Dual Targeting siRNA Agents

Abstract
The invention relates to dual targeting siRNA agents targeting a PCSK9 gene and a second gene, and methods of using dual targeting siRNA agents to inhibit expression of PCSK9 and to treat PCSK9 related disorders, e.g., hyperlipidemia.
Description
REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing with 4166 sequences submitted electronically as a text file named AYL108C4_sequencelisting.txt, created on Feb. 20, 2020, with a size of 1,351,680 bytes. The sequence listing is incorporated by reference.


FIELD OF THE INVENTION

The invention relates to a composition of two covalently linked siRNAs, e.g., a dual targeting siRNA agent. At least one siRNA is a dsRNA that targets a PCSK9 gene. The covalently linked siRNA agent is used in methods of inhibition of PCSK9 gene expression and methods of treatment of pathological conditions associated with PCSK9 gene expression, e.g., hyperlipidemia.


BACKGROUND OF THE INVENTION

Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine protease family. The other eight mammalian subtilisin proteases, PCSK1-PCSK8 (also called PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and S1P/SKI-1) are proprotein convertases that process a wide variety of proteins in the secretory pathway and play roles in diverse biological processes (Bergeron, F. (2000) J. Mol. Endocrinol. 24, 1-22, Gensberg, K., (1998) Semin. Cell Dev. Biol. 9, 11-17, Seidah, N. G. (1999) Brain Res. 848, 45-62, Taylor, N. A., (2003) FASEB J. 17, 1215-1227, and Zhou, A., (1999) J Biol. Chem. 274, 20745-20748). PCSK9 has been proposed to play a role in cholesterol metabolism. PCSK9 mRNA expression is down-regulated by dietary cholesterol feeding in mice (Maxwell, K. N., (2003) J. Lipid Res. 44, 2109-2119), up-regulated by statins in HepG2 cells (Dubuc, G., (2004) Arterioscler. Thromb. Vasc. Biol. 24, 1454-1459), and up-regulated in sterol regulatory element binding protein (SREBP) transgenic mice (Horton, J. D., (2003) Proc. Natl. Acad. Sci. USA 100, 12027-12032), similar to the cholesterol biosynthetic enzymes and the low-density lipoprotein receptor (LDLR). Furthermore, PCSK9 missense mutations have been found to be associated with a form of autosomal dominant hypercholesterolemia (Hchola3) (Abifadel, M., et al. (2003) Nat. Genet. 34, 154-156, Timms, K. M., (2004) Hum. Genet. 114, 349-353, Leren, T. P. (2004) Clin. Genet. 65, 419-422). PCSK9 may also play a role in determining LDL cholesterol levels in the general population, because single-nucleotide polymorphisms (SNPs) have been associated with cholesterol levels in a Japanese population (Shioji, K., (2004) J. Hum. Genet. 49, 109-114).


Autosomal dominant hypercholesterolemias (ADHs) are monogenic diseases in which patients exhibit elevated total and LDL cholesterol levels, tendon xanthomas, and premature atherosclerosis (Rader, D. J., (2003) J Clin. Invest. 111, 1795-1803). The pathogenesis of ADHs and a recessive form, autosomal recessive hypercholesterolemia (ARH) (Cohen, J. C., (2003) Curr. Opin. Lipidol. 14, 121-127), is due to defects in LDL uptake by the liver. ADH may be caused by LDLR mutations, which prevent LDL uptake, or by mutations in the protein on LDL, apolipoprotein B, which binds to the LDLR. ARH is caused by mutations in the ARH protein that are necessary for endocytosis of the LDLR-LDL complex via its interaction with clathrin. Therefore, if PCSK9 mutations are causative in Hchola3 families, it seems likely that PCSK9 plays a role in receptor-mediated LDL uptake.


Overexpression studies point to a role for PCSK9 in controlling LDLR levels and, hence, LDL uptake by the liver (Maxwell, K. N. (2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol. Chem. 279, 50630-50638). Adenoviral-mediated overexpression of mouse or human PCSK9 for 3 or 4 days in mice results in elevated total and LDL cholesterol levels; this effect is not seen in LDLR knockout animals (Maxwell, K. N. (2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol. Chem. 279, 50630-50638). In addition, PCSK9 overexpression results in a severe reduction in hepatic LDLR protein, without affecting LDLR mRNA levels, SREBP protein levels, or SREBP protein nuclear to cytoplasmic ratio.


Loss of function mutations in PCSK9 have been designed in mouse models (Rashid et al., (2005) PNAS, 102, 5374-5379), and identified in human individuals (Cohen et al. (2005) Nature Genetics 37:161-165). In both cases loss of PCSK9 function lead to lowering of total and LDLc cholesterol. In a retrospective outcome study over 15 years, loss of one copy of PCSK9 was shown to shift LDLc levels lower and to lead to an increased risk-benefit protection from developing cardiovascular heart disease (Cohen et al., (2006) N Engl. J. Med., 354:1264-1272).


X-box binding protein 1 (XBP-1) is a basic leucine zipper transcription factor that is involved in the cellular unfolded protein response (UPR). XBP-1 is known to be active in the endoplasmic reticulum (ER). The ER consists of a system of folded membranes and tubules in the cytoplasm of cells. Proteins and lipids are manufactured and processed in the ER. When unusual demands are placed on the ER, “ER stress” occurs. ER stress can be triggered by a viral infection, gene mutations, exposure to toxins, aggregation of improperly folded proteins or a shortage of intracellular nutrients. The result can be Type II diabetes, metabolic syndrome, a neurological disorder or cancer.


Two XBP-1 isoforms are known to exist in cells: spliced XBP-1S and unspliced XBP-1U. Both isoforms of XBP-1 bind to the 21-bp Tax-responsive element of the human T-lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR) in vitro and transactivate HTLV-1 transcription. HTLV-1 is associated with a rare form of blood dyscrasia known as Adult T-cell Leukemia/lymphoma (ATLL) and a myelopathy, tropical spastic paresis.


Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.


A description of siRNA targeting PCSK9 can be found in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161). Additional disclosure can be found in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487).


A description of siRNA targeting XPB-1 can be found in U.S. patent application Ser. No. 12/425,811 filed on Apr. 17, 2009 and published as US 2009-0275638.


Dual targeting siRNAs can be found in International patent application publication no. WO/2007/091269.


SUMMARY OF THE INVENTION

Described herein are dual targeting siRNA agent in which a first siRNA targeting PCSK9 is covalently joined to a second siRNA targeting a gene implicated in cholesterol metabolism, e.g., XBP-1. The two siRNAs are covalently linked via, e.g., a disulfide linker.


Accordingly one aspect of the invention is a dual targeting siRNA agent having a first dsRNA targeting a PCSK9 gene and a second dsRNA targeting a second gene, wherein the first dsRNA and the second dsRNA are linked with a covalent linker. The second gene is can be, e.g., XBP-1, PCSK9, PCSK5, ApoC3, SCAP, or MIG12. In one embodiment, the second gene is XBP-1. Each dsRNA is 30 nucleotides or less in length. In general, each strand of each dsRNA is 19-23 bases in length.


In one embodiment, the dual targeting siRNA agent comprising a first dsRNA AD-10792 targeting a PCSK9 gene and a second dsRNA AD-18038 targeting an XBP-1 gene, wherein AD-10792 sense strand and AD-18038 sense strand are covalently linked with a disulfide linker.


The first dsRNA of the dual targeting siRNA agent targets a PCSK9 gene. In one aspect, the first dsRNA includes at least 15 contiguous nucleotides of an antisense strand of one of Tables 1, 2, or 4-8, or includes an antisense strand of one of Tables 1, 2, or 4-8, or includes a sense strand and an antisense strand of one of Tables 1, 2, or 4-8. The first dsRNA can be AD-9680 or AD-10792.


In some embodiments, the second dsRNA target XBP-1. In one aspect, the second dsRNA includes at least 15 contiguous nucleotides of an antisense strand of one of Tables 3 or 9-13, or includes an antisense strand of one of Tables 3 or 9-13, or includes a sense strand and an antisense strand of one of Tables 3 or 9-13. For example, the second dsRNA can be AD-18038.


Either the first and second dsRNA can include at least one modified nucleotide, e.g., a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. In some embodiments, the first and second dsRNAs include “endo-light” modification with 2′-O-methyl modified nucleotides and nucleotides comprising a 5′-phosphorothioate group.


The first and second dsRNAs are linked with a covalent linker. In some embodiments, the linker is a disulfide linker. Various combinations of strands can be linked, e.g., the first and second dsRNA sense strands are covalently linked or, e.g., the first and second dsRNA antisense strands are covalently linked. In some embodiments, any of the dual targeting siRNA agents of the invention include a ligand.


Also included in the invention are isolated cells having and vectors encoding the dual targeting siRNA agent described herein.


In one aspect, administration of the dual targeting siRNA agent to a cell inhibits expression of the PCSK9 gene and the second gene at a level equivalent to inhibition of expression of both genes using administration of each siRNA individually. In another aspect, administration of the dual targeting siRNA agent to a subject results in a greater reduction of total serum cholesterol that that obtained by administration of each siRNA alone.


The invention also includes a pharmaceutical composition comprising the dual targeting siRNA agents described herein and a pharmaceutical carrier. In one embodiment, the pharmaceutical carrier is a lipid formulation, e.g., a lipid formulation including cationic lipid DLinDMA or cationic lipid XTC. Examples of lipid formulations described in (but not limited to) Table A, below. The lipid formulation can be XTC/DSPC/Cholesterol/PEG-DMG at % mol ratios of 50/10/38.5/1.5.


Another aspect of the invention includes methods of using the dual targeting siRNA agents described herein. In one embodiment, the invention is a method of inhibiting expression of the PCSK9 gene and a second gene in a cell, the method comprising (a) introducing into the cell the any of the dual targeting siRNA agents and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the PCSK9 gene and the second gene, thereby inhibiting expression of the PCSK9 gene and the second gene in the cell.


In another embodiment, the invention includes methods of treating a disorder mediated by PCSK9 expression with the step of administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical compositions described herein. In one aspect, the disorder is hyperlipidemia. In still another embodiment, the invention includes methods of reducing total serum cholesterol in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical compositions described herein.


The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.





DESCRIPTION OF THE DRAWINGS


FIG. 1A is a graph showing the effect on PCSK9 mRNA levels in primary mouse hepatocytes following treatment with a dual targeting siRNA, AD-23426. AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA. Lipo2000: control transfection agent only FIG. 1B is a graph showing the effect on XBP-1 mRNA levels in primary mouse hepatocytes following treatment with a dual targeting siRNA, AD-23426. AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA. Lipo2000: control transfection agent only.



FIG. 2 is a graph showing the effect on PCSK9 and XBP-1 mRNA levels in mice following treatment with a dual targeting siRNA, AD-23426. LNP09 (lipid) formulated siRNA was administered to mice as described. AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.



FIG. 3 is a graph showing the effect on serum cholesterol levels in mice following treatment with a dual targeting siRNA, AD-23426. LNP09 (lipid) formulated siRNA was administered to mice as described. AD-23426 was more effective at reducing serum cholesterol compared to each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.



FIG. 4A is a graph showing the effect on IFN-α in human PBMC following treatment with a dual targeting siRNA, AD-23426. FIG. 4B is a graph showing the effect on TNF-α in human PBMC following treatment with a dual targeting siRNA, AD-23426. DOTAP and LNP09 (lipid) formulated siRNAs was administered huPBMC as described below. AD-23426 did not induce IFN-α or TNF-α.





DETAILED DESCRIPTION OF THE INVENTION

The invention provides a solution to the problem of treating diseases that can be modulated by the down regulation of the PCSK9 gene, such as hyperlipidemia, by using dual targeting siRNA to silence the PCSK9 gene.


The invention provides compositions and methods for inhibiting the expression of the PCSK9 gene in a subject using two siRNA, e.g., a dual targeting siRNA. The invention also provides compositions and methods for treating pathological conditions and diseases, such as hyperlipidemia, that can be modulated by down regulating the expression of the PCSK9 gene.


The dual targeting siRNA agents target a PCSK9 gene and at least one other gene. The other gene can be another region of the PCSK9 gene, or can be another gene, e.g., XBP-1.


The dual targeting siRNA agents have the advantage of lower toxicity, lower off-target effects, and lower effective concentration compared to individual siRNAs.


The use of the dual targeting siRNA dsRNAs enables the targeted degradation of an mRNA that is involved in the regulation of the LDL receptor and circulating cholesterol levels. Using cell-based and animal assays it was demonstrated that inhibiting both a PCSK9 gene and an XBP-1 gene using a dual targeting siRNA is at least as effective at inhibiting their corresponding targets as the use of single siRNAs. It was also demonstrated that administration of a dual targeting siRNA results in a synergistic lowering of total serum cholesterol. Thus, reduction of total serum cholesterol is enhanced with a dual targeting siRNA compared to a single target siRNA.


Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.


“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.


The term “PCSK9” refers to the proprotein convertase subtilisin kexin 9 gene or protein (also known as FH3, HCHOLA3, NARC-1, NARC1). Examples of mRNA sequences to PCSK9 include but are not limited to the following: human: NM_174936; mouse: NM_153565, and rat: NM_199253. Additional examples of PCSK9 mRNA sequences are readily available using, e.g., GenBank.


The term “XBP-1” refers to—Box Protein 1, which is also known as Tax-responsive element-binding protein 5, TREBS, and XBP2. XBP-1 sequence can be found as NCBI GeneID:7494 and RefSeq ID number:NM_005080 (human) and NM_013842 (mouse). A dsRNA featured in the invention can target a specific XBP-1 isoform, e.g., the spliced form (XBP-1S) or the unspliced form (XBP-1U), or a dsRNA featured in the invention can target both isoforms by binding to a common region of the mRNA transcript.


The term “PCSK5” refers to the Proprotein convertase subtilisin/kexin type 5 gene, mRNA or protein belonging to the subtilisin-like proprotein convertase family.


The term “ApoC3” refers to the Apolipoprotein C-III protein gene, mRNA or protein, and is a very low density lipoprotein (VLDL).


The term “SCAP” refers to the SREBP cleavage-activating protein gene, mRNA or protein. SCAP is a regulatory protein that is required for the proteolytic cleavage of the sterol regulatory element binding protein (SREBP). Example of siRNA targeting SCAP are described in U.S. patent application Ser. No. 11/857,120, filed on Sep. 18, 2007, published as US 20090093426. This application and the siRNA sequences described therein are incorporated by reference for all purposes.


The term “MIG12” is a gene also known as TMSB10 and TB10 refers to the thymosin beta 10 gene. Example of siRNA targeting MIG12 are described International patent application no. PCT/US10/25444, filed on Feb. 25, 2010, published as WO/20XX This application and the siRNA sequences described therein are incorporated by reference for all purposes.


As used herein, the term “iRNA” refers to an agent that contains RNA and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. The term iRNA includes siRNA.


As described in more detail below, the term “siRNA” and “siRNA agent” refers to a dsRNA that mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.


A “double-stranded RNA” or “dsRNA,” as used herein, refers to an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA.


The term “dual targeting siRNA agent” refers to a composition of two siRNAs, e.g., two dsRNAs. One dsRNA includes an antisense strand with a first region of complementarity to a first target gene, e.g., PCSK9. The second dsRNA include an antisense strand with a second region of complementarity to a second target gene. In some embodiments, the first and second target genes are identical, e.g., both are PCSK9 and each dsRNA targets a different region of PCSK9. In other embodiments, the first and second target genes are different, e.g., the first dsRNA targets PCSK9 and the second dsRNA targets a different gene, e.g., XBP-1.


“Covalent linker” refers to a molecule for covalently joining two molecules, e.g., two dsRNAs. As described in more detail below, the term includes, e.g., a nucleic acid linker, a peptide linker, and the like and includes disulfide linkers.


The term “target gene” refers to a gene of interest, e.g., PCSK9 or a second gene, e.g., XBP-1, targeted by an siRNA of the invention for inhibition of expression.


As described in more detail below, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween.


As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.


As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.


Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.


“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.


The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.


As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of the target gene (e.g., an mRNA encoding PCSK9 or a second gene, e.g., XBP-1). For example, a polynucleotide is complementary to at least a part of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding PCSK9.


The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleotide, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.


In one aspect, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. However, it is self evident that under no circumstances is a double stranded DNA molecule encompassed by the term “iRNA.”


As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′ end, 3′ end or both ends of either an antisense or sense strand of a dsRNA. One or more of the nucleotides in the overhang can be replaced with a nucleoside thiophosphate.


The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.


The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.


The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.


As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817. These applications are incorporated herein by reference in their entirety.


“Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art.


As used herein, the term “modulate the expression of,” refers to at an least partial “inhibition” or partial “activation” of target gene expression in a cell treated with an iRNA composition as described herein compared to the expression of the target gene in an untreated cell.


The terms “activate,” “enhance,” “up-regulate the expression of,” “increase the expression of,” and the like, in so far as they refer to a target gene, herein refer to the at least partial activation of the expression of a target gene, as manifested by an increase in the amount of target mRNA, which may be isolated from or detected in a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression of a target gene is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).


In one embodiment, expression of a target gene is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA as described herein. In some embodiments, a target gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured in the invention. In some embodiments, expression of a target gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA as described herein. In some embodiments, the target gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000 fold or more in cells treated with an iRNA as described herein compared to the expression in an untreated cell. Activation of expression by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42, and in US20070111963 and US2005226848, each of which is incorporated herein by reference.


The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of,” and the like, in so far as they refer to a target gene, herein refer to the at least partial suppression of the expression of a target gene, as manifested by a reduction of the amount of target mRNA which may be isolated from or detected in a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression of target gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of










(

mRNA





in





control





cells

)

-

(

mRNA





in





treated





cells

)



(

mRNA





in





control





cells

)


·
100


%




Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to target gene expression, e.g., the amount of protein encoded by a target gene, or the number of cells displaying a certain phenotype, e.g., lack of or decreased cytokine production. In principle, target gene silencing may be determined in any cell expressing target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given iRNA inhibits the expression of the target gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.


For example, in certain instances, expression of a target gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% by administration of an iRNA featured in the invention. In some embodiments, a target gene is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA featured in the invention. In some embodiments, a target gene is suppressed by at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more by administration of an iRNA as described herein.


As used herein in the context of target gene expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by target expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by target expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition.


By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder.


As used herein, the phrase “therapeutically effective amount”” refers to an amount that provides a therapeutic benefit in the treatment or management of pathological processes mediated by target gene expression, e.g., PCSK9 and/or a second gene, e.g., XBP-1, or an overt symptom of pathological processes mediated target gene expression. The phrase “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the prevention of pathological processes mediated by target gene expression or an overt symptom of pathological processes mediated by target gene expression. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by target gene expression, the patient's history and age, the stage of pathological processes mediated by target gene expression, and the administration of other agents that inhibit pathological processes mediated by target gene expression.


As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an iRNA effective to produce the intended pharmacological or therapeutic result. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter.


The term “pharmaceutically carrier” refers to a carrier for administration of a therapeutic agent, e.g., a dual targeting siRNA agent. Carriers are described in more detail below, and include lipid formulations, e.g., LNP09 and SNALP formulations.


Double-Stranded Ribonucleic Acid (dsRNA)


Described herein are dual targeting siRNA agents, e.g., siRNAs that inhibit the expression of a PCSK9 gene and a second gene. The dual targeting siRNA agent includes two siRNA covalently linked via, e.g., a disulfide linker. The first siRNA targets a first region of a PCSK9 gene. The second siRNA targets a second gene, e.g., XBP-1, or, e.g., targets a second region of the PCSK9 gene.


The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Applied Biosystems, Inc. Further descriptions of synthesis are found below and in the examples.


Each siRNA is a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a target gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.


Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.


Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a “linker.”


Generally, the duplex structure of the siRNA, e.g., dsRNA, is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 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, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs.


The two siRNAs in the dual targeting siRNA agent can have duplex lengths that are identical or that differ.


The region of complementarity to the target sequence in an siRNA is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. The region of complementarity can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides. In some embodiments the target sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.


The two siRNAs in the dual targeting siRNA agent can have regions of complementarity that are identical in length or that differ in length.


Any of the dsRNA, e.g., siRNA as described herein may include one or more single-stranded nucleotide overhangs. In one embodiment, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, or 1 or 2 or 3 or 4 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. The two siRNAs in the dual targeting siRNA agent can have different or identical overhangs as described by location, length, and nucleotide.


The dual targeting siRNA agent includes at least a first siRNA targeting a first region of a PCSK9 gene. In one embodiment, a PCSK9 gene is a human PCSK9 gene. In another embodiment the PCSK9 gene is a mouse or a rat PCSK9 gene. Exemplary siRNA targeting PCSK9 are described in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161). Additional disclosure can be found in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487). The sequences of the target, sense, and antisense strands are incorporated by reference for all purposes.


Tables 1, 2, and 4-8 disclose sequences of the target, sense strands, and antisense strands of PCSK9 targeting siRNA.


In one embodiment the first siRNA is AD-9680. The dsRNA AD-9680 targets the human PCSK 9 gene at nucleotides 3530-3548 of a human PCSK9 gene (accession number NM_174936).









TABLE 1







AD-9680 siRNA sequences









Table 1: AD-9680
Sequence 5′ to 3′
SEQ ID NO:





Target sequence
UUCUAGACCUGUUUUGCUU
4142





Sense strand
UUCUAGACCUGUUUUGCUU
4143





Sense strand,
uucuAGAccuGuuuuGcuuTsT
4144


modified







Antisense strand
AAGCAAAACAGGUCUAGAA
4145





Antisense strand,
AAGcAAAAcAGGUCuAGAATsT
4146


modified









In another embodiment, the first siRNA is AD-10792. The dsRNA AD-10792 targets the PCSK9 gene at nucleotides 1091-1109 of a human PCSK9 gene (accession number NM_174936). AD-10792 is also complementary to rodent PCSK9.









TABLE 2







AD-10792 siRNA sequences











SEQ


Table 2: AD-10792
Sequence 5′ to 3′
ID NO:





Target sequence
GCCUGGAGUUUAUUCGGAA
4147





Sense strand
GCCUGGAGUUUAUUCGGAA
4148





Sense strand,
GccuGGAGuuuAuucGGAATsT
4149


modified







Antisense strand
UUCCGAAUAAACUCCAGGC
4150





Antisense strand,
UUCCGAAuAAACUCcAGGCTsT
4151


modified









The second siRNA of the dual targeting siRNA agent targets a second gene. In one embodiment, the second gene is PCSK9, and the second siRNA target a region of PCSK9 that is different from the region targeted by the first siRNA.


Alternatively, the second siRNA targets a different second gene. Examples include genes that interact with PCSK9 and/or are involved with lipid metabolism or cholesterol metabolism. For example, the second target gene can be XBP-1, PCSK5, ApoC3, SCAP, MIG12, HMG CoA Reductase, or IDOL (Inducible Degrader of the LDLR) and the like. In one embodiment, the second gene is a human gene. In another embodiment the second gene is a mouse or a rat gene.


In one embodiment, the second siRNA targets the XBP-1 gene. Exemplary siRNA targeting XBP-1 can be found in U.S. patent application Ser. No. 12/425,811 filed Apr. 17, 2009 (published as US 2009-0275638). The sequences of the target, sense, and antisense strands are incorporated by reference for all purposes.


Tables 3 and 9-13 disclose sequences of the target, sense strands, and antisense strands of XBP-1 targeting siRNA.


In one embodiment the first siRNA is AD-18038. The dsRNA AD-18038 targets the human XBP-1 gene at nucleotides 896-914 of a human XBP-1 gene (accession number NM_001004210).









TABLE 3







AD-18038 siRNA sequences









Table 3: AD-18038
Sequence 5′ to 3′
SEQ ID NO:





Target sequence
CACCCUGAAUUCAUUGUCU
4153





Sense strand
CACCCUGAAUUCAUUGUCU
4154





Sense strand, modified
cAcccuGAAuucAuuGucudTsdT
4155





Antisense strand
AGACAAUGAAUUCAGGGUG
4156





Antisense strand, modified
AGAcAAUGAAUUcAGGGUGdTsdT
4157









Additional dsRNA


A dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences in Tables 1-13, and differing in their ability to inhibit the expression of a target gene by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated according to the invention.


In addition, the RNAs provided in Tables 1-13 identify a site in the target gene transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of such sequences. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least 15 contiguous nucleotides from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a target gene.


While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, above represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.


Further, it is contemplated that for any sequence identified, e.g., in Tables 1-13, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.


An iRNA as described in Tables 1-13 can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described in Tables 1-13 contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of a PCSK9 gene, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a PCSK9 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a PCSK9 gene is important, especially if the particular region of complementarity in a PCSK9 gene is known to have polymorphic sequence variation within the population.


Covalent Linkage


The dual targeting siRNA agents of the invention include two siRNAs joined via a covalent linker. Covalent linkers are well-known to one of skill in the art and include, e.g., a nucleic acid linker, a peptide linker, and the like.


The covalent linker joins the two siRNAs. The covalent linker can join two sense strands, two antisense strands, one sense and one antisense strand, two sense strands and one antisense strand, two antisense strands and one sense strand, or two sense and two antisense strands.


The covalent linker can include RNA and/or DNA and/or a peptide. The linker can be single stranded, double stranded, partially single strands, or partially double stranded. In some embodiments the linker includes a disulfide bond. The linker can be cleavable or non-cleavable.


The covalent linker can be, e.g., dTsdTuu=(5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-3′-phosphate); rUsrU (a thiophosphate linker: 5′-uridyl-3′-thiophosphate-5′-uridyl-3′-phosphate); an rUrU linker; dTsdTaa (aadTsdT, 5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-adenyl-3′-phosphate-5′-adenyl-3′-phosphate); dTsdT (5′-2′deoxythyrnidyl-3′-thiophosphate-5′-2′ deoxythymidyl-3′-phosphate); dTsdTuu=uudTsdT=5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-3′-phosphate.


The covalent linker can be a polyRNA, such as poly(5′-adenyl-3′-phosphate—AAAAAAAA) or poly(5′-cytidyl-3′-phosphate-5′-uridyl-3′-phosphate—CUCUCUCU)), e.g., Xn single stranded poly RNA linker wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyRNA linker. The covalent linker can be a polyDNA, such as poly(5′-2′deoxythymidyl-3′-phosphate—TTTTTTTT), e.g., wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyDNA linker. a single stranded polyDNA linker wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyDNA linker.


The covalent linker can include a disulfide bond, optionally a bis-hexyl-disulfide linker. In one embodiment, the disulfide linker is




embedded image


The covalent linker can include a peptide bond, e.g., include amino acids. In one embodiment, the covalent linker is a 1-10 amino acid long linker, preferably comprising 4-5 amino acids, optionally X-Gly-Phe-Gly-Y wherein X and Y represent any amino acid.


The covalent linker can include HEG, a hexaethylenglycol linker.


Modifications


In yet another embodiment, at least one of the siRNA of the dual targeting siRNA agent is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in this invention include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.


Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.


Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, each of which is herein incorporated by reference


Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.


Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.


In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.


Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2—[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.


Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to Cm alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C1 to Cm lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below.


Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.


An iRNA may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.


Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.


The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).


Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.


Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2′-docosanoyl-uridine-3′-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in U.S. Provisional Patent Application No. 61/223,665 (“the '665 application”), filed Jul. 7, 2009, entitled “Oligonucleotide End Caps” and International patent application no. PCT/US10/41214, filed Jul. 7, 2010.


Ligands


Another modification of the RNA of an iRNA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).


In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.


Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.


Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.


Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.


Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.


The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.


In one ligand, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.


A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.


In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.


In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.


In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).


In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.


The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.


A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:4158). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:4159)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:4160)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:4161)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.


An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing αvβ3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).


A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).


Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which is herein incorporated by reference.


Chimeras


It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds. “Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.


Non-ligand groups


In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.


Delivery of iRNA


The delivery of an iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.


Direct Delivery


In general, any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.


Vector Encoded dsRNAs


In another aspect, the dsRNAs of the invention can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).


The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.


iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.


iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.


Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.


Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.


Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.


In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.


Adenoviruses are also contemplated for use in delivery of iRNAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.


Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.


Another preferred viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.


The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.


The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.


Pharmaceutical Compositions Containing iRNA


In one embodiment, the invention provides pharmaceutical compositions containing a dual targeting siRNA agent, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the siRNA is useful for treating a disease or disorder associated with the expression or activity of a target gene, such as pathological processes mediated by PCSK9 expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.


The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of the target genes. In general, a suitable dose of siRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg, or 50 mg/kg per single dose.


The pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.


The effect of a single dose of siRNA on PCSK9 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.


The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.


Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by PCSK9 expression. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a transgene expressing human PCSK9.


The present invention also includes pharmaceutical compositions and formulations that include the iRNA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.


The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).


Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.


Liposomal Formulations


There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.


Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.


In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.


Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.


Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.


Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.


Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis


Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).


Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).


One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).


Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).


Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).


Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).


Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.


A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.


Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.


Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).


If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.


If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.


If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.


If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.


The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).


Nucleic Acid Lipid Particles


In one embodiment, a dual targeting siRNA agent featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a nucleic acid-lipid particle, e.g., a SPLP, pSPLP, or SNALP. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. Nucleic acid-lipid particles, e.g., SNALPs, typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP”, which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683.


The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. For example, the mean diameter of the particles can be about 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 140 nm, 145 nm, or 150 nm.


In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.


In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. The lipid to dsRNA ratio can be about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 113:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1.


The nucleic acid lipid particles include a cationic lipid. The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC), (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyOdidodecan-2-ol (Tech G1, e.g., C12-200), or a mixture thereof.


The cationic lipid may comprise from about 10 mol % to about 70 mol % or about 40 mol % of the total lipid present in the particle. The cationic lipid may comprise 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol % of the total lipid present in the particle. The cationic lipid may comprise 57.1 mol % or 57.5 mol % of the total lipid present in the particle.


In one embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (XTC) can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.


In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±nm and a 0.027 siRNA/Lipid Ratio.


The nucleic acid lipid particle generally includes a non-cationic lipid. The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof.


The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle. The non-cationic lipid may be about 5 mol %, 6 mol %, 7 mol %, 7.5 mol %, 7.7 mol %, 8 mol %, 9 mol %, 10 mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol %, 15 mol %, 16 mol %, 17 mol %, 18 mol %, 19 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol %.


The nucleic acid lipid particle generally includes a conjugated lipid. The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C]8). The conjugated lipid can be PEG-DMG (PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000); PEG-DSG (PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000); or PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000).


The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0 17.0, 18, 19.0 or 20.0 mol % of the total lipid present in the particle.


In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle. For example, the nucleic acid-lipid particle further includes cholesterol at about 5 mol %, 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol %. The nucleic acid-lipid particle can include cholesterol at about 31.5 mol %, 34.4 mol %, 35 mol %, 38.5 mol %, or 40 mol % of the total lipid present in the particle.


LNP01


In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.




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LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.


Exemplary Nucleic Acid Lipid Particles


Additional exemplary lipid-dsRNA formulations are as follows:











TABLE A







cationic lipid/non-cationic




lipid/cholesterol/PEG-lipid conjugate



Cationic
Mol % ratios



Lipid
Lipid:siRNA ratio







SNALP
DLinDMA
DLinDMA/DPPC/Cholesterol/PEG-cDMA




(57.1/7.1/34.4/1.4)




lipid:siRNA~7:1


S-XTC
XTC
XTC/DPPC/Cholesterol/PEG-cDMA




57.1/7.1/34.4/1.4




lipid:siRNA~7:1


LNP05
XTC
XTC/DSPC/Cholesterol/PEG-DMG




57.5/7.5/31.5/3.5




lipid:siRNA~6:1


LNP06
XTC
XTC/DSPC/Cholesterol/PEG-DMG




57.5/7.5/31.5/3.5




lipid:siRNA~11:1


LNP07
XTC
XTC/DSPC/Cholesterol/PEG-DMG




60/7.5/31/1.5,




lipid:siRNA~6:1


LNP08
XTC
XTC/DSPC/Cholesterol/PEG-DMG




60/7.5/31/1.5,




lipid:siRNA—11:1


LNP09
XTC
XTC/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP10
ALN100
ALN100/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP11
MC3
MC-3/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP12
C12-200
C12-200/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP13
XTC
XTC/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 33:1


LNP14
MC3
MC3/DSPC/Chol/PEG-DMG




40/15/40/5




Lipid:siRNA: 11:1


LNP15
MC3
MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG




50/10/35/4.5/0.5




Lipid:siRNA: 11:1


LNP16
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP17
MC3
MC3/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP18
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 12:1


LNP19
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/35/5




Lipid:siRNA: 8:1


LNP20
MC3
MC3/DSPC/Chol/PEG-DPG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP22
XTC
XTC/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1









SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.


XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International patent application no. PCT/US 10/22614, filed Jan. 29, 2010, which are hereby incorporated by reference.


MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, which are hereby incorporated by reference.


ALN100, i.e., ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.


C12-200, i.e., Tech G1 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009, which is hereby incorporated by reference.


Synthesis of Cationic Lipids.


Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.


“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.


“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.


“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.


“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.


“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below.


Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.


The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.


“Halogen” means fluoro, chloro, bromo and iodo.


In some embodiments, the methods of the invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.


Synthesis of Formula A


In one embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A; XTC is a cationic lipid of formula A:




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where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring.


In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.




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Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.




embedded image


Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.


Synthesis of MC3 Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).


Synthesis of ALNY-100


Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:




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Synthesis of 515:


To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).


Synthesis of 516:


To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H]-232.3 (96.94%).


Synthesis of 517A and 517B:


The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (lx 50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude


517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]-266.3, [M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.


Synthesis of 518:


Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.


General Procedure for the Synthesis of Compound 519:


A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR □=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.


General Synthesis of Nucleic Acid Lipid Particles


Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.


Other Formulations


Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.


Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.


Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.


The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.


The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.


Additional Formulations


Emulsions


The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.


Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).


Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).


Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.


A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).


Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.


Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.


The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.


In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).


The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.


Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene olyel ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.


Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.


Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.


Penetration Enhancers


In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.


Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.


Surfactants:


In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).


Fatty Acids:


Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).


Bile Salts:


The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).


Chelating Agents:


Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).


Non-Chelating Non-Surfactants:


As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).


Agents that enhance uptake of iRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lotto et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.


Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.


Carriers


Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.


Excipients


In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).


Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.


Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.


Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.


Other Components


The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.


Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.


In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more biologic agents which function by a non-RNAi mechanism. Examples of such biologics include, biologics that target one or more of PD-1, PD-L1, or B7-H1 (CD80) (e.g., monoclonal antibodies against PD-1, PD-L1, or B7-H1), or one or more recombinant cytokines (e.g., IL6, IFN-γ, and TNF).


Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.


The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.


In addition to their administration, as discussed above, the dual targeting siRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by PCSK9 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.


Methods Using Dual Targeting siRNAs


In one aspect, the invention provides use of a dual targeting siRNA agent for inhibiting the expression of the PCSK9 gene in a mammal. The method includes administering a composition of the invention to the mammal such that expression of the target PCSK9 gene is decreased. In some embodiments, PCSK9 expression is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, or four weeks or longer. For example, in certain instances, expression of the PCSK9 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a dual targeting siRNA agent described herein. In some embodiments, the PCSK9 gene is suppressed by at least about 60%, 70%, or 80% by administration of the dual targeting siRNA agent. In some embodiments, the PCSK9 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide.


