGGGCGCGGGCUCCGGTT
3965

(598C) stab00

599
GCCGGAGCCCGCGCCCGGAGGCG
2602
36085
VEGF:617L21 antisense siNA
CCUCCGGGCGCGGGCUCCGTT
3966

(599C) stab00

600
CCGGAGCCCGCGCCCGGAGGCGG
2603
36086
VEGF:618L21 antisense siNA
GCCUCCGGGCGCGGGCUCCTT
3967

(600C) stab00

652
CACUGAAACUUUUCGUCCAACUU
2604
36087
VEGF:670L21 antisense siNA
GUUGGACGAAAAGUUUCAGTT
3968

(652C) stab00

653
ACUGAAACUUUUCGUCCAACUUC
2605
36088
VEGF:671L21 antisense siNA
AGUUGGACGAAAAGUUUCATT
3969

(653C) stab00

654
CUGAAACUUUUCGUCCAACUUCU
2606
36089
VEGF:672L21 antisense siNA
AAGUUGGACGAAAAGUUUCTT
3970

(654C) stab00

658
AACUUUUCGUCCAACUUCUGGGC
2607
36090
VEGF:676L21 antisense siNA
CCAGAAGUUGGACGAAAAGTT
3971

(658C) stab00

672
CUUCUGGGCUGUUCUCGCUUCGG
2608
36091
VEGF:690L21 antisense siNA
GAAGCGAGAACAGCCCAGATT
3972

(672C) stab00

674
UCUGGGCUGUUCUCGCUUCGGAG
2609
36092
VEGF:692L21 antisense siNA
CCGAAGCGAGAACAGCCCATT
3973

(674C) stab00

691
UCGGAGGAGCCGUGGUCCGCGCG
2610
36093
VEGF:709L21 antisense siNA
CGCGGACCACGGCUCCUCCTT
3974

(691C) stab00

692
CGGAGGAGCCGUGGUCCGCGCGG
2611
36094
VEGF:710L21 antisense siNA
GCGCGGACCACGGCUCCUCTT
3975

(692C) stab00

758
CCGGGAGGAGCCGCAGCCGGAGG
2612
36095
VEGF:776L21 antisense siNA
UCCGGCUGCGGCUCCUCCCTT
3976

(758C) stab00

759
CGGGAGGAGCCGCAGCCGGAGGA
2613
36096
VEGF:777121 antisense siNA
CUCCGGCUGCGGCUCCUCCTT
3977

(759C) stab00

760
GGGAGGAGCCGCAGCCGGAGGAG
2614
36097
VEGF:778L21 antisense siNA
CCUCCGGCUGCGGCUCCUCTT
3978

(760C) stab00

795
GAAGAGAAGGAAGAGGAGAGGGG
2615
36098
VEGF:813L21 antisense siNA
CCUCUCCUCUUCCUUCUCUTT
3979

(795C) stab00

886
GUGCUCCAGCCGCGCGCGCUCCC
2616
36099
VEGF:904L21 antisense siNA
GAGCGCGCGCGGCUGGAGCTT
3980

(886C) stab00

977
GCCCCACAGCCCGAGCCGGAGAG
2617
36100
VEGF:995L21 antisense siNA
CUCCGGCUCGGGCUGUGGGTT
3981

(977C) stab00

978
CCCCACAGCCCGAGCCGGAGAGG
2618
36101
VEGF:996L21 antisense siNA
UCUCCGGCUCGGGCUGUGGTT
3982

(978C) stab00

1038
ACCAUGAACUUUCUGCUGUCUUG
2619
36102
VEGF:1056L21 antisense siNA
AGACAGCAGAAAGUUCAUGTT
3983

(1038C) stab00

1043
GAACUUUCUGCUGUCUUGGGUGC
2620
36103
VEGF:1061L21 antisense siNA
ACCCAAGACAGCAGAAAGUTT
3984

(1043C) stab00

1049
UCUGCUGUCUUGGGUGCAUUGGA
2621
36104
VEGF:1067L21 antisense siNA
CAAUGCACCCAAGACAGCATT
3985

(1049C) stab00

1061
GGUGCAUUGGAGCCUUGCCUUGC
2622
36105
VEGF:1079L21 antisense siNA
AAGGCAAGGCUCCAAUGCATT
3986

(1061C) stab00

1072
GCCUUGCCUUGCUGCUCUACCUC
2623
36106
VEGF:1090L21 antisense siNA
GGUAGAGCAGCAAGGCAAGTT
3987

(1072C) stab00

1088
UCACCUCCACCAUGCCAAGUGGU
2624
36107
VEGF:1106L21 antisense siNA
CACUUGGCAUGGUGGAGGUTT
3988

(1088C) stab00

1089
CUCCUCCACCAUGCCAAGUGGUC
2625
36108
VEGF:1107L21 antisense siNA
CCACUUGGCAUGGUGGAGGTT
3989

(1089C) stab00

1095
CACCAUGCCAAGUGGUCCCAGGC
2626
36109
VEGF:1113L21 antisense siNA
CUGGGACCACUUGGCAUGGTT
3990

(1095C) stab00

1110
UCCCAGGCUGCACCCAUGGCAGA
2627
36110
VEGF:1128L21 antisense siNA
UGCCAUGGGUGCAGCCUGGTT
3991

(1110C) stab00

1175
AUUCUAUCAGCGCAGCUACUGCC
2628
36111
VEGF:1193L21 antisense siNA
CAGUAGCUGCGCUGAUAGATT
3992

(1175C) stab00

1220
CAUCUUCCAGGAGUACCCUGAUG
2629
36112
VEGF:1238L21 antisense siNA
UCAGGGUACUCCUGGAAGATT
3993

(1220C) stab00

1253
CAUCUUCAAGCCAUCCUGUGUGC
2630
36113
VEGF:1271L21 antisense siNA
ACACAGGAUGGCUUGAAGATT
3994

(1253C) stab00

1300
CUAAUGACGAGGGCCUGGAGUGU
2631
36114
VEGF:1318L21 antisense siNA
ACUCCAGGCCCUCGUCAUUTT
3995

(1300C) stab00

1309
CGGGCCUGGAGUGUGUGCCCACU
2632
36115
VEGF:1327L21 antisense siNA
UGGGCACACAOUCCAGGCCTT
3996

(1309C) stab00

1326
CCCACUGAGGAGUCCAACAUCAC
2633
36116
VEGF:1344L21 antisense siNA
GAUGUUGGACUOCUCAGUGTT
3997

(1326C) stab00

1338
UCCAACAUCACCAUGCAGAUUAU
2634
36117
VEGF:1356L21 antisense siNA
AAUCUGCAUGGUGAUGUUGTT
3998

(1338C) stab00

1342
ACAUCACCAUGCAGAUUAUGCGG
2635
36118
VEGF:1360L21 antisense siNA
GCAUAAUCUGCAUGGUGAUTT
3999

(1342C) stab00

1351
UGCAGAUUAUGCGGAUCAAACCU
2636
36119
VEGF:1369L21 antisense siNA
GUUUGAUCCGCAUAAUCUGTT
4000

(1351C) stab00

1352
GCAGAUUAUGCGGAUCAAACCUC
2637
36120
VEGF:1370L21 antisense siNA
GGUUUGAUCCGCAUAAUCUTT
4001

(1352C) stab00

1353
CAGAUUAUGCGGAUCAAACCUCA
2638
36121
VEGF:1371L21 antisense siNA
AGGUUUGAUCCGCAUAAUCTT
4002

(1353C) stab00

1389
AUAGGAGAGAUGAGCUUCCUACA
2639
36122
VEGF:1407L21 antisense siNA
UAGGAAGCUCAUCUCUCCUTT
4003

(1389C) stab00

1398
GAGAGCUUCCUACAGCACAACAA
2640
36123
VEGF:1416L21 antisense siNA
GUUGUGOUGUAGGAAGCUCTT
4004

(1398C) stab00

1401
AGCUUCCUACAGCACAACAAAUG
2641
36124
VEGF:1419L21 antisense siNA
UUUGUUGUGCUGUAGGAAGTT
4005

(1401C) stab00

1407
CCACAGCACAACAAAUGUGAAUG
2642
36125
VEGF:1425L21 antisense siNA
UUCACAUUUGUUGUGCUGUTT
4006

(1407C) stab00

1408
UACAGCACAACAAAUGUGAAUGC
2643
36126
VEGF:1426L21 antisense siNA
AUUCACAUUUGUUGUGCUGTT
4007

(1408C) stab00

1417
ACAAAUGUGAAUGCAGACCAAAG
2644
36127
VEGF:1435L21 antisense siNA
UUGGUCUGCAUUCACAUUUTT
4008

(1417C) stab00

1089
UACCUCOACCAUGCCAAGUGGUC
2645
37293
VEGF:1089U21 sense siNA
B ccuccAccAuGccAAGuGGTT B
4009

stab07

1090
ACCUCCACCAUGCCAAGUGGUCC
2646
37294
VEGF:1090U21 sense siNA
B cuccAccAuGccAAGuGGuTT B
4010

stab07

1095
CACCAUGCCAAGUGGUCCCAGGC
2626
37295
VEGF:1095U21 sense siNA
B ccAuGccAAGuGGucccAGTT B
4011

stab07

1096
ACCAUGCCAAGUGGUCCCAGGCU
2647
37296