The methods and compositions described herein can be used to treat diseases and conditions that can be modulated by down regulating PCSK9 gene expression. For example, the compositions described herein can be used to treat hyperlipidemia and other forms of lipid imbalance such as hypercholesterolemia, hypertriglyceridemia and the pathological conditions associated with these disorders such as heart and circulatory diseases


Therefore, the invention also relates to the use of a dual targeting siRNA agent for the treatment of a PCSK9-mediated disorder or disease. For example, a dual targeting siRNA agent is used for treatment of a hyperlipidemia.


The effect of the decreased PCSK9 gene preferably results in a decrease in LDLc (low density lipoprotein cholesterol) levels in the blood, and more particularly in the serum, of the mammal. In some embodiments, LDLc levels are decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.


The method includes administering a dual targeting siRNA agent to the subject to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, and airway (aerosol) administration. In some embodiments, the compositions are administered by intravenous infusion or injection.


The method includes administering a dual targeting siRNA agent, e.g., a dose sufficient to depress levels of PCSK9 mRNA for at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days; and optionally, administering a second single dose of dsRNA, wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the PCSK9 gene in a subject.


In one embodiment, doses of dual targeting siRNA agent are administered not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administrations can be maintained for one, two, three, or six months, or one year or longer.


In another embodiment, administration can be provided when Low Density Lipoprotein cholesterol (LDLc) levels reach or surpass a predetermined minimal level, such as greater than 70 mg/dL, 130 mg/dL, 150 mg/dL, 200 mg/dL, 300 mg/dL, or 400 mg/dL.


In general, the dual targeting siRNA agent does not activate the immune system, e.g., it does not increase cytokine levels, such as TNF-alpha or IFN-alpha levels. For example, when measured by an assay, such as an in vitro PBMC assay, such as described herein, the increase in levels of TNF-alpha or IFN-alpha, is less than 30%, 20%, or 10% of control cells treated with a control dsRNA, such as a dsRNA that does not target PCSK9.


For example, a subject can be administered a therapeutic amount of dual targeting siRNA agent, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dual targeting siRNA agent can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the dual targeting siRNA agent can reduce PCSK9 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.


Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.


A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given dual targeting siRNA agent drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.


Additional Agents


In further embodiments, administration of a dual targeting siRNA agent is administered in combination an additional therapeutic agent. The dual targeting siRNA agent and an additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.


Examples of additional therapeutic agents include those known to treat an agent known to treat a lipid disorders, such as hypercholesterolemia, atherosclerosis or dyslipidemia. For example, a dual targeting siRNA agent featured in the invention can be administered with, e.g., an HMG-CoA reductase inhibitor (e.g., a statin), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck & Co.'s Cozaar®), an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a gene-based therapy, a composite vascular protectant (e.g., AGI-1067, from Atherogenics), a glycoprotein inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor (e.g., S-8921, from Shionogi), a squalene synthase inhibitor, or a monocyte chemoattractant protein (MCP)-I inhibitor. Exemplary HMG-CoA reductase inhibitors include atorvastatin (Pfizer's Lipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck's Zocor®/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas), lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma's Liposcler), fluvastatin (Novartis' Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin (Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g., bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol), clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier's Lipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate (Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrants include, e.g., cholestyramine (Bristol-Myers Squibb's Questran® and Questran Light™), colestipol (e.g., Pharmacia's Colestid), and colesevelam (Genzyme/Sankyo's WelChol′). Exemplary niacin therapies include, e.g., immediate release formulations, such as Aventis' Nicobid, Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit. Niacin extended release formulations include, e.g., Kos Pharmaceuticals' Niaspan and Upsher-Smith's Slo-Niacin. Exemplary antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel (Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine (e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Other aspirin-like compounds useful in combination with a dsRNA targeting PCSK9 include, e.g., Asacard (slow-release aspirin, by Pharmacia) and Pamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplary angiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g., Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplary acyl CoA cholesterol acetyltransferase (ACAT) inhibitors include, e.g., avasimibe (Pfizer), eflucimibe (BioM{circumflex over (ε)}rieux Pierre Fabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito). Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer). Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics). Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer). Other exemplary cholesterol modulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433 (Wyeth-Ayerst). Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca). Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242) (AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson), GW-409544 (Ligand Pharniaceuticals/GlaxoSmithKline), GW-501516 (Ligand Pharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and Eli Lilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674 (Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin). Exemplary gene-based therapies include, e.g., AdGWEGF121.10 (GenVec), ApoAl (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics/Incyte, Aventis, Xenon). Exemplary Glycoprotein Ilb/IIIa inhibitors include, e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban (Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals). Exemplary squalene synthase inhibitors include, e.g., BMS-1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I inhibitor is, e.g., RS-504393 (Roche Bioscience). The anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivative Nyclin (Yamanouchi Pharmacuticals) are also appropriate for administering in combination with a dsRNA featured in the invention. Exemplary combination therapies suitable for administration with a dsRNA targeting PCSK9 include, e.g., advicor (Niacin/lovastatin from Kos Pharmaceuticals), amlodipine/atorvastatin (Pfizer), and ezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals). Agents for treating hypercholesterolemia, and suitable for administration in combination with a dsRNA targeting PCSK9 include, e.g., lovastatin, niacin Altoprev® Extended-Release Tablets (Andrx Labs), lovastatin Caduet® Tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor® Tablets (AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis), fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodium Lipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules (Gate), Niaspan Extended-Release Tablets (Kos), niacin Pravachol Tablets (Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott), fenofibrate Vytorin® 10/10 Tablets (Merck/Schering-Plough Pharmaceuticals), ezetimibe, simvastatin WelChol™ Tablets (Sankyo), colesevelam hydrochloride Zetia® Tablets (Schering), ezetimibe Zetia® Tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor® Tablets (Merck).


In one embodiment, a dual targeting siRNA agent is administered in combination with an ezetimibe/simvastatin combination (e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)).


In one embodiment, the dual targeting siRNA agent is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the dual targeting siRNA agent and the additional therapeutic agent are administered at the same time.


In another aspect, the invention features, a method of instructing an end user, e.g., a caregiver or a subject, on how to administer a dual targeting siRNA agent described herein. The method includes, optionally, providing the end user with one or more doses of the dual targeting siRNA agent, and instructing the end user to administer the dual targeting siRNA agent on a regimen described herein, thereby instructing the end user.


Identification of Patients


In one aspect, the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, or total cholesterol lowering. The method includes administering to the patient a dual targeting siRNA agent in an amount sufficient to lower the patient's LDL levels or ApoB levels, e.g., without substantially lowering HDL levels.


Genetic predisposition plays a role in the development of target gene associated diseases, e.g., hyperlipidemia. Therefore, a patient in need of a dual targeting siRNA agent can be identified by taking a family history, or, for example, screening for one or more genetic markers or variants. A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering a dual targeting siRNA agent. For example, a DNA test may also be performed on the patient to identify a mutation in the PCSK9 gene, before a PCSK9 dsRNA is administered to the patient.


Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.


EXAMPLES
Example 1. iRNA Synthesis

Source of Reagents


Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.


Oligonucleotide Synthesis.


All oligonucleotides are synthesized on an AKTAoligopilot synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 500 Å, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH3CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO-oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is used.


3′-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies. Conjugation of ligands to 5′-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid-support-bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes.


Deprotection I (Nucleobase Deprotection)


After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at 55° C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture is then reduced to −30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac.


Deprotection II (Removal of 2′-TBDMS Group)


The dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer until purification.


Analysis


The oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.


HPLC Purification


The ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC. The unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH3CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotidess are diluted in water to 150 μL and then pipetted into special vials for CGE and LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.


iRNA Preparation


For the general preparation of iRNA, equimolar amounts of sense and antisense strand are heated in 1×PBS at 95° C. for 5 min and slowly cooled to room temperature. Integrity of the duplex is confirmed by HPLC analysis.


Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table B.









TABLE B







Abbreviations of nucleotide monomers used in nucleic acid sequence


representation. It will be understood that these monomers, when present in


an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds.








Abbreviation
Nucleotide (s)





A
adenosine


C
cytidine


G
guanosine


U
uridine


N
any nucleotide (G, A, C, T or U)


a
2′-O-methyladenosine


c
2′-O-methylcytidine


g
2′-O-methylguanosine


u
2′-O-methyluridine


dT, T
2′-deoxythymidine


s
phosphorothioate linkage









Example 2. PCSK9 siRNA Design, Synthesis, and Screening

A description of the design, synthesis, and assays using PCSK9 siRNA can be found in detail in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161) and in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487). All are incorporated by reference in their entirety for all purposes.


The sequences of siRNA targeting a PCSK9 gene are described in Table 1 and Table 2 above, and Tables 4-8 below.


Example 3. XBP-1 siRNA Design, Synthesis, and Screening

A description of the design, synthesis, and assays using XBP-1 siRNA can be found in detail in U.S. patent application Ser. No. 12/425,811 filed on Apr. 17, 2009 and published as US 2009-0275638. This application is incorporated by reference in its entirety for all purposes.


The sequences of siRNA targeting a XBP-1 gene are described in Table 3 above, and Tables 9-13 below.


Example 4. A Dual Targeting siRNA Agent

A dual targeting siRNA agent was synthesized. The sense and antisense strands for AD-10792 (target gene is PCSK9, see Table 2)) and AD-18038 (target gene is XBP-1, see Table 3) were synthesized. The two sense strands were covalently bound using a disulfide linker “Q51” with the structure shown below.




embedded image


The resulting dual sense strand was hybridized to the corresponding antisense strands to create a 42 mer dual targeting siRNA agent “AD-23426” (SEQ ID NOS 4162-4165, respectively, in order of appearance):









GccuGGAGuuuAuucGGAAdTsdTQ51cAcccuGAAuucAuuGucudTsdT





dTsdTCGGAcCUCAAAuAAGCCUU dTsdTGUGGGAcUUAAGUAAcAGA






Example 5. Inhibition of PCSK9 and Xbp-1 mRNA Levels by the PCSK9-Xbp1 Dual Targeting siRNA in Primary Mouse Hepatocytes

Primary mouse hepatocytes were transfected with dual targeting AD-23426 or individual siRNAs (AD-10792 and AD-18038) in lipofectamine 2000 (Invitrogen protocol). 48 hours after transfection cells were harvested and lysed. PCSK9, Xbp-1 and GAPDH transcripts were measured via bDNA in cell lysates prepared according to manufacturer's protocol. PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase) and graphed.


As shown in FIG. 1, the dual targeting siRNA was at least as effective at inhibiting their corresponding target gene as the single siRNAs.


Example 6. Inhibition of PCSK9 and Xbp-1 mRNA Levels and Reduction of Total Serum Cholesterol by the PCSK9-Xbp1 Dual Targeting siRNA in Mice

The dual targeting AD-23426 was formulated in an LNP09 formulation: XTC/DSPC/Cholesterol/PEG-DMG in a % mol ratio of 50/10/38.5/1.5 with a lipid:siRNA ratio of about 10:1. The LNP09-AD-23426 was administered by tail vein injection into C57B6 mice at 6.0 mg/kg, 2.0 mg/kg and 0.6 mg/kg. LNP09 formulated single siRNAs (AD-10792 and AD-18038) were administered each at 3.0 mg/kg, 1.0 mg/kg and 0.3 mg/kg. Livers and plasma were harvested 72 hours post-injection (5 animals per group).


PCSK9, Xbp-1 and GAPDH transcript levels were measured via bDNA in livers prepared according to the manufacturer's protocol. PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase) and graphed. The results are shown in FIG. 2.


Total cholesterol was measure in serum according to manufacturer's instructions using a cholesterol kit from WAKO Tex.


The results demonstrate that the dual targeting siRNAs were at least as effective at inhibiting their corresponding target as single siRNAs in vivo. The results also show that the dual targeting construct has an additive effect compared to the single siRNAs at reducing total serum cholesterol.


Example 7: No Induction of IFN-α and TNF-α in HuPBMC

The effect of a dual targeting siRNA, AD-23426, on IFN-α and TNF-α in human PBMC was investigated.


Whole Blood anti-coagulated with Sodium Heparin was obtained from healthy donors at Research Blood Components, Inc (Boston, Mass.). Peripheral blood mononuclear cells (PBMC) were isolated by standard Ficoll-Hypaque density centrifugation. Isolated PBMC were seeded at 1×105 cells/well in 96 well plates and cultured in RPMI 1640 GlutaMax Medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic (Invitrogen). siRNAs were transfected using DOTAP Transfection Reagent (Roche Applied Science). DOTAP was first diluted in Opti-MEM (Invitrogen) for 5 minutes before mixing with an equal volume of Opti-MEM containing the siRNA. siRNA/transfection reagent complexes were incubated for 15 minutes at room temperature prior to being added to PBMC. siRNAs were transfected at final concentrations of 266 nM, 133 nM or 67 nM using 16 μg/ml, 8 μg/ml or 4 μg/ml DOTAP, respectively. The ratio of siRNA to DOTAP is 16.5 μmol/μg. Transfected PBMC were incubated at 37° C., 5% CO2 for 24 hrs after which supernatants were harvested and stored at −80° C. until analysis. Quantitative cytokine analysis was done using commercially available Instant ELISA Kits for IFN-α (BMS216INST) and TNF-α (BMS223INST); both from Bender MedSystems (Vienna, Austria).


LNP09 and DOTAP formulated siRNAs were administered. Control siRNAs were AD-1730, AD-1955, AD-6248, AD-18889, AD-5048, and AD-18221. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.


The results are shown in FIG. 4. AD-23426 did not induce production of IFN-α and TNF-α, similar to the result obtained with the single target gene siRNAs. As expected, unmodified siRNAs (AD-5048 and AD-18889) induced production of both IFN-α and TNF-α. These results demonstrate that a dual targeting siRNA does not induce an immune response.


Example 8. Reduction of Total Serum Cholesterol with PCSK9-Xbp1 Dual Targeting siRNA Humans

A human subject is treated with a pharmaceutical composition, e.g., a nucleic acid-lipid particle having a dual targeting siRNA agent.


At time zero, a suitable first dose of the pharmaceutical composition is subcutaneously administered to the subject. The composition is formulated as described herein. After a period of time, the subject's condition is evaluated, e.g., by measurement of total serum cholesterol. This measurement can be accompanied by a measurement of PCSK9 expression in said subject, and/or the products of the successful siRNA-targeting of PCSK9 mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.


After treatment, the subject's condition is compared to the condition existing prior to the treatment, or relative to the condition of a similarly afflicted but untreated subject.


Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the present disclosure which will allow them to practice this invention to the full scope of the claims hereinafter appended.