VEGF:1096U21 sense siNA
B cAuGccAAGuGGucccAGGTT B
4012

stab07

1097
CCAUGCCAAGUGGUCCCAGGCUG
2648
37297
VEGF:1097U21 sense siNA
B AuGccAAGuGGucccAGGcTT B
4013

stab07

1098
CAUGCCAAGUGGUCCCAGGCUGC
2649
37298
VEGF:1098U21 sense siNA
B uGccAAGuGGucccAGGcuTT B
4014

stab07

1099
AUGCCAAGUGGUCCCAGGCUGCA
2650
37299
VEGF:1099U21 sense siNA
B GccAAGuGGucccAGGcuGTT B
4015

stab07

1100
UGCCAAGUGGUCCCAGGCUGCAC
2651
37300
VEGF:1100U21 sense siNA
B ccAAGuGGucccAGGcuGcTT B
4016

stab07

1104
AAGUGGUCCCAGGCUGCACCCAU
2652
37301
VEGF:1104U21 sense siNA
B GuGGucccAGGcuGcAcccTT B
4017

stab07

1105
AGUGGUCCCAGGCUGCACCCAUG
2653
37302
VEGF:1105U21 sense siNA
B uGGucccAGGcuGcAcccATT B
4018

stab07

1208
GACCCUGGUGGACAUCUUCCAGG
2562
37303
VEGF:1208U21 sense siNA
B cccuGGuGGAcAucuuccATT B
4019

stab07

1424
UGAAUGCAGACCAAAGAAAGAUA
2654
37304
VEGF:1424U21 sense siNA
B AAuGcAGAccAAAGAAAGATT B
4020

stab07

1549
GCUCAGAGCGGAGAAAGCAUUUG
2655
37305
VEGF:1549U21 sense siNA
B ucAGAGcGGAGAAAGcAuuTT B
4021

stab07

1584
CCGCAGACGUGUAAAUGUUCCUG
2565
37306
VEGF:1584U21 sense siNA
B GcAGAcGuGuAAAuGuccTT B
4022

stab07

1585
CGCAGACGUGUAAAUGUUCCUGC
2566
37307
VEGF:1585U21 sense siNA
B cAGAcGuGuAAAuGuuccuTT B
4023

stab07

1589
GACGUGUAAAUGUUCCUGCAAAA
2567
37308
VEGF:1589U21 sense siNA
B cGuGuAAAuGuuccuGcAATT B
4024

stab07

1591
CGUGUAAAUGUUCCUGCAAAAAC
2554
37309
VEGF:1591U21 sense siNA
B uGuAAAuGuuccuGcAAAATT B
4025

stab07

1592
GUGUAAAUGUUCCUGCAAAAACA
2555
37310
VEGF:1592U21 sense siNA
B GuAAAuGuuccuGcAAAAATT B
4026

stab07

1593
UGUAAAUGUUCCUGCAAAAACAC
2556
37311
VEGF:1593U21 sense siNA
B uAAAuGuuccuGcAAAAAcTT B
4027

stab07

1594
GUAAAUGUUCCUGCAAAAACACA
2557
37312
VEGF:1594U21 sense siNA
B AAAuGuuccuGcAAAAAcATT B
4028

stab07

1595
UAAAUGUUCCUGCAAAAACACAG
2568
37313
VEGF:1595U21 sense siNA
B AAuGuuccuGcAAAAAcAcTT B
4029

stab07

1597
AAUGUUCCUGCAAAAACACAGAC
2656
37314
VEGF:1597U21 sense siNA
B uGuuccuGcAAAAAcAcAGTT B
4030

stab07

1598
AUGUUCCUGCAAAAACACAGACU
2657
37315
VEGF:1598U21 sense siNA
B GuuccuGcAAAAAcAcAGATT B
4031

stab07

1599
UGUUCCUGCAAAAACACAGACUC
2658
37316
VEGF:1599U21 sense siNA
B uuccuGcAAAAAcAcAGAcTT B
4032

stab07

1600
GUUCCUGCAAAAACACAGACUCG
2659
37317
VEGF:1600U21 sense siNA
B uccuGcAAAAAcAcAGACuTT B
4033

stab07

1604
CUGCAAAAACACAGACUCGCGUU
2558
37318
VEGF:1604U21 sense siNA
B GcAAAAAcAcAGAcucGcGTT B
4034

stab07

1605
UGCAAAAACACAGACUCGCGUUG
2660
37319
VEGF:1605U21 sense siNA
B cAAAAAcAcAGAcucGcGuTT B
4035

stab07

1608
AAAAACACAGACUCGCGUUGCAA
2661
37320
VEGF:1608U21 sense siNA
B AAAcAcAGAcucGcGuuGcTT B
4036

stab07

1612
ACACAGACUCGCGUUGCAAGGCG
2662
37321
VEGF:1612U21 sense siNA
B AcAGAcucGcGuuGcAAGGTT B
4037

stab07

1616
AGACUCGCGUUGCAAGGCGAGGC
2663
37322
VEGF:1616U21 sense siNA
B AcucGcGuuGcAAGGcGAGTT B
4038

stab07

1622
GCGUUGCAAGGCGAGGCAGCUUG
2664
37323
VEGF:1622U21 sense siNA
B GuuGcAAGGcGAGGcAGcuTT B
4039

stab07

1626
UGCAAGGCGAGGCAGCUUGAGUU
2665
37324
VEGF:1626U21 sense siNA
B cAAGGcGAGGcAGcuuGAGTT B
4040

stab07

1628
CAAGGCGAGGCAGCUUGAGUUAA
2666
37325
VEGF:1628U21 sense siNA
B AGGcGAGGcAGcuuGAGuuTT B
4041

stab07

1633
CGAGGCAGCUUGAGUUAAACGAA
2573
37326
VEGF:1633U21 sense siNA
B AGGcAGCuuGAGuuAAAcGTT B
4042

stab07

1634
GAGGCAGCUUGAGUUAAACGAAC
2574
37327
VEGF:1634U21 sense siNA
B GGcAGcuuGAGuuAAAcGATT B
4043

stab07

1635
AGGCAGCUUGAGUUAAACGAACG
2575
37328
VEGF:1635U21 sense siNA
B GcAGcuuGAGuuAAAcGAATT B
4044

stab07

1637
GCAGCUUGAGUUAAACGAACGUA
2559
37329
VEGF:1637U21 sense siNA
B AGcuuGAGuuAAAcGAAcGTT B
4045

stab07

1643
UGAGUUAAACGAACGUACUUGCA
2667
37330
VEGF:1643U21 sense siNA
B AGuuAAAcGAAcGuAcuuGTT B
4046

stab07

1645
AGUUAAACGAACGUACUUGCAGA
2668
37331
VEGF:1645U21 sense siNA
B uuAAAcGAAcGuAcuuGcATT B
4047

stab07

1646
GUUAAACGAACGUACUUGCAGAU
2669
37332
VEGF:1646U21 sense siNA
B uAAAcGAAcGuAcuuGcAGTT B
4048

stab07

1647
UUAAACGAACGUACUUGCAGAUG
2670
37333
VEGF:1647U21 sense siNA
B AAAcGAAcGuAcuuGcAGATT B
4049

stab07

1648
UAAACGAACGUACUUGCAGAUGU
2577
37334
VEGF:1648U21 sense siNA
B AAcGAAcGuAcuuGcAGAuTT B
4050

stab07

1655
ACGUACUUGCAGAUGUGACAAGC
2671
37335
VEGF:1655U21 sense siNA
B GuAcuuGcAGAuGuGAcAATT B
4051

stab07

1656
CGUACUUGCAGAUGUGACAAGCC
2560
37336
VEGF:1656U21 sense siNA
B uAcuuGcAGAuGuGAcAAGTT B
4052

stab07

1657
GUACUUGCAGAUGUGACAAGCCG
2672
37337
VEGF:1657U21 sense siNA
B AcuuGcAGAuGuGAcAAGcTT B
4053

stab07

1089
UACCUCCACCAUGCCAAGUGGUC
2645
37338
VEGF:1107L21 antisense
ccAcuuGGcAuGGuGGAGGTT
4054

siNA (1089C) stab26

1090
ACCUCCACCAUGCCAAGUGGUCC
2646
37339
VEGF:1108L21 antisense
ACCAcuuGGcAuGGuGGAGTT
4055

siNA (1090C) stab26

1095
CACCAUGCCAAGUGGUCCCAGGC
2626
37340
VEGF:1113L21 antisense
CUGGGAccAcuuGGcAuGGTT
4056

siNA (1095C) stab26

1096
ACCAUGCCAAGUGGUCCCAGGCU
2647
37341
VEGF:1114L21 antisense
CCUGGGAccAcuuGGcAuGTT
4057

siNA (1096C) stab26

1097
CCAUGCCAAGUGGUCCCAGGCUG
2648
37342
VEGF:1115L21 antisense
GCCuGGGAccAcuuGGcAuTT
4058

siNA (1097C) stab26

1098
CAUGCCAAGUGGUCCCAGGCUGC
2649
37343
VEGF:1116L21 antisense
AGCcuGGGAccAcuuGGcATT
4059

siNA (1098C) stab26

1099
AUGCCAAGUGGUCCCAGGCUGCA
2650
37344
VEGF:1117L21 antisense
CAGccuGGGAccAcuuGGcTT
4060

siNA (1099C) stab26

1100
UGCCAAGUGGUCCCAGGCUGCAC
2651
37345
VEGF:1118L21 antisense
GCAGccuGGGAccAcuuGGTT
4061

siNA (1100C) stab26

1104
AAGUGGUCCCAGGCUGCACCCAU
2652
37346
VEGF:1122L21 antisense
GGGuGcAGccuGGGAccAcTT
4062

siNA (1104C) stab26

1105
AGUGGUCCCAGGCUGCACCCAUG
2653
37347
VEGF:1123L21 antisense
UGGGuGcAGccuGGGAccATT
4063

siNA (1105C) stab26

1208
GACCCUGGUGGACAUCUUCCAGG
2562
37348
VEGF:1226L21 antisense
UGGAAGAuGuccAccAGGGTT
4064

siNA (1208C) stab26

1214
GGUGGACAUCUUCCAGGAGUACC
2542
37349
VEGF:1232L21 antisense
UACuccuGGAAGAuGuccATT
4065

siNA (1214C) stab26

1421
AUGUGAAUGCAGACCAAAGAAAG
2551
37350
VEGF:1439L21 antisense
UUCuuuGGucuGcAuucAcTT
4066

siNA (1421C) stab26

1423
GUGAAUGCAGACCAAAGAAAGAU
2552
37351
VEGF:1441L21 antisense
CUUucuuuGGucuGcAuucTT
4067

siNA (1423C) stab26

1424
UGAAUGCAGACCAAAGAAAGAUA
2654
37352
VEGF:1442L21 antisense
UCUuucuuuGGucuGcAuuTT
4068

siNA (1424C) stab26

1549
GCUCAGAGCGGAGAAAGCAUUUG
2655
37353
VEGF:1567L21 antisense
AAUGcuuucuccGcucuGATT
4069

siNA (1549C) stab26

1584
CCGCAGACGUGUAAAUGUUCCUG
2565
37354
VEGF:1602L21 antisense
GGAAcAuuuAcAcGucuGcTT
4070

siNA (1584C) stab26

1585
CGCAGACGUGUAAAUGUUCCUGC
2566
37355
VEGF:1603L21 antisense
AGGAAcAuuuAcAcGucuGTT
4071

siNA (1585C) stab26

1589
GACGUGUAAAUGUUCCUGCAAAA
2567
37356
VEGF:1607L21 antisense
UUGcAGGAAcAuuuAcAcGTT
4072

siNA (1589C) stab26

1591
CGUGUAAAUGUUCCUGCAAAAAC
2554
37357
VEGF:1609L21 antisense
UUUuGcAGGAAcAuuuAcATT
4073

siNA (1591C) stab26

1592
GUGUAAAUGUUCCUGCAAAAACA
2555
37358
VEGF:1610L21 antisense
UUUuuGcAGGAAcAuuuAcTT
4074

siNA (1592C) stab26

1593
UGUAAAUGUUCCUGCAAAAACAC
2556
37359
VEGF:1611L21 antisense
GUUuuuGcAGGAAcAuuuATT
4075

siNA (1593C) stab26

1594
GUAAAUGUUCCUGCAAAAACACA
2557
37360
VEGF:1612L21 antisense
UGUuuuuGcAGGAAcAuuuTT
4076

siNA (1594C) stab26

1595
UAAAUGUUCCUGCAAAAACACAG
2568
37361
VEGF:1613L21 antisense
GUGuuuuuGcAGGAACAuuTT
4077

siNA (1595C) stab26

1597
AAUGUUCCUGCAAAAACACAGAC
2656
37362
VEGF:1615L21 antisense
CUGuGuuuuuGcAGGAAcATT
4078

siNA (1597C) stab26

1598
AUGUUCCUGCAAAAACACAGACU
2657
37363
VEGF:1616L21 antisense
UCUGuGuuuuuGcAGGAAcTT
4079

siNA (1598C) stab26

1599
UGUUCCUGCAAAAACACAGACUC
2658
37364
VEGF:1617L21 antisense
GUCuGuGuuuuuGcAGGAATT
4080

siNA (1599C) stab26

1600
GUUCCUGCAAAAACACAGACUCG
2659
37365
VEGF:1618L21 antisense
AGUcuGuGuuuuuGcAGGATT
4081

siNA (1600C) stab26

1604
CUGCAAAAACACAGACUCGCGUU
2558
37366
VEGF:1622L21 antisense
CGCGAGucuGuGuuuuuGcTT
4082

siNA (1604C) stab26

1605
UGCAAAAACACAGACUCGCGUUG
2660
37367
VEGF:1623L21 antisense
ACGcGAGucuGuGuuuuuGTT
4083

siNA (1605C) stab26

1608
AAAAACACAGACUCGCGUUGCAA
2661
37368
VEGF:1626L21 antisense
GCAAcGcGAGucuGuGuuuTT
4084

siNA (1608C) stab26

1612
ACACAGACUCGCGUUGCAAGGCG
2662
37369
VEGF:1630L21 antisense
CCUuGcAAcGcGAGucuGuTT
4085

siNA (1612C) stab26

1616
AGACUCGCGUUGCAAGGCGAGGC
2663
37370
VEGF:1634L21 antisense
CUCGccuuGcAAcGcGAGuTT
4086

siNA (1616C) stab26

1622
GCGUUGCAAGGCGAGGCAGCUUG
2664
37371
VEGF:1640L21 antisense
AGCuGccucGccuuGcAAcTT
4087

siNA (1622C) stab26

1626
UGCAAGGCGAGGCAGCUUGAGUU
2665
37372
VEGF:1644L21 antisense
CUCAAGcuGccucGccuuGTT
4088

siNA (1626C) stab26

1628
CAAGGCGAGGCAGCUUGAGUUAA
2666
37373
VEGF:1646L21 antisense
AACucAAGcuGccucGccuTT
4089

siNA (1628C) stab26

1633
CGAGGCAGCUUGAGUUAAACGAA
2573
37374
VEGF:1651L21 antisense
CGUuuAAcucAAGcuGccuTT
4090

siNA (1633C) stab26

1634
GAGGCAGCUUGAGUUAAACGAAC
2574
37375
VEGF:1652L21 antisense
UCGuuuAAcucAAGcuGccTT
4091

siNA (1634C) stab26

1635
AGGCAGCUUGAGUUAAACGAACG
2575
37376
VEGF:1653L21 antisense
UUCGuuuAAcucAAGcuGcTT
4092

siNA (1635C) stab26

1636
GGCAGCUUGAGUUAAACGAACGU
2576
37377
VEGF:1654L21 antisense
GUUcGuuuAAcucAAGcuGTT
4093

siNA (1636C) stab26

1637
GCAGCUUGAGUUAAACGAACGUA
2559
37378
VEGF:1655L21 antisense
CGUucGuuuAAcucAAGcuTT
4094

siNA (1637C) stab26

1643
UGAGUUAAACGAACGUACUUGCA
2667
37379
VEGF:1661L21 antisense
CAAGuAcGuucGuuuAAcuTT
4095

siNA (1643C) stab26

1645
AGUUAAACGAACGUACUUGCAGA
2668
37380
VEGF:1663L21 antisense
UGCAAGuAcGuucGuuuAATT
4096

siNA (1645C) stab26

1646
GUUAAACGAACGUACUUGCAGAU
2669
37381
VEGF:1664L21 antisense
CUGcAAGuAcGuucGuuuATT
4097

siNA (1646C) stab26

1647
UUAAACGAACGUACUUGCAGAUG
2670
37382
VEGF:1665L21 antisense
UCUGcAAGuAcGuucGuuuTT
4098

siNA (1647C) stab26

1648
UAAACGAACGUACUUGCAGAUGU
2577
37383
VEGF:1666L21 antisense
AUCuGcAAGuAcGuucGuuTT
4099

siNA (1648C) stab26

1655
ACGUACUUGCAGAUGUGACAAGC
2671
37384
VEGF:1673L21 antisense
UUGucAcAucuGcAAGuAcTT
4100

siNA (1655C) stab26

1656
CGUACUUGCAGAUGUGACAAGCC
2560
37385
VEGF:1674L21 antisense
CUUGucAcAucuGcAAGuATT
4101

siNA (1656C) stab26

1657
GUACUUGCAGAUGUGACAAGCCG
2672
37386
VEGF:1675L21 antisense
GCUuGucAcAucuGcAAGuTT
4102

siNA (1657C) stab26

1562
AAAGCAUUUGUUUGUACAAGAUC
2581
37575
VEGF:1562U21 sense siNA
B AGcAuuuGuuuGuAcAAGATT B
4103

stab07

1562
AAAGCAUUUGUUUGUACAAGAUC
2581
37577
VEGF:1580121 antisense siNA
UCUuGuAcAAAcAAAuGcuTT
4104

(1562C) stab26

1215
GUGGACAUCUUCCAGGAGUACCC
2543
37789
VEGF:1233121 antisense siNA
GUACUccuGGAAGAuGuccTT
4105

(1215C) stab26

VEGF/VEGFR multifunctional siNA

1501
ACCUCACUGCCACUCUAAUUGUC
2673
34692
F/K bf-1a siNA stab00
CAAUUAGAGUGGCAGUGAGCAAA
4106

CCUCACUGCCACUCUAAUUGUCA

[FLT1:1519121 (1501C)-14
GTT

+KDR:503U21]