TABLE 4







Sequences of siRNA targeted to PCSK9














SEQ
Antisense
SEQ



*Target
Sense strand (5′-3′)1
ID NO:
strand (5′-3′)1
ID NO:
Duplex #















 2-20
AGCGACGUCGAGGCGCUCATT
   1
UGAGCGCCUCGAC
   2
AD-15220





GUCGCUTT







15-33
CGCUCAUGGUUGCAGGCGGTT
   3
CCGCCUGCAACCA
   4
AD-15275





UGAGCGTT







16-34
GCUCAUGGUUGCAGGCGGGTT
   5
CCCGCCUGCAACC
   6
AD-15301





AUGAGCTT







30-48
GCGGGCGCCGCCGUUCAGUTT
   7
ACUGAACGGCGGC
   8
AD-15276





GCCCGCTT







31-49
CGGGCGCCGCCGUUCAGUUTT
   9
AACUGAACGGCGG
  10
AD-15302





CGCCCGTT







32-50
GGGCGCCGCCGUUCAGUUCTT
  11
GAACUGAACGGCG
  12
AD-15303





GCGCCCTT







40-58
CCGUUCAGUUCAGGGUCUGTT
  13
CAGACCCUGAACU
  14
AD-15221





GAACGGTT







43-61
UUCAGUUCAGGGUCUGAGCTT
  15
GCUCAGACCCUGA
  16
AD-15413





ACUGAATT







 82-100
GUGAGACUGGCUCGGGCGGTT
  17
CCGCCCGAGCCAG
  18
AD-15304





UCUCACTT







100-118
GGCCGGGACGCGUCGUUGCTT
  19
GCAACGACGCGUC
  20
AD-15305





CCGGCCTT







101-119
GCCGGGACGCGUCGUUGCATT
  21
UGCAACGACGCGU
  22
AD-15306





CCCGGCTT







102-120
CCGGGACGCGUCGUUGCAGTT
  23
CUGCAACGACGCG
  24
AD-15307





UCCCGGTT







105-123
GGACGCGUCGUUGCAGCAGTT
  25
CUGCUGCAACGAC
  26
AD-15277





GCGUCCTT







135-153
UCCCAGCCAGGAUUCCGCGTsT
  27
CGCGGAAUCCUGG
  28
AD-9526





CUGGGATsT







135-153
ucccAGccAGGAuuccGcGTsT
  29
CGCGGAAUCCUGG
  30
AD-9652





CUGGGATsT







136-154
CCCAGCCAGGAUUCCGCGCTsT
  31
GCGCGGAAUCCUG
  32
AD-9519





GCUGGGTsT







136-154
cccAGccAGGAuuccGcGcTsT
  33
GCGCGGAAUCCUG
  34
AD-9645





GCUGGGTsT







138-156
CAGCCAGGAUUCCGCGCGCTsT
  35
GCGCGCGGAAUCC
  36
AD-9523





UGGCUGTsT







138-156
cAGccAGGAuuccGcGcGcTsT
  37
GCGCGCGGAAUCC
  38
AD-9649





UGGCUGTsT







185-203
AGCUCCUGCACAGUCCUCCTsT
  39
GGAGGACUGUGCA
  40
AD-9569





GGAGCUTsT







185-203
AGcuccuGcAcAGuccuccTsT
  41
GGAGGACUGUGcA
  42
AD-9695





GGAGCUTsT







205-223
CACCGCAAGGCUCAAGGCGTT
  43
CGCCUUGAGCCUU
  44
AD-15222





GCGGUGTT







208-226
CGCAAGGCUCAAGGCGCCGTT
  45
CGGCGCCUUGAGC
  46
AD-15278





CUUGCGTT







210-228
CAAGGCUCAAGGCGCCGCCTT
  47
GGCGGCGCCUUGA
  48
AD-15178





GCCUUGTT







232-250
GUGGACCGCGCACGGCCUCTT
  49
GAGGCCGUGCGCG
  50
AD-15308





GUCCACTT







233-251
UGGACCGCGCACGGCCUCUTT
  51
AGAGGCCGUGCGC
  52
AD-15223





GGUCCATT







234-252
GGACCGCGCACGGCCUCUATT
  53
UAGAGGCCGUGCG
  54
AD-15309





CGGUCCTT







235-253
GACCGCGCACGGCCUCUAGTT
  55
CUAGAGGCCGUGC
  56
AD-15279





GCGGUCTT







236-254
ACCGCGCACGGCCUCUAGGTT
  57
CCUAGAGGCCGUG
  58
AD-15194





CGCGGUTT







237-255
CCGCGCACGGCCUCUAGGUTT
  59
ACCUAGAGGCCGU
  60
AD-15310





GCGCGGTT







238-256
CGCGCACGGCCUCUAGGUCTT
  61
GACCUAGAGGCCG
  62
AD-15311





UGCGCGTT







239-257
GCGCACGGCCUCUAGGUCUTT
  63
AGACCUAGAGGCC
  64
AD-15392





GUGCGCTT







240-258
CGCACGGCCUCUAGGUCUCTT
  65
GAGACCUAGAGGC
  66
AD-15312





CGUGCGTT







248-266
CUCUAGGUCUCCUCGCCAGTT
  67
CUGGCGAGGAGAC
  68
AD-15313





CUAGAGTT







249-267
UCUAGGUCUCCUCGCCAGGTT
  69
CCUGGCGAGGAGA
  70
AD-15280





CCUAGATT







250-268
CUAGGUCUCCUCGCCAGGATT
  71
UCCUGGCGAGGAG
  72
AD-15267





ACCUAGTT







252-270
AGGUCUCCUCGCCAGGACATT
  73
UGUCCUGGCGAGG
  74
AD-15314





AGACCUTT







258-276
CCUCGCCAGGACAGCAACCTT
  75
GGUUGCUGUCCUG
  76
AD-15315





GCGAGGTT







300-318
CGUCAGCUCCAGGCGGUCCTsT
  77
GGACCGCCUGGAG
  78
AD-9624





CUGACGTsT







300-318
cGucAGcuccAGGcGGuccTsT
  79
GGACCGCCUGGAG
  80
AD-9750





CUGACGTsT







301-319
GUCAGCUCCAGGCGGUCCUTsT
  81
AGGACCGCCUGGA
  82
AD-9623





GCUGACTsT







301-319
GucAGcuccAGGcGGuccuTsT
  83
AGGACCGCCUGGA
  84
AD-9749





GCUGACTsT







370-388
GGCGCCCGUGCGCAGGAGGTT
  85
CCUCCUGCGCACG
  86
AD-15384





GGCGCCTT







408-426
GGAGCUGGUGCUAGCCUUGTsT
  87
CAAGGCUAGCACC
  88
AD-9607





AGCUCCTsT







408-426
GGAGcuGGuGcuAGccuuGTsT
  89
cAAGGCuAGcACc
  90
AD-9733





AGCUCCTsT







411-429
GCUGGUGCUAGCCUUGCGUTsT
  91
ACGCAAGGCUAGC
  92
AD-9524





ACCAGCTsT







411-429
GcuGGuGcuAGccuuGcGuTsT
  93
ACGcAAGGCuAGc
  94
AD-9650





ACcAGCTsT







412-430
CUGGUGCUAGCCUUGCGUUTsT
  95
AACGCAAGGCUAG
  96
AD-9520





CACCAGTsT







412-430
CUGGUGCUAGCCUUGCGUUTsT
  97
AACGCAAGGCUAG
  98
AD-9520





CACCAGTsT







412-430
cuGGuGcuAGccuuGcGuuTsT
  99
AACGcAAGGCuAG
 100
AD-9646





cACcAGTsT







416-434
UGCUAGCCUUGCGUUCCGATsT
 101
UCGGAACGCAAGG
 102
AD-9608





CUAGCATsT







416-434
uGcuAGccuuGcGuuccGATsT
 103
UCGGAACGcAAGG
 104
AD-9734





CuAGcATsT







419-437
UAGCCUUGCGUUCCGAGGATsT
 105
UCCUCGGAACGCA
 106
AD-9546





AGGCUATsT







419-437
uAGccuuGcGuuccGAGGATsT
 107
UCCUCGGAACGcA
 108
AD-9672





AGGCuATsT







439-457
GACGGCCUGGCCGAAGCACTT
 109
GUGCUUCGGCCAG
 110
AD-15385





GCCGUCTT







447-465
GGCCGAAGCACCCGAGCACTT
 111
GUGCUCGGGUGCU
 112
AD-15393





UCGGCCTT







448-466
GCCGAAGCACCCGAGCACGTT
 113
CGUGCUCGGGUGC
 114
AD-15316





UUCGGCTT







449-467
CCGAAGCACCCGAGCACGGTT
 115
CCGUGCUCGGGUG
 116
AD-15317





CUUCGGTT







458-476
CCGAGCACGGAACCACAGCTT
 117
GCUGUGGUUCCGU
 118
AD-15318





GCUCGGTT







484-502
CACCGCUGCGCCAAGGAUCTT
 119
GAUCCUUGGCGCA
 120
AD-15195





GCGGUGTT







486-504
CCGCUGCGCCAAGGAUCCGTT
 121
CGGAUCCUUGGCG
 122
AD-15224





CAGCGGTT







487-505
CGCUGCGCCAAGGAUCCGUTT
 123
ACGGAUCCUUGGC
 124
AD-15188





GCAGCGTT







489-507
CUGCGCCAAGGAUCCGUGGTT
 125
CCACGGAUCCUUG
 126
AD-15225





GCGCAGTT







500-518
AUCCGUGGAGGUUGCCUGGTT
 127
CCAGGCAACCUCC
 128
AD-15281





ACGGAUTT







509-527
GGUUGCCUGGCACCUACGUTT
 129
ACGUAGGUGCCAG
 130
AD-15282





GCAACCTT







542-560
AGGAGACCCACCUCUCGCATT
 131
UGCGAGAGGUGGG
 132
AD-15319





UCUCCUTT







543-561
GGAGACCCACCUCUCGCAGTT
 133
CUGCGAGAGGUGG
 134
AD-15226





GUCUCCTT







544-562
GAGACCCACCUCUCGCAGUTT
 135
ACUGCGAGAGGUG
 136
AD-15271





GGUCUCTT







549-567
CCACCUCUCGCAGUCAGAGTT
 137
CUCUGACUGCGAG
 138
AD-15283





AGGUGGTT







552-570
CCUCUCGCAGUCAGAGCGCTT
 139
GCGCUCUGACUGC
 140
AD-15284





GAGAGGTT







553-571
CUCUCGCAGUCAGAGCGCATT
 141
UGCGCUCUGACUG
 142
AD-15189





CGAGAGTT







554-572
UCUCGCAGUCAGAGCGCACTT
 143
GUGCGCUCUGACU
 144
AD-15227





GCGAGATT







555-573
CUCGCAGUCAGAGCGCACUTsT
 145
AGUGCGCUCUGAC
 146
AD-9547





UGCGAGTsT







555-573
cucGcAGucAGAGcGcAcuTsT
 147
AGUGCGCUCUGAC
 148
AD-9673





UGCGAGTsT







558-576
GCAGUCAGAGCGCACUGCCTsT
 149
GGCAGUGCGCUCU
 150
AD-9548





GACUGCTsT







558-576
GcAGucAGAGcGcAcuGccTsT
 151
GGcAGUGCGCUCU
 152
AD-9674





GACUGCTsT







606-624
GGGAUACCUCACCAAGAUCTsT
 153
GAUCUUGGUGAGG
 154
AD-9529





UAUCCCTsT







606-624
GGGAuAccucAccAAGAucTsT
 155
GAUCUUGGUGAGG
 156
AD-9655





uAUCCCTsT







659-677
UGGUGAAGAUGAGUGGCGATsT
 157
UCGCCACUCAUCU
 158
AD-9605





UCACCATsT







659-677
uGGuGAAGAuGAGuGGcGATsT
 159
UCGCcACUcAUCU
 160
AD-9731





UcACcATsT







663-681
GAAGAUGAGUGGCGACCUGTsT
 161
CAGGUCGCCACUC
 162
AD-9596





AUCUUCTsT







663-681
GAAGAuGAGuGGcGAccuGTsT
 163
cAGGUCGCcACUc
 164
AD-9722





AUCUUCTsT







704-722
CCCAUGUCGACUACAUCGATsT
 165
UCGAUGUAGUCGA
 166
AD-9583





CAUGGGTsT







704-722
cccAuGucGAcuAcAucGATsT
 167
UCGAUGuAGUCGA
 168
AD-9709





cAUGGGTsT







718-736
AUCGAGGAGGACUCCUCUGTsT
 169
CAGAGGAGUCCUC
 170
AD-9579





CUCGAUTsT







718-736
AucGAGGAGGAcuccucuGTsT
 171
cAGAGGAGUCCUC
 172
AD-9705





CUCGAUTsT







758-776
GGAACCUGGAGCGGAUUACTT
 173
GUAAUCCGCUCCA
 174
AD-15394





GGUUCCTT







759-777
GAACCUGGAGCGGAUUACCTT
 175
GGUAAUCCGCUCC
 176
AD-15196





AGGUUCTT







760-778
AACCUGGAGCGGAUUACCCTT
 177
GGGUAAUCCGCUC
 178
AD-15197





CAGGUUTT







777-795
CCCUCCACGGUACCGGGCGTT
 179
CGCCCGGUACCGU
 180
AD-15198





GGAGGGTT







782-800
CACGGUACCGGGCGGAUGATsT
 181
UCAUCCGCCCGGU
 182
AD-9609





ACCGUGTsT







782-800
cAcGGuAccGGGcGGAuGATsT
 183
UcAUCCGCCCGGu
 184
AD-9735





ACCGUGTsT







783-801
ACGGUACCGGGCGGAUGAATsT
 185
UUCAUCCGCCCGG
 186
AD-9537





UACCGUTsT







783-801
AcGGuAccGGGcGGAuGAATsT
 187
UUcAUCCGCCCGG
 188
AD-9663





uACCGUTsT







784-802
CGGUACCGGGCGGAUGAAUTsT
 189
AUUCAUCCGCCCG
 190
AD-9528





GUACCGTsT







784-802
cGGuAccGGGcGGAuGAAuTsT
 191
AUUcAUCCGCCCG
 192
AD-9654





GuACCGTsT







785-803
GGUACCGGGCGGAUGAAUATsT
 193
UAUUCAUCCGCCC
 194
AD-9515





GGUACCTsT







785-803
GGuAccGGGcGGAuGAAuATsT
 195
uAUUcAUCCGCCC
 196
AD-9641





GGuACCTsT







786-804
GUACCGGGCGGAUGAAUACTsT
 197
GUAUUCAUCCGCC
 198
AD-9514





CGGUACTsT







786-804
GuAccGGGcGGAuGAAuAcTsT
 199
GuAUUcAUCCGCC
 200
AD-9640





CGGuACTsT







788-806
ACCGGGCGGAUGAAUACCATsT
 201
UGGUAUUCAUCCG
 202
AD-9530





CCCGGUTsT







788-806
AccGGGcGGAuGAAuAccATsT
 203
UGGuAUUcAUCCG
 204
AD-9656





CCCGGUTsT







789-807
CCGGGCGGAUGAAUACCAGTsT
 205
CUGGUAUUCAUCC
 206
AD-9538





GCCCGGTsT







789-807
ccGGGcGGAuGAAuAccAGTsT
 207
CUGGuAUUcAUCC
 208
AD-9664





GCCCGGTsT







825-843
CCUGGUGGAGGUGUAUCUCTsT
 209
GAGAUACACCUCC
 210
AD-9598





ACCAGGTsT







825-843
ccuGGuGGAGGuGuAucucTsT
 211
GAGAuAcACCUCc
 212
AD-9724





ACcAGGTsT







826-844
CUGGUGGAGGUGUAUCUCCTsT
 213
GGAGAUACACCUC
 214
AD-9625





CACCAGTsT







826-844
cuGGuGGAGGuGuAucuccTsT
 215
GGAGAuAcACCUC
 216
AD-9751





cACcAGTsT







827-845
UGGUGGAGGUGUAUCUCCUTsT
 217
AGGAGAUACACCU
 218
AD-9556





CCACCATsT







827-845
uGGuGGAGGuGuAucuccuTsT
 219
AGGAGAuAcACCU
 220
AD-9682





CcACcATsT







828-846
GGUGGAGGUGUAUCUCCUATsT
 221
UAGGAGAUACACC
 222
AD-9539





UCCACCTsT







828-846
GGuGGAGGuGuAucuccuATsT
 223
uAGGAGAuAcACC
 224
AD-9665





UCcACCTsT







831-849
GGAGGUGUAUCUCCUAGACTsT
 225
GUCUAGGAGAUAC
 226
AD-9517





ACCUCCTsT







831-849
GGAGGuGuAucuccuAGAcTsT
 227
GUCuAGGAGAuAc
 228
AD-9643





ACCUCCTsT







833-851
AGGUGUAUCUCCUAGACACTsT
 229
GUGUCUAGGAGAU
 230
AD-9610





ACACCUTsT







833-851
AGGuGuAucuccuAGAcAcTsT
 231
GUGUCuAGGAGAu
 232
AD-9736





AcACCUTsT







833-851
AfgGfuGfuAfuCfuCfcUfaG
 233
P*gUfgUfcUfaG
 234
AD-14681



faCfaCfTsT

fgAfgAfuAfcAf







cCfuTsT







833-851
AGGUfGUfAUfCfUfCfCfUfA
 235
GUfGUfCfUfAGG
 236
AD-14691



GACfACfTsT

AGAUfACfACfCf







UfTsT







833-851
AgGuGuAuCuCcUaGaCaCTsT
 237
P*gUfgUfcUfaG
 238
AD-14701





fgAfgAfuAfcAf







cCfuTsT







833-851
AgGuGuAuCuCcUaGaCaCTsT
 239
GUfGUfCfUfAGG
 240
AD-14711





AGAUfACfACfCf







UfTsT







833-851
AfgGfuGfuAfuCfuCfcUfaG
 241
GUGUCuaGGagAU
 242
AD-14721



faCfaCfTsT

ACAccuTsT







833-851
AGGUfGUfAUfCfUfCfCfUfA
 243
GUGUCuaGGagAU
 244
AD-14731



GACfACfTsT

ACAccuTsT







833-851
AgGuGuAuCuCcUaGaCaCTsT
 245
GUGUCuaGGagAU
 246
AD-14741





ACAccuTsT







833-851
GfcAfcCfcUfcAfuAfgGfcC
 247
P*uCfcAfgGfcC
 248
AD-15087



fuGfgAfTsT

fuAfuGfaGfgGf







uGfcTsT







833-851
GCfACfCfCfUfCfAUfAGGCf
 249
UfCfCfAGGCfCf
 250
AD-15097



CfUfGGATsT

UfAUfGAGGGUfG







CfTsT







833-851
GcAcCcUcAuAgGcCuGgATsT
 251
P*uCfcAfgGfcC
 252
AD-15107





fuAfuGfaGfgGf







uGfcTsT







833-851
GcAcCcUcAuAgGcCuGgATsT
 253
UfCfCfAGGCfCf
 254
AD-15117





UfAUfGAGGGUfG







CfTsT







833-851
GfcAfcCfcUfcAfuAfgGfcC
 255
UCCAGgcCUauGA
 256
AD-15127



fuGfgAfTsT

GGGugcTsT







833-851
GCfACfCfCfUfCfAUfAGGCf
 257
UCCAGgcCUauGA
 258
AD-15137



CfUfGGATsT

GGGugcTsT







833-851
GcAcCcUcAuAgGcCuGgATsT
 259
UCCAGgcCUauGA
 260
AD-15147





GGGugcTsT







836-854
UGUAUCUCCUAGACACCAGTsT
 261
CUGGUGUCUAGGA
 262
AD-9516





GAUACATsT







836-854
uGuAucuccuAGAcAccAGTsT
 263
CUGGUGUCuAGGA
 264
AD-9642





GAuAcATsT







840-858
UCUCCUAGACACCAGCAUATsT
 265
UAUGCUGGUGUCU
 266
AD-9562





AGGAGATsT







840-858
ucuccuAGAcAccAGcAuATsT
 267
uAUGCUGGUGUCu
 268
AD-9688





AGGAGATsT







840-858
UfcUfcCfuAfgAfcAfcCfaG
 269
P*uAfuGfcUfgG
 270
AD-14677



fcAfuAfTsT

fuGfuCfuAfgGf







aGfaTsT







840-858
UfCfUfCfCfUfAGACfACfCf
 271
UfAUfGCfUfGGU
 272
AD-14687



AGCfAUfATsT

fGUfCfUfAGGAG







ATsT







840-858
UcUcCuAgAcAcCaGcAuATsT
 273
P*uAfuGfcUfgG
 274
AD-14697





fuGfuCfuAfgGf







aGfaTsT







840-858
UcUcCuAgAcAcCaGcAuATsT
 275
UfAUfGCfUfGGU
 276
AD-14707





fGUfCfUfAGGAG







ATsT







840-858
UfcUfcCfuAafAfcAfcCfaG
 277
UAUGCugGUguCU
 278
AD-14717



fcAfuAfTsT

AGGagaTsT







840-858
UfCfUfCfCfUfAGACfACfCf
 279
UAUGCugGUguCU
 280
AD-14727



AGCfAUfATsT

AGGagaTsT







840-858
UcUcCuAgAcAcCaGcAuATsT
 281
UAUGCugGUguCU
 282
AD-14737





AGGagaTsT







840-858
AfgGfcCfuGfgAfgUfuUfaU
 283
P*cCfgAfaUfaA
 284
AD-15083



fuCfgGfTsT

faCfuCfcAfgGf







cCfuTsT







840-858
AGGCfCfUfGGAGUfUfUfAUf
 285
CfCfGAAUfAAAC
 286
AD-15093



UfCfGGTsT

fUfCfCfAGGCfC







fUfTsT







840-858
AgGcCuGgAgUuUaUuCgGTsT
 287
P*cCfgAfaUfaA
 288
AD-15103





faCfuCfcAfgGf







cCfuTsT







840-858
AgGcCuGgAgUuUaUuCgGTsT
 289
CfCfGAAUfAAAC
 290
AD-15113





fUfCfCfAGGCfC







fUfTsT







840-858
AfgGfcCfuGfgAfgUfuUfaU
 291
CCGAAuaAAcuCC
 292
AD-15123



fuCfgGfTsT

AGGccuTsT







840-858
AGGCfCfUfGGAGUfUfUfAUf
 293
CCGAAuaAAcuCC
 294
AD-15133



UfCfGGTsT

AGGccuTsT







840-858
AgGcCuGgAgUuUaUuCgGTsT
 295
CCGAAuaAAcuCC
 296
AD-15143





AGGccuTsT







841-859
CUCCUAGACACCAGCAUACTsT
 297
GUAUGCUGGUGUC
 298
AD-9521





UAGGAGTsT







841-859
cuccuAGAcAccAGcAuAcTsT
 299
GuAUGCUGGUGUC
 300
AD-9647





uAGGAGTsT







842-860
UCCUAGACACCAGCAUACATsT
 301
UGUAUGCUGGUGU
 302
AD-9611





CUAGGATsT







842-860
uccuAGAcAccAGcAuAcATsT
 303
UGuAUGCUGGUGU
 304
AD-9737





CuAGGATsT







843-861
CCUAGACACCAGCAUACAGTsT
 305
CUGUAUGCUGGUG
 306
AD-9592





UCUAGGTsT







843-861
ccuAGAcAccAGcAuAcAGTsT
 307
CUGuAUGCUGGUG
 308
AD-9718





UCuAGGTsT







847-865
GACACCAGCAUACAGAGUGTsT
 309
CACUCUGUAUGCU
 310
AD-9561





GGUGUCTsT







847-865
GAcAccAGcAuAcAGAGuGTsT
 311
cACUCUGuAUGCU
 312
AD-9687





GGUGUCTsT







855-873
CAUACAGAGUGACCACCGGTsT
 313
CCGGUGGUCACUC
 314
AD-9636





UGUAUGTsT







855-873
cAuAcAGAGuGAccAccGGTsT
 315
CCGGUGGUcACUC
 316
AD-9762





UGuAUGTsT







860-878
AGAGUGACCACCGGGAAAUTsT
 317
AUUUCCCGGUGGU
 318
AD-9540





CACUCUTsT







860-878
AGAGuGAccAccGGGAAAuTsT
 319
AUUUCCCGGUGGU
 320
AD-9666





cACUCUTsT







861-879
GAGUGACCACCGGGAAAUCTsT
 321
GAUUUCCCGGUGG
 322
AD-9535





UCACUCTsT







861-879
GAGuGAccAccGGGAAAucTsT
 323
GAUUUCCCGGUGG
 324
AD-9661





UcACUCTsT







863-881
GUGACCACCGGGAAAUCGATsT
 325
UCGAUUUCCCGGU
 326
AD-9559





GGUCACTsT







863-881
GuGAccAccGGGAAAucGATsT
 327
UCGAUUUCCCGGU
 328
AD-9685





GGUcACTsT







865-883
GACCACCGGGAAAUCGAGGTsT
 329
CCUCGAUUUCCCG
 330
AD-9533





GUGGUCTsT







865-883
GAccAccGGGAAAucGAGGTsT
 331
CCUCGAUUUCCCG
 332
AD-9659





GUGGUCTsT







866-884
ACCACCGGGAAAUCGAGGGTsT
 333
CCCUCGAUUUCCC
 334
AD-9612





GGUGGUTsT







866-884
AccAccGGGAAAucGAGGGTsT
 335
CCCUCGAUUUCCC
 336
AD-9738





GGUGGUTsT







867-885
CCACCGGGAAAUCGAGGGCTsT
 337
GCCCUCGAUUUCC
 338
AD-9557





CGGUGGTsT







867-885
ccAccGGGAAAucGAGGGcTsT
 339
GCCCUCGAUUUCC
 340
AD-9683





CGGUGGTsT







875-893
AAAUCGAGGGCAGGGUCAUTsT
 341
AUGACCCUGCCCU
 342
AD-9531





CGAUUUTsT







875-893
AAAucGAGGGcAGGGucAuTsT
 343
AUGACCCUGCCCU
 344
AD-9657





CGAUUUTsT







875-893
AfaAfuCfgAfgGfgCfaGfgG
 345
P*aUfgAfcCfcU
 346
AD-14673



fuCfaUfTsT

fgCfcCfuCfgAf







uUfuTsT







875-893
AAAUfCfGAGGGCfAGGGUfCf
 347
AUfGACfCfCfUf
 348
AD-14683



AUfTsT

GCfCfCfUfCfGA







UfUfUfTsT







875-893
AaAuCgAgGgCaGgGuCaUTsT
 349
P*aUfgAfcCfcU
 350
AD-14693





fgCfcCfuCfgAf







uUfuTsT







875-893
AaAuCgAgGgCaGgGuCaUTsT
 351
AUfGACfCfCfUf
 352
AD-14703





GCfCfCfUfCfGA







UfUfUfTsT







875-893
AfaAfuCfgAfgGfgCfaGfgG
 353
AUGACccUGccCU
 354
AD-14713



fuCfaUfTsT

CGAuuuTsT







875-893
AAAUfCfGAGGGCfAGGGUfCf
 355
AUGACccUGccCU
 356
AD-14723



AUfTsT

CGAuuuTsT







875-893
AaAuCgAgGgCaGgGuCaUTsT
 357
AUGACccUGccCU
 358
AD-14733





CGAuuuTsT







875-893
CfgGfcAfcCfcUfcAfuAfgG
 359
P*cAfgGfcCfuA
 360
AD-15079



fcCfuGfTsT

fuGfaGfgGfuGf







cCfgTsT







875-893
CfGGCfACfCfCfUfCfAUfAG
 361
CfAGGCfCfUfAU
 362
AD-15089



GCfCfUfGTsT

fGAGGGUfGCfCf







GTsT







875-893
CgGcAcCcUcAuAgGcCuGTsT
 363
P*cAfgGfcCfuA
 364
AD-15099





fuGfaGfgGfuGf







cCfgTsT







875-893
CgGcAcCcUcAuAgGcCuGTsT
 365
CfAGGCfCfUfAU
 366
AD-15109





fGAGGGUfGCfCf







GTsT







875-893
CfgGfcAfcCfcUfcAfuAfgG
 367
CAGGCcuAUgaGG
 368
AD-15119



fcCfuGfTsT

GUGccgTsT







875-893
CfGGCfACfCfCfUfCfAUfAG
 369
CAGGCcuAUgaGG
 370
AD-15129



GCfCfUfGTsT

GUGccgTsT







875-893
CgGcAcCcUcAuAgGcCuGTsT
 371
CAGGCcuAUgaGG
 372
AD-15139





GUGccgTsT







877-895
AUCGAGGGCAGGGUCAUGGTsT
 373
CCAUGACCCUGCC
 374
AD-9542





CUCGAUTsT







877-895
AucGAGGGcAGGGucAuGGTsT
 375
CcAUGACCCUGCC
 376
AD-9668





CUCGAUTsT







878-896
cGAGGGcAGGGucAuGGucTsT
 377
GACcAUGACCCUG
 378
AD-9739





CCCUCGTsT







880-898
GAGGGCAGGGUCAUGGUCATsT
 379
UGACCAUGACCCU
 380
AD-9637





GCCCUCTsT







880-898
GAGGGcAGGGucAuGGucATsT
 381
UGACcAUGACCCU
 382
AD-9763





GCCCUCTsT







882-900
GGGCAGGGUCAUGGUCACCTsT
 383
GGUGACCAUGACC
 384
AD-9630





CUGCCCTsT







882-900
GGGcAGGGucAuGGucAccTsT
 385
GGUGACcAUGACC
 386
AD-9756





CUGCCCTsT







885-903
CAGGGUCAUGGUCACCGACTsT
 387
GUCGGUGACCAUG
 388
AD-9593





ACCCUGTsT







885-903
cAGGGucAuGGucAccGAcTsT
 389
GUCGGUGACcAUG
 390
AD-9719





ACCCUGTsT







886-904
AGGGUCAUGGUCACCGACUTsT
 391
AGUCGGUGACCAU
 392
AD-9601





GACCCUTsT







886-904
AGGGucAuGGucAccGAcuTsT
 393
AGUCGGUGACcAU
 394
AD-9727





GACCCUTsT







892-910
AUGGUCACCGACUUCGAGATsT
 395
UCUCGAAGUCGGU
 396
AD-9573





GACCAUTsT







892-910
AuGGucAccGAcuucGAGATsT
 397
UCUCGAAGUCGGU
 398
AD-9699





GACcAUTsT







899-917
CCGACUUCGAGAAUGUGCCTT
 399
GGCACAUUCUCGA
 400
AD-15228





AGUCGGTT







921-939
GGAGGACGGGACCCGCUUCTT
 401
GAAGCGGGUCCCG
 402
AD-15395





UCCUCCTT







993-1011
CAGCGGCCGGGAUGCCGGCTsT
 403
GCCGGCAUCCCGG
 404
AD-9602





CCGCUGTsT







 993-1011
cAGcGGccGGGAuGccGGcTsT
 405
GCCGGcAUCCCGG
 406
AD-9728





CCGCUGTsT







1020-1038
GGGUGCCAGCAUGCGCAGCTT
 407
GCUGCGCAUGCUG
 408
AD-15386





GCACCCTT







1038-1056
CCUGCGCGUGCUCAACUGCTsT
 409
GCAGUUGAGCACG
 410
AD-9580





CGCAGGTsT







1038-1056
ccuGcGcGuGcucAAcuGcTsT
 411
GcAGUUGAGcACG
 412
AD-9706





CGcAGGTsT







1040-1058
UGCGCGUGCUCAACUGCCATsT
 413
UGGCAGUUGAGCA
 414
AD-9581





CGCGCATsT







1040-1058
uGcGcGuGcucAAcuGccATsT
 415
UGGcAGUUGAGcA
 416
AD-9707





CGCGcATsT







1042-1060
CGCGUGCUCAACUGCCAAGTsT
 417
CUUGGCAGUUGAG
 418
AD-9543





CACGCGTsT







1042-1060
cGcGuGcucAAcuGccAAGTsT
  419
CUUGGcAGUUGAG
 420
AD-9669





cACGCGTsT







1053-1071
CUGCCAAGGGAAGGGCACGTsT
 421
CGUGCCCUUCCCU
 422
AD-9574





UGGCAGTsT







1053-1071
cuGccAAGGGAAGGGcAcGTsT
 423
CGUGCCCUUCCCU
 424
AD-9700





UGGcAGTsT







1057-1075
CAAGGGAAGGGCACGGUUATT
 425
UAACCGUGCCCUU
 426
AD-15320





CCCUUGTT







1058-1076
AAGGGAAGGGCACGGUUAGTT
 427
CUAACCGUGCCCU
 428
AD-15321





UCCCUUTT







1059-1077
AGGGAAGGGCACGGUUAGCTT
 429
GCUAACCGUGCCC
 430
AD-15199





UUCCCUTT







1060-1078
GGGAAGGGCACGGUUAGCGTT
 431
CGCUAACCGUGCC
 432
AD-15167





CUUCCCTT







1061-1079
GGAAGGGCACGGUUAGCGGTT
 433
CCGCUAACCGUGC
 434
AD-15164





CCUUCCTT







1062-1080
GAAGGGCACGGUUAGCGGCTT
 435
GCCGCUAACCGUG
 436
AD-15166





CCCUUCTT







1063-1081
AAGGGCACGGUUAGCGGCATT
 437
UGCCGCUAACCGU
 438
AD-15322





GCCCUUTT







1064-1082
AGGGCACGGUUAGCGGCACTT
 439
GUGCCGCUAACCG
 440
AD-15200





UGCCCUTT







1068-1086
CACGGUUAGCGGCACCCUCTT
 441
GAGGGUGCCGCUA
 442
AD-15213





ACCGUGTT







1069-1087
ACGGUUAGCGGCACCCUCATT
 443
UGAGGGUGCCGCU
 444
AD-15229





AACCGUTT







1072-1090
GUUAGCGGCACCCUCAUAGTT
 445
CUAUGAGGGUGCC
 446
AD-15215





GCUAACTT







1073-1091
UUAGCGGCACCCUCAUAGGTT
 447
CCUAUGAGGGUGC
 448
AD-15214





CGCUAATT







1076-1094
GCGGCACCCUCAUAGGCCUTsT
 449
AGGCCUAUGAGGG
 450
AD-9315





UGCCGCTsT







1079-1097
GCACCCUCAUAGGCCUGGATsT
 451
UCCAGGCCUAUGA
 452
AD-9326





GGGUGCTsT







1085-1103
UCAUAGGCCUGGAGUUUAUTsT
 453
AUAAACUCCAGGC
 454
AD-9318





CUAUGATsT







1090-1108
GGCCUGGAGUUUAUUCGGATsT
 455
UCCGAAUAAACUC
 456
AD-9323





CAGGCCTsT







1091-1109
GCCUGGAGUUUAUUCGGAATsT
 457
UUCCGAAUAAACU
 458
AD-9314





CCAGGCTsT







1091-1109
GccuGGAGuuuAuucGGAATsT
 459
UUCCGAAuAAACU
 460
AD-10792





CcAGGCTsT







1091-1109
GccuGGAGuuuAuucGGAATsT
 461
UUCCGAAUAACUC
 462
AD-10796





CAGGCTsT







1093-1111
CUGGAGUUUAUUCGGAAAATsT
 463
UUUUCCGAAUAAA
 464
AD-9638





CUCCAGTsT







1093-1111
cuGGAGuuuAuucGGAAAATsT
 465
UUUUCCGAAuAAA
 466
AD-9764





CUCcAGTsT







1095-1113
GGAGUUUAUUCGGAAAAGCTsT
 467
GCUUUUCCGAAUA
 468
AD-9525





AACUCCTsT







1095-1113
GGAGuuuAuucGGAAAAGcTsT
 469
GCUUUUCCGAAuA
 470
AD-9651





AACUCCTsT







1096-1114
GAGUUUAUUCGGAAAAGCCTsT
 471
GGCUUUUCCGAAU
 472
AD-9560





AAACUCTsT







1096-1114
GAGuuuAuucGGAAAAGccTsT
 473
GGCUUUUCCGAAu
 474
AD-9686





AAACUCTsT







1100-1118
UUAUUCGGAAAAGCCAGCUTsT
 475
AGCUGGCUUUUCC
 476
AD-9536





GAAUAATsT







1100-1118
uuAuucGGAAAAGccAGcuTsT
 477
AGCUGGCUUUUCC
 478
AD-9662





GAAuAATsT







1154-1172
CCCUGGCGGGUGGGUACAGTsT
 479
CUGUACCCACCCG
 480
AD-9584





CCAGGGTsT







1154-1172
cccuGGcGGGuGGGuAcAGTsT
 481
CUGuACCcACCCG
 482
AD-9710





CcAGGGTsT







1155-1173
CCUGGCGGGUGGGUACAGCTT
 483
GCUGUACCCACCC
 484
AD-15323





GCCAGGTT







1157-1175
UGGCGGGUGGGUACAGCCGTsT
 485
CGGCUGUACCCAC
 486
AD-9551





CCGCCATsT







1157-1175
uGGcGGGuGGGuAcAGccGTsT
 487
CGGCUGuACCcAC
 488
AD-9677





CCGCcATsT







1158-1176
GGCGGGUGGGUACAGCCGCTT
 489
GCGGCUGUACCCA
 490
AD-15230





CCCGCCTT







1162-1180
GGUGGGUACAGCCGCGUCCTT
 491
GGACGCGGCUGUA
 492
AD-15231





CCCACCTT







1164-1182
UGGGUACAGCCGCGUCCUCTT
 493
GAGGACGCGGCUG
 494
AD-15285





UACCCATT







1172-1190
GCCGCGUCCUCAACGCCGCTT
 495
GCGGCGUUGAGGA
 496
AD-15396





CGCGGCTT







1173-1191
CCGCGUCCUCAACGCCGCCTT
 497
GGCGGCGUUGAGG
 498
AD-15397





ACGCGGTT







1216-1234
GUCGUGCUGGUCACCGCUGTsT
 499
CAGCGGUGACCAG
 500
AD-9600





CACGACTsT







1216-1234
GucGuGcuGGucAccGcuGTsT
 501
cAGCGGUGACcAG
 502
AD-9726





cACGACTsT







1217-1235
UCGUGCUGGUCACCGCUGCTsT
 503
GCAGCGGUGACCA
 504
AD-9606





GCACGATsT







1217-1235
ucGuGcuGGucAccGcuGcTsT
 505
GcAGCGGUGACcA
 506
AD-9732





GcACGATsT







1223-1241
UGGUCACCGCUGCCGGCAATsT
 507
UUGCCGGCAGCGG
 508
AD-9633





UGACCATsT







1223-1241
uGGucAccGcuGccGGcAATsT
 509
UUGCCGGcAGCGG
 510
AD-9759





UGACcATsT







1224-1242
GGUCACCGCUGCCGGCAACTsT
 511
GUUGCCGGCAGCG
 512
AD-9588





GUGACCTsT







1224-1242
GGucAccGcuGccGGcAAcTsT
 513
GUUGCCGGcAGCG
 514
AD-9714





GUGACCTsT







1227-1245
CACCGCUGCCGGCAACUUCTsT
 515
GAAGUUGCCGGCA
 516
AD-9589





GCGGUGTsT







1227-1245
cAccGcuGccGGcAAcuucTsT
 517
GAAGUUGCCGGcA
 518
AD-9715





GCGGUGTsT







1229-1247
CCGCUGCCGGCAACUUCCGTsT
 519
CGGAAGUUGCCGG
 520
AD-9575





CAGCGGTsT







1229-1247
ccGcuGccGGcAAcuuccGTsT
 521
CGGAAGUUGCCGG
 522
AD-9701





cAGCGGTsT







1230-1248
CGCUGCCGGCAACUUCCGGTsT
 523
CCGGAAGUUGCCG
 524
AD-9563





GCAGCGTsT







1230-1248
cGcuGccGGcAAcuuccGGTsT
 525
CCGGAAGUUGCCG
 526
AD-9689





GcAGCGTsT







1231-1249
GCUGCCGGCAACUUCCGGGTsT
 527
CCCGGAAGUUGCC
 528
AD-9594





GGCAGCTsT







1231-1249
GcuGccGGcAAcuuccGGGTsT
 529
CCCGGAAGUUGCC
 530
AD-9720





GGcAGCTsT







1236-1254
CGGCAACUUCCGGGACGAUTsT
 531
AUCGUCCCGGAAG
 532
AD-9585





UUGCCGTsT







1236-1254
cGGcAAcuuccGGGAcGAuTsT
 533
AUCGUCCCGGAAG
 534
AD-9711





UUGCCGTsT







1237-1255
GGCAACUUCCGGGACGAUGTsT
 535
CAUCGUCCCGGAA
 536
AD-9614





GUUGCCTsT







1237-1255
GGcAAcuuccGGGAcGAuGTsT
 537
cAUCGUCCCGGAA
 538
AD-9740





GUUGCCTsT







1243-1261
UUCCGGGACGAUGCCUGCCTsT
 539
GGCAGGCAUCGUC
 540
AD-9615





CCGGAATsT







1243-1261
uuccGGGAcGAuGccuGccTsT
 541
GGcAGGcAUCGUC
 542
AD-9741





CCGGAATsT







1248-1266
GGACGAUGCCUGCCUCUACTsT
 543
GUAGAGGCAGGCA
 544
AD-9534





UCGUCCTsT







1248-1266
GGACGAUGCCUGCCUCUACTsT
 545
GUAGAGGCAGGCA
 546
AD-9534





UCGUCCTsT







1248-1266
GGAcGAuGccuGccucuAcTsT
 547
GuAGAGGcAGGcA
 548
AD-9660





UCGUCCTsT







1279-1297
GCUCCCGAGGUCAUCACAGTT
 549
CUGUGAUGACCUC
 550
AD-15324





GGGAGCTT







1280-1298
CUCCCGAGGUCAUCACAGUTT
 551
ACUGUGAUGACCU
 552
AD-15232





CGGGAGTT







1281-1299
UCCCGAGGUCAUCACAGUUTT
 553
AACUGUGAUGACC
 554
AD-15233





UCGGGATT







1314-1332
CCAAGACCAGCCGGUGACCTT
 555
GGUCACCGGCUGG
 556
AD-15234





UCUUGGTT







1315-1333
CAAGACCAGCCGGUGACCCTT
 557
GGGUCACCGGCUG
 558
AD-15286





GUCUUGTT







1348-1366
ACCAACUUUGGCCGCUGUGTsT
 559
CACAGCGGCCAAA
 560
AD-9590





GUUGGUTsT







1348-1366
AccAAcuuuGGccGcuGuGTsT
 561
cAcAGCGGCcAAA
 562
AD-9716





GUUGGUTsT







1350-1368
CAACUUUGGCCGCUGUGUGTsT
 563
CACACAGCGGCCA
 564
AD-9632





AAGUUGTsT







1350-1368
cAAcuuuGGccGcuGuGuGTsT
 565
cAcAcAGCGGCcA
 566
AD-9758





AAGUUGTsT







1360-1378
CGCUGUGUGGACCUCUUUGTsT
 567
CAAAGAGGUCCAC
 568
AD-9567





ACAGCGTsT







1360-1378
cGcuGuGuGGAccucuuuGTsT
  569
cAAAGAGGUCcAc
 570
AD-9693





AcAGCGTsT







1390-1408
GACAUCAUUGGUGCCUCCATsT
 571
UGGAGGCACCAAU
 572
AD-9586





GAUGUCTsT







1390-1408
GAcAucAuuGGuGccuccATsT
 573
UGGAGGcACcAAU
 574
AD-9712





GAUGUCTsT







1394-1412
UCAUUGGUGCCUCCAGCGATsT
 575
UCGCUGGAGGCAC
 576
AD-9564





CAAUGATsT







1394-1412
ucAuuGGuGccuccAGcGATsT
 577
UCGCUGGAGGcAC
 578
AD-9690





cAAUGATsT







1417-1435
AGCACCUGCUUUGUGUCACTsT
 579
GUGACACAAAGCA
 580
AD-9616





GGUGCUTsT







1417-1435
AGcAccuGcuuuGuGucAcTsT
 581
GUGAcAcAAAGcA
 582
AD-9742





GGUGCUTsT







1433-1451
CACAGAGUGGGACAUCACATT
 583
UGUGAUGUCCCAC
 584
AD-15398





UCUGUGTT







1486-1504
AUGCUGUCUGCCGAGCCGGTsT
 585
CCGGCUCGGCAGA
 586
AD-9617





CAGCAUTsT







1486-1504
AuGcuGucuGccGAGccGGTsT
 587
CCGGCUCGGcAGA
 588
AD-9743





cAGcAUTsT







1491-1509
GUCUGCCGAGCCGGAGCUCTsT
 589
GAGCUCCGGCUCG
 590
AD-9635





GCAGACTsT







1491-1509
GucuGccGAGccGGAGcucTsT
 591
GAGCUCCGGCUCG
 592
AD-9761





GcAGACTsT







1521-1539
GUUGAGGCAGAGACUGAUCTsT
 593
GAUCAGUCUCUGC
 594
AD-9568





CUCAACTsT







1521-1539
GuuGAGGcAGAGAcuGAucTsT
 595
GAUcAGUCUCUGC
 596
AD-9694





CUcAACTsT







1527-1545
GCAGAGACUGAUCCACUUCTsT
 597
GAAGUGGAUCAGU
 598
AD-9576





CUCUGCTsT







1527-1545
GcAGAGAcuGAuccAcuucTsT
 599
GAAGUGGAUcAGU
 600
AD-9702





CUCUGCTsT







1529-1547
AGAGACUGAUCCACUUCUCTsT
 601
GAGAAGUGGAUCA
 602
AD-9627





GUCUCUTsT







1529-1547
AGAGAcuGAuccAcuucucTsT
 603
GAGAAGUGGAUcA
 604
AD-9753





GUCUCUTsT







1543-1561
UUCUCUGCCAAAGAUGUCATsT
 605
UGACAUCUUUGGC
 606
AD-9628





AGAGAATsT







1543-1561
uucucuGccAAAGAuGucATsT
 607
UGAcAUCUUUGGc
 608
AD-9754





AGAGAATsT







1545-1563
CUCUGCCAAAGAUGUCAUCTsT
 609
GAUGACAUCUUUG
 610
AD-9631





GCAGAGTsT







1545-1563
cucuGccAAAGAuGucAucTsT
 611
GAUGAcAUCUUUG
 612
AD-9757





GcAGAGTsT







1580-1598
CUGAGGACCAGCGGGUACUTsT
 613
AGUACCCGCUGGU
 614
AD-9595





CCUCAGTsT







1580-1598
cuGAGGAccAGcGGGuAcuTsT
 615
AGuACCCGCUGGU
 616
AD-9721





CCUcAGTsT







1581-1599
UGAGGACCAGCGGGUACUGTsT
 617
CAGUACCCGCUGG
 618
AD-9544





UCCUCATsT







1581-1599
uGAGGAccAGcGGGuAcuGTsT
 619
cAGuACCCGCUGG
 620
AD-9670





UCCUcATsT







1666-1684
ACUGUAUGGUCAGCACACUTT
 621
AGUGUGCUGACCA
 622
AD-15235





UACAGUTT







1668-1686
UGUAUGGUCAGCACACUCGTT
 623
CGAGUGUGCUGAC
 624
AD-15236





CAUACATT







1669-1687
GUAUGGUCAGCACACUCGGTT
 625
CCGAGUGUGCUGA
 626
AD-15168





CCAUACTT







1697-1715
GGAUGGCCACAGCCGUCGCTT
 627
GCGACGGCUGUGG
 628
AD-15174





CCAUCCTT







1698-1716
GAUGGCCACAGCCGUCGCCTT
 629
GGCGACGGCUGUG
 630
AD-15325





GCCAUCTT







1806-1824
CAAGCUGGUCUGCCGGGCCTT
 631
GGCCCGGCAGACC
 632
AD-15326





AGCUUGTT







1815-1833
CUGCCGGGCCCACAACGCUTsT
 633
AGCGUUGUGGGCC
 634
AD-9570





CGGCAGTsT







1815-1833
cuGccGGGcccAcAAcGcuTsT
 635
AGCGUUGUGGGCC
 636
AD-9696





CGGcAGTsT







1816-1834
UGCCGGGCCCACAACGCUUTsT
 637
AAGCGUUGUGGGC
 638
AD-9566





CCGGCATsT







1816-1834
uGccGGGcccAcAAcGcuuTsT
 639
AAGCGUUGUGGGC
 640
AD-9692





CCGGcATsT







1818-1836
CCGGGCCCACAACGCUUUUTsT
 641
AAAAGCGUUGUGG
 642
AD-9532





GCCCGGTsT







1818-1836
ccGGGcccAcAAcGcuuuuTsT
 643
AAAAGCGUUGUGG
 644
AD-9658





GCCCGGTsT







1820-1838
GGGCCCACAACGCUUUUGGTsT
 645
CCAAAAGCGUUGU
 646
AD-9549





GGGCCCTsT







1820-1838
GGGcccAcAAcGcuuuuGGTsT
 647
CcAAAAGCGUUGU
 648
AD-9675





GGGCCCTsT







1840-1858
GGUGAGGGUGUCUACGCCATsT
 649
UGGCGUAGACACC
 650
AD-9541





CUCACCTsT







1840-1858
GGuGAGGGuGucuAcGccATsT
 651
UGGCGuAGAcACC
 652
AD-9667





CUcACCTsT







1843-1861
GAGGGUGUCUACGCCAUUGTsT
 653
CAAUGGCGUAGAC
 654
AD-9550





ACCCUCTsT







1843-1861
GAGGGuGucuAcGccAuuGTsT
 655
cAAUGGCGuAGAc
 656
AD-9676





ACCCUCTsT







1861-1879
GCCAGGUGCUGCCUGCUACTsT
 657
GUAGCAGGCAGCA
 658
AD-9571





CCUGGCTsT







1861-1879
GccAGGuGcuGccuGcuAcTsT
 659
GuAGcAGGcAGcA
 660
AD-9697





CCUGGCTsT







1862-1880
CCAGGUGCUGCCUGCUACCTsT
 661
GGUAGCAGGCAGC
 662
AD-9572





ACCUGGTsT







1862-1880
ccAGGuGcuGccuGcuAccTsT
 663
GGuAGcAGGcAGc
 664
AD-9698





ACCUGGTsT







2008-2026
ACCCACAAGCCGCCUGUGCTT
 665
GCACAGGCGGCUU
 666
AD-15327





GUGGGUTT







2023-2041
GUGCUGAGGCCACGAGGUCTsT
 667
GACCUCGUGGCCU
 668
AD-9639





CAGCACTsT







2023-2041
GuGcuGAGGccAcGAGGucTsT
 669
GACCUCGUGGCCU
 670
AD-9765





cAGcACTsT







2024-2042
UGCUGAGGCCACGAGGUCATsT
 671
UGACCUCGUGGCC
 672
AD-9518





UCAGCATsT







2024-2042
UGCUGAGGCCACGAGGUCATsT
 673
UGACCUCGUGGCC
 674
AD-9518





UCAGCATsT







2024-2042
uGcuGAGGccAcGAGGucATsT
 675
UGACCUCGUGGCC
 676
AD-9644





UcAGcATsT







2024-2042
UfgCfuGfaGfgCfcAfcGfaG
 677
P*uGfaCfcUfcG
 678
AD-14672



fgUfcAfTsT

fuGfgCfcUfcAf







gCfaTsT







2024-2042
UfGCfUfGAGGCfCfACfGAGG
 679
UfGACfCfUfCfG
 680
AD-14682



UfCfATsT

UfGGCfCfUfCfA







GCfATsT







2024-2042
UgCuGaGgCcAcGaGgUcATsT
 681
P*uGfaCfcUfcG
 682
AD-14692





fuGfgCfcUfcAf







gCfaTsT







2024-2042
UgCuGaGgCcAcGaGgUcATsT
 683
UfGACfCfUfCfG
 684
AD-14702





UfGGCfCfUfCfA







GCfATsT







2024-2042
UfgCfuGfaGfgCfcAfcGfaG
 685
UGACCucGUggCC
 686
AD-14712



fgUfcAfTsT

UCAgcaTsT







2024-2042
UfGCfUfGAGGCfCfACfGAGG
 687
UGACCucGUggCC
 688
AD-14722



UfCfATsT

UCAgcaTsT







2024-2042
UgCuGaGgCcAcGaGgUcATsT
 689
UGACCucGUggCC
 690
AD-14732





UCAgcaTsT







2024-2042
GfuGfgUfcAfgCfgGfcCfgG
 691
P*cAfuCfcCfgG
 692
AD-15078



fgAfuGfTsT

fcCfgCfuGfaCf







cAfcTsT







2024-2042
GUfGGUfCfAGCfGGCfCfGGG
 693
CfAUfCfCfCfGG
 694
AD-15088



AUfGTsT

CfCfGCfUfGACf







CfACfTsT







2024-2042
GuGgUcAgCgGcCgGgAuGTsT
 695
P*cAfuCfcCfgG
 696
AD-15098





fcCfgCfuGfaCf







cAfcTsT







2024-2042
GuGgUcAgCgGcCgGgAuGTsT
 697
CfAUfCfCfCfGG
 698
AD-15108





CfCfGCfUfGACf







CfACfTsT







2024-2042
GfuGfgUfcAfgCfgGfcCfgG
 699
CAUCCcgGCcgCU
 700
AD-15118



fgAfuGfTsT

GACcacTsT







2024-2042
GUfGGUfCfAGCfGGCfCfGGG
 701
CAUCCcgGCcgCU
 702
AD-15128



AUfGTsT

GACcacTsT







2024-2042
GuGgUcAgCgGcCgGgAuGTsT
 703
CAUCCcgGCcgCU
 704
AD-15138





GACcacTsT







2030-2048
GGCCACGAGGUCAGCCCAATT
 705
UUGGGCUGACCUC
 706
AD-15237





GUGGCCTT







2035-2053
CGAGGUCAGCCCAACCAGUTT
 707
ACUGGUUGGGCUG
 708
AD-15287





ACCUCGTT







2039-2057
GUCAGCCCAACCAGUGCGUTT
 709
ACGCACUGGUUGG
 710
AD-15238





GCUGACTT







2041-2059
CAGCCCAACCAGUGCGUGGTT
 711
CCACGCACUGGUU
 712
AD-15328





GGGCUGTT







2062-2080
CACAGGGAGGCCAGCAUCCTT
 713
GGAUGCUGGCCUC
 714
AD-15399





CCUGUGTT







2072-2090
CCAGCAUCCACGCUUCCUGTsT
 715
CAGGAAGCGUGGA
 716
AD-9582





UGCUGGTsT







2072-2090
ccAGcAuccAcGcuuccuGTsT
 717
cAGGAAGCGUGGA
 718
AD-9708





UGCUGGTsT







2118-2136
AGUCAAGGAGCAUGGAAUCTsT
 719
GAUUCCAUGCUCC
 720
AD-9545





UUGACUTsT







2118-2136
AGucAAGGAGcAuGGAAucTsT
 721
GAUUCcAUGCUCC
 722
AD-9671





UUGACUTsT







2118-2136
AfgUfcAfaGfgAfgCfaUfgG
 723
P*gAfuUfcCfaU
 724
AD-14674



faAfuCfTsT

fgCfuCfcUfuGf







aCfuTsT







2118-2136
AGUfCfAAGGAGCfAUfGGAAU
 725
GAUfUfCfCfAUf
 726
AD-14684



fCfTsT

GCfUfCfCfUfUf







GACfUfTsT







2118-2136
AgUcAaGgAgCaUgGaAuCTsT
 727
P*gAfuUfcCfaU
 728
AD-14694





fgCfuCfcUfuGf







aCfuTsT







2118-2136
AgUcAaGgAgCaUgGaAuCTsT
 729
GAUfUfCfCfAUf
 730
AD-14704





GCfUfCfCfUfUf







GACfUfTsT







2118-2136
AfgUfcAfaGfgAfgCfaUfgG
 731
GAUUCcaUGcuCC
 732
AD-14714



faAfuCfTsT

UUGacuTsT







2118-2136
AGUfCfAAGGAGCfAUfGGAAU
 733
GAUUCcaUGcuCC
 734
AD-14724



fCfTsT

UUGacuTsT







2118-2136
AgUcAaGgAgCaUgGaAuCTsT
 735
GAUUCcaUGcuCC
 736
AD-14734





UUGacuTsT







2118-2136
GfcGfgCfaCfcCfuCfaUfaG
 737
P*aGfgCfcUfaU
 738
AD-15080



fgCfcUfTsT

fgAfgGfgUfgCf







cGfcTsT







2118-2136
GCfGGCfACfCfCfUfCfAUfA
 739
AGGCfCfUfAUfG
 740
AD-15090



GGCfCfUfTsT

AGGGUfGCfCfGC







fTsT







2118-2136
GcGgCaCcCuCaUaGgCcUTsT
 741
P*aGfgCfcUfaU
 742
AD-15100





fgAfgGfgUfgCf







cGfcTsT







2118-2136
GcGgCaCcCuCaUaGgCcUTsT
 743
AGGCfCfUfAUfG
 744
AD-15110





AGGGUfGCfCfGC







fTsT







2118-2136
GfcGfgCfaCfcCfuCfaUfaG
 745
AGGCCuaUGagGG
 746
AD-15120



fgCfcUfTsT

UGCcgcTsT







2118-2136
GCfGGCfACfCfCfUfCfAUfA
 747
AGGCCuaUGagGG
 748
AD-15130



GGCfCfUfTsT

UGCcgcTsT







2118-2136
GcGgCaCcCuCaUaGgCcUTsT
 749
AGGCCuaUGagGG
 750
AD-15140





UGCcgcTsT







2122-2140
AAGGAGCAUGGAAUCCCGGTsT
 751
CCGGGAUUCCAUG
 752
AD-9522





CUCCUUTsT







2122-2140
AAGGAGcAuGGAAucccGGTsT
 753
CCGGGAUUCcAUG
 754
AD-9648





CUCCUUTsT







2123-2141
AGGAGCAUGGAAUCCCGGCTsT
 755
GCCGGGAUUCCAU
 756
AD-9552





GCUCCUTsT







2123-2141
AGGAGcAuGGAAucccGGcTsT
 757
GCCGGGAUUCcAU
 758
AD-9678





GCUCCUTsT







2125-2143
GAGCAUGGAAUCCCGGCCCTsT
 759
GGGCCGGGAUUCC
 760
AD-9618





AUGCUCTsT







2125-2143
GAGcAuGGAAucccGGcccTsT
 761
GGGCCGGGAUUCc
 762
AD-9744





AUGCUCTsT







2230-2248
GCCUACGCCGUAGACAACATT
 763
UGUUGUCUACGGC
 764
AD-15239





GUAGGCTT







2231-2249
CCUACGCCGUAGACAACACTT
 765
GUGUUGUCUACGG
 766
AD-15212





CGUAGGTT







2232-2250
CUACGCCGUAGACAACACGTT
 767
CGUGUUGUCUACG
 768
AD-15240





GCGUAGTT







2233-2251
UACGCCGUAGACAACACGUTT
 769
ACGUGUUGUCUAC
 770
AD-15177





GGCGUATT







2235-2253
CGCCGUAGACAACACGUGUTT
 771
ACACGUGUUGUCU
 772
AD-15179





ACGGCGTT







2236-2254
GCCGUAGACAACACGUGUGTT
 773
CACACGUGUUGUC
 774
AD-15180





UACGGCTT







2237-2255
CCGUAGACAACACGUGUGUTT
 775
ACACACGUGUUGU
 776
AD-15241





CUACGGTT







2238-2256
CGUAGACAACACGUGUGUATT
 777
UACACACGUGUUG
 778
AD-15268





UCUACGTT







2240-2258
UAGACAACACGUGUGUAGUTT
 779
ACUACACACGUGU
 780
AD-15242





UGUCUATT







2241-2259
AGACAACACGUGUGUAGUCTT
 781
GACUACACACGUG
 782
AD-15216





UUGUCUTT







2242-2260
GACAACACGUGUGUAGUCATT
 783
UGACUACACACGU
 784
AD-15176





GUUGUCTT







2243-2261
ACAACACGUGUGUAGUCAGTT
 785
CUGACUACACACG
 786
AD-15181





UGUUGUTT







2244-2262
CAACACGUGUGUAGUCAGGTT
 787
CCUGACUACACAC
 788
AD-15243





GUGUUGTT







2247-2265
CACGUGUGUAGUCAGGAGCTT
 789
GCUCCUGACUACA
 790
AD-15182





CACGUGTT







2248-2266
ACGUGUGUAGUCAGGAGCCTT
 791
GGCUCCUGACUAC
 792
AD-15244





ACACGUTT







2249-2267
CGUGUGUAGUCAGGAGCCGTT
 793
CGGCUCCUGACUA
 794
AD-15387





CACACGTT







2251-2269
UGUGUAGUCAGGAGCCGGGTT
 795
CCCGGCUCCUGAC
 796
AD-15245





UACACATT







2257-2275
GUCAGGAGCCGGGACGUCATsT
 797
UGACGUCCCGGCU
 798
AD-9555





CCUGACTsT







2257-2275
GucAGGAGccGGGAcGucATsT
 799
UGACGUCCCGGCU
 800
AD-9681





CCUGACTsT







2258-2276
UCAGGAGCCGGGACGUCAGTsT
 801
CUGACGUCCCGGC
 802
AD-9619





UCCUGATsT







2258-2276
ucAGGAGccGGGAcGucAGTsT
 803
CUGACGUCCCGGC
 804
AD-9745





UCCUGATsT







2259-2277
CAGGAGCCGGGACGUCAGCTsT
 805
GCUGACGUCCCGG
 806
AD-9620





CUCCUGTsT







2259-2277
cAGGAGccGGGAcGucAGcTsT
 807
GCUGACGUCCCGG
 808
AD-9746





CUCCUGTsT







2263-2281
AGCCGGGACGUCAGCACUATT
 809
UAGUGCUGACGUC
 810
AD-15288





CCGGCUTT







2265-2283
CCGGGACGUCAGCACUACATT
 811
UGUAGUGCUGACG
 812
AD-15246





UCCCGGTT







2303-2321
CCGUGACAGCCGUUGCCAUTT
 813
AUGGCAACGGCUG
 814
AD-15289





UCACGGTT







2317-2335
GCCAUCUGCUGCCGGAGCCTsT
 815
GGCUCCGGCAGCA
 816
AD-9324





GAUGGCTsT







2375-2393
CCCAUCCCAGGAUGGGUGUTT
 817
ACACCCAUCCUGG
 818
AD-15329





GAUGGGTT







2377-2395
CAUCCCAGGAUGGGUGUCUTT
 819
AGACACCCAUCCU
 820
AD-15330





GGGAUGTT







2420-2438
AGCUUMAAAUGGUUCCGATT
 821
UCGGAACCAUUUU
 822
AD-15169





AAAGCUTT







2421-2439
GCUUUAAAAUGGUUCCGACTT
 823
GUCGGAACCAUUU
 824
AD-15201





UAAAGCTT







2422-2440
CUUMAAAUGGUUCCGACUTT
 825
AGUCGGAACCAUU
 826
AD-15331





UUAAAGTT







2423-2441
UUUAAAAUGGUUCCGACUUTT
 827
AAGUCGGAACCAU
 828
AD-15190





UUUAAATT







2424-2442
UUAAAAUGGUUCCGACUUGTT
 829
CAAGUCGGAACCA
 830
AD-15247





UUUUAATT







2425-2443
UAAAAUGGUUCCGACUUGUTT
 831
ACAAGUCGGAACC
 832
AD-15248





AUUUUATT







2426-2444
AAAAUGGUUCCGACUUGUCTT
 833
GACAAGUCGGAAC
 834
AD-15175





CAUUUUTT







2427-2445
AAAUGGUUCCGACUUGUCCTT
 835
GGACAAGUCGGAA
 836
AD-15249





CCAUUUTT







2428-2446
AAUGGUUCCGACUUGUCCCTT
 837
GGGACAAGUCGGA
 838
AD-15250





ACCAUUTT







2431-2449
GGUUCCGACUUGUCCCUCUTT
 839
AGAGGGACAAGUC
 840
AD-15400





GGAACCTT







2457-2475
CUCCAUGGCCUGGCACGAGTT
 841
CUCGUGCCAGGCC
 842
AD-15332





AUGGAGTT







2459-2477
CCAUGGCCUGGCACGAGGGTT
 843
CCCUCGUGCCAGG
 844
AD-15388





CCAUGGTT







2545-2563
GAACUCACUCACUCUGGGUTT
 845
ACCCAGAGUGAGU
 846
AD-15333





GAGUUCTT







2549-2567
UCACUCACUCUGGGUGCCUTT
 847
AGGCACCCAGAGU
 848
AD-15334





GAGUGATT







2616-2634
UUUCACCAUUCAAACAGGUTT
 849
ACCUGUUUGAAUG
 850
AD-15335





GUGAAATT







2622-2640
CAUUCAAACAGGUCGAGCUTT
 851
AGCUCGACCUGUU
 852
AD-15183





UGAAUGTT







2623-2641
AUUCAAACAGGUCGAGCUGTT
 853
CAGCUCGACCUGU
 854
AD-15202





UUGAAUTT







2624-2642
UUCAAACAGGUCGAGCUGUTT
 855
ACAGCUCGACCUG
 856
AD-15203





UUUGAATT







2625-2643
UCAAACAGGUCGAGCUGUGTT
 857
CACAGCUCGACCU
 858
AD-15272





GUUUGATT







2626-2644
CAAACAGGUCGAGCUGUGCTT
 859
GCACAGCUCGACC
 860
AD-15217





UGUUUGTT







2627-2645
AAACAGGUCGAGCUGUGCUTT
 861
AGCACAGCUCGAC
 862
AD-15290





CUGUUUTT







2628-2646
AACAGGUCGAGCUGUGCUCTT
 863
GAGCACAGCUCGA
 864
AD-15218





CCUGUUTT







2630-2648
CAGGUCGAGCUGUGCUCGGTT
 865
CCGAGCACAGCUC
 866
AD-15389





GACCUGTT







2631-2649
AGGUCGAGCUGUGCUCGGGTT
 867
CCCGAGCACAGCU
 868
AD-15336





CGACCUTT







2633-2651
GUCGAGCUGUGCUCGGGUGTT
 869
CACCCGAGCACAG
 870
AD-15337





CUCGACTT







2634-2652
UCGAGCUGUGCUCGGGUGCTT
 871
GCACCCGAGCACA
 872
AD-15191





GCUCGATT







2657-2675
AGCUGCUCCCAAUGUGCCGTT
 873
CGGCACAUUGGGA
 874
AD-15390





GCAGCUTT







2658-2676
GCUGCUCCCAAUGUGCCGATT
 875
UCGGCACAUUGGG
 876
AD-15338





AGCAGCTT







2660-2678
UGCUCCCAAUGUGCCGAUGTT
 877
CAUCGGCACAUUG
 878
AD-15204





GGAGCATT







2663-2681
UCCCAAUGUGCCGAUGUCCTT
 879
GGACAUCGGCACA
 880
AD-15251





UUGGGATT







2665-2683
CCAAUGUGCCGAUGUCCGUTT
 881
ACGGACAUCGGCA
 882
AD-15205





CAUUGGTT







2666-2684
CAAUGUGCCGAUGUCCGUGTT
 883
CACGGACAUCGGC
 884
AD-15171





ACAUUGTT







2667-2685
AAUGUGCCGAUGUCCGUGGTT
 885
CCACGGACAUCGG
 886
AD-15252





CACAUUTT







2673-2691
CCGAUGUCCGUGGGCAGAATT
 887
UUCUGCCCACGGA
 888
AD-15339





CAUCGGTT







2675-2693
GAUGUCCGUGGGCAGAAUGTT
 889
CAUUCUGCCCACG
 890
AD-15253





GACAUCTT







2678-2696
GUCCGUGGGCAGAAUGACUTT
 891
AGUCAUUCUGCCC
 892
AD-15340





ACGGACTT







2679-2697
UCCGUGGGCAGAAUGACUUTT
 893
AAGUCAUUCUGCC
 894
AD-15291





CACGGATT







2683-2701
UGGGCAGAAUGACUUDUAUTT
 895
AUAAAAGUCAUUC
 896
AD-15341





UGCCCATT







2694-2712
ACUUUUAUUGAGCUCUUGUTT
 897
ACAAGAGCUCAAU
 898
AD-15401





AAAAGUTT







2700-2718
AUUGAGCUCUUGUUCCGUGTT
 899
CACGGAACAAGAG
 900
AD-15342





CUCAAUTT







2704-2722
AGCUCUUGUUCCGUGCCAGTT
 901
CUGGCACGGAACA
 902
AD-15343





AGAGCUTT







2705-2723
GCUCUUGUUCCGUGCCAGGTT
 903
CCUGGCACGGAAC
 904
AD-15292





AAGAGCTT







2710-2728
UGUUCCGUGCCAGGCAUUCTT
 905
GAAUGCCUGGCAC
 906
AD-15344





GGAACATT







2711-2729
GUUCCGUGCCAGGCAUUCATT
 907
UGAAUGCCUGGCA
 908
AD-15254





CGGAACTT







2712-2730
UUCCGUGCCAGGCAUUCAATT
 909
UUGAAUGCCUGGC
 910
AD-15345





ACGGAATT







2715-2733
CGUGCCAGGCAUUCAAUCCTT
 911
GGAUUGAAUGCCU
 912
AD-15206





GGCACGTT







2716-2734
GUGCCAGGCAUUCAAUCCUTT
 913
AGGAUUGAAUGCC
 914
AD-15346





UGGCACTT







2728-2746
CAAUCCUCAGGUCUCCACCTT
 915
GGUGGAGACCUGA
 916
AD-15347





GGAUUGTT







2743-2761
CACCAAGGAGGCAGGAUUCTsT
 917
GAAUCCUGCCUCC
 918
AD-9577





UUGGUGTsT







2743-2761
cAccAAGGAGGcAGGAuucTsT
 919
GAAUCCUGCCUCC
 920
AD-9703





UUGGUGTsT







2743-2761
CfaCfcAfaGfgAfgGfcAfgG
 921
P*gAfaUfcCfuG
 922
AD-14678



faUfuCfTsT

fcCfuCfcUfuGf







gUfgTsT







2743-2761
CfACfCfAAGGAGGCfAGGAUf
 923
GAAUfCfCfUfGC
 924
AD-14688



UfCfTsT

fCfUfCfCfUfUf







GGUfGTsT







2743-2761
CaCcAaGgAgGcAgGaUuCTsT
925
P*gAfaUfcCfuG
 926
AD-14698





fcCfuCfcUfuGf







gUfgTsT







2743-2761
CaCcAaGgAgGcAgGaUuCTsT
 927
GAAUfCfCfUfGC
 928
AD-14708





fCfUfCfCfUfUf







GGUfGTsT







2743-2761
CfaCfcAfaGfgAfgGfcAfgG
 929
GAAUCcuGCcuCC
 930
AD-14718



faUfuCfTsT

UUGgugTsT







2743-2761
CfACfCfAAGGAGGCfAGGAUf
 931
GAAUCcuGCcuCC
 932
AD-14728



UfCfTsT

UUGgugTsT







2743-2761
CaCcAaGgAgGcAgGaUuCTsT
 933
GAAUCcuGCcuCC
 934
AD-14738





UUGgugTsT







2743-2761
GfgCfcUfgGfaGfuUfuAfuU
 935
P*uCfcGfaAfuA
 936
AD-15084



fcGfgAfTsT

faAfcUfcCfaGf







gCfcTsT







2743-2761
GGCfCfUfGGAGUfUfUfAUfU
 937
UfCfCfGAAUfAA
 938
AD-15094



fCfGGATsT

ACfUfCfCfAGGC







fCfTsT







2743-2761
GgCcUgGaGuUuAuUcGgATsT
 939
P*uCfcGfaAfuA
 940
AD-15104





faAfcUfcCfaGf







gCfcTsT







2743-2761
GgCcUgGaGuUuAuUcGgATsT
 941
UfCfCfGAAUfAA
 942
AD-15114





ACfUfCfCfAGGC







fCfTsT







2743-2761
GfgCfcUfgGfaGfuUfuAfuU
 943
UCCGAauAAacUC
 944
AD-15124



fcGfgAfTsT

CAGgccTsT







2743-2761
GGCfCfUfGGAGUfUfUfAUfU
 945
UCCGAauAAacUC
 946
AD-15134



fCfGGATsT

CAGgccTsT







2743-2761
GgCcUgGaGuUuAuUcGgATsT
 947
UCCGAauAAacUC
 948
AD-15144





CAGgccTsT







2753-2771
GCAGGAUUCUUCCCAUGGATT
 949
UCCAUGGGAAGAA
 950
AD-15391





UCCUGCTT







2794-2812
UGCAGGGACAAACAUCGUUTT
 951
AACGAUGUUUGUC
 952
AD-15348





CCUGCATT







2795-2813
GCAGGGACAAACAUCGUUGTT
 953
CAACGAUGUUUGU
 954
AD-15349





CCCUGCTT







2797-2815
AGGGACAAACAUCGUUGGGTT
 955
CCCAACGAUGUUU
 956
AD-15170





GUCCCUTT







2841-2859
CCCUCAUCUCCAGCUAACUTT
 957
AGUUAGCUGGAGA
 958
AD-15350





UGAGGGTT







2845-2863
CAUCUCCAGCUAACUGUGGTT
 959
CCACAGUUAGCUG
 960
AD-15402





GAGAUGTT







2878-2896
GCUCCCUGAUUAAUGGAGGTT
 961
CCUCCAUUAAUCA
 962
AD-15293





GGGAGCTT







2881-2899
CCCUGAUUAAUGGAGGCUUTT
 963
AAGCCUCCAUUAA
 964
AD-15351





UCAGGGTT







2882-2900
CCUGAUUAAUGGAGGCUUATT
 965
UAAGCCUCCAUUA
 966
AD-15403





AUCAGGTT







2884-2902
UGAUUAAUGGAGGCUUAGCTT
 967
GCUAAGCCUCCAU
 968
AD-15404





UAAUCATT







2885-2903
GAUUAAUGGAGGCUUAGCUTT
 969
AGCUAAGCCUCCA
 970
AD-15207





UUAAUCTT







2886-2904
AUUAAUGGAGGCUUAGCUUTT
 971
AAGCUAAGCCUCC
 972
AD-15352





AUUAAUTT







2887-2905
UUAAUGGAGGCUUAGCUUUTT
 973
AAAGCUAAGCCUC
 974
AD-15255





CAUUAATT







2903-2921
UUUCUGGAUGGCAUCUAGCTsT
 975
GCUAGAUGCCAUC
 976
AD-9603





CAGAAATsT







2903-2921
uuucuGGAuGGcAucuAGcTsT
 977
GCuAGAUGCcAUC
 978
AD-9729





cAGAAATsT







2904-2922
UUCUGGAUGGCAUCUAGCCTsT
 979
GGCUAGAUGCCAU
 980
AD-9599





CCAGAATsT







2904-2922
uucuGGAuGGcAucuAGccTsT
 981
GGCuAGAUGCcAU
 982
AD-9725





CcAGAATsT







2905-2923
UCUGGAUGGCAUCUAGCCATsT
 983
UGGCUAGAUGCCA
 984
AD-9621





UCCAGATsT







2905-2923
ucuGGAuGGcAucuAGccATsT
 985
UGGCuAGAUGCcA
 986
AD-9747





UCcAGATsT







2925-2943
AGGCUGGAGACAGGUGCGCTT
 987
GCGCACCUGUCUC
 988
AD-15405





CAGCCUTT







2926-2944
GGCUGGAGACAGGUGCGCCTT
 989
GGCGCACCUGUCU
 990
AD-15353





CCAGCCTT







2927-2945
GCUGGAGACAGGUGCGCCCTT
 991
GGGCGCACCUGUC
 992
AD-15354





UCCAGCTT







2972-2990
UUCCUGAGCCACCUUUACUTT
 993
AGUAAAGGUGGCU
 994
AD-15406





CAGGAATT







2973-2991
UCCUGAGCCACCUUUACUCTT
 995
GAGUAAAGGUGGC
 996
AD-15407





UCAGGATT







2974-2992
CCUGAGCCACCUUUACUCUTT
 997
AGAGUAAAGGUGG
 998
AD-15355





CUCAGGTT







2976-2994
UGAGCCACCUUUACUCUGCTT
 999
GCAGAGUAAAGGU
1000
AD-15356





GGCUCATT







2978-2996
AGCCACCUUUACUCUGCUCTT
1001
GAGCAGAGUAAAG
1002
AD-15357





GUGGCUTT







2981-2999
CACCUUUACUCUGCUCUAUTT
1003
AUAGAGCAGAGUA
1004
AD-15269





AAGGUGTT







2987-3005
UACUCUGCUCUAUGCCAGGTsT
1005
CCUGGCAUAGAGC
1006
AD-9565





AGAGUATsT







2987-3005
uAcucuGcucuAuGccAGGTsT
1007
CCUGGcAuAGAGc
1008
AD-9691





AGAGuATsT







2998-3016
AUGCCAGGCUGUGCUAGCATT
1009
UGCUAGCACAGCC
1010
AD-15358





UGGCAUTT







3003-3021
AGGCUGUGCUAGCAACACCTT
1011
GGUGUUGCUAGCA
1012
AD-15359





CAGCCUTT







3006-3024
CUGUGCUAGCAACACCCAATT
1013
UUGGGUGUUGCUA
1014
AD-15360





GCACAGTT







3010-3028
GCUAGCAACACCCAAAGGUTT
1015
ACCUUUGGGUGUU
1016
AD-15219





GCUAGCTT







3038-3056
GGAGCCAUCACCUAGGACUTT
1017
AGUCCUAGGUGAU
1018
AD-15361





GGCUCCTT







3046-3064
CACCUAGGACUGACUCGGCTT
1019
GCCGAGUCAGUCC
1020
AD-15273





UAGGUGTT







3051-3069
AGGACUGACUCGGCAGUGUTT
1021
ACACUGCCGAGUC
1022
AD-15362





AGUCCUTT







3052-3070
GGACUGACUCGGCAGUGUGTT
1023
CACACUGCCGAGU
1024
AD-15192





CAGUCCTT







3074-3092
UGGUGCAUGCACUGUCUCATT
1025
UGAGACAGUGCAU
1026
AD-15256





GCACCATT







3080-3098
AUGCACUGUCUCAGCCAACTT
1027
GUUGGCUGAGACA
1028
AD-15363





GUGCAUTT







3085-3103
CUGUCUCAGCCAACCCGCUTT
1029
AGCGGGUUGGCUG
1030
AD-15364





AGACAGTT







3089-3107
CUCAGCCAACCCGCUCCACTsT
1031
GUGGAGCGGGUUG
1032
AD-9604





GCUGAGTsT







3089-3107
cucAGccAAcccGcuccAcTsT
1033
GUGGAGCGGGUUG
1034
AD-9730





GCUGAGTsT







3093-3111
GCCAACCCGCUCCACUACCTsT
1035
GGUAGUGGAGCGG
1036
AD-9527





GUUGGCTsT







3093-3111
GccAAcccGcuccAcuAccTsT
1037
GGuAGUGGAGCGG
1038
AD-9653





GUUGGCTsT







3096-3114
AACCCGCUCCACUACCCGGTT
1039
CCGGGUAGUGGAG
1040
AD-15365





CGGGUUTT







3099-3117
CCGCUCCACUACCCGGCAGTT
1041
CUGCCGGGUAGUG
1042
AD-15294





GAGCGGTT







3107-3125
CUACCCGGCAGGGUACACATT
1043
UGUGUACCCUGCC
1044
AD-15173





GGGUAGTT







3108-3126
UACCCGGCAGGGUACACAUTT
1045
AUGUGUACCCUGC
1046
AD-15366





CGGGUATT







3109-3127
ACCCGGCAGGGUACACAUUTT
1047
AAUGUGUACCCUG
1048
AD-15367





CCGGGUTT







3110-3128
CCCGGCAGGGUACACAUUCTT
1049
GAAUGUGUACCCU
1050
AD-15257





GCCGGGTT







3112-3130
CGGCAGGGUACACAUUCGCTT
1051
GCGAAUGUGUACC
1052
AD-15184





CUGCCGTT







3114-3132
GCAGGGUACACAUUCGCACTT
1053
GUGCGAAUGUGUA
1054
AD-15185





CCCUGCTT







3115-3133
CAGGGUACACAUUCGCACCTT
1055
GGUGCGAAUGUGU
1056
AD-15258





ACCCUGTT







3116-3134
AGGGUACACAUUCGCACCCTT
1057
GGGUGCGAAUGUG
1058
AD-15186





UACCCUTT







3196-3214
GGAACUGAGCCAGAAACGCTT
1059
GCGUUUCUGGCUC
1060
AD-15274





AGUUCCTT







3197-3215
GAACUGAGCCAGAAACGCATT
1061
UGCGUUUCUGGCU
1062
AD-15368





CAGUUCTT







3198-3216
AACUGAGCCAGAAACGCAGTT
1063
CUGCGUUUCUGGC
1064
AD-15369





UCAGUUTT







3201-3219
UGAGCCAGAAACGCAGAUUTT
1064
AAUCUGCGUUUCU
1066
AD-15370





GGCUCATT







3207-3225
AGAAACGCAGAUUGGGCUGTT
1067
CAGCCCAAUCUGC
1068
AD-15259





GUUUCUTT







3210-3228
AACGCAGAUUGGGCUGGCUTT
1069
AGCCAGCCCAAUC
1070
AD-15408





UGCGUUTT







3233-3251
AGCCAAGCCUCUUCUUACUTsT
1071
AGUAAGAAGAGGC
1072
AD-9597





UUGGCUTsT







3233-3251
AGccAAGccucuucuuAcuTsT
1073
AGuAAGAAGAGGC
1074
AD-9723





UUGGCUTsT







3233-3251
AfgCfcAfaGfcCfuCfuUfcU
1075
P*aGfuAfaGfaA
1076
AD-14680



fuAfcUfTsT

fgAfgGfcUfuGf







gCfuTsT







3233-3251
AGCfCfAAGCfCfUfCfUfUfC
1077
AGUfAAGAAGAGG
1078
AD-14690



fUfUfACfUfTsT

CfUfUfGGCfUfT







sT







3233-3251
AgCcAaGcCuCuUcUuAcUTsT
1079
P*aGfuAfaGfaA
1080
AD-14700





fgAfgGfcUfuGf







gCfuTsT







3233-3251
AgCcAaGcCuCuUcUuAcUTsT
1081
AGUfAAGAAGAGG
1082
AD-14710





CfUfUfGGCfUfT







sT







3233-3251
AfgCfcAfaGfcCfuCfuUfcU
1083
AGUAAgaAGagGC
1084
AD-14720



fuAfcUfTsT

UUGgcuTsT







3233-3251
AGCfCfAAGCfCfUfCfUfUfC
1085
AGUAAgaAGagGC
1086
AD-14730



fUfUfACfUfTsT

UUGgcuTsT







3233-3251
AgCcAaGcCuCuUcUuAcUTsT
1087
AGUAAgaAGagGC
1088
AD-14740





UUGgcuTsT







3233-3251
UfgGfuUfcCfcUfgAfgGfaC
1089
P*gCfuGfgUfcC
1090
AD-15086



fcAfgCfTsT

fuCfaGfgGfaAf







cCfaTsT







3233-3251
UfGGUfUfCfCfCfUfGAGGAC
1091
GCfUfGGUfCfCf
1092
AD-15096



fCfAGCfTsT

UfCfAGGGAACfC







fATsT







3233-3251
UgGuUcCcUgAgGaCcAgCTsT
1093
P*gCfuGfgUfcC
1094
AD-15106





fuCfaGfgGfaAf







cCfaTsT







3233-3251
UgGuUcCcUgAgGaCcAgCTsT
1095
GCfUfGGUfCfCf
1096
AD-15116





UfCfAGGGAACfC







fATsT







3233-3251
UfgGfuUfcCfcUfgAfgGfaC
1097
GCUGGucCUcaGG
1098
AD-15126



fcAfgCfTsT

GAAccaTsT







3233-3251
UfGGUfUfCfCfCfUfGAGGAC
1099
GCUGGucCUcaGG
1100
AD-15136



fCfAGCfTsT

GAAccaTsT







3233-3251
UgGuUcCcUgAgGaCcAgCTsT
1101
GCUGGucCUcaGG
1102
AD-15146





GAAccaTsT







3242-3260
UCUUCUUACUUCACCCGGCTT
1103
GCCGGGUGAAGUA
1104
AD-15260





AGAAGATT







3243-3261
CUUCUUACUUCACCCGGCUTT
1105
AGCCGGGUGAAGU
1106
AD-15371





AAGAAGTT







3244-3262
UUCUUACUUCACCCGGCUGTT
1107
CAGCCGGGUGAAG
1108
AD-15372





UAAGAATT







3262-3280
GGGCUCCUCAUUUUUACGGTT
1109
CCGUAAAAAUGAG
1110
AD-15172





GAGCCCTT







3263-3281
GGCUCCUCAUUUUUACGGGTT
1111
CCCGUAAAAAUGA
1112
AD-15295





GGAGCCTT







3264-3282
GCUCCUCAUUUUUACGGGUTT
1113
ACCCGUAAAAAUG
1114
AD-15373





AGGAGCTT







3265-3283
CUCCUCAUUUUUACGGGUATT
1115
UACCCGUAAAAAU
1116
AD-15163





GAGGAGTT







3266-3284
UCCUCAUUUUUACGGGUAATT
1117
UUACCCGUAAAAA
1118
AD-15165





UGAGGATT







3267-3285
CCUCAUUUUUACGGGUAACTT
1119
GUUACCCGUAAAA
1120
AD-15374





AUGAGGTT







3268-3286
CUCAUUUUUACGGGUAACATT
1121
UGUUACCCGUAAA
1122
AD-15296





AAUGAGTT







3270-3288
CAUUUUUACGGGUAACAGUTT
1123
ACUGUUACCCGUA
1124
AD-15261





AAAAUGTT







3271-3289
AUUUUUACGGGUAACAGUGTT
1125
CACUGUUACCCGU
1126
AD-15375





AAAAAUTT







3274-3292
UUUACGGGUAACAGUGAGGTT
1127
CCUCACUGUUACC
1128
AD-15262





CGUAAATT







3308-3326
CAGACCAGGAAGCUCGGUGTT
1129
CACCGAGCUUCCU
1130
AD-15376





GGUCUGTT







3310-3328
GACCAGGAAGCUCGGUGAGTT
1131
CUCACCGAGCUUC
1132
AD-15377





CUGGUCTT







3312-3330
CCAGGAAGCUCGGUGAGUGTT
1133
CACUCACCGAGCU
1134
AD-15409





UCCUGGTT







3315-3333
GGAAGCUCGGUGAGUGAUGTT
1135