1502
CCUCACUGCCACUCUAAUUGUCA
2674
34693
F/K bf-2a siNA stab00
ACAAUUAGAGUGGCAGUGAGCAAA
4107

CCUCACUGCCACUCUAAUUGUCA

[FLT1:1520121 (1502C)-13
GTT

+KDR:503U21]

1503
CUCACUGCCACUCUAAUUGUCAA
2675
34694
F/K bf-3a siNA stab00
GACAAUUAGAGUGGCAGUGAGCAA
4108

CCUCACUGCCACUCUAAUUGUCA

[FLT1:1521121 (1503C)-12
AGTT

+KDR:503U21]

3646
AAAGCAUUUGUUUGUACAAGAUC
2676
34695
V/F bf-1a siNA stab00
UGUGCCAGCAGUCCAGCAUUUGUU
4109

UCAUGCUGGACUGCUGGCACAGA

(FLT1:3664L19 (3646C)-5
UGUACAAGATT

+VEGF:1562U21]

5353
AGAGAGACGGGGUCAGAGAGAGC
2677
34696
V/F bf-2a siNA stab00
UUGGUAUAGAGACGGGGUCAGAGA
4110

AAGACCCCGUCUCUAUACCAACC

[FLT1:5371L19 (5353C)-12
GATT

+VEGF:360U21]

1501
ACCUCACUGCCACUCUAAUUGUC
2678
34697
F/K bf-1b siNA stab00
CUUUGCUCACUGCCACUCUAAUU
4111

UCAGAGUGGCAGUGAGCAAAGGG

[KDR:521L21 (503C)-14
GTT

+FLT1:1501U21]

1502
CCUCACUGCCACUCUAAUUGUCA
2679
34698
F/K bf-2b siNA stab00
CUUUGCUCACUGCCACUCUAAUU
4112

UCAGAGUGGCAGUGAGCAAAGGG

[KDR:521L21 (503C)-13
GUTT

+FLT1:1502U21]

1503
CUCACUGCCACUCUAAUUGUCAA
2680
34699
F/K bf-3b siNA stab00
CUUUGCUCACUGCCACUCUAAUU
4113

UCAGAGUGGCAGUGAGCAAAGGG

[KDR:521L21 (503C)-12
GUCTT

+FLT1:1503U21]

3646
AAAGCAUUUGUUUGUACAAGAUC
2676
34700
V/F bf-1b siNA stab00
UCUUGUACAAACAAAUGCUGGACU
4114

UCAUGCUGGACUGCUGGCACAGA

[VEGF:1580L19 (1562C)-5
GCUGGCACATT

+FLT1:3646U21]

5353
AGAGAGACGGGGUCAGAGAGAGC
2677
34701
V/F bf-2b siNA stab00
UCUCUCUGACCCCGUCUCUAUACC
4115

AAGACCCCGUCUCUAUACCAACC

[VEGF:378L21 (360C)-12
AATT

+FLT1:5353U21]

3646
AAUGUGAAUGCAGACCAAAGAAA
2681
34702
V/F bf-3a siNA stab00
UGUGCCAGCAGUCCAGCATT
4116

UCAUGCUGGACUGCUGGCACAGA

[FLT1:3664L19 (3646C)
UGUGAAUGCAGACCAAAGATT

+VEGF1420:U21]

3646
AAUGUGAAUGCAGACCAAAGAAA
2681
34703
V/F bf-3b siNA stab00
UCUUUGGUCUGCAUUCACA
4117

UCAUGCUGGACUGCUGGCACAGA

[VEGF1438:L19 (1420C) +
AUGCUGGACUGCUGGCACATT

FLT1:3646U21]

3648
AAUGUGAAUGCAGACCAAAGAAA
2681
34704
V/F bf-4a siNA stab00
UGUGCCAGCAGUCCAGC
4118

UCAUGCUGGACUGCUGGCACAGA

[FLT1:3664L17 (3648C) +
UGAAUGCAGACCAAAGATT

VEGFI422:U19]

3648
AAUGUGAAUGCAGACCAAAGAAA
2681
34705
V/F bf-4b siNA stab00
UCUUUGGUCUGCAUUCA
4119

UCAUGCUGGACUGCUGGCACAGA

[VEGF1438:L17 (1422C) +
GCUGGACUGCUGGCACATT

FLT1:3648U199

3646
AAUGUGAAUGCAGACCAAAGAAA
2681
34706
V/F bf-5a siNA stab00
UGUGCCAGCAGUCCAGCAU
4120

UCAUGCUGGACUGCUGGCACAGA

[FLT1:3664L19 (3646C) +
GAAUGCAGACCAAAGAAAGTT

VEGF1423:U199

3646
AAUGUGAAUGCAGACCAAAGAAA
2681
34707
V/F bf-5b siNA stab00
CUUUCUUUGGUCUGCAUUC
4121

UCAUGCUGGACUGCUGGCACAGA

[VEGF1441:L19 (1420C) +
AUGCUGGACUGCUGGCACATT

FLT1:3646U21]

3646
AUGUGAAUGCAGACCAAAGAAAG
2682
34708
V/F bf-6a siNA stab00
UGUGCCAGCAGUCCAGCAU
4122

UCAUGCUGGACUGCUGGCACAGA

[FLT1:3664L19 (3646C) +
GUGAAUGCAGACCAAAGAATT

VEGF1421:U21]

3646
AUGUGAAUGCAGACCAAAGAAAG
2682
34709
V/F bf-6b siNA stab00
UUCUUUGGUCUGCAUUCAC
4123

UCAUGCUGGACUGCUGGCACAGA

[VEGF1439:L19 (1421C) +
AUGCUGGACUGCUGGCACATT

FLT1:3646U21]

1215
GUGGACAUCUUCCAGGAGUACCC
2683
36408
V/F bf-L-03 siNA stab00
GGACAUCUUCCAGGAGUACTT L
4124

CUGAACUGAGUUUAAAAGGCACC

[VEGF:1215U21 o18S
GAACUGAGUUUAAAAGGCATT

FLT1:346U21]

1421
AUGUGAAUGCAGACCAAAGAAAG
2684
36409
V/F bf-L-02 siNA stab00
GUGAAUGCAGACCAAAGAATT L
4125

CUGAACUGAGUUUAAAAGGCACC

[VEGF:1421 U21 o18S
GAACUGAGUUUAAAAGGCATT

FLT1:346U21]

3854
UUUGAGCAUGGAAGAGGAUUCUG
2685
36411
F/K bf-L-04 siNA stab00
UGAGCAUGGAAGAGGAUUCTT L
4126

CUGAACUGAGUUUAAAAGGCACC

[KDR:3854U21 o18S
GAACUGAGUUUAAAAGGCATT

FLT1:346U21]

346
CUGAACUGAGUUUAAAAGGCACC
2686
36416
V/F bf-L-01 siNA stab00
GAACUGAGUUUAAAAGGCATT L
4127

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:346U21 o18S
GUGAAUGCAGACCAAAGAATT

VEGF:1421U21]

3646
UCAUGCUGGACUGCUGGCACAGA
2687
36425
V/F bf-L-05 siNA stab00
AUGCUGGACUGCUGGCACATT L
4128

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:3646U21 o18S
GUGAAUGCAGACCAAAGAATT

VEGF:1421U21]

3646
UCAUGCUGGACUGCUGGCACAGA
2687
36426
V/F bf-L-06 siNA stab00
AUGCUGGACUGCUGGCACATT W
4129

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:3646U21 c12S
GUGAAUGCAGACCAAAGAATT

VEGF:1421U21]

3646
UCAUGCUGGACUGCUGGCACAGA
2687
36427
V/F bf-L-07 siNA stab00
AUGCUGGACUGCUGGCACATT Y
4130

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:3646U21 o9S
GUGAAUGCAGACCAAAGAATT

VEGF:1421U21]

3646
UCAUGCUGGACUGCUGGCACAGA
2687
36428
V/F bf-L-08 siNA stab00
AUGCUGGACUGCUGGCACATT Z
4131

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:3646U21 c3S
GUGAAUGCAGACCAAAGAATT

VEGF:1421U21]

3646
UCAUGCUGGACUGCUGGCACAGA
2687
36429
V/F bf-L-09 siNA stab00
AUGCUGGACUGCUGGCACATT LL
4132

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:3646U21 2x o18S
GUGAAUGCAGACCAAAGAATT

VEGF:1421U21]

162
UCCCUCUUCUUUUUUCUUAAACA
2688
37537
V/K bf-1a siNA stab00
UUUAAGAAAAAAGAAGAGGAAGCUC
4133

AGAAGAAGAGGAAGCUCCUGAAG

[VEGF:180L21 (162C)-9 +
CUGATT

KDR:3263U21]

164
CCUCUUCUUUUUUCUUAAACAUU
2689
37538
V/F bf-7a siNA stab00
UGUUUAAGAAAAAAGAAGAAGGAAA
4134

UCAAAGAAGAAGGAAACAGAAUC

[VEGF:182L21 (164C)-8 +
CAGAATT

FLT1:594U21]

202
AUUGUUUCUCGUUUUAAUUUAUU
2690
37539
V/F bf-8a siNA stab00
UAAAUUAAAACGAGAAACAUUC
4135

AGCGAGAAACAUUCUUUUAUCUG

[VEGF:220L21 (202C)-9 +
UUUUAUCTT

FLT1:3323U21]