CAUCACUCACCGA
1136
AD-15378





GCUUCCTT







3324-3342
GUGAGUGAUGGCAGAACGATT
1137
UCGUUCUGCCAUC
1138
AD-15410





ACUCACTT







3326-3344
GAGUGAUGGCAGAACGAUGTT
1139
CAUCGUUCUGCCA
1140
AD-15379





UCACUCTT







3330-3348
GAUGGCAGAACGAUGCCUGTT
1141
CAGGCAUCGUUCU
1142
AD-15187





GCCAUCTT







3336-3354
AGAACGAUGCCUGCAGGCATT
1143
UGCCUGCAGGCAU
1144
AD-15263





CGUUCUTT







3339-3357
ACGAUGCCUGCAGGCAUGGTT
1145
CCAUGCCUGCAGG
1146
AD-15264





CAUCGUTT







3348-3366
GCAGGCAUGGAACUUUUUCTT
1147
GAAAAAGUUCCAU
1148
AD-15297





GCCUGCTT







3356-3374
GGAACUUUUUCCGUUAUCATT
1149
UGAUAACGGAAAA
1150
AD-15208





AGUUCCTT







3357-3375
GAACUUUUUCCGUUAUCACTT
1151
GUGAUAACGGAAA
1152
AD-15209





AAGUUCTT







3358-3376
AACUUUUUCCGUUAUCACCTT
1153
GGUGAUAACGGAA
1154
AD-15193





AAAGUUTT







3370-3388
UAUCACCCAGGCCUGAUUCTT
1155
GAAUCAGGCCUGG
1156
AD-15380





GUGAUATT







3378-3396
AGGCCUGAUUCACUGGCCUTT
1157
AGGCCAGUGAAUC
1158
AD-15298





AGGCCUTT







3383-3401
UGAUUCACUGGCCUGGCGGTT
1159
CCGCCAGGCCAGU
1160
AD-15299





GAAUCATT







3385-3403
AUUCACUGGCCUGGCGGAGTT
1161
CUCCGCCAGGCCA
1162
AD-15265





GUGAAUTT







3406-3424
GCUUCUAAGGCAUGGUCGGTT
1163
CCGACCAUGCCUU
1164
AD-15381





AGAAGCTT







3407-3425
CUUCUAAGGCAUGGUCGGGTT
1165
CCCGACCAUGCCU
1166
AD-15210





UAGAAGTT







3429-3447
GAGGGCCAACAACUGUCCCTT
1167
GGGACAGUUGUUG
1168
AD-15270





GCCCUCTT







3440-3458
ACUGUCCCUCCUUGAGCACTsT
1169
GUGCUCAAGGAGG
1170
AD-9591





GACAGUTsT







3440-3458
AcuGucccuccuuGAGcAcTsT
1171
GUGCUcAAGGAGG
1172
AD-9717





GAcAGUTsT







3441-3459
CUGUCCCUCCUUGAGCACCTsT
1173
GGUGCUCAAGGAG
1174
AD-9622





GGACAGTsT







3441-3459
cuGucccuccuuGAGcAccTsT
1175
GGUGCUcAAGGAG
1176
AD-9748





GGAcAGTsT







3480-3498
ACAUUUAUCUUUUGGGUCUTsT
1177
AGACCCAAAAGAU
1178
AD-9587





AAAUGUTsT







3480-3498
AcAuuuAucuuuuGGGucuTsT
1179
AGACCcAAAAGAu
1180
AD-9713





AAAUGUTsT







3480-3498
AfcAfuUfuAfuCfuUfuUfgG
1181
P*aGfaCfcCfaA
1182
AD-14679



fgUfcUfTsT

faAfgAfuAfaAf







uGfuTsT







3480-3498
ACfAUfUfUfAUfCfUfUfUfU
1183
AGACfCfCfAAAA
1184
AD-14689



fGGGUfCfUfTsT

GAUfAAAUfGUfT







sT







3480-3498
AcAuUuAuCuUuUgGgUcUTsT
1185
P*aGfaCfcCfaA
1186
AD-14699





faAfgAfuAfaAf







uGfuTsT







3480-3498
AcAuUuAuCuUuUgGgUcUTsT
1187
AGACfCfCfAAAA
1188
AD-14709





GAUfAAAUfGUfT







sT







3480-3498
AfcAfuUfuAfuCfuUfuUfgG
1189
AGACCcaAAagAU
1190
AD-14719



fgUfcUfTsT

AAAuguTsT







3480-3498
ACfAUfUfUfAUfCfUfUfUfU
1191
AGACCcaAAagAU
1192
AD-14729



fGGGUfCfUfTsT

AAAuguTsT







3480-3498
AcAuUuAuCuUuUgGgUcUTsT
1193
AGACCcaAAagAU
1194
AD-14739





AAAuguTsT







3480-3498
GfcCfaUfcUfgCfuGfcCfgG
1195
P*gGfcUfcCfgG
1196
AD-15085



faGfcCfTsT

fcAfgCfaGfaUf







gGfcTsT







3480-3498
GCfCfAUfCfUfGCfUfGCfCf
1197
GGCfUfCfCfGGC
1198
AD-15095



GGAGCfCfTsT

fAGCfAGAUfGGC







fTsT







3480-3498
GcCaUcUgCuGcCgGaGcCTsT
1199
P*gGfcUfcCfgG
1200
AD-15105





fcAfgCfaGfaUf







gGfcTsT







3480-3498
GcCaUcUgCuGcCgGaGcCTsT
1201
GGCfUfCfCfGGC
1202
AD-15115





fAGCfAGAUfGGC







fTsT







3480-3498
GfcCfaUfcUfgCfuGfcCfgG
1203
GGCUCauGCagCA
1204
AD-15125



faGfcCfTsT

GAUggcTsT







3480-3498
GCfCfAUfCfUfGCfUfGCfCf
1205
GGCUCauGCagCA
1206
AD-15135



GGAGCfCfTsT

GAUggcTsT







3480-3498
GcCaUcUgCuGcCgGaGcCTsT
1207
GGCUCauGCagCA
1208
AD-15145





GAUggcTsT







3481-3499
CAUUUAUCUUUUGGGUCUGTsT
1209
CAGACCCAAAAGA
1210
AD-9578





UAAAUGTsT







3481-3499
cAuuuAucuuuuGGGucuGTsT
1211
cAGACCcAAAAGA
1212
AD-9704





uAAAUGTsT







3485-3503
UAUCUUUUGGGUCUGUCCUTsT
1213
AGGACAGACCCAA
1214
AD-9558





AAGAUATsT







3485-3503
uAucuuuuGGGucuGuccuTsT
1215
AGGAcAGACCcAA
1216
AD-9684





AAGAuATsT







3504-3522
CUCUGUUGCCUUUUUACAGTsT
1217
CUGUAAAAAGGCA
1218
AD-9634





ACAGAGTsT







3504-3522
cucuGuuGccuuuuuAcAGTsT
1219
CUGuAAAAAGGcA
1220
AD-9760





AcAGAGTsT







3512-3530
CCUUUUUACAGCCAACUUUTT
1221
AAAGUUGGCUGUA
1222
AD-15411





AAAAGGTT







3521-3539
AGCCAACUUUUCUAGACCUTT
1223
AGGUCUAGAAAAG
1224
AD-15266





UUGGCUTT







3526-3544
ACUUUUCUAGACCUGUUUUTT
1225
AAAACAGGUCUAG
1226
AD-15382





AAAAGUTT







3530-3548
UUCUAGACCUGUUUUGCUUTsT
1227
AAGCAAAACAGGU
1228
AD-9554





CUAGAATsT







3530-3548
uucuAGAccuGuuuuGcuuTsT
1229
AAGcAAAAcAGGU
1230
AD-9680





CuAGAATsT







3530-3548
UfuCfuAfgAfcCfuGfuUfuU
1231
P*aAfgCfaAfaA
1232
AD-14676



fgCfuUfTsT

fcAfgGfuCfuAf







gAfaTsT







3530-3548
UfUfCfUfAGACfCfUfGUfUf
1233
AAGCfAAAACfAG
1234
AD-14686



UfUfGCfUfUfTsT

GUfCfUfAGAATs







T







3530-3548
UuCuAgAcCuGuUuUgCuUTsT
1235
P*aAfgCfaAfaA
1236
AD-14696





fcAfgGfuCfuAf







gAfaTsT







3530-3548
UuCuAgAcCuGuUuUgCuUTsT
1237
AAGCfAAAACfAG
1238
AD-14706





GUfCfUfAGAATs







T







3530-3548
UfuCfuAfgAfcCfuGfuUfuU
1239
AAGcAaaACagGU
1240
AD-14716



ffCfuUfTsT

CUAgaaTsT







3530-3548
UfUfCfUfAGACfCfUfGUfUf
1241
AAGcAaaACagGU
1242
AD-14726



UfUfGCfUfUfTsT

CUAgaaTsT







3530-3548
UuCuAgAcCuGuUuUgCuUTsT
1243
AAGcAaaACagGU
1244
AD-14736





CUAgaaTsT







3530-3548
CfaUfaGfgCfcUfgGfaGfuU
1245
P*aAfuAfaAfcU
1246
AD-15082



fuAfuUfTsT

fcCfaGfgCfcUf







aUfgTsT







3530-3548
CfAUfAGGCfCfUfGGAGUfUf
1247
AAUfAAACfUfCf
1248
AD-15092



UfAUfUfTsT

CfAGGCfCfUfAU







fGTsT







3530-3548
CaUaGgCcUgGaGuUuAuUTsT
1249
P*aAfuAfaAfcU
1250
AD-15102





fcCfaGfgCfcUf







aUfgTsT







3530-3548
CaUaGgCcUgGaGuUuAuUTsT
1251
AAUfAAACfUfCf
1252
AD-15112





CfAGGCfCfUfAU







fGTsT







3530-3548
CfaUfaGfgCfcUfgGfaGfuU
1253
AAUAAacUCcaGG
1254
AD-15122



fuAfuUfTsT

CCUaugTsT







3530-3548
CfAUfAGGCfCfUfGGAGUfUf
1255
AAUAAacUCcaGG
1256
AD-15132



UfAUfUfTsT

CCUaugTsT







3530-3548
CaUaGgCcUgGaGuUuAuUTsT
1257
AAUAAacUCcaGG
1258
AD-15142





CCUaugTsT







3531-3549
UCUAGACCUGUUUUGCUUUTsT
1259
AAAGCAAAACAGG
1260
AD-9553





UCUAGATsT







3531-3549
ucuAGAccuGuuuuGcuuuTsT
1261
AAAGcAAAAcAGG
1262
AD-9679





UCuAGATsT







3531-3549
UfcUfaGfaCfcUfgUfuUfuG
1263
P*aAfaGfcAfaA
1264
AD-14675



fcUfuUfTsT

faCfaGfgUfcUf







aGfaTsT







3531-3549
UfCfUfAGACfCfUfGUfUfUf
1265
AAAGCfAAAACfA
1266
AD-14685



UfGCfUfUfUfTsT

GGUfCfUfAGATs







T







3531-3549
UcUaGaCcUgUuUuGcUuUTsT
1267
P*aAfaGfcAfaA
1268
AD-14695





faCfaGfgUfcUf







aGfaTsT







3531-3549
UcUaGaCcUgUuUuGcUuUTsT
1269
AAAGCfAAAACfA
1270
AD-14705





GGUfCfUfAGATs







T







3531-3549
UfcUfaGfaCfcUfgUfuUfuG
1271
AAAGCaaAAcaGG
1272
AD-14715



fcUfuUfTsT

UCUagaTsT







3531-3549
UfCfUfAGACfCfUfGUfUfUf
1273
AAAGCaaAAcaGG
1274
AD-14725



UfGCfUfUfUfTsT

UCUagaTsT







3531-3549
UcUaGaCcUgUuUuGcUuUTsT
1275
AAAGCaaAAcaGG
1276
AD-14735





UCUagaTsT







3531-3549
UfcAfuAfgGfcCfuGfgAfgU
1277
P*aUfaAfaCfuC
1278
AD-15081



fuUfaUfTsT

fcAfgGfcCfuAf







uGfaTsT







3531-3549
UfCfAUfAGGCfCfUfGGAGUf
1279
AUfAAACfUfCfC
1280
AD-15091



UfUfAUfTsT

fAGGCfCfUfAUf







GATsT







3531-3549
UcAuAgGcCuGgAgUuUaUTsT
1281
P*aUfaAfaCfuC
1282
AD-15101





fcAfgGfcCfuAf







uGfaTsT







3531-3549
UcAuAgGcCuGgAgUuUaUTsT
1283
AUfAAACfUfCfC
1284
AD-15111





fAGGCfCfUfAUf







GATsT







3531-3549
UfcAfuAfgGfcCfuGfgAfgU
1285
AUAAAcuCCagGC
1286
AD-15121



fuUfaUfTsT

CUAugaTsT







3531-3549
UfCfAUfAGGCfCfUfGGAGUf
1287
AUAAAcuCCagGC
1288
AD-15131



UfUfAUfTsT

CUAugaTsT







3531-3549
UcAuAgGcCuGgAgUuUaUTsT
1289
AUAAAcuCCagGC
1290
AD-15141





CUAugaTsT







3557-3575
UGAAGAUAUUUAUUCUGGGTsT
1291
CCCAGAAUAAAUA
1292
AD-9626





UCUUCATsT







3557-3575
uGAAGAuAuuuAuucuGGGTsT
1293
CCcAGAAuAAAuA
1294
AD-9752





UCUUcATsT







3570-3588
UCUGGGUUUUGUAGCAUUUTsT
1295
AAAUGCUACAAAA
1296
AD-9629





CCCAGATsT







3570-3588
ucuGGGuuuuGuAGcAuuuTsT
1297
AAAUGCuAcAAAA
1298
AD-9755





CCcAGATsT







3613-3631
AUAAAAACAAACAAACGUUTT
1299
AACGUUUGUUUGU
1300
AD-15412





UUUUAUTT







3617-3635
AAACAAACAAACGUUGUCCTT
1301
GGACAACGUUUGU
1302
AD-15211





UUGUUUTT







3618-3636
AACAAACAAACGUUGUCCUTT
1303
AGGACAACGUUUG
1304
AD-15300





UUUGUUTT





*Target: target in human PCSK9 gene, access. # NM_174936


U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a g: corresponding


2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro


ribonucleotide; where nucleotides are written in sequence, they are connected by


3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by


3′-O-5′-O phosphorodiester groups; unless denoted by prefix “P*“, oligonucleotides


are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides


bear 3′-OH on the 3′-most nucleotide.













TABLE 5







Sequences of modified dsRNA targeted to PCSK9












Sense strand sequence
SEQ ID
Antisense-strand
SEQ ID


Duplex #
(5′-3′)1
NO:
sequence (5′-3′)1
NO: 





AD-10792
GccuGGAGuuuAuucGGAATsT
1305
UUCCGAAuAAACUCcAGGCTsT
1306





AD-10793
GccuGGAGuuuAuucGGAATsT
1307
uUcCGAAuAAACUccAGGCTsT
1308





AD-10796
GccuGGAGuuuAuucGGAATsT
1309
UUCCGAAUAAACUCCAGGCTsT
1310





AD-12038
GccuGGAGuuuAuucGGAATsT
1311
UUCCGAAUAAACUCCAGGCTsT
1312





AD-12039
GccuGGAGuuuAuucGGAATsT
1313
UUCCGAAUAAACUCCAGGCTsT
1314





AD-12040
GccuGGAGuuuAuucGGAATsT
1315
UUcCGAAUAAACUCCAGGCTsT
1316





AD-12041
GccuGGAGuuuAuucGGAATsT
1317
UUCCGAAUAAACUCCAGGCTsT
1318





AD-12042
GCCUGGAGUUUAUUCGGAATsT
1319
UUCCGAAUAAACUCCAGGCTsT
1320





AD-12043
GCCUGGAGUUUAUUCGGAATsT
1321
UUCCGAAUAAACUCCAGGCTsT
1322





AD-12044
GCCUGGAGUUUAUUCGGAATsT
1323
UUCCGAAUAAACUCCAGGCTsT
1324





AD-12045
GCCUGGAGUUUAUUCGGAATsT
1325
UUCCGAAUAAACUCCAGGCTsT
1326





AD-12046
GccuGGAGuuuAuucGGAA
1327
UUCCGAAUAAACUCCAGGCscsu
1328





AD-12047
GccuGGAGuuuAuucGGAAA
1329
UUUCCGAAUAAACUCCAGGCscsu
1330





AD-12048
GccuGGAGuuuAuuGGAAAA
1331
UUUUCCGAAUAAACUCCAGGCscsu
1332





AD-12049
GccuGGAGuuuAuucGGAAAAG
1333
CUUUUCCGAAUAAACUCCAGGCscsu
1334





AD-12050
GccuGGAGuuuAuucGGAATTab
1335
UUCCGAAUAAACUCCAGGCTTab
1336





AD-12051
GccuGGAGuuuAuucGGAAATTab
1337
UUUCCGAAUAAACUCCAGGCTTab
1338





AD-12052
GccuGGAGuuuAuucGGAAAATTab
1339
UUUUCCGAAUAAACUCCAGGCTTab
1340





AD-12053
GccuGGAGuuuAuucGGAAAAGTTab
1341
CUUUUCCGAAUAAACUCCAGGCTTab
1342





AD-12054
GCCUGGAGUUUAUUCGGAATsT
1343
UUCCGAAUAAACUCCAGGCscsu
1344





AD-12055
GccuGGAGuuuAuucGGAATsT
1345
UUCCGAAUAAACUCCAGGCscsu
1346





AD-12056
GcCuGgAgUuUaUuCgGaA
1347
UUCCGAAUAAACUCCAGGCTTab
1348





AD-12057
GcCuGgAgUuUaUuCgGaA
1349
UUCCGAAUAAACUCCAGGCTsT
1350





AD-12058
GcCuGgAgUuUaUuCgGaA
1351
UUCCGAAUAAACUCCAGGCTsT
1352





AD-12059
GcCuGgAgUuUaUuCgGaA
1353
uUcCGAAuAAACUccAGGCTsT
1354





AD-12060
GcCuGgAgUuUaUuCgGaA
1355
UUCCGaaUAaaCUCCAggc
1356





AD-12061
GcCuGgnAgUuUaUuCgGaATsT
1357
UUCCGaaUAaaCUCCAggcTsT
1358





AD-12062
GcCuGgAgUuUaUuCgGaATTab
1359
UUCCGaaUAaaCUCCAggcTTab
1360





AD-12063
GcCuGgAgUuUaUuCgGaA
1361
UUCCGaaUAaaCUCCAggcscsu
1362





AD-12064
GcCuGgnAgUuUaUuCgGaATsT
1363
UUCCGAAuAAACUCcAGGCTsT
1364





AD-12065
GcCuGgAgUuUaUuCgGaATTab
1365
UUCCGAAuAAACUCcAGGCTTab
1366





AD-12066
GcCuGgAgUuUaUuCgGaA
1367
UUCCGAAuAAACUCcAGGCscsu
1368





AD-12067
GcCuGgnAgUuUaUuCgGaATsT
1369
UUCCGAAUAAACUCCAGGCTsT
1370





AD-12068
GcCuGgAgUuUaUuCgGaATTab
1371
UUCCGAAUAAACUCCAGGCTTab
1372





AD-12069
GcCuGgAgUuUaUuCgGaA
1373
UUCCGAAUAAACUCCAGGCScsu
1374





AD-12338
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf
1375
P*uUfcCfgAfaUfaAfaCf
1376





uCfcAfgGfc






AD-12339
GcCuGgAgUuUaUuCgGaA
1377
P*uUfcCfgAfaUfaAfaCf
1378





uCfcAfgGfc






AD-12340
GccuGGAGuuuAuucGGAA
1379
P*uUfcCfgAfaUfaAfaCf
1380





uCfcAfgGfc






AD-12341
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT
1381
P*uUfcCfgAfaUfaAfaCf
1382





uCfcAfgGfcTsT






AD-12342
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT
1383
UUCCGAAuAAACUCcAGGCTsT
1384





AD-12343
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT
1385
uUcCGAAuAAACUccAGGCTsT
1386





AD-12344
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT
1387
UUCCGAAUAAACUCCAGGCTsT
1388





AD-12345
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT
1389
UUCCGAAUAAACUCCAGGCScsu
1390





AD-12346
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT
1391
UUCCGaaUAaaCUCCAggcscsu
1392





AD-12347
GCCUGGAGUUUAUUCGGAATsT
1393
P*uUfcCfgAfaUfaAfaCf
1394





uCfcAfgGfcTsT






AD-12348
GccuGGAGuuuAuucGGAATsT
1395
P*uUfcCfgAfaUfaAfaCf
1396





uCfcAfgGfcTsT






AD-12349
GcCuGgnAgUuUaUuCgGaATsT
1397
P*uUfcCfgAfaUfaAfaCf
1398





uCfcAfgGfcTsT






AD-12350
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTTab
1399
P*uUfcCfgAfaUfaAfaCf
1400





uCfcAfgGfcTTab






AD-12351
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf
1401
P*uUfcCfgAfaUfaAfaCf
1402





uCfcAfgGfcsCfsu






AD-12352
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf
1403
UUCCGaaUAaaCUCCAggcs
1404





csu






AD-12354
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf
1405
UUCCGAAUAAACUCCAGGCs
1406





csu






AD-12355
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf
1407
UUCCGAAUAAACUCCAGGCT
1408





sT






AD-12356
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf
1409
uUcCGAAuAAACUccAGGCT
1410





sT






AD-12357
GmocCmouGmogAm02gUmouUmoaUmo
1411
UUCCGaaUAaaCUCCAggc
1412



uCmogGmoaA








AD-12358
GmocCmouGmogAm02gUmouUmoaUmo
1413
P*uUfcCfgAfaUfaAfaCf
1414



uCmogGmoaA

uCfcAfgGfc






AD-12359
GmocCmouGmogAm02gUmouUmoaUmo
1415
P*uUfcCfgAfaUfaAfaCf
1416



uCmogGmoaA

uCfcAfgGfcsCfsu






AD-12360
GmocCmouGmogAm02gUmouUmoaUmo
1417
UUCCGAAUAAACUCCAGGCS
1418



uCmogGmoaA

csu






AD-12361
GmocCmouGmogAm02gUmouUmoaUmo
1419
UUCCGAAuAAACUCcAGGCT
1420



uCmogGmoaA

sT






AD-12362
GmocCmouGmogAm02gUmouUmoaUmo
1421
uUcCGAAuAAACUccAGGCT
1422



uCmogGmoaA

sT






AD-12363
GmocCmouGmogAm02gUmouUmoaUmo
1423
UUCCGaaUAaaCUCCAggcs
1424



uCmogGmoaA

csu






AD-12364
GmocCmouGmogAmogUmouUmoaUmou
1425
UUCCGaaUAaaCUCCAggcT
1426



CmogGmoaATsT

sT






AD-12365
GmocCmouGmogAmogUmouUmoaUmou
1427
UUCCGAAuAAACUCcAGGCT
1428



CmogGmoaATsT

sT






AD-12366
GmocCmouGmogAmogUmouUmoaUmou
1429
UUCCGAAUAAACUCCAGGCT
1430



CmogGmoaATsT

sT






AD-12367
GmocmocmouGGAGmoumoumouAmoum
1431
UUCCGaaUAaaCUCCAggcT
1432



oumocGGAATsT

sT






AD-12368
GmocmocmouGGAGmoumoumouAmoum
1433
UUCCGAAuAAACUCcAGGCT
1434



oumocGGAATsT

sT






AD-12369
GmocmocmouGGAGmoumoumouAmoum
1435
UUCCGAAUAAACUCCAGGCT
1436



oumocGGAATsT

sT






AD-12370
GmocmocmouGGAGmoumoumouAmoum
1437
P*UfUfCfCfGAAUfAAACf
1438



oumocGGAATsT

UfCfCfAGGCfTsT






AD-12371
GmocmocmouGGAGmoumoumouAmoum
1439
P*UfUfCfCfGAAUfAAACf
1440



oumocGGAATsT

UfCfCfAGGCfsCfsUf






AD-12372
GmocmocmouGGAGmoumoumouAmoum
1441
P*uUfcCfgAfaUfaAfaCf
1442



oumocGGAATsT

uCfcAfgGfcsCfsu






AD-12373
GmocmocmouGGAGmoumoumouAmoum
1443
UUCCGAAUAAACUCCAGGCT
1444



oumocGGAATsT

sT






AD-12374
GCfCfUfGGAGUfUfUfAUfUfCfGGAA
1445
UfUfCfCfGAAUfAAACfUf
1446



TsT

CfCfAGGCfTsT






AD-12375
GCfCfUfGGAGUfUfUfAUfUfCfGGAA
1447
UUCCGAAUAAACUCCAGGCT
1448



TsT

sT






AD-12377
GCfCfUfGGAGUfUfUfAUfUfCfGGAA
1449
uUcCGAAuAAACUccAGGCT
1450



TsT

sT






AD-12378
GCfCfUfGGAGUfUfUfAUfUfCfGGAA
1451
UUCCGaaUAaaCUCCAggcs
1452



TsT

csu






AD-12379
GCfCfUfGGAGUfUfUfAUfUfCfGGAA
1453
UUCCGAAUAAACUCCAGGCS
1454



TsT

csu






AD-12380
GCfCfUfGGAGUfUfUfAUfUfCfGGAA
1455
P*uUfcCfgAfaUfaAfaCf
1456



TsT

uCfcAfgGfcsCfsu






AD-12381
GCfCfUfGGAGUfUfUfAUfUfCfGGAA
1457
P*uUfcCfgAfaUfaAfaCf
1458



TsT

uCfcAfgGfcTsT






AD-12382
GCfCfUfGGAGUfUfUfAUfUfCfGGAA
1459
P*UfUfCfCfGAAUfAAACf
1460



TsT

UfCfCfAGGCfTsT






AD-12383
GCCUGGAGUUUAUUCGGAATST
1461
P*UfUfCfCfGAAUfAAACf
1462





UfCfCfAGGCfTsT






AD-12384
GccuGGAGuuuAuucGGAATsT
1463
P*UfUfCfCfGAAUfAAACf
1464





UfCfCfAGGCfTsT






AD-12385
GcCuGgnAgUuUaUuCgGaATsT
1465
P*UfUfCfCfGAAUfAAACf
1466





UfCfCfAGGCfTsT






AD-12386
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf
1467
P*UfUfCfCfGAAUfAAACf
1468





UfCfCfAGGCfTsT






AD-12387
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA
1469
UfUfCfCfGAAUfAAACfUf
1470





CfCfAGGCfsCfsUf






AD-12388
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA
1471
P*uUfcCfgAfaUfaAfaCf
1472





uCfcAfgGfc






AD-12389
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA
1473
P*uUfcCfgAfaUfaAfaCf
1474





uCfcAfgGfcsCfsu






AD-12390
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA
1475
UUCCGAAUAAACUCCAGGCS
1476





csu






AD-12391
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA
1477
UUCCGaaUAaaCUCCAggc
1478





AD-12392
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA
1479
UUCCGAAUAAACUCCAGGCT
1480





sT






AD-12393
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA
1481
UUCCGAAuAAACUCcAGGCT
1482





sT






AD-12394
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA
1483
uUcCGAAuAAACUccAGGCT
1484





sT






AD-12395
GmocCmouGmogAmogUmouUmoaUmou
1485
P*UfUfCfCfGAAUfAAACf
1486



CmogGmoaATsT

UfCfCfAGGCfsCfsUf






AD-12396
GmocCmouGmogAm02gUmouUmoaUmo
1487
P*UfUfCfCfGAAUfAAACf
1488



uCmogGmoaA

UfCfCfAGGCfsCfsUf






AD-12397
GfcCfuGfgAfgUfuUfaUfuCfgGfaAf
1489
P*UfUfCfCfGAAUfAAACf
1490





UfCfCfAGGCfsCfsUf






AD-12398
GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTsT
1491
P*UfUfCfCfGAAUfAAACf
1492





UfCfCfAGGCfsCfsUf






AD-12399
GcCuGgnAgUuUaUuCgGaATsT
1493
P*UfUfCfCfGAAUfAAACf
1494





UfCfCfAGGCfsCfsUf






AD-12400
GCCUGGAGUUUAUUCGGAATST
1495
P*UfUfCfCfGAAUfAAACf
1496





UfCfCfAGGCfsCfsUf






AD-12401
GccuGGAGuuuAuucGGAATsT
1497
P*UfUfCfCfGAAUfAAACf
1498





UfCfCfAGGCfsCfsUf






AD-12402
GccuGGAGuuuAuucGGAA
1499
P*UfUfCfCfGAAUfAAACf
1500





UfCfCfAGGCfsCfsUf






AD-12403
GCfCfUfGGAGGUfUfUfAUfUfCfGGAA
1501
P*UfUfCfCfGAAUfAAACf
1502





UfCfCfAGGCfsCfsUf






AD-9314
GCCUGGAGUUUAUUCGGAATST
1503
UUCCGAAUAAACUCCAGGCT
1504





sT






AD-10794
ucAuAGGccuGGAGuuuAudTsdT
1525
AuAAACUCcAGGCCuAUGAd
1526





TsdT






AD-10795
ucAuAGGccuGGAGuuuAudTsdT
1527
AuAAACUccAGGcCuAuGAd
1528





TsdT






AD-10797
ucAuAGGccuGGAGuuuAudTsdT
1529
AUAAACUCCAGGCCUAUGAd
1530





TsdT 





U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with inteijected “s” are connected by 3′-O-5′-O phosphorothiodiester groups; unless denoted by prefix “P*”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide.













TABLE 6 







dsRNA targeted to PCSK9: mismatches and modifications










Duplex #
Strand
SEQ ID NO:
Sequence (5′ to 3′) 





AD-9680
S
1531
uucuAGAccuGuuuuGcuudTsdT



AS
1532
AAGcAAAAcAGGUCuAGAAdTsdT





AD-3267
S
1535
uucuAGAcCuGuuuuGcuuTsT



AS
1536
AAGcAAAAcAGGUCuAGAATsT





AD-3268
S
1537
uucuAGAccUGuuuuGcuuTsT



AS
1538
AAGcAAAAcAGGUCuAGAATsT





AD-3269
S
1539
uucuAGAcCUGuuuuGcuuTsT



AS
1540
AAGcAAAAcAGGUCuAGAATsT





AD-3270
S
1541
uucuAGAcY1uGuuuuGcuuTsT



AS
1542
AAGcAAAAcAGGUCuAGAATsT





AD-3271
S
1543
uucuAGAcY1UGuuuuGcuuTsT



AS
1544
AAGcAAAAcAGGUCuAGAATsT





AD-3272
S
1545
uucuAGAccYIGuuuuGcuuTsT



AS
1546
AAGcAAAAcAGGUCuAGAATsT





AD-3273
S
1547
uucuAGAcCY1GuuuuGcuuTsT



AS
1548
AAGcAAAAcAGGUCuAGAATsT





AD-3274
S
1549
uucuAGAccuY1uuuuGcuuTsT



AS
1550
AAGcAAAAcAGGUCuAGAATsT





AD-3275
S
1551
uucuAGAcCUY1uuuuGcuuTsT



AS
1552
AAGcAAAAcAGGUCuAGAATsT





AD-14676
S
1553
UfuCfuAfgAfcCfuGfuUfuUfgCfuUfTsT



AS
1554
P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT





AD-3276
S
1555
UfuCfuAfgAfcCuGfuUfuUfgCfuUfTsT



AS
1556
P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT





AD-3277
S
1557
UfuCfuAfgAfcCfUGfuUfuUfgCfuUfTsT



AS
1558
P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT





AD-3278
S
1559
UfuCfuAfgAfcCUGfuUfuUfgCfuUfTsT



AS
1560
P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT





AD-3279
S
1561
UfuCfuAfgAfcY1uGfuUfuUfgCfuUfTsT



AS
1562
P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT





AD-3280
S
1563
UfuCfuAfgAfcY1UGfuUfuUfgCfuUfTsT



AS
1564
P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT





AD-3281
S
1565
UfuCfuAfgAfcCfY1GfuUfuUfgCfuUfTsT



AS
1566
P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT





AD-3282
S
1567
UfuCfuAfgAfcCY1GfuUfuUfgCfuUfTsT



AS
1568
P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT





AD-3283
S
1569
UfuCfuAfgAfcCfuY1uUfuUfgCfuUfTsT



AS
1570
P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT





AD-3284
S
1571
UfuCfuAfgAfcCUY1uUfuUfgCfuUfTsT



AS
1572
P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT





AD-10792
S
459
GccuGGAGuuuAuucGGAATsT



AS
460
UUCCGAAuAAACUCcAGGCTsT





AD-3254
S
1573
GccuGGAGuYIuAuucGGAATsT



AS
1574
UUCCGAAuAAACUCcAGGCTsT





AD-3255
S
1575
GccuGGAGUY1uAuucGGAATsT



AS
1576
UUCCGAAuAAACUCcAGGCTsT 





Strand: S/Sense; AS/Antisense; U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af. Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; Y1 corresponds to DFT difluorotoluyl ribo(or deoxyribo)nucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by 3′-O-5 -O phosphorothiodiester groups; unless denoted by prefix “P*”, oligonucleotides are devoid of a 5′-phospltate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide













TABLE 7 







Sequences of unmodified siRNA flanking AD-9680















SEQ


Duplex #
Strand
Sequence (5′ to 3′)
*Target
ID NO:





AD-22169-b1
sense
CAGCCAACUUUUCUAGACCdTsdT
3520
1577



antis
GGUCUAGAAAAGUUGGCUGdTsdT
3520
1578





AD-22170-b1
sense
AGCCAACUUUUCUAGACCUdTsdT
3521
1579



antis
AGGUCUAGAAAAGUUGGCUdTsdT
3521
1580





AD-22171-b1
sense
GCCAACUUUUCUAGACCUGdTsdT
3522
1581



antis
CAGGUCUAGAAAAGUUGGCdTsdT
3522
1582





AD-22172-b1
sense
CCAACUUUUCUAGACCUGUdTsdT
3523
1583



antis
ACAGGUCUAGAAAAGUUGGdTsdT
3523
1584





AD-22173-b1
sense
CAACUUUUCUAGACCUGUUdTsdT
3524
1585



antis
AACAGGUCUAGAAAAGUUGdTsdT
3524
1586





AD-22174-b1
sense
AACUUUUCUAGACCUGUUUdTsdT
3525
1587



antis
AAACAGGUCUAGAAAAGUUdTsdT
3525
1588





AD-22175-b1
sense
ACUUUUCUAGACCUGUUUUdTsdT
3526
1589



antis
AAAACAGGUCUAGAAAAGUdTsdT
3526
1590





AD-22176-b1
sense
CUUUUCUAGACCUGUUUUGdTsdT
3527
1591



antis
CAAAACAGGUCUAGAAAAGdTsdT
3527
1592





AD-22177-b1
sense
UUUUCUAGACCUGUUUUGCdTsdT
3528
1593



antis
GCAAAACAGGUCUAGAAAAdTsdT
3528
1594





AD-22178-b1
sense
UUUCUAGACCUGUUUUGCUdTsdT
3529
1595



antis
AGCAAAACAGGUCUAGAAAdTsdT
3529
1596





AD-22179-b1
sense
UCUAGACCUGUUUUGCUUUdTsdT
3531
1597



antis
AAAGCAAAACAGGUCUAGAdTsdT
3531
1598





AD-22180-b1
sense
CUAGACCUGUUUUGCUUUUdTsdT
3532
1599



antis
AAAAGCAAAACAGGUCUAGdTsdT
3532
1600





AD-22181-b1
sense
UAGACCUGUUUUGCUUUUGdTsdT
3533
1601



antis
CAAAAGCAAAACAGGUCUAdTsdT
3533
1602





AD-22182-b1
sense
AGACCUGUUUUGCUUUUGUdTsdT
3534
1603



antis
ACAAAAGCAAAACAGGUCUdTsdT
3534
1604





AD-22183-b1
sense
GACCUGUUUUGCUUUUGUAdTsdT
3535
1605



antis
UACAAAAGCAAAACAGGUCdTsdT
3535
1606





AD-22184-b1
sense
ACCUGUUUUGCUUUUGUAAdTsdT
3536
1607



antis
UUACAAAAGCAAAACAGGUdTsdT
3536
1608





AD-22185-b1
sense
CCUGUUUUGCUUUUGUAACdTsdT
3537
1609



antis
GUUACAAAAGCAAAACAGGdTsdT
3537
1610





AD-22186-b1
sense
CUGUUUUGCUUUUGUAACUdTsdT
3538
1611



antis
AGUUACAAAAGCAAAACAGdTsdT
3538
1612





AD-22187-b1
sense
UGUUUUGCUUUUGUAACUUdTsdT
3539
1613



antis
AAGUUACAAAAGCAAAACAdTsdT
3539
1614





AD-22188-b1
sense
GUUUUGCUUUUGUAACUUGdTsdT
3540
1615



antis
CAAGUUACAAAAGCAAAACdTsdT
3540
1616





AD-22189-b1
sense
UUUUGCUUUUGUAACUUGAdTsdT
3541
1617



antis
UCAAGUUACAAAAGCAAAAdTsdT
3541
1618





AD-22190-b1
sense
UUUGCUUUUGUAACUUGAAdTsdT
3542
1619



antis
UUCAAGUUACAAAAGCAAAdTsdT
3542
1620





AD-22191-b1
sense
UUGCUUUUGUAACUUGAAGdTsdT
3543
1621



antis
CUUCAAGUUACAAAAGCAAdTsdT
3543
1622





AD-22192-b1
sense
UGCUUUUGUAACUUGAAGAdTsdT
3544
1623



antis
UCUUCAAGUUACAAAAGCAdTsdT
3544
1624





AD-22193-b1
sense
GCUUUUGUAACUUGAAGAUdTsdT
3545
1625



antis
AUCUUCAAGUUACAAAAGCdTsdT
3545
1626





AD-22194-b1
sense
CUUUUGUAACUUGAAGAUAdTsdT
3546
1627



antis
UAUCUUCAAGUUACAAAAGdTsdT
3546
1628





AD-22195-b1
sense
UUUUGUAACUUGAAGAUAUdTsdT
3547
1629



antis
AUAUCUUCAAGUUACAAAAdTsdT
3547
1630





AD-22196-b1
sense
UUUGUAACUUGAAGAUAUUdTsdT
3548
1631



antis
AAUAUCUUCAAGUUACAAAdTsdT
3548
1632





AD-22197-b1
sense
UUGUAACUUGAAGAUAUUUdTsdT
3549
1633



antis
AAAUAUCUUCAAGUUACAAdTsdT
3549
1634





AD-22198-b1
sense
UGUAACUUGAAGAUAUUUAdTsdT
3550
1635



antis
UAAAUAUCUUCAAGUUACAdTsdT
3550
1636





AD-22199-b1
sense
GUAACUUGAAGAUAUUUAUdTsdT
3551
1637



antis
AUAAAUAUCUUCAAGUUACdTsdT
3551
1638





AD-22200-b1
sense
UAACUUGAAGAUAUUUAUUdTsdT
3552
1639



antis
AAUAAAUAUCUUCAAGUUAdTsdT
3552
1640





AD-22201-b1
sense
AACUUGAAGAUAUUUAUUCdTsdT
3553
1641



antis
GAAUAAAUAUCUUCAAGUUdTsdT
3553
1642





AD-22202-b1
sense
ACUUGAAGAUAUUUAUUCUdTsdT
3554
1643



antis
AGAAUAAAUAUCUUCAAGUdTsdT
3554
1644





AD-22203-b1
sense
CUUGAAGAUAUUUAUUCUGdTsdT
3555
1645



antis
CAGAAUAAAUAUCUUCAAGdTsdT
3555
1646





AD-22204-b1
sense
UUGAAGAUAUUUAUUCUGGdTsdT
3556
1647



antis
CCAGAAUAAAUAUCUUCAAdTsdT
3556
1648





AD-22205-b1
sense
UGAAGAUAUUUAUUCUGGGdTsdT
3557
1649



antis
CCCAGAAUAAAUAUCUUCAdTsdT
3557
1650





AD-22206-b1
sense
GAAGAUAUUUAUUCUGGGUdTsdT
3558
1651



antis
ACCCAGAAUAAAUAUCUUCdTsdT
3558
1652 





*Target: target in human PCSK9 gene, access, # NM_174936


U, C, A, G: corresponding ribonucleotide; dT: deoxythymidine; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups: nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups.