237
UCCCCACUUGAAUCGGGCCGACG
2691
37540
V/F bf-9a siNA stab00
UCGGCCCGAUUCAAGUGGGCCU
4136

GAUCAAGUGGGCCUUGGAUCGCU

[VEGF:255L21 (237C)-9 +
UGGAUCGTT

FLT1:5707U21]

238
CCCCACUUGAAUCGGGCCGACGG
2692
37541
V/F bf-10a siNA stab00
GUCGGCCCGAUUCAAGUGGCCA
4137

UUUUCAAGUGGCCAGAGGCAUGG

[VEGF:256L21 (238C)-9 +
GAGGCAUTT

FLT1:3260U21]

338
CUCCAGAGAGAAGUCGAGGAAGA
2693
37542
V/K bf-2a siNA stab00
UUCCUCGACUUCUCUCUGGUUG
4138

GGUCUCUCUGGUUGUGUAUGUCC

[VEGF:356L21 (338C)-9 +
UGUAUGUTT

KDR:1541U21]

360
AGAGAGACGGGGUCAGAGAGAGC
2694
37543
V/F bf-11a siNA stab00
UCUCUCUGACCCCGUCUCU
4139

AGACCCCGUCUCUAUACCAACCA

[VEGF:378L21 (360C)-11 +
AUACCAACTT

FLT1:5354U21'

484
GCAGCUGACCAGUCGCGCUGACG
2695
37544
V/F bf-12a siNA stab00
UCAGCGCGACUGGUCAGCUACUGG
4140

CAUGGUCAGCUACUGGGACACCG

[VEGF:502L21 (484C)-9 +
GACACTT

FLT1:251U21]

654
CUGAAACUUUUCGUCCAACUUCU
2696
37545
V/F bf-13a siNA stab00
AAGUUGGACGAAAAGUUUCCACUU
4141

AAAAAAGUUUCCACUUGACACUU

[VEGF:672L21 (654C)-9 +
GACACTT

FLT1:758U21]

978
CCCCACAGCCCGAGCCGGAGAGG
2697
37546
V/F bf-14a siNA stab00
UCUCCGGCUCGGGCUGUGG
4142

UUGCUGUGGGAAAUCUUCUCCUU

[VEGF:996L21 (978C)-7 +
GAAAUCUUCUCCTT

FLT1:3513U21]

1038
ACCAUGAACUUUCUGCUGUCUUG
2698
37547
V/F bf-15a siNA stab00
AGACAGCAGAAAGUUCAUGA
4143

UCAAGUUCAUGAGCCUGGAAAGA

[VEGF:1056L21 (1038C)-9 +
GCCUGGAAATT

FLT1:3901U21]

1095
CACCAUGCCAAGUGGUCCCAGGC
2699
37548
V/K bf-3a siNA stab00
CUGGGACCACUUGGCAUGG
4144

AGGGCAUGGAGUUCUUGGCAUCG

[VEGF:1113L21 (1095C)-7 +
AGUUCUUGGCAUTT

KDR:3346U21]

1253
CAUCUUCAAGCCAUCCUGUGUGC
2700
37549
V/K bf-4a saNA stab00
ACACAGGAUGGCUUGAAGAU
4145

UGUUGAAGAUGGGAAGGAUUUGC

[VEGF:1271L21 (1253C)-7 +
GGGAAGGAUUUTT

KDR:4769U21]

1351
UGCAGAUUAUGCGGAUCAAACCU
2701
37550
V/F bf-16a siNA stab00
GUUUGAUCCGCAUAAUCU
4146

AACGCAUAAUCUGGGACAGUAGA

[VEGF:1369L21 (1351C)-11 +
GGGACAGUATT

FLT1:796U21]

1352
GCAGAUUAUGCGGAUCAAACCUC
2702
37551
V/F bf-17a siNA stab00
GGUUUGAUCCGCAUAAUC
4147

AACGCAUAAUCUGGGACAGUAGA

[VEGF:1370L21 (1352C)-10 +
UGGGACAGUATT

FLT1:796U21]

1389
AUAGGAGAGAUGAGCUUCCUACA
2703
37552
V/K bf-5a siNA stab00
UAGGAAGCUCAUCUCUCCUG
4148

UAAUCUCUCCUGUGGAUUCCUAC

[VEGF:1407L21 (1389C)-9 +
UGGAUUCCUTT

KDR:1588U210]

1401
AGCUUCCUACAGCACAACAAAUG
2704
37553
V/F bf-18a siNA stab00
UUUGUUGUGCUGUAGGA
4149

UCAGGAAGCUCUGAUGAUGUCAG

[VEGF:1419L21 (1401C)-6 +
AGCUCUGAUGAUGUCTT

FLT1:3864U211

1408
UACAGCACAACAAAUGUGAAUGC
2705
37554
V/K bf-6a siNA stab00
AUUCACAUUUGUUGUGCUG
4150

UCGUUGUGCUGUUUCUGACUCCU

[VEGF:1426L21 (1408C)-9 +
UUUCUGACUCTT

KDR:5038U21]

1417
ACAAAUGUGAAUGCAGACCAAAG
2706
37555
V/K bf-7a siNA stab00
UUGGUCUGCAUUCACAUUU
4151

CUAUUCACAUUUUGUAUCAGUAU

[VEGF:1435L21 (1417C)-10 +
UGUAUCAGUTT

KDR:5737U21]

162
UCCCUCUUCUUUUUUCUUAAACA
2688
37556
V/K bf-1b siNA stab00
UCAGGAGCUUCCUCUUCUUU
4152

AGAAGAAGAGGAAGCUCCUGAAG

[KDR:3281L21 (3263C)-9 +
UUUCUUAAATT

VEGF:162U21]

164
CCUCUUCUUUUUUCUUAAACAUU
2689
37557
V/F bf-7b siNA stab00
UUCUGUUUCCUUCUUCUU
4153

UCAAAGAAGAAGGAAACAGAAUC

[FLT1:612L21 (594C)-8 +
UUUUCUUAAACATT

VEGF:164U21]

202
AUUGUUUCUCGUUUUAAUUUAUU
2690
37558
V/F bf-8b siNA stab00
GAUAAAAGAAUGUUUCU
4154

AGCGAGAAACAUUCUUUUAUCUG

[FLT1:3341121 (3323C)-9 +
CGUUUUAAUUUATT

VEGF:202U21]

237
UCCCCACUUGAAUCGGGCCGACG
2691
37559
V/F bf-9b siNA stab00
CGAUCCAAGGCCCACUUG
4155

GAUCAAGUGGGCCUUGGAUCGCU

[FLT1:5725121 (5707C)-9 +
AAUCGGGCCGATT

VEGF:237U21]

238
CCCCACUUGAAUCGGGCCGACGG
2692
37560
V/F bf-10b siNA stab00
AUGCCUCUGGCCAC
4156

UUUUCAAGUGGCCAGAGGCAUGG

[FLT1 :3278121 (3260C)-9
UUGAAUCGGGCCGACTT

VEGF:238U21]

338
CUCCAGAGAGAAGUCGAGGAAGA
2693
37561
V/K bf-2b siNA stab00
ACAUACACAACCAGAGAGA
4157

GGUCUCUCUGGUUGUGUAUGUCC

[KDR:1559121 (1541C)-9 +
AGUCGAGGAATT

VEGF:338U21]

360
AGAGAGACGGGGUCAGAGAGAGC
2694
37562
V/F bf-11b siNA stab00
GUUGGUAUAGAGACG
4158

AGACCCCGUCUCUAUACCAACCA

[FLT1 :5372121 (5354C)-11 +
GGGUCAGAGAGATT

VEGF:360U21]

484
GCAGCUGACCAGUCGCGCUGACG
2695
37563
V/F bf-12b siNA stab00
GUGUCCCAGUAGCUGA
4159

CAUGGUCAGCUACUGGGACACCG

[FLT1:269121 (251C)-9 +
CCAGUCGCGCUGATT

VEGF:484U21]

654
CUGAAACUUUUCGUCCAACUUCU
2696
37564
V/F bf-13b siNA stab00
GUGUCAAGUGGAAACUU
4160

AAAAAAGUUUCCACUUGACACUU

[FLT1:776121 (758C)-9 +
UUCGUCCAACUUTT

VEGF:654U21]

978
CCCCACAGCCCGAGCCGGAGAGG
2697
37565
V/F bf-14b siNA stab00
GGAGAAGAUUUCCCACAG
4161

UUGCUGUGGGAAAUCUUCUCCUU

[FLT1:3531121 (3513C)-7 +
CCCGAGCCGGAGATT

VEGF:978U21]

1038
ACCAUGAACUUUCUGCUGUCUUG
2698
37566
V/F bf-15b siNA stab00
UUUCCAGGCUCAUGAAC
4162

UCAAGUUCAUGAGCCUGGAAAGA

[FLT1:3919121 (3901C)-9 +
UUUCUGCUGUCUTT

VEGF:1038U21]

1095
CACCAUGCCAAGUGGUCCCAGGC
2699
37567
V/K bf-3b siNA stab00
AUGCCAAGAACUCCAUG
4163

AGGGCAUGGAGUUCUUGGCAUCG

[KDR:3364121 (3346C)-7 +
CCAAGUGGUCCCAGTT

VEGF:1095U21]