TABLE 8 







Sequences of modified siRNA flanking AD-9680











Duplex #
Strand
Sequence (5′ to 3′)
*Target
SEQ ID NO: 





AD-22098-b1
sense
cAGccAAcuuuucuAGAccdTsdT
3520
1653



antis
GGUCuAGAAAAGUUGGCUGdTsdT
3520
1654





AD-22099-b1
sense
AGccAAcuuuucuAGAccudTsdT
3521
1655



antis
AGGUCuAGAAAAGUUGGCUdTsdT
3521
1656





AD-22100-b1
sense
GccAAcuuuucuAGAccuGdTsdT
3522
1657



antis
cAGGUCuAGAAAAGUUGGCdTsdT
3522
1658





AD-22101-b1
sense
ccAAcuuuucuAGAccuGudTsdT
3523
1659



antis
AcAGGUCuAGAAAAGUUGGdTsdT
3523
1660





AD-22102-b1
sense
cAAcuuuucuAGAccuGuudTsdT
3524
1661



antis
AAcAGGUCuAGAAAAGUUGdTsdT
3524
1662





AD-22103-b1
sense
AAcuuuucuAGAccuGuuudTsdT
3525
1663



antis
AAAcAGGUCuAGAAAAGUUdTsdT
3525
1664





AD-22104-b1
sense
AcuuuucuAGAccuGuuuudTsdT
3526
1665



antis
AAAAcAGGUCuAGAAAAGUdTsdT
3526
1666





AD-22105-b1
sense
cuuuucuAGAccuGuuuuGdTsdT
3527
1667



antis
cAAAAcAGGUCuAGAAAAGdTsdT
3527
1668





AD-22106-b1
sense
uuuucuAGAccuGuuuuGcdTsdT
3528
1669



antis
GcAAAAcAGGUCuAGAAAAdTsdT
3528
1670





AD-22107-b1
sense
uuucuAGAccuGuuuuGcudTsdT
3529
1671



antis
AGcAAAAcAGGUCuAGAAAdTsdT
3529
1672





AD-22108-b1
sense
ucuAGAccuGuuuuGcuuudTsdT
3531
1673



antis
AAAGcAAAAcAGGUCuAGAdTsdT
3531
1674





AD-22109-b1
sense
cuAGAccuGuuuuGcuuuudTsdT
3532
1675



antis
AAAAGcAAAAcAGGUCuAGdTsdT
3532
1676





AD-22110-b1
sense
uAGAccuGuuuuGcuuuuGdTsdT
3533
1677



antis
cAAAAGcAAAAcAGGUCuAdTsdT
3533
1678





AD-22111-b1
sense
AGAccuGuuuuGcuuuuGudTsdT
3534
1679



antis
AcAAAAGcAAAAcAGGUCUdTsdT
3534
1680





AD-22112-b1
sense
GAccuGuuuuGcuuuuGuAdTsdT
3535
1681



antis
uAcAAAAGcAAAAcAGGUCdTsdT
3535
1682





AD-22113-b1
sense
AccuGuuuuGcuuuuGuAAdTsdT
3536
1683



antis
UuAcAAAAGcAAAAcAGGUdTsdT
3536
1684





AD-22114-b1
sense
ccuGuuuuGcuuuuGuAAcdTsdT
3537
1685



antis
GUuAcAAAAGcAAAAcAGGdTsdT
3537
1686





AD-22115-b1
sense
cuGuuuuGcuuuuGuAAcudTsdT
3538
1687



antis
AGUuAcAAAAGcAAAAcAGdTsdT
3538
1688



sense
uGuuuuGcuuuuGuAAcuudTsdT
3539
1689



antis
AAGUuAcAAAAGcAAAAcAdTsdT
3539
1690





AD-22116-b1
sense
GuuuuGcuuuuGuAAcuuGdTsdT
3540
1691



antis
cAAGUuAcAAAAGcAAAACdTsdT
3540
1692





AD-22117-b1
sense
uuuuGcuuuuGuAAcuuGAdTsdT
3541
1693



antis
UcAAGUuAcAAAAGcAAAAdTsdT
3541
1694





AD-22118-b1
sense
uuuGcuuuuGuAAcuuGAAdTsdT
3542
1695



antis
UUcAAGUuAcAAAAGcAAAdTsdT
3542
1696





AD-22119-b1
sense
uuGcuuuuGuAAcuuGAAGdTsdT
3543
1697



antis
CUUcAAGUuAcAAAAGcAAdTsdT
3543
1698





AD-22120-b1
sense
uGcuuuuGuAAcuuGAAGAdTsdT
3544
1699



antis
UCUUcAAGUuAcAAAAGcAdTsdT
3544
1700





AD-22121-b1
sense
GcuuuuGuAAcuuGAAGAudTsdT
3545
1701



antis
AUCUUcAAGUuAcAAAAGCdTsdT
3545
1702





AD-22122-b1
sense
cuuuuGuAAcuuGAAGAuAdTsdT
3546
1703



antis
uAUCUUcAAGUuAcAAAAGdTsdT
3546
1704





AD-22123-b1
sense
uuuuGuAAcuuGAAGAuAudTsdT
3547
1705



antis
AuAUCUUcAAGUuAcAAAAdTsdT
3547
1706





AD-22124-b1
sense
uuuGuAAcuuGAAGAuAuudTsdT
3548
1707



antis
AAuAUCUUcAAGUuAcAAAdTsdT
3548
1708





AD-22125-b1
sense
uuGuAAcuuGAAGAuAuuudTsdT
3549
1709



antis
AAAuAUCUUcAAGUuAcAAdTsdT
3549
1710





AD-22126-b1
sense
uGuAAcuuGAAGAuAuuuAdTsdT
3550
1711



antis
uAAAuAUCUUcAAGUuAcAdTsdT
3550
1712





AD-22127-b1
sense
GuAAcuuGAAGAuAuuuAudTsdT
3551
1713



antis
AuAAAuAUCUUcAAGUuACdTsdT
3551
1714





AD-22128-b1
sense
uAAcuuGAAGAuAuuuAuudTsdT
3552
1715



antis
AAuAAAuAUCUUcAAGUuAdTsdT
3552
1716





AD-22129-b1
sense
AAcuuGAAGAuAuuuAuucdTsdT
3553
1717



antis
GAAuAAAuAUCUUcAAGUUdTsdT
3553
1718





AD-22130-b1
sense
AcuuGAAGAuAuuuAuucudTsdT
3554
1719



antis
AGAAuAAAuAUCUUcAAGUdTsdT
3554
1720





AD-22131-b1
sense
cuuGAAGAuAuuuAuucuGdTsdT
3555
1721



antis
cAGAAuAAAuAUCUUcAAGdTsdT
3555
1722





AD-22132-b1
sense
uuGAAGAuAuuuAuucuGGdTsdT
3556
1723



antis
CcAGAAuAAAuAUCUUcAAdTsdT
3556
1724





AD-22133-b1
sense
uGAAGAuAuuuAuucuGGGdTsdT
3557
1725



antis
CCcAGAAuAAAuAUCUUcAdTsdT
3557
1726





AD-22134-b1
sense
GAAGAuAuuuAuucuGGGudTsdT
3558
1727



antis
ACCcAGAAuAAAuAUCUUCdTsdT
3558
1728 





*Target: 5′ nutlcoetide of target sequence in human PCSK9 gene, access # NM_174936


U, C, A, G: corresponding ribonucleotide; dT: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; Y1 corresponds to DFT difluorotoluyl ribo(or deoxyribo) nucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups; unless denoted by prefix “P*”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide













TABLE 9







Sequences of XBP-1 dsRNAs


*Target refers to target gene and location of target sequence. NM_001004210


is the gene for rat XBP-1. XM_001103095 is the sequence for Macacamulatta


(rhesus monkey) XBP-1.












SEQ ID

SEQ ID



Target*
NO
sense (5′-3′)
NO
antisense (5′-3′)





NM_001004210
1729
CCCAGCUGAUUAGUGUCUA
1753
UAGACACUAAUCAGCUGGG


1128-1146









NM_001004210
1730
CCAGCUGAUUAGUGUCUAA
1754
UUAGACACUAAUCAGCUGG


1129-1147









NM_001004210
1731
CUCCCAGAGGUCUACCCAG
1755
CUGGGUAGACCUCUGGGAG


677-695









NM_001004210
1732
GAUCACCCUGAAUUCAUUG
1756
CAAUGAAUUCAGGGUGAUC


893-911









NM_001004210
1733
UCACCCUGAAUUCAUUGUC
1757
GACAAUGAAUUCAGGGUGA


895-913









NM_001004210
1734
CCCCAGCUGAUUAGUGUCU
1758
AGACACUAAUCAGCUGGGG


1127-1145









NM_001004210
1735
AUCACCCUGAAUUCAUUGU
1759
ACAAUGAAUUCAGGGUGAU


894-912









NM_001004210
1736
CAUUUAUUUAAAACUACCC
1760
GGGUAGUUUUAAAUAAAUG


1760-1778









NM_001004210
1737
ACUGAAAAACAGAGUAGCA
1761
UGCUACUCUGUUUUUCAGU


215-233









NM_001004210
1738
CCAUUUAUUUAAAACUACC
1762
GGUAGUUUUAAAUAAAUGG


1759-1777









NM_001004210
1739
UUGAGAACCAGGAGUUAAG
1763
CUUAACUCCUGGUUCUCAA


367-385









NM_001004210
1740
CACCCUGAAUUCAUUGUCU
1764
AGACAAUGAAUUCAGGGUG


896-914









NM_001004210
1741
AACUGAAAAACAGAGUAGC
1765
GCUACUCUGUUUUUCAGUU


214-232









NM_001004210
1742
CUGAAAAACAGAGUAGCAG
1766
CUGCUACUCUGUUUUUCAG


216-234









XM_001103095
1743
AGAAAAUCAGCUUUUACGA
1767
UCGUAAAAGCUGAUUUUCU


387-405









XM_001103095
1744
UCCCCAGCUGAUUAGUGUC
1768
GACACUAAUCAGCUGGGGA


1151-1169









XM_001103095
1745
UACUUAUUAUGUAAGGGUC
1769
GACCCUUACAUAAUAAGUA


1466-1484









XM_001103095
1746
UAUCUUAAAAGGGUGGUAG
1770
CUACCACCCUUUUAAGAUA


1435-1453









XM_001103095
1747
CCAUGGAUUCUGGCGGUAU
1771
AUACCGCCAGAAUCCAUGG


577-595









XM_001103095
1748
UUAAUGAACUAAUUCGUUU
1772
AAACGANUUAGUUCAUUAA


790-808









XM_001103095
1749
AGGGUCAUUAGACAAAUGU
1773
ACAUUUGUCUAAUGACCCU


1479-1497









XM_001103095
1750
UGAACUAAUUCGUUUUGAC
1774
GUCAAAACGAAUUAGUUCA


794-812









XM_001103095
1751
UUCCCCAGCUGAUUAGUGU
1775
ACACUAAUCAGCUGGGGAA


1150-1168









XM_001103095
1752
UAUGUAAGGGUCAUUAGAC
1776
GUCUAAUGACCCUUACAUA


1473-1491
















TABLE 10







Target gene name and target sequence location for


dsRNA targeting XBP-1


*Target refers to target gene and location of


target sequence. NM_001004210 is the gene for rat


XBP-1. XM_001103095 is the sequence for Macaca



mulatta (rhesus monkey) XBP-1.









Duplex 
Target gene and location


#
of target sequence





D18027
NM_001004210_1128-1146





D18028
NM_001004210_1129-1147





D18029
NM_001004210_677-695





D18030
NM_001004210_893-911





D18031
NM_001004210_895-913





D18032
NM_001004210_1127-1145





D18033
NM_001004210_894-912





D18034
NM_001004210_1760-1778





AD18035
NM_001004210_215-233





AD18036
NM_001004210_1759-1777





AD18037
NM_001004210_367-385





AD18038
NM_001004210_896-914





AD18039
NM_001004210_214-232





AD18040
NM_001004210_216-234





AD18041
XM_001103095_387-405





AD18042
XM_001103095_1151-1169





AD18043
XM_001103095_1466-1484





AD18044
XM_001103095_1435-1453





AD18045
XM_001103095_577-595





AD18046
XM_001103095_790-808





AD18047
XM_001103095_1479-1497





AD18048
XM_001103095_794-812





AD18049
XM_001103095_1150-1168





AD18050
XM_001103095_1473-1491
















TABLE 11







Sequences of dsRNA targeting XBP-1, with Endolight chemistry modifications


U, C, A, G: corresponding ribonucleotide; dT: deoxythymidine; u, c, a, g:


corresponding 2′-O-methyl ribonucleotide; where nucleotides are written in


sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with


interjected ″s″ are connected by 3′-O-5′-O phosphorothiodiester groups.












SEQ

SEQ




ID

ID



Duplex #
NO
Sense (5′-3′)
NO
Antisense (5′-3′)





AD18027
4166
cccAGcuGAuuAGuGucuAdTsdT
1800
uAGAcACuAAUcAGCUGGGdTsdT





AD18028
1777
ccAGcuGAuuAGuGucuAAdTsdT
1801
UuAGAcACuAAUcAGCUGGdTsdT





AD18029
1778
cucccAGAGGucuAcccAGdTsdT
1802
CUGGGuAGACCUCUGGGAGdTsdT





AD18030
1779
GAucAcccuGAAuucAuuGdTsdT
1803
cAAUGAAUUcAGGGUGAUCdTsdT





AD18031
1780
ucAcccuGAAuucAuuGucdTsdT
1804
GAcAAUGAAUUcAGGGUGAdTsdT





AD18032
1781
ccccAGcuGAuuAGuGucudTsdT
1805
AGAcACuAAUcAGCUGGGGdTsdT





AD18033
1782
AucAcccuGAAuucAuuGudTsdT
1806
AcAAUGAAUUcAGGGUGAUdTsdT





AD18034
1783
cAuuuAuuuAAAAcuAcccdTsdT
1807
GGGuAGUUUuAAAuAAAUGdTsdT





AD18035
1784
AcuGAAAAAcAGAGuAGcAdTsdT
1808
UGCuACUCUGUUUUUcAGUdTsdT





AD18036
1785
ccAuuuAuuuAAAAcuAccdTsdT
1809
GGuAGUUUuAAAuAAAUGGdTsdT





AD18037
1786
uuGAGAAccAGGAGuuAAGdTsdT
1810
CUuAACUCCUGGUUCUcAAdTsdT





AD18038
1787
cAcccuGAAuucAuuGucudTsdT
1811
AGAcAAUGAAUUcAGGGUGdTsdT





AD18039
1788
AAcuGAAAAAcAGAGuAGcdTsdT
1812
GCuACUCUGUUUUUcAGUUdTsdT





AD18040
1789
cuGAAAAAcAGAGuAGcAGdTsdT
1813
CUGCuACUCUGUUUUUcAGdTsdT





AD18041
1790
AGAAAAucAGcuuuuAcGAdTsdT
1814
UCGuAAAAGCUGAUUUUCUdTsdT





AD18042
1791
uccccAGcuGAuuAGuGucdTsdT
1815
GAcACuAAUcAGCUGGGGAdTsdT





AD18043
1792
uAcuuAuuAuGuAAGGGucdTsdT
1816
GACCCUuAcAuAAuAAGuAdTsdT





AD18044
1793
uAucuuAAAAGGGuGGuAGdTsdT
1817
CuACcACCCUUUuAAGAuAdTsdT





AD18045
1794
ccAuGGAuucuGGcGGuAudTsdT
1818
AuACCGCcAGAAUCcAUGGdTsdT





AD18046
1795
uuAAuGAAcuAAuucGuuudTsdT
1819
AAACGAAUuAGUUcAUuAAdTsdT





AD18047
1796
AGGGucAuuAGAcAAAuGudTsdT
1820
AcAUUUGUCuAAUGACCCUdTsdT





AD18048
1797
uGAAcuAAuucGuuuuGAcdTsdT
1821
GUcAAAACGAAUuAGUUcAdTsdT





AD18049
1798
uuccccAGcuGAuuAGuGudTsdT
1822
AcACuAAUcAGCUGGGGAAdTsdT





AD18050
1799
uAuGuAAGGGucAuuAGAcdTsdT
1823
GUCuAAUGACCCUuAcAuAdTsdT
















TABLE 12







Sequences of dsRNA targeting both human and rhesus monkey XBP-1.


*Target refers location of target sequence in NM_005080 (human XBP-1 mRNA).


Sense and antisense sequences are described with optional dinucleotide (NN)


overhangs.