1253
CAUCUUCAAGCCAUCCUGUGUGC
2700
37568
V/K bf-4b siNA stab00
AAAUCCUUCCCAUCUUCA
4164

UGUUGAAGAUGGGAAGGAUUUGC

[KDR:4787121 (4769C)-7 +
AGCCAUCCUGUGUTT

VEGF:1253U21]

1351
UGCAGAUUAUGCGGAUCAAACCU
2701
37569
V/F bf-16b siNA stab00
UACUGUCCCAGAUUAUG
4165

AACGCAUAAUCUGGGACAGUAGA

[FLT1:814121 (796C)-11 +
CGGAUCAAACTT

VEGF:1351U21]

1352
GCAGAUUAUGCGGAUCAAACCUC
2702
37570
V/F bf-17b siNA stab00
UACUGUCCCAGAUUAUGCG
4166

AACGCAUAAUCUGGGACAGUAGA

[FLT1:814121 (796C)-10 +
GAUCAAACCTT

VEGF:1352U21]

1389
AUAGGAGAGAUGAGCUUCCUACA
2703
37571
V/K bf-5b siNA stab00
AGGAAUCCACAGGAGAGAUGA
4167

UAAUCUCUCCUGUGGAUUCCUAC

[KDR:1606L21 (1588C)-9 +
GCUUCCUATT

VEGF:1389U21]

1401
AGCUUCCUACAGCACAACAAAUG
2704
37572
V/F bf-18b siNA stab00
GACAUCAUCAGAGCUUCCUACAGC
4168

UCAGGAAGCUCUGAUGAUGUCAG

[FLT1:3882L21 (3864C)-6 +
ACAACAAATT

VEGF:1401U21]

1408
UACAGCACAACAAAUGUGAAUGC
2705
37573
V/K bf-6b siNA stab00
GAGUCAGAAACAGCACAACAAA
4169

UCGUUGUGCUGUUUCUGACUCCU

[KDR:5056L21 (5038C)-9 +
UGUGAAUTT

VEGF:1408U21]

1417
ACAAAUGUGAAUGCAGACCAAAG
2706
37574
V/K bf-7b siNA stab00
ACUGAUACAAAAUGUGAAU
4170

CUAUUCACAUUUUGUAUCAGUAU

[KDR:5755L21 (5737C)-10 +
GCAGACCAATT

VEGF:1417U21]

3646
AAAGCAUUUGUUUGUACAAGAUC
2676
37578
V/F bf-1a siNA stab07/26
UGUGccAGcAGuccAGcAu

UCAUGCUGGACUGCUGGCACAGA

[FLT1:3664L19 (3646C)-5 +

AGcAuuuGuuuGuAcAAGATT B
4171

VEGF:1562U21]

3646
AAAGCAUUUGUUUGUACAAGAUC
2676
37579
V/F bf-1b siNA stab07/26
UCUuGuAcAAAcAAAuGcu
4172

UCAUGCUGGACUGCUGGCACAGA

[VEGF:1580L19 (1562C)-5 +

AuGcuGGAcuGcuGGcAcATT B

FLT1:3646U21]

1215
GUGGACAUCUUCCAGGAGUACCC
2683
37777
V/F bf-L-03 siNA stab07
B GGAcAucuuccAGGAGuAcTT L

CUGAACUGAGUUUAAAAGGCACC

[VEGF:1215U21 o18S

GAAcuGAGuuuAAAAGGcATT B
4173

FLT1:346U21]

1421
AUGUGAAUGCAGACCAAAGAAAG
2684
37778
V/F bf-L-02 siNA stab07
B GuGAAuGcAGAccAAAGAATT L
4174

CUGAACUGAGUUUAAAAGGCACC

[VEGF:1421U21 o18S

GAAcuGAGuuuAAAAGGcATT B

FLT1:346U21]

1421
CUGAACUGAGUUUAAAAGGCACC
2686
37779
V/F bf-L-01 siNA stab07
B GAAcuGAGuuuAAAAGGcATT L
4175

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:346U21 o18S

GuGAAuGcAGAccAAAGAATT B

VEGF:1421U21]

1421
UCAUGCUGGACUGCUGGCACAGA
2687
37780
V/F bf-L-05 siNA stab07
B AuGcuGGAcuGcuGGcAcATT L
4176

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:3646U21 o18S

GuGAAuGcAGAccAAAGAATT B

VEGF:1421U21]

1421
UCAUGCUGGACUGCUGGCACAGA
2687
37783
V/F bf-L-05 siNA stab00
AUGCUGGACUGCUGGCACATT
4177

AUGUGAAUGCAGACCAAAGAAAG

[FLT1 :3646U21 10nt
GAUCATCGTA

VEGF:1421U21]
GUGAAUGCAGACCAAAGAATT

1421
UCAUGCUGGACUGCUGGCACAGA
2687
37784
V/F bf-L-05 siNA stab00
AUGCUGGACUGCUGGCACATT
4178

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:3646U21 6nt
GAUCAT GUGAAUGCAGACCA

AAGAATT

1421
UCAUGCUGGACUGCUGGCACAGA
2687
37785
V/F bf-L-05 siNA stab00
AUGCUGGACUGCUGGCACATT GAU
4179

AUGUGAAUGCAGACCAAAGAAAG

VEGF:1421U21]
GUGAAUGCAGACCAAAGAATT

[FLT1:3646U21 3nt

1421
UCAUGCUGGACUGCUGGCACAGA
2687
37786
V/F bf-L-05 siNA stab00
AUGCUGGACUGCUGGCACATT
4180

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:3646U21 no linker
GUGAAUGCAGACCAAAGAATT

VEGF:1421U21]

1421
AUGUGAAUGCAGACCAAAGAAAG
2682
37787
V/F bf-6a siNA stab07/26
UGUGccAGcAGuccAGcAuTT
4181

UCAUGCUGGACUGCUGGCACAGA

[FLT1:3664L19 (3646C) +

GuGAAuGcAGAccAAAGAATT B

VEGF1421:U21]

1421
AUGUGAAUGCAGACCAAAGAAAG
2682
37788
V/F bf-6b siNA stab07/26
UUCuuuGGucuGcAuucAcTT
4182

UCAUGCUGGACUGCUGGCACAGA

[VEGF1 439:L19 (1421C) +

AuGcuGGAcuGcuGGcAcATT B

FLT1:3646U21]

346
CUGAACUGAGUUUAAAAGGCACC
2686
38287
V/F bf-L-10a siNA stab09
B GAACUGAGUUUAAAAGGCATT L
4183

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:346U21 o18S
GUGAAUGCAGACCAAAGAATT B

VEGF:1421U21]

346
CUGAACUGAGUUUAAAAGGCACC
2686
38288
V/F bf-L-11a siNA stab09
B GAACUGAGUUUAAAAGGCA
4184

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:346U21 +
GUGAAUGCAGACCAAAGAA B

VEGF:1421U21]

346
CUGAACUGAGUUUAAAAGGCACC
2686
38289
V/F bf-L-11b siNA stab00
UUCUUUGGUCUGCAUUCAC
4185

AUGUGAAUGCAGACCAAAGAAAG

[VEGF:1439L21 (1421C) +
UGCCUUUUAAACUCAGUUC

FLT1:364L21 (346C)]

346
CUGAACUGAGUUUAAAAGGCACC
2686
38369
V/F bf-L-26a siNA stab22
UGCCUUUUAAACUCAGUUC
4186

AUGUGAAUGCAGACCAAAGAAAG

[FLT1:364L21 siNA (346C) +
GUGAAUGCAGACCAAAGAAU B

VEGF:1421U21]

346
CUGAACUGAGUUUAAAAGGCACC
2686
38370
V/F bf-L-26b siNA stab22
UUCUUUGGUCUGCAUUCAC
4187

AUGUGAAUGCAGACCAAAGAAAG

[VEGF:1439L21 siNA
GAACUGAGUUUAAAAGGCATT B

(1421C) + FLT1:346U21

siNA]

VEGF/VEGFR DFO siNA

349
AACUGAGUUUAAAAGGCACCCAG
2289
32718
FLT1:367L21 siRNA (349C) v1
pGGGUGCCUUUUAAACUC
2810

5′p palindrome
GAGUUUAAAAG B

349
AACUGAGUUUAAAAGGCACCCAG
2289
32719
FLT1:367L21 siRNA (349C) v2
pGGGUGCCUUUUAAACUCAG
2811

5′p palindrome
GAGUUUAAAAG B

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
32720
FLT1:2967L21 siRNA (2949C)
pCAUCAGAGGCCCUCCUUGC
2812

vi 5′p palindrome
AAGGAGGGCCUCU B

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
32721
FLT1:2967L21 siRNA (2949C)
pCAUCAGAGGCCCUCCUU
2813

v2 5′p palindrome
AAGGAGGGCCUCUG B

2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
32722
FLT1:2967L21 siRNA (2949C)
pCAUCAGAGGCCCUCCU
2814

v3 5′p palindrome
AGGAGGGCCUCUG B

354
AGUUUAAAAGGCACCCAGCACAUC
2707
32805
FLT1:372L21 siRNA (354C)
pGUGCUGGGUGCCUUUUAAA
4188

v1 5′p palindrome
AGGCACCCAGC B

354
AGUUUAAAAGGCACCCAGCACAUC
2707
32806
FLT1:372121 siRNA (354C)
pGUGCUGGGUGCCUUUAAA
4189

v2 5′p palindrome
GGCACCCAGC B

354
AGUUUAAAAGGCACCCAGCACAUC
2707
32807
FLT1:372121 siRNA (354C)
pGUGCUGGGUGCCUU
4190

v3 5′p palindrome
AAGGCACCCAGC B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32808
FLT1:1247L21 siRNA (1229C)
pAAUGCUUUAUCAUAUAUAU
4191

v1 5′p palindrome
GAUAAAGC B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32809
FLT1:1247L21 siRNA (1229C)
pAAUGCUUUAUCAUAUAU
4192

v2 5′p palindrome
GAUAAAGC B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32810
FLT1:1247L21 sIRNA (1229C)
pAAUGCUUUAUCAUAU
4193

v3 5′p palindrome
GAUAAAGC B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32811
FLT1:1247L21 siRNA (1229C)
pAAUGCUUUAUCAUAU
4194

v4 5′p palindrome
GAUAAAGCA B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32812
FLT1:1247L21 siRNA (1229C)
pAAUGCUUUAUCAUAUAU
4195

v5 5′p palindrome
GAUAAAGCAUU B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32813
FLT1:1247L21 siRNA (1229C)
pAAUGCUUUAUCAUAU
4196

v6 5′p palindrome
GAUAAAGCAUU B

349
AACUGAGUUUAAAAGGCACCCAG
2289
33056
FLT1:367L21 siRNA (349C)
pGGGUGCCUUUUAAACUCAG
4197

v3 5′p palindrome
GAGUUUAAAAGG B

349
AACUGAGUUUAAAAGGCACCCAG
2289
33057
FLT1:367L21 siRNA (349C)
pGGGUGCCUUUUAAACUC
4198

v4 5′p palindrome
GAGUUUAAAAGGCA B

349
AACUGAGUUUAAAAGGCACCCAG
2289
33058
FLT1:367L21 siRNA (349C)
pGGGUGCCUUUUAAACU
4199

v5 5′p palindrome
AGUUUAAAAGG B

349
AACUGAGUUUAAAAGGCACCCAG
2289
33059
FLT1:367L21 siRNA (349C)
pGGGUGCCUUUUAAACU
4200

v6 5′p palindrome
AGUUUAAAAGGC B

349
AACUGAGUUUAAAAGGCACCCAG
2289
33060
FLT1:367L21 siRNA (349C)
pGGGUGCCUUUUAAACU
4201

v7 5′p palindrome
AGUUUAAAAGGCA B

349
AACUGAGUUUAAAAGGCACCCAG
2289
33061
FLT1:367L21 siRNA (349C)
pGGGUGCCUUUUAAACU
4202

v8 5′p palindrome
AGUUUAAAAGGCAC B

349
AACUGAGUUUAAAAGGCACCCAG
2289
33062
FLT1:367L21 siRNA (349C)
pGGGUGCCUUUUAAAC
4203

v9 5′p palindrome
GUUUAAAAGGC B

349
AACUGAGUUUAAAAGGCACCCAG
2289
33063
FLT1:367L21 sIRNA (349C)
pGGGUGCCUUUUAAAC
4204

v10 5′p palindrome
GUUUAAAAGGCA B

349
AACUGAGUUUAAAAGGCACCCAG
2289
33064
FLT1:367L21 siRNA (349C)
pGGGUGCCUUUUAAAC
4205

v11 5′p palindrome
GUUUAAAAGGCAC B

354
AGUUUAAAAGGCACCCAGCACAU
2316
34092
FLT1:371L18 siRNA (354C)
pUGCUGGGUGCCUUUUAAA
4206

v4 5′p palindrome
AGGCACCCAGC B

354
AGUUUAAAAGGCACCCAGCACAU
2316
34093
FLT1:370L17 siRNA (354C)
pGCUGGGUGCCUUUUAAA
4207

v5 5′p palindrome
AGGCACCCAGC B

354
AGUUUAAAAGGCACCCAGCACAU
2316
34094
FLT1:370L17 siRNA (354C)
pGCUGGGUGCCUUUUAAA
4208

v6 5′p palindrome
AGGCACCCAGCT B

354
AGUUUAAAAGGCACCCAGCACAU
2316
34095
FLT1:370L17 siRNA (354C)
pGCUGGGUGCCUUUUAAA
4209

v7 5′p palindrome
AGGCACCCAG B

354
AGUUUAAAAGGCACCCAGCACAU
2316
34096
FLT1:369L16 siRNA (354C)
pCUGGGUGCCUUUU
4210

v8 5′p palindrome
AAAAGGCACCCAG B

354
AGUUUAAAAGGCACCCAGCACAU
2316
34097
FLT1:369L16 siRNA (354C)
pCUGGGUGCCUUUUAAA
4211

v9 5′p palindrome
AGGCACCCA B

354
AGUUUAAAAGGCACCCAGCACAU
2316
34098
FLT1:368L15 siRNA (354C)
pUGGGUGCCUUUUAAA
4212

v10 5′p palindrome
AGGCACCCA B

354
AGUUUAAAAGGCACCCAGCACAU
2316
34099
FLT1:368L15 siRNA (354C)
pUGGGUGCCUUUUAAA
4213

v11 5′p palindrome
AGGCACCCAT B

354
AGUUUAAAAGGCACCCAGCACAU
2316
34100
FLT1:368L15 siRNA (354C)
pUGGGUGCCUUUUAAA
4214

v12 5′p palindrome
AGGCACCCAU B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34101
FLT1:1247L21 siRNA (1229C)
pUGCUUUAUCAUAUAUAU
4215

v14 5′p palindrome
GAUAAAGCA B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34102
FLT1:1247L21 siRNA (1229C)
pUGCUUUAUCAUAUAUAU
4216

v15 5′p palindrome
GAUAAAGC B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34103
FLT1:1247L21 siRNA (1229C)
pGCUUUAUCAUAUAUAU
4217

v16 5′p palindrome
GAUAAAGC B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34104
FLT1:1247L17 siRNA (1229C)
AAUGCUUUAUCAUAUAU
4218

v5 palindrome
GAUAAAGCAUU B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34105
FLT1:1247L17 siRNA (1229C)
pAAUGCUUUAUCAUAUAU
4219

v7 5′p palindrome
GAUAAAGCAUUT B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34106
FLT1:1247L17 siRNA (1229C)
pAAUGCUUUAUCAUAUAU
4220

v8 5′p palindrome
GAUAAAGCAUUTT B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34107
FLT1:1247L17 siRNA (1229C)
pAAUGCUUUAUCAUAUAU
4221

v9 5′p palindrome
GAUAAAGCAU B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34108
FLT1:1247L16 siRNA (1229C)
pAUGCUUUAUCAUAUAU
4222

v10 5′p palindrome
GAUAAAGCAU B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34109
FLT1:1247L16 siRNA (1229C)
pAUGCUUUAUCAUAUAU
4223

v11 5′p palindrome
GAUAAAGCAUT B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34110
FLT1:1247L16 siRNA (1229C)
pAUGCUUUAUCAUAUAU
4224

v12 5′p palindrome
GAUAAAGCAUTT B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34111
FLT1:1247L16 siRNA (1229C)
pAUGCUUUAUCAUAUAU
4225

v13 5′p palindrome
GAUAAAGCA B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34112
FLT1:1247L17 siRNA (1229C)
pAAUGCUUUAUOAUAUAU
4226

v14 5′p palindrome
CUAUAAGCAUU B

1229
GOAUAUAUAUGAUAAAGCAUUCA
2708
34113
FLT1:1247L17 siRNA (1229C)
pAAUGCUUUUAGUUAUAU
4227

v15 5′p palindrome
GAUAAAGCAUU B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34114
FLT1:1247L17 siRNA (1229C)
pAAUCOUUAAUCUUAUUU
4228

v16 5′p palindrome
GAUAAAGCAUU B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34115
FLT1:1247L17 siRNA (1229C)
pAAuGcuuuAucAuAuAu
4229

v17 5′p palindrome
GAuAAAGcAuu B

1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34116
FLT1:1247L17 siRNA (1229C)
pAAuGcuuuAucAuAuAu
4230

v18 5′p palindrome
GAuAAAGcAuu B

Uppercase = ribonucleotide

u,c = 2′-deoxy-2′-fluoro U,C

T = thymidine

B = inverted deoxy abasic

s = phosphorothioate linkage

A = deoxy Adenosine

G = deoxy Guanosine

G = 2′-O-methyl Guanosine

A = 2′-O-methyl Adenosine

X = 3′-deoxy T

X = nitroindole

Z = nitropyrrole

T = thymidine

t = L-thymidine

u = L-uridine

D = inverted thymidine

L = 5′amino mod-C5 TFA ( from W.W.)