SEQ ID

SEQ


Target*
sense (5′-3′)
NO
antisense (5′-3′)
ID NO





 100-118
CUGCUUCUGUCGGGGCAGCNN
1824
GCUGCCCCGACAGAAGCAGNN
28





1011-1029
GAGCUGGGUAUCUCAAAUCNN
1825
GAUUUGAGAUACCCAGCUCNN
28





 101-119
UGCUUCUGUCGGGGCAGCCNN
1826
GGCUGCCCCGACAGAAGCANN
28





1012-1030
AGCUGGGUAUCUCAAAUCUNN
1827
AGAUUUGAGAUACCCAGCUNN
28





1013-1031
GCUGGGUAUCUCAAAUCUGNN
1828
CAGAUUUGAGAUACCCAGCNN
28





1014-1032
CUGGGUAUCUCAAAUCUGCNN
1829
GCAGAUUUGAGAUACCCAGNN
28





1015-1033
UGGGUAUCUCAAAUCUGCUNN
1830
AGCAGAUUUGAGAUACCCANN
28





1016-1034
GGGUAUCUCAAAUCUGCUUNN
1831
AAGCAGAUUUGAGAUACCCNN
28





1017-1035
GGUAUCUCAAAUCUGCUUUNN
1832
AAAGCAGAUUUGAGAUACCNN
28





1018-1036
GUAUCUCAAAUCUGCUUUCNN
1833
GAAAGCAGAUUUGAGAUACNN
28





1019-1037
UAUCUCAAAUCUGCUUUCANN
1834
UGAAAGCAGAUUUGAGAUANN
28





1020-1038
AUCUCAAAUCUGCUUUCAUNN
1835
AUGAAAGCAGAUUUGAGAUNN
28





1021-1039
UCUCAAAUCUGCUUUCAUCNN
1836
GAUGAAAGCAGAUUUGAGANN
28





 102-120
GCUUCUGUCGGGGCAGCCCNN
1837
GGGCUGCCCCGACAGAAGCNN
28





1022-1040
CUCAAAUCUGCUUUCAUCCNN
1838
GGAUGAAAGCAGAUUUGAGNN
28





1023-1041
UCAAAUCUGCUUUCAUCCANN
1839
UGGAUGAAAGCAGAUUUGANN
28





1024-1042
CAAAUCUGCUUUCAUCCAGNN
1840
CUGGAUGAAAGCAGAUUUGNN
28





1025-1043
AAAUCUGCUUUCAUCCAGCNN
1841
GCUGGAUGAAAGCAGAUUUNN
29





1026-1044
AAUCUGCUUUCAUCCAGCCNN
1842
GGCUGGAUGAAAGCAGAUUNN
29





1027-1045
AUCUGCUUUCAUCCAGCCANN
1843
UGGCUGGAUGAAAGCAGAUNN
29





1028-1046
UCUGCUUUCAUCCAGCCACNN
1844
GUGGCUGGAUGAAAGCAGANN
29





1029-1047
CUGCUUUCAUCCAGCCACUNN
1845
AGUGGCUGGAUGAAAGCAGNN
29





1030-1048
UGCUUUCAUCCAGCCACUGNN
1846
CAGUGGCUGGAUGAAAGCANN
29





1031-1049
GCUUUCAUCCAGCCACUGCNN
1847
GCAGUGGCUGGAUGAAAGCNN
29





 103-121
CUUCUGUCGGGGCAGCCCGNN
1848
CGGGCUGCCCCGACAGAAGNN
29





1032-1050
CUUUCAUCCAGCCACUGCCNN
1849
GGCAGUGGCUGGAUGAAAGNN
29





1033-1051
UUUCAUCCAGCCACUGCCCNN
1850
GGGCAGUGGCUGGAUGAAANN
29





 104-122
UUCUGUCGGGGCAGCCCGCNN
1851
GCGGGCUGCCCCGACAGAANN
29





 105-123
UCUGUCGGGGCAGCCCGCCNN
1852
GGCGGGCUGCCCCGACAGANN
29





1056-1074
CCAUCUUCCUGCCUACUGGNN
1853
CCAGUAGGCAGGAAGAUGGNN
29





1057-1075
CAUCUUCCUGCCUACUGGANN
1854
UCCAGUAGGCAGGAAGAUGNN
29





1058-1076
AUCUUCCUGCCUACUGGAUNN
1855
AUCCAGUAGGCAGGAAGAUNN
29





1059-1077
UCUUCCUGCCUACUGGAUGNN
1856
CAUCCAGUAGGCAGGAAGANN
29





1060-1078
CUUCCUGCCUACUGGAUGCNN
1857
GCAUCCAGUAGGCAGGAAGNN
29





1061-1079
UUCCUGCCUACUGGAUGCUNN
1858
AGCAUCCAGUAGGCAGGAANN
29





 106-124
CUGUCGGGGCAGCCCGCCUNN
1859
AGGCGGGCUGCCCCGACAGNN
29





1062-1080
UCCUGCCUACUGGAUGCUUNN
1860
AAGCAUCCAGUAGGCAGGANN
29





1063-1081
CCUGCCUACUGGAUGCUUANN
1861
UAAGCAUCCAGUAGGCAGGNN
29





1064-1082
CUGCCUACUGGAUGCUUACNN
1862
GUAAGCAUCCAGUAGGCAGNN
29





1065-1083
UGCCUACUGGAUGCUUACANN
1863
UGUAAGCAUCCAGUAGGCANN
29





1066-1084
GCCUACUGGAUGCUUACAGNN
1864
CUGUAAGCAUCCAGUAGGCNN
29





1067-1085
CCUACUGGAUGCUUACAGUNN
1865
ACUGUAAGCAUCCAGUAGGNN
29





1068-1086
CUACUGGAUGCUUACAGUGNN
1866
CACUGUAAGCAUCCAGUAGNN
29





1069-1087
UACUGGAUGCUUACAGUGANN
1867
UCACUGUAAGCAUCCAGUANN
29





1070-1088
ACUGGAUGCUUACAGUGACNN
1868
GUCACUGUAAGCAUCCAGUNN
29





1071-1089
CUGGAUGCUUACAGUGACUNN
1869
AGUCACUGUAAGCAUCCAGNN
29





 107-125
UGUCGGGGCAGCCCGCCUCNN
1870
GAGGCGGGCUGCCCCGACANN
29





1072-1090
UGGAUGCUUACAGUGACUGNN
1871
CAGUCACUGUAAGCAUCCANN
29





1073-1091
GGAUGCUUACAGUGACUGUNN
1872
ACAGUCACUGUAAGCAUCCNN
29





1074-1092
GAUGCUUACAGUGACUGUGNN
1873
CACAGUCACUGUAAGCAUCNN
29





1075-1093
AUGCUUACAGUGACUGUGGNN
1874
CCACAGUCACUGUAAGCAUNN
29





1076-1094
UGCUUACAGUGACUGUGGANN
1875
UCCACAGUCACUGUAAGCANN
29





1077-1095
GCUUACAGUGACUGUGGAUNN
1876
AUCCACAGUCACUGUAAGCNN
29





1078-1096
CUUACAGUGACUGUGGAUANN
1877
UAUCCACAGUCACUGUAAGNN
29





 108-126
GUCGGGGCAGCCCGCCUCCNN
1878
GGAGGCGGGCUGCCCCGACNN
29





 109-127
UCGGGGCAGCCCGCCUCCGNN
1879
CGGAGGCGGGCUGCCCCGANN
29





 110-128
CGGGGCAGCCCGCCUCCGCNN
1880
GCGGAGGCGGGCUGCCCCGNN
29





 111-129
GGGGCAGCCCGCCUCCGCCNN
1881
GGCGGAGGCGGGCUGCCCCNN
29





1116-1134
UUCAGUGACAUGUCCUCUCNN
1882
GAGAGGACAUGUCACUGAANN
29





 112-130
GGGCAGCCCGCCUCCGCCGNN
1883
CGGCGGAGGCGGGCUGCCCNN
29





 113-131
GGCAGCCCGCCUCCGCCGCNN
1884
GCGGCGGAGGCGGGCUGCCNN
29





1136-1154
GCUUGGUGUAAACCAUUCUNN
1885
AGAAUGGUUUACACCAAGCNN
29





1137-1155
CUUGGUGUAAACCAUUCUUNN
1886
AAGAAUGGUUUACACCAAGNN
29





1138-1156
UUGGUGUAAACCAUUCUUGNN
1887
CAAGAAUGGUUUACACCAANN
29





1139-1157
UGGUGUAAACCAUUCUUGGNN
1888
CCAAGAAUGGUUUACACCANN
29





1140-1158
GGUGUAAACCAUUCUUGGGNN
1889
CCCAAGAAUGGUUUACACCNN
29





1141-1159
GUGUAAACCAUUCUUGGGANN
1890
UCCCAAGAAUGGUUUACACNN
29





 114-132
GCAGCCCGCCUCCGCCGCCNN
1891
GGCGGCGGAGGCGGGCUGCNN
29





1142-1160
UGUAAACCAUUCUUGGGAGNN
1892
CUCCCAAGAAUGGUUUACANN
29





1143-1161
GUAAACCAUUCUUGGGAGGNN
1893
CCUCCCAAGAAUGGUUUACNN
29





1144-1162
UAAACCAUUCUUGGGAGGANN
1894
UCCUCCCAAGAAUGGUUUANN
29





1145-1163
AAACCAUUCUUGGGAGGACNN
1895
GUCCUCCCAAGAAUGGUUUNN
29





1146-1164
AACCAUUCUUGGGAGGACANN
1896
UGUCCUCCCAAGAAUGGUUNN
29





1147-1165
ACCAUUCUUGGGAGGACACNN
1897
GUGUCCUCCCAAGAAUGGUNN
29





1148-1166
CCAUUCUUGGGAGGACACUNN
1898
AGUGUCCUCCCAAGAAUGGNN
29





1149-1167
CAUUCUUGGGAGGACACUUNN
1899
AAGUGUCCUCCCAAGAAUGNN
29





1150-1168
AUUCUUGGGAGGACACUUUNN
1900
AAAGUGUCCUCCCAAGAAUNN
29





1151-1169
UUCUUGGGAGGACACUUUUNN
1901
AAAAGUGUCCUCCCAAGAANN
29





 115-133
CAGCCCGCCUCCGCCGCCGNN
1902
CGGCGGCGGAGGCGGGCUGNN
29





1152-1170
UCUUGGGAGGACACUUUUGNN
1903
CAAAAGUGUCCUCCCAAGANN
29





1153-1171
CUUGGGAGGACACUUUUGCNN
1904
GCAAAAGUGUCCUCCCAAGNN
29





1154-1172
UUGGGAGGACACUUUUGCCNN
1905
GGCAAAAGUGUCCUCCCAANN
29





1155-1173
UGGGAGGACACUUUUGCCANN
1906
UGGCAAAAGUGUCCUCCCANN
29





1156-1174
GGGAGGACACUUUUGCCAANN
1907
UUGGCAAAAGUGUCCUCCCNN
29





1157-1175
GGAGGACACUUUUGCCAAUNN
1908
AUUGGCAAAAGUGUCCUCCNN
29





1158-1176
GAGGACACUUUUGCCAAUGNN
1909
CAUUGGCAAAAGUGUCCUCNN
29





1159-1177
AGGACACUUUUGCCAAUGANN
1910
UCAUUGGCAAAAGUGUCCUNN
29





1160-1178
GGACACUUUUGCCAAUGAANN
1911
UUCAUUGGCAAAAGUGUCCNN
29





1161-1179
GACACUUUUGCCAAUGAACNN
1912
GUUCAUUGGCAAAAGUGUCNN
29





 116-134
AGCCCGCCUCCGCCGCCGGNN
1913
CCGGCGGCGGAGGCGGGCUNN
29





1162-1180
ACACUUUUGCCAAUGAACUNN
1914
AGUUCAUUGGCAAAAGUGUNN
29





 117-135
GCCCGCCUCCGCCGCCGGANN
1915
UCCGGCGGCGGAGGCGGGCNN
29





 118-136
CCCGCCUCCGCCGCCGGAGNN
1916
CUCCGGCGGCGGAGGCGGGNN
29





1182-1200
UUUCCCCAGCUGAUUAGUGNN
1917
CACUAAUCAGCUGGGGAAANN
29





1183-1201
UUCCCCAGCUGAUUAGUGUNN
1918
ACACUAAUCAGCUGGGGAANN
29





1184-1202
UCCCCAGCUGAUUAGUGUCNN
1919
GACACUAAUCAGCUGGGGANN
29





1185-1203
CCCCAGCUGAUUAGUGUCUNN
1920
AGACACUAAUCAGCUGGGGNN
29





1186-1204
CCCAGCUGAUUAGUGUCUANN
1921
UAGACACUAAUCAGCUGGGNN
29





1187-1205
CCAGCUGAUUAGUGUCUAANN
1922
UUAGACACUAAUCAGCUGGNN
29





1188-1206
CAGCUGAUUAGUGUCUAAGNN
1923
CUUAGACACUAAUCAGCUGNN
29





1189-1207
AGCUGAUUAGUGUCUAAGGNN
1924
CCUUAGACACUAAUCAGCUNN
29





1190-1208
GCUGAUUAGUGUCUAAGGANN
1925
UCCUUAGACACUAAUCAGCNN
29





1191-1209
CUGAUUAGUGUCUAAGGAANN
1926
UUCCUUAGACACUAAUCAGNN
29





 119-137
CCGCCUCCGCCGCCGGAGCNN
1927
GCUCCGGCGGCGGAGGCGGNN
29





1192-1210
UGAUUAGUGUCUAAGGAAUNN
1928
AUUCCUUAGACACUAAUCANN
29





1193-1211
GAUUAGUGUCUAAGGAAUGNN
1929
CAUUCCUUAGACACUAAUCNN
29





1194-1212
AUUAGUGUCUAAGGAAUGANN
1930
UCAUUCCUUAGACACUAAUNN
29





1195-1213
UUAGUGUCUAAGGAAUGAUNN
1931
AUCAUUCCUUAGACACUAANN
29





1196-1214
UAGUGUCUAAGGAAUGAUCNN
1932
GAUCAUUCCUUAGACACUANN
29





1197-1215
AGUGUCUAAGGAAUGAUCCNN
1933
GGAUCAUUCCUUAGACACUNN
29





1198-1216
GUGUCUAAGGAAUGAUCCANN
1934
UGGAUCAUUCCUUAGACACNN
29





 120-138
CGCCUCCGCCGCCGGAGCCNN
1935
GGCUCCGGCGGCGGAGGCGNN
29





 121-139
GCCUCCGCCGCCGGAGCCCNN
1936
GGGCUCCGGCGGCGGAGGCNN
29





1218-1236
UACUGUUGCCCUUUUCCUUNN
1937
AAGGAAAAGGGCAACAGUANN
29





1219-1237
ACUGUUGCCCUUUUCCUUGNN
1938
CAAGGAAAAGGGCAACAGUNN
29





1220-1238
CUGUUGCCCUUUUCCUUGANN
1939
UCAAGGAAAAGGGCAACAGNN
29





1221-1239
UGUUGCCCUUUUCCUUGACNN
1940
GUCAAGGAAAAGGGCAACANN
29





 122-140
CCUCCGCCGCCGGAGCCCCNN
1941
GGGGCUCCGGCGGCGGAGGNN
30





1222-1240
GUUGCCCUUUUCCUUGACUNN
1942
AGUCAAGGAAAAGGGCAACNN
30





1223-1241
UUGCCCUUUUCCUUGACUANN
1943
UAGUCAAGGAAAAGGGCAANN
30





1224-1242
UGCCCUUUUCCUUGACUAUNN
1944
AUAGUCAAGGAAAAGGGCANN
30





1225-1243
GCCCUUUUCCUUGACUAUUNN
1945
AAUAGUCAAGGAAAAGGGCNN
30





1226-1244
CCCUUUUCCUUGACUAUUANN
1946
UAAUAGUCAAGGAAAAGGGNN
30





1227-1245
CCUUUUCCUUGACUAUUACNN
1947
GUAAUAGUCAAGGAAAAGGNN
30





1228-1246
CUUUUCCUUGACUAUUACANN
1948
UGUAAUAGUCAAGGAAAAGNN
30





1229-1247
UUUUCCUUGACUAUUACACNN
1949
GUGUAAUAGUCAAGGAAAANN
30





1230-1248
UUUCCUUGACUAUUACACUNN
1950
AGUGUAAUAGUCAAGGAAANN
30





1231-1249
UUCCUUGACUAUUACACUGNN
1951
CAGUGUAAUAGUCAAGGAANN
30





 123-141
CUCCGCCGCCGGAGCCCCGNN
1952
CGGGGCUCCGGCGGCGGAGNN
30





1232-1250
UCCUUGACUAUUACACUGCNN
1953
GCAGUGUAAUAGUCAAGGANN
30





1233-1251
CCUUGACUAUUACACUGCCNN
1954
GGCAGUGUAAUAGUCAAGGNN
30





1234-1252
CUUGACUAUUACACUGCCUNN
1955
AGGCAGUGUAAUAGUCAAGNN
30





1235-1253
UUGACUAUUACACUGCCUGNN
1956
CAGGCAGUGUAAUAGUCAANN
30





1236-1254
UGACUAUUACACUGCCUGGNN
1957
CCAGGCAGUGUAAUAGUCANN
30





1237-1255
GACUAUUACACUGCCUGGANN
1958
UCCAGGCAGUGUAAUAGUCNN
30





1238-1256
ACUAUUACACUGCCUGGAGNN
1959
CUCCAGGCAGUGUAAUAGUNN
30





1239-1257
CUAUUACACUGCCUGGAGGNN
1960
CCUCCAGGCAGUGUAAUAGNN
30





1240-1258
UAUUACACUGCCUGGAGGANN
1961
UCCUCCAGGCAGUGUAAUANN
30





1241-1259
AUUACACUGCCUGGAGGAUNN
1962
AUCCUCCAGGCAGUGUAAUNN
30





 124-142
UCCGCCGCCGGAGCCCCGGNN
1963
CCGGGGCUCCGGCGGCGGANN
30





1242-1260
UUACACUGCCUGGAGGAUANN
1964
UAUCCUCCAGGCAGUGUAANN
30





1243-1261
UACACUGCCUGGAGGAUAGNN
1965
CUAUCCUCCAGGCAGUGUANN
30





1244-1262
ACACUGCCUGGAGGAUAGCNN
1966
GCUAUCCUCCAGGCAGUGUNN
30





1245-1263
CACUGCCUGGAGGAUAGCANN
1967
UGCUAUCCUCCAGGCAGUGNN
30





1246-1264
ACUGCCUGGAGGAUAGCAGNN
1968
CUGCUAUCCUCCAGGCAGUNN
30





 125-143
CCGCCGCCGGAGCCCCGGCNN
1969
GCCGGGGCTCCGGCGGCGGNN
30





 126-144
CGCCGCCGGAGCCCCGGCCNN
1970
GGCCGGGGCTCCGGCGGCGNN
30





 127-145
GCCGCCGGAGCCCCGGCCGNN
1971
CGGCCGGGGCTCCGGCGGCNN
30





1280-1298
CUUCAUUCAAAAAGCCAAANN
1972
UUUGGCUUUUUGAAUGAAGNN
30





1281-1299
UUCAUUCAAAAAGCCAAAANN
1973
UUUUGGCUUUUUGAAUGAANN
30





 128-146
CCGCCGGAGCCCCGGCCGGNN
1974
CCGGCCGGGGCTCCGGCGGNN
30





1282-1300
UCAUUCAAAAAGCCAAAAUNN
1975
AUUUUGGCUUUUUGAAUGANN
30





1283-1301
CAUUCAAAAAGCCAAAAUANN
1976
UAUUUUGGCUUUUUGAAUGNN
30





1284-1302
AUUCAAAAAGCCAAAAUAGNN
1977
CUAUUUUGGCUUUUUGAAUNN
30





1285-1303
UUCAAAAAGCCAAAAUAGANN
1978
UCUAUUUUGGCUUUUUGAANN
30





1286-1304
UCAAAAAGCCAAAAUAGAGNN
1979
CUCUAUUUUGGCUUUUUGANN
30





1287-1305
CAAAAAGCCAAAAUAGAGANN
1980
UCUCUAUUUUGGCUUUUUGNN
30





1288-1306
AAAAAGCCAAAAUAGAGAGNN
1981
CUCUCUAUUUUGGCUUUUUNN
30





1289-1307
AAAAGCCAAAAUAGAGAGUNN
1982
ACUCUCUAUUUUGGCUUUUNN
30





1290-1308
AAAGCCAAAAUAGAGAGUANN
1983
UACUCUCUAUUUUGGCUUUNN
30





 129-147
CGCCGGAGCCCCGGCCGGCNN
1984
GCCGGCCGGGGCTCCGGCGNN
30





 130-148
GCCGGAGCCCCGGCCGGCCNN
1985
GGCCGGCCGGGGCTCCGGCNN
30





1310-1328
ACAGUCCUAGAGAAUUCCUNN
1986
AGGAAUUCUCUAGGACUGUNN
30





 131-149
CCGGAGCCCCGGCCGGCCANN
1987
TGGCCGGCCGGGGCTCCGGNN
30





 132-150
CGGAGCCCCGGCCGGCCAGNN
1988
CTGGCCGGCCGGGGCTCCGNN
30





1330-1348
UAUUUGUUCAGAUCUCAUANN
1989
UAUGAGAUCUGAACAAAUANN
30





1331-1349
AUUUGUUCAGAUCUCAUAGNN
1990
CUAUGAGAUCUGAACAAAUNN
30





 133-151
GGAGCCCCGGCCGGCCAGGNN
1991
CCTGGCCGGCCGGGGCTCCNN
30





1332-1350
UUUGUUCAGAUCUCAUAGANN
1992
UCUAUGAGAUCUGAACAAANN
30





1333-1351
UUGUUCAGAUCUCAUAGAUNN
1993
AUCUAUGAGAUCUGAACAANN
30





1334-1352
UGUUCAGAUCUCAUAGAUGNN
1994
CAUCUAUGAGAUCUGAACANN
30





1335-1353
GUUCAGAUCUCAUAGAUGANN
1995
UCAUCUAUGAGAUCUGAACNN
30





 134-152
GAGCCCCGGCCGGCCAGGCNN
1996
GCCTGGCCGGCCGGGGCTCNN
30





 135-153
AGCCCCGGCCGGCCAGGCCNN
1997
GGCCTGGCCGGCCGGGGCTNN
30





 136-154
GCCCCGGCCGGCCAGGCCCNN
1998
GGGCCTGGCCGGCCGGGGCNN
30





1365-1383
UGUCUUUUGACAUCCAGCANN
1999
UGCUGGAUGUCAAAAGACANN
30





1366-1384
GUCUUUUGACAUCCAGCAGNN
2000
CUGCUGGAUGUCAAAAGACNN
30





1367-1385
UCUUUUGACAUCCAGCAGUNN
2001
ACUGCUGGAUGUCAAAAGANN
30





1368-1386
CUUUUGACAUCCAGCAGUCNN
2002
GACUGCUGGAUGUCAAAAGNN
30





1369-1387
UUUUGACAUCCAGCAGUCCNN
2003
GGACUGCUGGAUGUCAAAANN
30





1370-1388
UUUGACAUCCAGCAGUCCANN
2004
UGGACUGCUGGAUGUCAAANN
30





1371-1389
UUGACAUCCAGCAGUCCAANN
2005
UUGGACUGCUGGAUGUCAANN
30





 137-155
CCCCGGCCGGCCAGGCCCUNN
2006
AGGGCCUGGCCGGCCGGGGNN
30





 138-156
CCCGGCCGGCCAGGCCCUGNN
2007
CAGGGCCUGGCCGGCCGGGNN
30





1391-1409
GUAUUGAGACAUAUUACUGNN
2008
CAGUAAUAUGUCUCAAUACNN
30





 139-157
CCGGCCGGCCAGGCCCUGCNN
2009
GCAGGGCCUGGCCGGCCGGNN
30





 140-158
CGGCCGGCCAGGCCCUGCCNN
2010
GGCAGGGCCUGGCCGGCCGNN
30





 141-159
GGCCGGCCAGGCCCUGCCGNN
2011
CGGCAGGGCCUGGCCGGCCNN
30





1414-1432
UAAGAAAUAUUACUAUAAUNN
2012
AUUAUAGUAAUAUUUCUUANN
30





1415-1433
AAGAAAUAUUACUAUAAUUNN
2013
AAUUAUAGUAAUAUUUCUUNN
30





1416-1434
AGAAAUAUUACUAUAAUUGNN
2014
CAAUUAUAGUAAUAUUUCUNN
30





1417-1435
GAAAUAUUACUAUAAUUGANN
2015
UCAAUUAUAGUAAUAUUUCNN
30





1418-1436
AAAUAUUACUAUAAUUGAGNN
2016
CUCAAUUAUAGUAAUAUUUNN
30





1419-1437
AAUAUUACUAUAAUUGAGANN
2017
UCUCAAUUAUAGUAAUAUUNN
30





1420-1438
AUAUUACUAUAAUUGAGAANN
2018
UUCUCAAUUAUAGUAAUAUNN
30





1421-1439
UAUUACUAUAAUUGAGAACNN
2019
GUUCUCAAUUAUAGUAAUANN
30





 142-160
GCCGGCCAGGCCCUGCCGCNN
2020
GCGGCAGGGCCUGGCCGGCNN
30





1422-1440
AUUACUAUAAUUGAGAACUNN
2021
AGUUCUCAAUUAUAGUAAUNN
30





1423-1441
UUACUAUAAUUGAGAACUANN
2022
UAGUUCUCAAUUAUAGUAANN
30





1424-1442
UACUAUAAUUGAGAACUACNN
2023
GUAGUUCUCAAUUAUAGUANN
30





1425-1443
ACUAUAAUUGAGAACUACANN
2024
UGUAGUUCUCAAUUAUAGUNN
30





1426-1444
CUAUAAUUGAGAACUACAGNN
2025
CUGUAGUUCUCAAUUAUAGNN
30





1427-1445
UAUAAUUGAGAACUACAGCNN
2026
GCUGUAGUUCUCAAUUAUANN
30





1428-1446
AUAAUUGAGAACUACAGCUNN
2027
AGCUGUAGUUCUCAAUUAUNN
30





1429-1447
UAAUUGAGAACUACAGCUUNN
2028
AAGCUGUAGUUCUCAAUUANN
30





1430-1448
AAUUGAGAACUACAGCUUUNN
2029
AAAGCUGUAGUUCUCAAUUNN
30





1431-1449
AUUGAGAACUACAGCUUUUNN
2030
AAAAGCUGUAGUUCUCAAUNN
30





 143-161
CCGGCCAGGCCCUGCCGCUNN
2031
AGCGGCAGGGCCUGGCCGGNN
30





1432-1450
UUGAGAACUACAGCUUUUANN
2032
UAAAAGCUGUAGUUCUCAANN
30





1433-1451
UGAGAACUACAGCUUUUAANN
2033
UUAAAAGCUGUAGUUCUCANN
30





1434-1452
GAGAACUACAGCUUUUAAGNN
2034
CUUAAAAGCUGUAGUUCUCNN
30





1435-1453
AGAACUACAGCUUUUAAGANN
2035
UCUUAAAAGCUGUAGUUCUNN
30





1436-1454
GAACUACAGCUUUUAAGAUNN
2036
AUCUUAAAAGCUGUAGUUCNN
30





1437-1455
AACUACAGCUUUUAAGAUUNN
2037
AAUCUUAAAAGCUGUAGUUNN
30





1438-1456
ACUACAGCUUUUAAGAUUGNN
2038
CAAUCUUAAAAGCUGUAGUNN
30





1439-1457
CUACAGCUUUUAAGAUUGUNN
2039
ACAAUCUUAAAAGCUGUAGNN
30





1440-1458
UACAGCUUUUAAGAUUGUANN
2040
UACAAUCUUAAAAGCUGUANN
30





1441-1459
ACAGCUUUUAAGAUUGUACNN
2041
GUACAAUCUUAAAAGCUGUNN
31





 144-162
CGGCCAGGCCCUGCCGCUCNN
2042
GAGCGGCAGGGCCUGGCCGNN
31





1442-1460
CAGCUUUUAAGAUUGUACUNN
2043
AGUACAAUCUUAAAAGCUGNN
31





1443-1461
AGCUUUUAAGAUUGUACUUNN
2044
AAGUACAAUCUUAAAAGCUNN
31





1444-1462
GCUUUUAAGAUUGUACUUUNN
2045
AAAGUACAAUCUUAAAAGCNN
31





1445-1463
CUUUUAAGAUUGUACUUUUNN
2046
AAAAGUACAAUCUUAAAAGNN
31





1446-1464
UUUUAAGAUUGUACUUUUANN
2047
UAAAAGUACAAUCUUAAAANN
31





1447-1465
UUUAAGAUUGUACUUUUAUNN
2048
AUAAAAGUACAAUCUUAAANN
31





1448-1466
UUAAGAUUGUACUUUUAUCNN
2049
GAUAAAAGUACAAUCUUAANN
31





1449-1467
UAAGAUUGUACUUUUAUCUNN
2050
AGAUAAAAGUACAAUCUUANN
31





1450-1468
AAGAUUGUACUUUUAUCUUNN
2051
AAGAUAAAAGUACAAUCUUNN
31





1451-1469
AGAUUGUACUUUUAUCUUANN
2052
UAAGAUAAAAGUACAAUCUNN
31





 145-163
GGCCAGGCCCUGCCGCUCANN
2053
UGAGCGGCAGGGCCUGGCCNN
31





1452-1470
GAUUGUACUUUUAUCUUAANN
2054
UUAAGAUAAAAGUACAAUCNN
31





1453-1471
AUUGUACUUUUAUCUUAAANN
2055
UUUAAGAUAAAAGUACAAUNN
31





1454-1472
UUGUACUUUUAUCUUAAAANN
2056
UUUUAAGAUAAAAGUACAANN
31





1455-1473
UGUACUUUUAUCUUAAAAGNN
2057
CUUUUAAGAUAAAAGUACANN
31





1456-1474
GUACUUUUAUCUUAAAAGGNN
2058
CCUUUUAAGAUAAAAGUACNN
31





1457-1475
UACUUUUAUCUUAAAAGGGNN
2059
CCCUUUUAAGAUAAAAGUANN
31





1458-1476
ACUUUUAUCUUAAAAGGGUNN
2060
ACCCUUUUAAGAUAAAAGUNN
31





1459-1477
CUUUUAUCUUAAAAGGGUGNN
2061
CACCCUUUUAAGAUAAAAGNN
31





1460-1478
UUUUAUCUUAAAAGGGUGGNN
2062
CCACCCUUUUAAGAUAAAANN
31





1461-1479
UUUAUCUUAAAAGGGUGGUNN
2063
ACCACCCUUUUAAGAUAAANN
31





 146-164
GCCAGGCCCUGCCGCUCAUNN
2064
AUGAGCGGCAGGGCCUGGCNN
31





1462-1480
UUAUCUUAAAAGGGUGGUANN
2065
UACCACCCUUUUAAGAUAANN
31





1463-1481
UAUCUUAAAAGGGUGGUAGNN
2066
CUACCACCCUUUUAAGAUANN
31





1464-1482
AUCUUAAAAGGGUGGUAGUNN
2067
ACUACCACCCUUUUAAGAUNN
31





1465-1483
UCUUAAAAGGGUGGUAGUUNN
2068
AACUACCACCCUUUUAAGANN
31





1466-1484
CUUAAAAGGGUGGUAGUUUNN
2069
AAACUACCACCCUUUUAAGNN
31





 147-165
CCAGGCCCUGCCGCUCAUGNN
2070
CAUGAGCGGCAGGGCCUGGNN
31





 148-166
CAGGCCCUGCCGCUCAUGGNN
2071
CCAUGAGCGGCAGGGCCUGNN
31





1486-1504
CCCUAAAAUACUUAUUAUGNN
2072
CAUAAUAAGUAUUUUAGGGNN
31





1487-1505
CCUAAAAUACUUAUUAUGUNN
2073
ACAUAAUAAGUAUUUUAGGNN
31





1488-1506
CUAAAAUACUUAUUAUGUANN
2074
UACAUAAUAAGUAUUUUAGNN
31





1489-1507
UAAAAUACUUAUUAUGUAANN
2075
UUACAUAAUAAGUAUUUUANN
31





1490-1508
AAAAUACUUAUUAUGUAAGNN
2076
CUUACAUAAUAAGUAUUUUNN
31





1491-1509
AAAUACUUAUUAUGUAAGGNN
2077
CCUUACAUAAUAAGUAUUUNN
31





 149-167
AGGCCCUGCCGCUCAUGGUNN
2078
ACCAUGAGCGGCAGGGCCUNN
31





1492-1510
AAUACUUAUUAUGUAAGGGNN
2079
CCCUUACAUAAUAAGUAUUNN
31





1493-1511
AUACUUAUUAUGUAAGGGUNN
2080
ACCCUUACAUAAUAAGUAUNN
31





1494-1512
UACUUAUUAUGUAAGGGUCNN
2081
GACCCUUACAUAAUAAGUANN
31





1495-1513
ACUUAUUAUGUAAGGGUCANN
2082
UGACCCUUACAUAAUAAGUNN
31





1496-1514
CUUAUUAUGUAAGGGUCAUNN
2083
AUGACCCUUACAUAAUAAGNN
31





1497-1515
UUAUUAUGUAAGGGUCAUUNN
2084
AAUGACCCUUACAUAAUAANN
31





1498-1516
UAUUAUGUAAGGGUCAUUANN
2085
UAAUGACCCUUACAUAAUANN
31





1499-1517
AUUAUGUAAGGGUCAUUAGNN
2086
CUAAUGACCCUUACAUAAUNN
31





1500-1518
UUAUGUAAGGGUCAUUAGANN
2087
UCUAAUGACCCUUACAUAANN
31





1501-1519
UAUGUAAGGGUCAUUAGACNN
2088
GUCUAAUGACCCUUACAUANN
31





 150-168
GGCCCUGCCGCUCAUGGUGNN
2089
CACCAUGAGCGGCAGGGCCNN
31





1502-1520
AUGUAAGGGUCAUUAGACANN
2090
UGUCUAAUGACCCUUACAUNN
31





1503-1521
UGUAAGGGUCAUUAGACAANN
2091
UUGUCUAAUGACCCUUACANN
31





1504-1522
GUAAGGGUCAUUAGACAAANN
2092
UUUGUCUAAUGACCCUUACNN
31





1505-1523
UAAGGGUCAUUAGACAAAUNN
2093
AUUUGUCUAAUGACCCUUANN
31





1506-1524
AAGGGUCAUUAGACAAAUGNN
2094
CAUUUGUCUAAUGACCCUUNN
31





1507-1525
AGGGUCAUUAGACAAAUGUNN
2095
ACAUUUGUCUAAUGACCCUNN
31





1508-1526
GGGUCAUUAGACAAAUGUCNN
2096
GACAUUUGUCUAAUGACCCNN
31





1509-1527
GGUCAUUAGACAAAUGUCUNN
2097
AGACAUUUGUCUAAUGACCNN
31





1510-1528
GUCAUUAGACAAAUGUCUUNN
2098
AAGACAUUUGUCUAAUGACNN
31





1511-1529
UCAUUAGACAAAUGUCUUGNN
2099
CAAGACAUUUGUCUAAUGANN
31





 151-169
GCCCUGCCGCUCAUGGUGCNN
2100
GCACCAUGAGCGGCAGGGCNN
31





1512-1530
CAUUAGACAAAUGUCUUGANN
2101
UCAAGACAUUUGUCUAAUGNN
31





1513-1531
AUUAGACAAAUGUCUUGAANN
2102
UUCAAGACAUUUGUCUAAUNN
31





1514-1532
UUAGACAAAUGUCUUGAAGNN
2103
CUUCAAGACAUUUGUCUAANN
31





1515-1533
UAGACAAAUGUCUUGAAGUNN
2104
ACUUCAAGACAUUUGUCUANN
31





1516-1534
AGACAAAUGUCUUGAAGUANN
2105
UACUUCAAGACAUUUGUCUNN
31





1517-1535
GACAAAUGUCUUGAAGUAGNN
2106
CUACUUCAAGACAUUUGUCNN
31





1518-1536
ACAAAUGUCUUGAAGUAGANN
2107
UCUACUUCAAGACAUUUGUNN
31





 152-170
CCCUGCCGCUCAUGGUGCCNN
2108
GGCACCAUGAGCGGCAGGGNN
31





 153-171
CCUGCCGCUCAUGGUGCCANN
2109
UGGCACCAUGAGCGGCAGGNN
31





1541-1559
GAAUUUAUGAAUGGUUCUUNN
2110
AAGAACCAUUCAUAAAUUCNN
31





 154-172
CUGCCGCUCAUGGUGCCAGNN
2111
CUGGCACCAUGAGCGGCAGNN
31





1542-1560
AAUUUAUGAAUGGUUCUUUNN
2112
AAAGAACCAUUCAUAAAUUNN
31





1543-1561
AUUUAUGAAUGGUUCUUUANN
2113
UAAAGAACCAUUCAUAAAUNN
31





1544-1562
UUUAUGAAUGGUUCUUUAUNN
2114
AUAAAGAACCAUUCAUAAANN
31





1545-1563
UUAUGAAUGGUUCUUUAUCNN
2115
GAUAAAGAACCAUUCAUAANN
31





1546-1564
UAUGAAUGGUUCUUUAUCANN
2116
UGAUAAAGAACCAUUCAUANN
31





1547-1565
AUGAAUGGUUCUUUAUCAUNN
2117
AUGAUAAAGAACCAUUCAUNN
31





1548-1566
UGAAUGGUUCUUUAUCAUUNN
2118
AAUGAUAAAGAACCAUUCANN
31





1549-1567
GAAUGGUUCUUUAUCAUUUNN
2119
AAAUGAUAAAGAACCAUUCNN
31





1550-1568
AAUGGUUCUUUAUCAUUUCNN
2120
GAAAUGAUAAAGAACCAUUNN
31





1551-1569
AUGGUUCUUUAUCAUUUCUNN
2121
AGAAAUGAUAAAGAACCAUNN
31





 155-173
UGCCGCUCAUGGUGCCAGCNN
2122
GCUGGCACCAUGAGCGGCANN
31





1552-1570
UGGUUCUUUAUCAUUUCUCNN
2123
GAGAAAUGAUAAAGAACCANN
31





1553-1571
GGUUCUUUAUCAUUUCUCUNN
2124
AGAGAAAUGAUAAAGAACCNN
31





1554-1572
GUUCUUUAUCAUUUCUCUUNN
2125
AAGAGAAAUGAUAAAGAACNN
31





1555-1573
UUCUUUAUCAUUUCUCUUCNN
2126
GAAGAGAAAUGAUAAAGAANN
31





1556-1574
UCUUUAUCAUUUCUCUUCCNN
2127
GGAAGAGAAAUGAUAAAGANN
31





1557-1575
CUUUAUCAUUUCUCUUCCCNN
2128
GGGAAGAGAAAUGAUAAAGNN
31





1558-1576
UUUAUCAUUUCUCUUCCCCNN
2129
GGGGAAGAGAAAUGAUAAANN
31





1559-1577
UUAUCAUUUCUCUUCCCCCNN
2130
GGGGGAAGAGAAAUGAUAANN
31





1560-1578
UAUCAUUUCUCUUCCCCCUNN
2131
AGGGGGAAGAGAAAUGAUANN
31





1561-1579
AUCAUUUCUCUUCCCCCUUNN
2132
AAGGGGGAAGAGAAAUGAUNN
31





 156-174
GCCGCUCAUGGUGCCAGCCNN
2133
GGCUGGCACCAUGAGCGGCNN
31





1562-1580
UCAUUUCUCUUCCCCCUUUNN
2134
AAAGGGGGAAGAGAAAUGANN
31





1563-1581
CAUUUCUCUUCCCCCUUUUNN
2135
AAAAGGGGGAAGAGAAAUGNN
31





1564-1582
AUUUCUCUUCCCCCUUUUUNN
2136
AAAAAGGGGGAAGAGAAAUNN
31





1565-1583
UUUCUCUUCCCCCUUUUUGNN
2137
CAAAAAGGGGGAAGAGAAANN
31





1566-1584
UUCUCUUCCCCCUUUUUGGNN
2138
CCAAAAAGGGGGAAGAGAANN
31





1567-1585
UCUCUUCCCCCUUUUUGGCNN
2139
GCCAAAAAGGGGGAAGAGANN
31





1568-1586
CUCUUCCCCCUUUUUGGCANN
2140
UGCCAAAAAGGGGGAAGAGNN
31





1569-1587
UCUUCCCCCUUUUUGGCAUNN
2141
AUGCCAAAAAGGGGGAAGANN
32





1570-1588
CUUCCCCCUUUUUGGCAUCNN
2142
GAUGCCAAAAAGGGGGAAGNN
32





1571-1589
UUCCCCCUUUUUGGCAUCCNN
2143
GGAUGCCAAAAAGGGGGAANN
32





 157-175
CCGCUCAUGGUGCCAGCCCNN
2144
GGGCUGGCACCAUGAGCGGNN
32





1572-1590
UCCCCCUUUUUGGCAUCCUNN
2145
AGGAUGCCAAAAAGGGGGANN
32





1573-1591
CCCCCUUUUUGGCAUCCUGNN
2146
CAGGAUGCCAAAAAGGGGGNN
32





1574-1592
CCCCUUUUUGGCAUCCUGGNN
2147
CCAGGAUGCCAAAAAGGGGNN
32





1575-1593
CCCUUUUUGGCAUCCUGGCNN
2148
GCCAGGAUGCCAAAAAGGGNN
32





1576-1594
CCUUUUUGGCAUCCUGGCUNN
2149
AGCCAGGAUGCCAAAAAGGNN
32





1577-1595
CUUUUUGGCAUCCUGGCUUNN
2150
AAGCCAGGAUGCCAAAAAGNN
32





1578-1596
UUUUUGGCAUCCUGGCUUGNN
2151
CAAGCCAGGAUGCCAAAAANN
32





1579-1597
UUUUGGCAUCCUGGCUUGCNN
2152
GCAAGCCAGGAUGCCAAAANN
32





1580-1598
UUUGGCAUCCUGGCUUGCCNN
2153
GGCAAGCCAGGAUGCCAAANN
32





1581-1599
UUGGCAUCCUGGCUUGCCUNN
2154
AGGCAAGCCAGGAUGCCAANN
32





 158-176
CGCUCAUGGUGCCAGCCCANN
2155
UGGGCUGGCACCAUGAGCGNN
32





1582-1600
UGGCAUCCUGGCUUGCCUCNN
2156
GAGGCAAGCCAGGAUGCCANN
32





1583-1601
GGCAUCCUGGCUUGCCUCCNN
2157
GGAGGCAAGCCAGGAUGCCNN
32





1584-1602
GCAUCCUGGCUUGCCUCCANN
2158
UGGAGGCAAGCCAGGAUGCNN
32





1585-1603
CAUCCUGGCUUGCCUCCAGNN
2159
CUGGAGGCAAGCCAGGAUGNN
32





1586-1604
AUCCUGGCUUGCCUCCAGUNN
2160
ACUGGAGGCAAGCCAGGAUNN
32





1587-1605
UCCUGGCUUGCCUCCAGUUNN
2161
AACUGGAGGCAAGCCAGGANN