L = hegS = hexethelyne glycol spacer; spacer-18 (Glen Research 10-1918-xx)

W = C12 spacer, spacer C12 (Glen Research 10-1928-xx)

Y = tetraethelyne glycol spacer; spacer 9 (Glen Research 10-1909-xx)

Z = C3 spacer, spacer C3 (Glen Research 10-1913-xx)

p = terminal phosphate

I = rI = ribo inosine (Glen Res #10-3044-xx)

U = 3′-O-AAethyl Uridine

Gyl = glyceryl

TABLE IV

Non-limiting examples of Stabilization Chemistries

for chemically modified siNA constructs

Chemistry
pyrimidine
Purine
cap
p = S
Strand

“Stab 00”
Ribo
Ribo
TT at 3′-

S/AS

ends

“Stab 1”
Ribo
Ribo
—
5 at 5′-end
S/AS

1 at 3′-end

“Stab 2”
Ribo
Ribo
—
All
Usually

linkages
AS

“Stab 3”
2′-fluoro
Ribo
—
4 at 5′-end
Usually S

4 at 3′-end

“Stab 4”
2′-fluoro
Ribo
5′ and 3′-
—
Usually S

ends

“Stab 5”
2′-fluoro
Ribo
—
1 at 3′-end
Usually

AS

“Stab 6”
2′-O-
Ribo
5′ and 3′-
—
Usually S

Methyl

ends

“Stab 7”
2′-fluoro
2′-deoxy
5′ and 3′-
—
Usually S

ends

“Stab 8”
2′-fluoro
2′-O-
—
1 at 3′-end
S/AS

Methyl

“Stab 9”
Ribo
Ribo
5′ and 3′-
—
Usually S

ends

“Stab 10”
Ribo
Ribo
—
1 at 3′-end
Usually

AS

“Stab 11”
2′-fluoro
2′-deoxy
—
1 at 3′-end
Usually

AS

“Stab 12”
2′-fluoro
LNA
5′ and 3′-

Usually S

ends

“Stab 13”
2′-fluoro
LNA

1 at 3′-end
Usually

AS

“Stab 14”
2′-fluoro
2′-deoxy

2 at 5′-end
Usually

1 at 3′-end
AS

“Stab 15”
2′-deoxy
2′-deoxy

2 at 5′-end
Usually

1 at 3′-end
AS

“Stab 16”
Ribo
2′-O-
5′ and 3′-

Usually S

Methyl
ends

“Stab 17”
2′-O-
2′-O-
5′ and 3′-

Usually S

Methyl
Methyl
ends

“Stab 18”
2′-fluoro
2′-O-
5′ and 3′-

Usually S

Methyl
ends

“Stab 19”
2′-fluoro
2′-O-
3′-end

S/AS

Methyl

“Stab 20”
2′-fluoro
2′-deoxy
3′-end

Usually

AS

“Stab 21”
2′-fluoro
Ribo
3′-end

Usually

AS

“Stab 22”
Ribo
Ribo
3′-end

Usually

AS

“Stab 23”
2′-fluoro*
2′-deoxy*
5′ and 3′-

Usually S

ends

“Stab 24”
2′-fluoro*
2′-O-
—
1 at 3′-end
S/AS

Methyl*

“Stab 25”
2′-fluoro*
2′-O-
—
1 at 3′-end
S/AS

Methyl*

“Stab 26”
2′-fluoro*
2′-O-
—

S/AS

Methyl*

“Stab 27”
2′-fluoro*
2′-O-
3′-end

S/AS

Methyl*

“Stab 28”
2′-fluoro*
2′-O-
3′-end

S/AS

Methyl*

“Stab 29”
2′-fluoro*
2′-O-

1 at 3′-end
S/AS

Methyl*

“Stab 30”
2′-fluoro*
2′-O-

S/AS

Methyl*

“Stab 31”
2′-fluoro*
2′-O-
3′-end

S/AS

Methyl*

“Stab 32”
2′-fluoro
2′-O-

S/AS

Methyl*

“Stab 33”
2′-fluoro
2′-deoxy*
5′ and 3′-
—
Usually S

ends

CAP = any terminal cap, see for example FIG. 10.

All Stab 00-33 chemistries can comprise 3′-terminal thymidine (TT) residues

All Stab 00-33 chemistries typically comprise about 21 nucleotides, but can vary as described herein.

S = sense strand

AS = antisense strand

*Stab 23 had a single ribonucleotide adjacent to 3′-CAP

*Stab 24 and Stab 28 have a single ribonucleotide at 5′-terminus

*Stab 25, Stab 26, and Stab 27 have three ribonucleotides at 5′-terminus

*Stab 29, Stab 30, Stab 31, and Stab 33 any purine at first three nucleotide positions from 5′-terminus are ribonucleotides

p = phosphorothioate linkage

TABLE V

A. 2.5 μmol Synthesis Cycle ABI 394 Instrument

Wait Time*
Wait Time*
Wait Time*

Reagent
Equivalents
Amount
DNA
2′-O-methyl
RNA

Phosphoramidites
6.5
163
μL

45
sec
2.5
min
7.5
min

S-Ethyl Tetrazole
23.8
238
μL

45
sec
2.5
min
7.5
min

Acetic Anhydride
100
233
μL

5
sec
5
sec
5
sec

N-Methyl Imidazole
186
233
μL

5
sec
5
sec
5
sec

TCA
176
2.3
mL

21
sec
21
sec
21
sec

Iodine
11.2
1.7
mL

45
sec
45
sec
45
sec

Beaucage
12.9
645
μL

100
sec
300
sec
300
sec

Acetonitrile
NA
6.67
mL

NA
NA
NA

B. 0.2 μmol Synthesis Cycle ABI 394 Instrument

Wait Time*
Wait Time*
Wait Time*

Reagent
Equivalents
Amount
DNA
2′-O-methyl
RNA

Phosphoramidites
15
31
μL

45
sec
233
sec
465
sec

S-Ethyl Tetrazole
38.7
31
μL

45
sec
233
min
465
min

Acetic Anhydride
655
124
μL

5
sec
5
sec
5
sec

N-Methyl Imidazole
1245
124
μL

5
sec
5
sec
5
sec

TCA
700
732
μL

10
sec
10
sec
10
sec

Iodine
20.6
244
μL

15
sec
15
sec
15
sec

Beaucage
7.7
232
μL

100
sec
300
sec
300
sec

Acetonitrile
NA
2.64
mL

NA
NA
NA

C. 0.2 μmol Synthesis Cycle 96 well Instrument

Equivalents: DNA/
Amount: DNA/2′-O-
Wait Time*
Wait Time*
Wait Time*

Reagent
2′-O-methyl/Ribo
methyl/Ribo
DNA
2′-O-methyl
Ribo

Phosphoramidites
22/33/66
40/60/120
μL
60
sec
180
sec
360
sec

S-Ethyl Tetrazole
70/105/210
40/60/120
μL
60
sec
180
min
360
sec

Acetic Anhydride
265/265/265
50/50/50
μL
10
sec
10
sec
10
sec

N-Methyl Imidazole
502/502/502
50/50/50
μL
10
sec
10
sec
10
sec

TCA
238/475/475
250/500/500
μL
15
sec
15
sec
15
sec

Iodine
6.8/6.8/6.8
80/80/80
μL
30
sec
30
sec
30
sec

Beaucage
34/51/51
80/120/120

100
sec
200
sec
200
sec

Acetonitrile
NA
1150/1150/1150
μL
NA
NA
NA

*Wait time does not include contact time during delivery.

*Tandem synthesis utilizes double coupling of linker molecule

Number	Date	Country
60393796	Jul 2002	US
60399348	Jul 2002	US
60358580	Feb 2002	US
60358580	Feb 2002	US
60363124	Mar 2002	US
60363124	Mar 2002	US
60386782	Jun 2002	US
60386782	Jun 2002	US
60406784	Aug 2002	US
60406784	Aug 2002	US
60408378	Sep 2002	US
60408378	Sep 2002	US
60409493	Sep 2002	US
60409493	Sep 2002	US
60440129	Jan 2003	US
60440129	Jan 2003	US
60292217	May 2001	US
60362016	Mar 2002	US
60306883	Jul 2001	US
60311865	Aug 2001	US
60543480	Feb 2004	US

	Number	Date	Country
Parent	10844076	May 2004	US
Child	10944611	Sep 2004	US
Parent	10831620	Apr 2004	US
Child	10844076	May 2004	US
Parent	10764957	Jan 2004	US
Child	10831620	Apr 2004	US
Parent	10670011	Sep 2003	US
Child	10764957	Jan 2004	US
Parent	10665255	Sep 2003	US
Child	10670011	Sep 2003	US
Parent	10664767	Sep 2003	US
Child	10665255	Sep 2003	US
Parent	PCT/US03/05022	Feb 2003	US
Child	10664767	Sep 2003	US
Parent	PCT/US04/16390	May 2004	US
Child	10944611	Sep 2004	US
Parent	10826966	Apr 2004	US
Child	PCT/US04/16390	May 2004	US
Parent	10757803	Jan 2004	US
Child	10826966	Apr 2004	US
Parent	10720448	Nov 2003	US
Child	10757803	Jan 2004	US
Parent	10693059	Oct 2003	US
Child	10720448	Nov 2003	US
Parent	10444853	May 2003	US
Child	10693059	Oct 2003	US
Parent	PCT/US03/05346	Feb 2003	US
Child	10444853	May 2003	US
Parent	PCT/US03/05028	Feb 2003	US
Child	10444853	May 2003	US
Parent	PCT/US04/13456	Apr 2004	US
Child	10944611	Sep 2004	US
Parent	10780447	Feb 2004	US
Child	PCT/US04/13456	Apr 2004	US
Parent	10427160	Apr 2003	US
Child	10780447	Feb 2004	US
Parent	PCT/US02/15876	May 2002	US
Child	10427160	Apr 2003	US
Parent	10727780	Dec 2003	US
Child	10944611	Sep 2004	US

RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

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International Classifications

Abstract

Description

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Parent Case Info

Provisional Applications (21)

Continuation in Parts (20)