32





1588-1606
CCUGGCUUGCCUCCAGUUUNN
2162
AAACUGGAGGCAAGCCAGGNN
32





1589-1607
CUGGCUUGCCUCCAGUUUUNN
2163
AAAACUGGAGGCAAGCCAGNN
32





1590-1608
UGGCUUGCCUCCAGUUUUANN
2164
UAAAACUGGAGGCAAGCCANN
32





1591-1609
GGCUUGCCUCCAGUUUUAGNN
2165
CUAAAACUGGAGGCAAGCCNN
32





 159-177
GCUCAUGGUGCCAGCCCAGNN
2166
CUGGGCUGGCACCAUGAGCNN
32





1592-1610
GCUUGCCUCCAGUUUUAGGNN
2167
CCUAAAACUGGAGGCAAGCNN
32





1593-1611
CUUGCCUCCAGUUUUAGGUNN
2168
ACCUAAAACUGGAGGCAAGNN
32





1594-1612
UUGCCUCCAGUUUUAGGUCNN
2169
GACCUAAAACUGGAGGCAANN
32





1595-1613
UGCCUCCAGUUUUAGGUCCNN
2170
GGACCUAAAACUGGAGGCANN
32





 160-178
CUCAUGGUGCCAGCCCAGANN
2171
UCUGGGCUGGCACCAUGAGNN
32





 161-179
UCAUGGUGCCAGCCCAGAGNN
2172
CUCUGGGCUGGCACCAUGANN
32





1615-1633
UUAGUUUGCUUCUGUAAGCNN
2173
GCUUACAGAAGCAAACUAANN
32





1616-1634
UAGUUUGCUUCUGUAAGCANN
2174
UGCUUACAGAAGCAAACUANN
32





1617-1635
AGUUUGCUUCUGUAAGCAANN
2175
UUGCUUACAGAAGCAAACUNN
32





 162-180
CAUGGUGCCAGCCCAGAGANN
2176
UCUCUGGGCUGGCACCAUGNN
32





 163-181
AUGGUGCCAGCCCAGAGAGNN
2177
CUCUCUGGGCUGGCACCAUNN
32





1639-1657
GAACACCUGCUGAGGGGGCNN
2178
GCCCCCUCAGCAGGUGUUCNN
32





1640-1658
AACACCUGCUGAGGGGGCUNN
2179
AGCCCCCUCAGCAGGUGUUNN
32





1641-1659
ACACCUGCUGAGGGGGCUCNN
2180
GAGCCCCCUCAGCAGGUGUNN
32





 164-182
UGGUGCCAGCCCAGAGAGGNN
2181
CCUCUCUGGGCUGGCACCANN
32





1642-1660
CACCUGCUGAGGGGGCUCUNN
2182
AGAGCCCCCUCAGCAGGUGNN
32





1643-1661
ACCUGCUGAGGGGGCUCUUNN
2183
AAGAGCCCCCUCAGCAGGUNN
32





1644-1662
CCUGCUGAGGGGGCUCUUUNN
2184
AAAGAGCCCCCUCAGCAGGNN
32





1645-1663
CUGCUGAGGGGGCUCUUUCNN
2185
GAAAGAGCCCCCUCAGCAGNN
32





1646-1664
UGCUGAGGGGGCUCUUUCCNN
2186
GGAAAGAGCCCCCUCAGCANN
32





1647-1665
GCUGAGGGGGCUCUUUCCCNN
2187
GGGAAAGAGCCCCCUCAGCNN
32





1648-1666
CUGAGGGGGCUCUUUCCCUNN
2188
AGGGAAAGAGCCCCCUCAGNN
32





1649-1667
UGAGGGGGCUCUUUCCCUCNN
2189
GAGGGAAAGAGCCCCCUCANN
32





1650-1668
GAGGGGGCUCUUUCCCUCANN
2190
UGAGGGAAAGAGCCCCCUCNN
32





 165-183
GGUGCCAGCCCAGAGAGGGNN
2191
CCCUCUCUGGGCUGGCACCNN
32





 166-184
GUGCCAGCCCAGAGAGGGGNN
2192
CCCCUCUCUGGGCUGGCACNN
32





1670-1688
GUAUACUUCAAGUAAGAUCNN
2193
GAUCUUACUUGAAGUAUACNN
32





1671-1689
UAUACUUCAAGUAAGAUCANN
2194
UGAUCUUACUUGAAGUAUANN
32





 167-185
UGCCAGCCCAGAGAGGGGCNN
2195
GCCCCUCUCUGGGCUGGCANN
32





1672-1690
AUACUUCAAGUAAGAUCAANN
2196
UUGAUCUUACUUGAAGUAUNN
32





1673-1691
UACUUCAAGUAAGAUCAAGNN
2197
CUUGAUCUUACUUGAAGUANN
32





1674-1692
ACUUCAAGUAAGAUCAAGANN
2198
UCUUGAUCUUACUUGAAGUNN
32





1675-1693
CUUCAAGUAAGAUCAAGAANN
2199
UUCUUGAUCUUACUUGAAGNN
32





1676-1694
UUCAAGUAAGAUCAAGAAUNN
2200
AUUCUUGAUCUUACUUGAANN
32





1677-1695
UCAAGUAAGAUCAAGAAUCNN
2201
GAUUCUUGAUCUUACUUGANN
32





1678-1696
CAAGUAAGAUCAAGAAUCUNN
2202
AGAUUCUUGAUCUUACUUGNN
32





1679-1697
AAGUAAGAUCAAGAAUCUUNN
2203
AAGAUUCUUGAUCUUACUUNN
32





1680-1698
AGUAAGAUCAAGAAUCUUUNN
2204
AAAGAUUCUUGAUCUUACUNN
32





1681-1699
GUAAGAUCAAGAAUCUUUUNN
2205
AAAAGAUUCUUGAUCUUACNN
32





1682-1700
UAAGAUCAAGAAUCUUUUGNN
2206
CAAAAGAUUCUUGAUCUUANN
32





1683-1701
AAGAUCAAGAAUCUUUUGUNN
2207
ACAAAAGAUUCUUGAUCUUNN
32





1684-1702
AGAUCAAGAAUCUUUUGUGNN
2208
CACAAAAGAUUCUUGAUCUNN
32





1685-1703
GAUCAAGAAUCUUUUGUGANN
2209
UCACAAAAGAUUCUUGAUCNN
32





1686-1704
AUCAAGAAUCUUUUGUGAANN
2210
UUCACAAAAGAUUCUUGAUNN
32





1687-1705
UCAAGAAUCUUUUGUGAAANN
2211
UUUCACAAAAGAUUCUUGANN
32





1707-1725
UAUAGAAAUUUACUAUGUANN
2212
UACAUAGUAAAUUUCUAUANN
32





1708-1726
AUAGAAAUUUACUAUGUAANN
2213
UUACAUAGUAAAUUUCUAUNN
32





1709-1727
UAGAAAUUUACUAUGUAAANN
2214
UUUACAUAGUAAAUUUCUANN
32





1710-1728
AGAAAUUUACUAUGUAAAUNN
2215
AUUUACAUAGUAAAUUUCUNN
32





1711-1729
GAAAUUUACUAUGUAAAUGNN
2216
CAUUUACAUAGUAAAUUUCNN
32





1712-1730
AAAUUUACUAUGUAAAUGCNN
2217
GCAUUUACAUAGUAAAUUUNN
32





1713-1731
AAUUUACUAUGUAAAUGCUNN
2218
AGCAUUUACAUAGUAAAUUNN
32





1714-1732
AUUUACUAUGUAAAUGCUUNN
2219
AAGCAUUUACAUAGUAAAUNN
32





1715-1733
UUUACUAUGUAAAUGCUUGNN
2220
CAAGCAUUUACAUAGUAAANN
32





1716-1734
UUACUAUGUAAAUGCUUGANN
2221
UCAAGCAUUUACAUAGUAANN
32





1717-1735
UACUAUGUAAAUGCUUGAUNN
2222
AUCAAGCAUUUACAUAGUANN
32





1718-1736
ACUAUGUAAAUGCUUGAUGNN
2223
CAUCAAGCAUUUACAUAGUNN
32





1719-1737
CUAUGUAAAUGCUUGAUGGNN
2224
CCAUCAAGCAUUUACAUAGNN
32





1720-1738
UAUGUAAAUGCUUGAUGGANN
2225
UCCAUCAAGCAUUUACAUANN
32





1721-1739
AUGUAAAUGCUUGAUGGAANN
2226
UUCCAUCAAGCAUUUACAUNN
32





1722-1740
UGUAAAUGCUUGAUGGAAUNN
2227
AUUCCAUCAAGCAUUUACANN
32





1723-1741
GUAAAUGCUUGAUGGAAUUNN
2228
AAUUCCAUCAAGCAUUUACNN
32





1724-1742
UAAAUGCUUGAUGGAAUUUNN
2229
AAAUUCCAUCAAGCAUUUANN
32





1725-1743
AAAUGCUUGAUGGAAUUUUNN
2230
AAAAUUCCAUCAAGCAUUUNN
32





1726-1744
AAUGCUUGAUGGAAUUUUUNN
2231
AAAAAUUCCAUCAAGCAUUNN
32





1727-1745
AUGCUUGAUGGAAUUUUUUNN
2232
AAAAAAUUCCAUCAAGCAUNN
32





1728-1746
UGCUUGAUGGAAUUUUUUCNN
2233
GAAAAAAUUCCAUCAAGCANN
32





1729-1747
GCUUGAUGGAAUUUUUUCCNN
2234
GGAAAAAAUUCCAUCAAGCNN
32





1730-1748
CUUGAUGGAAUUUUUUCCUNN
2235
AGGAAAAAAUUCCAUCAAGNN
32





1731-1749
UUGAUGGAAUUUUUUCCUGNN
2236
CAGGAAAAAAUUCCAUCAANN
32





1732-1750
UGAUGGAAUUUUUUCCUGCNN
2237
GCAGGAAAAAAUUCCAUCANN
32





1733-1751
GAUGGAAUUUUUUCCUGCUNN
2238
AGCAGGAAAAAAUUCCAUCNN
32





1734-1752
AUGGAAUUUUUUCCUGCUANN
2239
UAGCAGGAAAAAAUUCCAUNN
32





1735-1753
UGGAAUUUUUUCCUGCUAGNN
2240
CUAGCAGGAAAAAAUUCCANN
32





1736-1754
GGAAUUUUUUCCUGCUAGUNN
2241
ACUAGCAGGAAAAAAUUCCNN
33





1737-1755
GAAUUUUUUCCUGCUAGUGNN
2242
CACUAGCAGGAAAAAAUUCNN
33





1738-1756
AAUUUUUUCCUGCUAGUGUNN
2243
ACACUAGCAGGAAAAAAUUNN
33





1739-1757
AUUUUUUCCUGCUAGUGUANN
2244
UACACUAGCAGGAAAAAAUNN
33





1740-1758
UUUUUUCCUGCUAGUGUAGNN
2245
CUACACUAGCAGGAAAAAANN
33





1741-1759
UUUUUCCUGCUAGUGUAGCNN
2246
GCUACACUAGCAGGAAAAANN
33





1742-1760
UUUUCCUGCUAGUGUAGCUNN
2247
AGCUACACUAGCAGGAAAANN
33





1743-1761
UUUCCUGCUAGUGUAGCUUNN
2248
AAGCUACACUAGCAGGAAANN
33





1744-1762
UUCCUGCUAGUGUAGCUUCNN
2249
GAAGCUACACUAGCAGGAANN
33





1745-1763
UCCUGCUAGUGUAGCUUCUNN
2250
AGAAGCUACACUAGCAGGANN
33





1746-1764
CCUGCUAGUGUAGCUUCUGNN
2251
CAGAAGCUACACUAGCAGGNN
33





1747-1765
CUGCUAGUGUAGCUUCUGANN
2252
UCAGAAGCUACACUAGCAGNN
33





1748-1766
UGCUAGUGUAGCUUCUGAANN
2253
UUCAGAAGCUACACUAGCANN
33





1749-1767
GCUAGUGUAGCUUCUGAAANN
2254
UUUCAGAAGCUACACUAGCNN
33





1750-1768
CUAGUGUAGCUUCUGAAAGNN
2255
CUUUCAGAAGCUACACUAGNN
33





1751-1769
UAGUGUAGCUUCUGAAAGGNN
2256
CCUUUCAGAAGCUACACUANN
33





1752-1770
AGUGUAGCUUCUGAAAGGUNN
2257
ACCUUUCAGAAGCUACACUNN
33





1753-1771
GUGUAGCUUCUGAAAGGUGNN
2258
CACCUUUCAGAAGCUACACNN
33





1754-1772
UGUAGCUUCUGAAAGGUGCNN
2259
GCACCUUUCAGAAGCUACANN
33





1755-1773
GUAGCUUCUGAAAGGUGCUNN
2260
AGCACCUUUCAGAAGCUACNN
33





1756-1774
UAGCUUCUGAAAGGUGCUUNN
2261
AAGCACCUUUCAGAAGCUANN
33





1757-1775
AGCUUCUGAAAGGUGCUUUNN
2262
AAAGCACCUUUCAGAAGCUNN
33





1758-1776
GCUUCUGAAAGGUGCUUUCNN
2263
GAAAGCACCUUUCAGAAGCNN
33





1777-1795
UCCAUUUAUUUAAAACUACNN
2264
GUAGUUUUAAAUAAAUGGANN
33





1778-1796
CCAUUUAUUUAAAACUACCNN
2265
GGUAGUUUUAAAUAAAUGGNN
33





1779-1797
CAUUUAUUUAAAACUACCCNN
2266
GGGUAGUUUUAAAUAAAUGNN
33





1780-1798
AUUUAUUUAAAACUACCCANN
2267
UGGGUAGUUUUAAAUAAAUNN
33





1781-1799
UUUAUUUAAAACUACCCAUNN
2268
AUGGGUAGUUUUAAAUAAANN
33





1782-1800
UUAUUUAAAACUACCCAUGNN
2269
CAUGGGUAGUUUUAAAUAANN
33





1783-1801
UAUUUAAAACUACCCAUGCNN
2270
GCAUGGGUAGUUUUAAAUANN
33





1784-1802
AUUUAAAACUACCCAUGCANN
2271
UGCAUGGGUAGUUUUAAAUNN
33





1785-1803
UUUAAAACUACCCAUGCAANN
2272
UUGCAUGGGUAGUUUUAAANN
33





1786-1804
UUAAAACUACCCAUGCAAUNN
2273
AUUGCAUGGGUAGUUUUAANN
33





1787-1805
UAAAACUACCCAUGCAAUUNN
2274
AAUUGCAUGGGUAGUUUUANN
33





1788-1806
AAAACUACCCAUGCAAUUANN
2275
UAAUUGCAUGGGUAGUUUUNN
33





1789-1807
AAACUACCCAUGCAAUUAANN
2276
UUAAUUGCAUGGGUAGUUUNN
33





1790-1808
AACUACCCAUGCAAUUAAANN
2277
UUUAAUUGCAUGGGUAGUUNN
33





1791-1809
ACUACCCAUGCAAUUAAAANN
2278
UUUUAAUUGCAUGGGUAGUNN
33





1792-1810
CUACCCAUGCAAUUAAAAGNN
2279
CUUUUAAUUGCAUGGGUAGNN
33





1793-1811
UACCCAUGCAAUUAAAAGGNN
2280
CCUUUUAAUUGCAUGGGUANN
33





1794-1812
ACCCAUGCAAUUAAAAGGUNN
2281
ACCUUUUAAUUGCAUGGGUNN
33





1795-1813
CCCAUGCAAUUAAAAGGUANN
2282
UACCUUUUAAUUGCAUGGGNN
33





1796-1814
CCAUGCAAUUAAAAGGUACNN
2283
GUACCUUUUAAUUGCAUGGNN
33





1797-1815
CAUGCAAUUAAAAGGUACANN
2284
UGUACCUUUUAAUUGCAUGNN
33





1798-1816
AUGCAAUUAAAAGGUACAANN
2285
UUGUACCUUUUAAUUGCAUNN
33





1799-1817
UGCAAUUAAAAGGUACAAUNN
2286
AUUGUACCUUUUAAUUGCANN
33





1800-1818
GCAAUUAAAAGGUACAAUGNN
2287
CAUUGUACCUUUUAAUUGCNN
33





1801-1819
CAAUUAAAAGGUACAAUGCNN
2288
GCAUUGUACCUUUUAAUUGNN
33





1802-1820
AAUUAAAAGGUACAAUGCANN
2289
UGCAUUGUACCUUUUAAUUNN
33





 187-205
AGCCCGGAGGCAGCGAGCGNN
2290
CGCTCGCTGCCTCCGGGCTNN
33





 188-206
GCCCGGAGGCAGCGAGCGGNN
2291
CCGCTCGCTGCCTCCGGGCNN
33





 189-207
CCCGGAGGCAGCGAGCGGGNN
2292
CCCGCTCGCTGCCTCCGGGNN
33





 190-208
CCGGAGGCAGCGAGCGGGGNN
2293
CCCCGCTCGCTGCCTCCGGNN
33





 191-209
CGGAGGCAGCGAGCGGGGGNN
2294
CCCCCGCTCGCTGCCTCCGNN
33





 192-210
GGAGGCAGCGAGCGGGGGGNN
2295
CCCCCCGCTCGCTGCCTCCNN
33





 193-211
GAGGCAGCGAGCGGGGGGCNN
2296
GCCCCCCGCTCGCTGCCTCNN
33





 194-212
AGGCAGCGAGCGGGGGGCUNN
2297
AGCCCCCCGCUCGCUGCCUNN
33





 195-213
GGCAGCGAGCGGGGGGCUGNN
2298
CAGCCCCCCGCUCGCUGCCNN
33





 196-214
GCAGCGAGCGGGGGGCUGCNN
2299
GCAGCCCCCCGCUCGCUGCNN
33





 197-215
CAGCGAGCGGGGGGCUGCCNN
2300
GGCAGCCCCCCGCUCGCUGNN
33





 198-216
AGCGAGCGGGGGGCUGCCCNN
2301
GGGCAGCCCCCCGCUCGCUNN
33





 199-217
GCGAGCGGGGGGCUGCCCCNN
2302
GGGGCAGCCCCCCGCUCGCNN
33





 200-218
CGAGCGGGGGGCUGCCCCANN
2303
UGGGGCAGCCCCCCGCUCGNN
33





 201-219
GAGCGGGGGGCUGCCCCAGNN
2304
CUGGGGCAGCCCCCCGCUCNN
33





 202-220
AGCGGGGGGCUGCCCCAGGNN
2305
CCUGGGGCAGCCCCCCGCUNN
33





 203-221
GCGGGGGGCUGCCCCAGGCNN
2306
GCCUGGGGCAGCCCCCCGCNN
33





 204-222
CGGGGGGCUGCCCCAGGCGNN
2307
CGCCUGGGGCAGCCCCCCGNN
33





 205-223
GGGGGGCUGCCCCAGGCGCNN
2308
GCGCCUGGGGCAGCCCCCCNN
33





 206-224
GGGGGCUGCCCCAGGCGCGNN
2309
CGCGCCUGGGGCAGCCCCCNN
33





 207-225
GGGGCUGCCCCAGGCGCGCNN
2310
GCGCGCCUGGGGCAGCCCCNN
33





 208-226
GGGCUGCCCCAGGCGCGCANN
2311
UGCGCGCCUGGGGCAGCCCNN
33





 209-227
GGCUGCCCCAGGCGCGCAANN
2312
UUGCGCGCCUGGGGCAGCCNN
33





 210-228
GCUGCCCCAGGCGCGCAAGNN
2313
CUUGCGCGCCUGGGGCAGCNN
33





 211-229
CUGCCCCAGGCGCGCAAGCNN
2314
GCUUGCGCGCCUGGGGCAGNN
33





 212-230
UGCCCCAGGCGCGCAAGCGNN
2315
CGCUUGCGCGCCUGGGGCANN
33





 247-265
CUGAGCCCCGAGGAGAAGGNN
2316
CCUUCUCCUCGGGGCUCAGNN
33





 248-266
UGAGCCCCGAGGAGAAGGCNN
2317
GCCUUCUCCUCGGGGCUCANN
33





 249-267
GAGCCCCGAGGAGAAGGCGNN
2318
CGCCTTCTCCTCGGGGCTCNN
33





 250-268
AGCCCCGAGGAGAAGGCGCNN
2319
GCGCCTTCTCCTCGGGGCTNN
33





 251-269
GCCCCGAGGAGAAGGCGCUNN
2320
AGCGCCUUCUCCUCGGGGCNN
33





 252-270
CCCCGAGGAGAAGGCGCUGNN
2321
CAGCGCCUUCUCCUCGGGGNN
33





 253-271
CCCGAGGAGAAGGCGCUGANN
2322
UCAGCGCCUUCUCCUCGGGNN
33





 254-272
CCGAGGAGAAGGCGCUGAGNN
2323
CUCAGCGCCUUCUCCUCGGNN
33





 255-273
CGAGGAGAAGGCGCUGAGGNN
2324
CCUCAGCGCCUUCUCCUCGNN
33





 256-274
GAGGAGAAGGCGCUGAGGANN
2325
UCCUCAGCGCCUUCUCCUCNN
33





 257-275
AGGAGAAGGCGCUGAGGAGNN
2326
CUCCUCAGCGCCUUCUCCUNN
33





 258-276
GGAGAAGGCGCUGAGGAGGNN
2327
CCUCCUCAGCGCCUUCUCCNN
33





 259-277
GAGAAGGCGCUGAGGAGGANN
2328
UCCUCCUCAGCGCCUUCUCNN
33





 260-278
AGAAGGCGCUGAGGAGGAANN
2329
UUCCUCCUCAGCGCCUUCUNN
33





 261-279
GAAGGCGCUGAGGAGGAAANN
2330
UUUCCUCCUCAGCGCCUUCNN
33





 262-280
AAGGCGCUGAGGAGGAAACNN
2331
GUUUCCUCCUCAGCGCCUUNN
33





 263-281
AGGCGCUGAGGAGGAAACUNN
2332
AGUUUCCUCCUCAGCGCCUNN
33





 264-282
GGCGCUGAGGAGGAAACUGNN
2333
CAGUUUCCUCCUCAGCGCCNN
33





 265-283
GCGCUGAGGAGGAAACUGANN
2334
UCAGUUUCCUCCUCAGCGCNN
33





 266-284
CGCUGAGGAGGAAACUGAANN
2335
UUCAGUUUCCUCCUCAGCGNN
33





 267-285
GCUGAGGAGGAAACUGAAANN
2336
UUUCAGUUUCCUCCUCAGCNN
33





 268-286
CUGAGGAGGAAACUGAAAANN
2337
UUUUCAGUUUCCUCCUCAGNN
33





 269-287
UGAGGAGGAAACUGAAAAANN
2338
UUUUUCAGUUUCCUCCUCANN
33





 270-288
GAGGAGGAAACUGAAAAACNN
2339
GUUUUUCAGUUUCCUCCUCNN
33





 271-289
AGGAGGAAACUGAAAAACANN
2340
UGUUUUUCAGUUUCCUCCUNN
33





 272-290
GGAGGAAACUGAAAAACAGNN
2341
CUGUUUUUCAGUUUCCUCCNN
34





 273-291
GAGGAAACUGAAAAACAGANN
2342
UCUGUUUUUCAGUUUCCUCNN
34





 274-292
AGGAAACUGAAAAACAGAGNN
2343
CUCUGUUUUUCAGUUUCCUNN
34





 275-293
GGAAACUGAAAAACAGAGUNN
2344
ACUCUGUUUUUCAGUUUCCNN
34





 276-294
GAAACUGAAAAACAGAGUANN
2345
UACUCUGUUUUUCAGUUUCNN
34





 277-295
AAACUGAAAAACAGAGUAGNN
2346
CUACUCUGUUUUUCAGUUUNN
34





 278-296
AACUGAAAAACAGAGUAGCNN
2347
GCUACUCUGUUUUUCAGUUNN
34





 279-297
ACUGAAAAACAGAGUAGCANN
2348
UGCUACUCUGUUUUUCAGUNN
34





 280-298
CUGAAAAACAGAGUAGCAGNN
2349
CUGCUACUCUGUUUUUCAGNN
34





 281-299
UGAAAAACAGAGUAGCAGCNN
2350
GCUGCUACUCUGUUUUUCANN
34





 282-300
GAAAAACAGAGUAGCAGCUNN
2351
AGCUGCUACUCUGUUUUUCNN
34





 283-301
AAAAACAGAGUAGCAGCUCNN
2352
GAGCUGCUACUCUGUUUUUNN
34





 284-302
AAAACAGAGUAGCAGCUCANN
2353
UGAGCUGCUACUCUGUUUUNN
34





 285-303
AAACAGAGUAGCAGCUCAGNN
2354
CUGAGCUGCUACUCUGUUUNN
34





 286-304
AACAGAGUAGCAGCUCAGANN
2355
UCUGAGCUGCUACUCUGUUNN
34





 287-305
ACAGAGUAGCAGCUCAGACNN
2356
GUCUGAGCUGCUACUCUGUNN
34





 288-306
CAGAGUAGCAGCUCAGACUNN
2357
AGUCUGAGCUGCUACUCUGNN
34





 289-307
AGAGUAGCAGCUCAGACUGNN
2358
CAGUCUGAGCUGCUACUCUNN
34





 290-308
GAGUAGCAGCUCAGACUGCNN
2359
GCAGUCUGAGCUGCUACUCNN
34





 291-309
AGUAGCAGCUCAGACUGCCNN
2360
GGCAGUCUGAGCUGCUACUNN
34





 292-310
GUAGCAGCUCAGACUGCCANN
2361
UGGCAGUCUGAGCUGCUACNN
34





 293-311
UAGCAGCUCAGACUGCCAGNN
2362
CUGGCAGUCUGAGCUGCUANN
34





 294-312
AGCAGCUCAGACUGCCAGANN
2363
UCUGGCAGUCUGAGCUGCUNN
34





 295-313
GCAGCUCAGACUGCCAGAGNN
2364
CUCUGGCAGUCUGAGCUGCNN
34





 296-314
CAGCUCAGACUGCCAGAGANN
2365
UCUCUGGCAGUCUGAGCUGNN
34





 297-315
AGCUCAGACUGCCAGAGAUNN
2366
AUCUCUGGCAGUCUGAGCUNN
34





 298-316
GCUCAGACUGCCAGAGAUCNN
2367
GAUCUCUGGCAGUCUGAGCNN
34





 299-317
CUCAGACUGCCAGAGAUCGNN
2368
CGAUCUCUGGCAGUCUGAGNN
34





 300-318
UCAGACUGCCAGAGAUCGANN
2369
UCGAUCUCUGGCAGUCUGANN
34





 301-319
CAGACUGCCAGAGAUCGAANN
2370
UUCGAUCUCUGGCAGUCUGNN
34





 302-320
AGACUGCCAGAGAUCGAAANN
2371
UUUCGAUCUCUGGCAGUCUNN
34





 303-321
GACUGCCAGAGAUCGAAAGNN
2372
CUUUCGAUCUCUGGCAGUCNN
34





 304-322
ACUGCCAGAGAUCGAAAGANN
2373
UCUUUCGAUCUCUGGCAGUNN
34





 305-323
CUGCCAGAGAUCGAAAGAANN
2374
UUCUUUCGAUCUCUGGCAGNN
34





 325-343
GCUCGAAUGAGUGAGCUGGNN
2375
CCAGCUCACUCAUUCGAGCNN
34





 326-344
CUCGAAUGAGUGAGCUGGANN
2376
UCCAGCUCACUCAUUCGAGNN
34





 327-345
UCGAAUGAGUGAGCUGGAANN
2377
UUCCAGCUCACUCAUUCGANN
34





 328-346
CGAAUGAGUGAGCUGGAACNN
2378
GUUCCAGCUCACUCAUUCGNN
34





 329-347
GAAUGAGUGAGCUGGAACANN
2379
UGUUCCAGCUCACUCAUUCNN
34





 330-348
AAUGAGUGAGCUGGAACAGNN
2380
CUGUUCCAGCUCACUCAUUNN
34





 331-349
AUGAGUGAGCUGGAACAGCNN
2381
GCUGUUCCAGCUCACUCAUNN
34





 332-350
UGAGUGAGCUGGAACAGCANN
2382
UGCUGUUCCAGCUCACUCANN
34





 333-351
GAGUGAGCUGGAACAGCAANN
2383
UUGCUGUUCCAGCUCACUCNN
34





 334-352
AGUGAGCUGGAACAGCAAGNN
2384
CUUGCUGUUCCAGCUCACUNN
34





 335-353
GUGAGCUGGAACAGCAAGUNN
2385
ACUUGCUGUUCCAGCUCACNN
34





 336-354
UGAGCUGGAACAGCAAGUGNN
2386
CACUUGCUGUUCCAGCUCANN
34





 337-355
GAGCUGGAACAGCAAGUGGNN
2387
CCACUUGCUGUUCCAGCUCNN
34





 338-356
AGCUGGAACAGCAAGUGGUNN
2388
ACCACUUGCUGUUCCAGCUNN
34





 339-357
GCUGGAACAGCAAGUGGUANN
2389
UACCACUUGCUGUUCCAGCNN
34





 340-358
CUGGAACAGCAAGUGGUAGNN
2390
CUACCACUUGCUGUUCCAGNN
34





 341-359
UGGAACAGCAAGUGGUAGANN
2391
UCUACCACUUGCUGUUCCANN
34





 342-360
GGAACAGCAAGUGGUAGAUNN
2392
AUCUACCACUUGCUGUUCCNN
34





 343-361
GAACAGCAAGUGGUAGAUUNN
2393
AAUCUACCACUUGCUGUUCNN
34





 344-362
AACAGCAAGUGGUAGAUUUNN
2394
AAAUCUACCACUUGCUGUUNN
34





 345-363
ACAGCAAGUGGUAGAUUUANN
2395
UAAAUCUACCACUUGCUGUNN
34





 346-364
CAGCAAGUGGUAGAUUUAGNN
2396
CUAAAUCUACCACUUGCUGNN
34





 347-365
AGCAAGUGGUAGAUUUAGANN
2397
UCUAAAUCUACCACUUGCUNN
34





 348-366
GCAAGUGGUAGAUUUAGAANN
2398
UUCUAAAUCUACCACUUGCNN
34





 349-367
CAAGUGGUAGAUUUAGAAGNN
2399
CUUCUAAAUCUACCACUUGNN
34





 350-368
AAGUGGUAGAUUUAGAAGANN
2400
UCUUCUAAAUCUACCACUUNN
34





 351-369
AGUGGUAGAUUUAGAAGAANN
2401
UUCUUCUAAAUCUACCACUNN
34





 352-370
GUGGUAGAUUUAGAAGAAGNN
2402
CUUCUUCUAAAUCUACCACNN
34





 353-371
UGGUAGAUUUAGAAGAAGANN
2403
UCUUCUUCUAAAUCUACCANN
34





 354-372
GGUAGAUUUAGAAGAAGAGNN
2404
CUCUUCUUCUAAAUCUACCNN
34





 355-373
GUAGAUUUAGAAGAAGAGANN
2405
UCUCUUCUUCUAAAUCUACNN
34





 356-374
UAGAUUUAGAAGAAGAGAANN
2406
UUCUCUUCUUCUAAAUCUANN
34





 357-375
AGAUUUAGAAGAAGAGAACNN
2407
GUUCUCUUCUUCUAAAUCUNN
34





 358-376
GAUUUAGAAGAAGAGAACCNN
2408
GGUUCUCUUCUUCUAAAUCNN
34





 359-377
AUUUAGAAGAAGAGAACCANN
2409
UGGUUCUCUUCUUCUAAAUNN
34





 360-378
UUUAGAAGAAGAGAACCAANN
2410
UUGGUUCUCUUCUUCUAAANN
34





 361-379
UUAGAAGAAGAGAACCAAANN
2411
UUUGGUUCUCUUCUUCUAANN
34





 362-380
UAGAAGAAGAGAACCAAAANN
2412
UUUUGGUUCUCUUCUUCUANN
34





 363-381
AGAAGAAGAGAACCAAAAANN
2413
TTTTTGGTTCTCTTCTTCTNN
34





 364-382
GAAGAAGAGAACCAAAAACNN
2414
GTTTTTGGTTCTCTTCTTCNN
34





 365-383
AAGAAGAGAACCAAAAACUNN
2415
AGUUUUUGGUUCUCUUCUUNN
34





 366-384
AGAAGAGAACCAAAAACUUNN
2416
AAGUUUUUGGUUCUCUUCUNN
34





 367-385
GAAGAGAACCAAAAACUUUNN
2417
AAAGUUUUUGGUUCUCUUCNN
34





 368-386
AAGAGAACCAAAAACUUUUNN
2418
AAAAGUUUUUGGUUCUCUUNN
34





 369-387
AGAGAACCAAAAACUUUUGNN
2419
CAAAAGUUUUUGGUUCUCUNN
34





 370-388
GAGAACCAAAAACUUUUGCNN
2420
GCAAAAGUUUUUGGUUCUCNN
34





 371-389
AGAACCAAAAACUUUUGCUNN
2421
AGCAAAAGUUUUUGGUUCUNN
34





 372-390
GAACCAAAAACUUUUGCUANN
2422
UAGCAAAAGUUUUUGGUUCNN
34





 373-391
AACCAAAAACUUUUGCUAGNN
2423
CUAGCAAAAGUUUUUGGUUNN
34





 374-392
ACCAAAAACUUUUGCUAGANN
2424
UCUAGCAAAAGUUUUUGGUNN
34





 375-393
CCAAAAACUUUUGCUAGAANN
2425
UUCUAGCAAAAGUUUUUGGNN
34





 376-394
CAAAAACUUUUGCUAGAAANN
2426
UUUCUAGCAAAAGUUUUUGNN
34





 377-395
AAAAACUUUUGCUAGAAAANN
2427
UUUUCUAGCAAAAGUUUUUNN
34





 378-396
AAAACUUUUGCUAGAAAAUNN
2428
AUUUUCUAGCAAAAGUUUUNN
34





 379-397
AAACUUUUGCUAGAAAAUCNN
2429
GAUUUUCUAGCAAAAGUUUNN
34





 380-398
AACUUUUGCUAGAAAAUCANN
2430
UGAUUUUCUAGCAAAAGUUNN
34





 381-399
ACUUUUGCUAGAAAAUCAGNN
2431
CUGAUUUUCUAGCAAAAGUNN
34





 382-400
CUUUUGCUAGAAAAUCAGCNN
2432
GCUGAUUUUCUAGCAAAAGNN
34





 383-401
UUUUGCUAGAAAAUCAGCUNN
2433
AGCUGAUUUUCUAGCAAAANN
34





 384-402
UUUGCUAGAAAAUCAGCUUNN
2434
AAGCUGAUUUUCUAGCAAANN
34





 385-403
UUGCUAGAAAAUCAGCUUUNN
2435
AAAGCUGAUUUUCUAGCAANN
34





 386-404
UGCUAGAAAAUCAGCUUUUNN
2436
AAAAGCUGAUUUUCUAGCANN
34





 387-405
GCUAGAAAAUCAGCUUUUANN
2437
UAAAAGCUGAUUUUCUAGCNN
34





 388-406
CUAGAAAAUCAGCUUUUACNN
2438
GUAAAAGCUGAUUUUCUAGNN
34





 389-407
UAGAAAAUCAGCUUUUACGNN
2439
CGUAAAAGCUGAUUUUCUANN
34





 390-408
AGAAAAUCAGCUUUUACGANN
2440
UCGUAAAAGCUGAUUUUCUNN
34





 391-409
GAAAAUCAGCUUUUACGAGNN
2441
CUCGUAAAAGCUGAUUUUCNN
35





 392-410
AAAAUCAGCUUUUACGAGANN
2442
UCUCGUAAAAGCUGAUUUUNN
35





 393-411
AAAUCAGCUUUUACGAGAGNN
2443
CUCUCGUAAAAGCUGAUUUNN
35





 394-412
AAUCAGCUUUUACGAGAGANN
2444
UCUCUCGUAAAAGCUGAUUNN
35





 395-413
AUCAGCUUUUACGAGAGAANN
2445
UUCUCUCGUAAAAGCUGAUNN
35





 396-414
UCAGCUUUUACGAGAGAAANN
2446
UUUCUCUCGUAAAAGCUGANN
35





 397-415
CAGCUUUUACGAGAGAAAANN
2447
UUUUCUCUCGUAAAAGCUGNN
35





 398-416
AGCUUUUACGAGAGAAAACNN
2448
GUUUUCUCUCGUAAAAGCUNN
35





 399-417
GCUUUUACGAGAGAAAACUNN
2449
AGUUUUCUCUCGUAAAAGCNN
35





 400-418
CUUUUACGAGAGAAAACUCNN
2450
GAGUUUUCUCUCGUAAAAGNN
35





 401-419
UUUUACGAGAGAAAACUCANN
2451
UGAGUUUUCUCUCGUAAAANN
35





 421-439
GGCCUUGUAGUUGAGAACCNN
2452
GGUUCUCAACUACAAGGCCNN
35





 422-440
GCCUUGUAGUUGAGAACCANN
2453
UGGUUCUCAACUACAAGGCNN
35





 423-441
CCUUGUAGUUGAGAACCAGNN
2454
CUGGUUCUCAACUACAAGGNN
35





 424-442
CUUGUAGUUGAGAACCAGGNN
2455
CCUGGUUCUCAACUACAAGNN
35





 425-443
UUGUAGUUGAGAACCAGGANN
2456
UCCUGGUUCUCAACUACAANN
35





 426-444
UGUAGUUGAGAACCAGGAGNN
2457
CUCCUGGUUCUCAACUACANN
35





 427-445
GUAGUUGAGAACCAGGAGUNN
2458
ACUCCUGGUUCUCAACUACNN
35





 428-446
UAGUUGAGAACCAGGAGUUNN
2459
AACUCCUGGUUCUCAACUANN
35





 429-447
AGUUGAGAACCAGGAGUUANN
2460
UAACUCCUGGUUCUCAACUNN
35





 430-448
GUUGAGAACCAGGAGUUAANN
2461
UUAACUCCUGGUUCUCAACNN
35





 431-449
UUGAGAACCAGGAGUUAAGNN
2462
CUUAACUCCUGGUUCUCAANN
35





 432-450
UGAGAACCAGGAGUUAAGANN
2463
UCUUAACUCCUGGUUCUCANN
35





 433-451
GAGAACCAGGAGUUAAGACNN
2464
GUCUUAACUCCUGGUUCUCNN
35





 434-452
AGAACCAGGAGUUAAGACANN
2465
UGUCUUAACUCCUGGUUCUNN
35





 435-453
GAACCAGGAGUUAAGACAGNN
2466
CUGUCUUAACUCCUGGUUCNN
35





 436-454
AACCAGGAGUUAAGACAGCNN
2467
GCUGUCUUAACUCCUGGUUNN
35





 437-455
ACCAGGAGUUAAGACAGCGNN
2468
CGCUGUCUUAACUCCUGGUNN
35





 438-456
CCAGGAGUUAAGACAGCGCNN
2469
GCGCUGUCUUAACUCCUGGNN
35





  44-62
GAGCUAUGGUGGUGGUGGCNN
2470
GCCACCACCACCAUAGCUCNN
35





  45-63
AGCUAUGGUGGUGGUGGCANN
2471
UGCCACCACCACCAUAGCUNN
35





 458-476
UGGGGAUGGAUGCCCUGGUNN
2472
ACCAGGGCAUCCAUCCCCANN
35





 459-477
GGGGAUGGAUGCCCUGGUUNN
2473
AACCAGGGCAUCCAUCCCCNN
35





 460-478
GGGAUGGAUGCCCUGGUUGNN
2474
CAACCAGGGCAUCCAUCCCNN
35





 461-479
GGAUGGAUGCCCUGGUUGCNN
2475
GCAACCAGGGCAUCCAUCCNN
35





 462-480
GAUGGAUGCCCUGGUUGCUNN
2476
AGCAACCAGGGCAUCCAUCNN
35





  46-64
GCUAUGGUGGUGGUGGCAGNN
2477
CUGCCACCACCACCAUAGCNN
35





  47-65
CUAUGGUGGUGGUGGCAGCNN
2478
GCUGCCACCACCACCAUAGNN
35





 482-500
AAGAGGAGGCGGAAGCCAANN
2479
TTGGCTTCCGCCTCCTCTTNN
35





 483-501
AGAGGAGGCGGAAGCCAAGNN
2480
CTTGGCTTCCGCCTCCTCTNN
35





 484-502
GAGGAGGCGGAAGCCAAGGNN
2481
CCTTGGCTTCCGCCTCCTCNN
35





 485-503
AGGAGGCGGAAGCCAAGGGNN
2482
CCCTTGGCTTCCGCCTCCTNN
35





 486-504
GGAGGCGGAAGCCAAGGGGNN
2483
CCCCTTGGCTTCCGCCTCCNN
35





  48-66
UAUGGUGGUGGUGGCAGCCNN
2484
GGCUGCCACCACCACCAUANN
35





 487-505
GAGGCGGAAGCCAAGGGGANN
2485
TCCCCTTGGCTTCCGCCTCNN
35





 488-506
AGGCGGAAGCCAAGGGGAANN
2486
TTCCCCTTGGCTTCCGCCTNN
35





 489-507
GGCGGAAGCCAAGGGGAAUNN
2487
AUUCCCCUUGGCUUCCGCCNN
35





 490-508
GCGGAAGCCAAGGGGAAUGNN
2488
CAUUCCCCUUGGCUUCCGCNN
35





  49-67
AUGGUGGUGGUGGCAGCCGNN
2489
CGGCUGCCACCACCACCAUNN
35





  50-68
UGGUGGUGGUGGCAGCCGCNN
2490
GCGGCUGCCACCACCACCANN
35





 510-528
AGUGAGGCCAGUGGCCGGGNN
2491
CCCGGCCACUGGCCUCACUNN
35





 511-529
GUGAGGCCAGUGGCCGGGUNN
2492
ACCCGGCCACUGGCCUCACNN
35





 512-530
UGAGGCCAGUGGCCGGGUCNN
2493
GACCCGGCCACUGGCCUCANN
35





 513-531
GAGGCCAGUGGCCGGGUCUNN
2494
AGACCCGGCCACUGGCCUCNN
35





 514-532
AGGCCAGUGGCCGGGUCUGNN
2495
CAGACCCGGCCACUGGCCUNN
35





 515-533
GGCCAGUGGCCGGGUCUGCNN
2496
GCAGACCCGGCCACUGGCCNN
35





 516-534
GCCAGUGGCCGGGUCUGCUNN
2497
AGCAGACCCGGCCACUGGCNN
35





 517-535
CCAGUGGCCGGGUCUGCUGNN
2498
CAGCAGACCCGGCCACUGGNN
35





 518-536
CAGUGGCCGGGUCUGCUGANN
2499
UCAGCAGACCCGGCCACUGNN
35





 519-537
AGUGGCCGGGUCUGCUGAGNN
2500
CUCAGCAGACCCGGCCACUNN
35





 520-538
GUGGCCGGGUCUGCUGAGUNN
2501
ACUCAGCAGACCCGGCCACNN
35





 521-539
UGGCCGGGUCUGCUGAGUCNN
2502
GACUCAGCAGACCCGGCCANN
35





 522-540
GGCCGGGUCUGCUGAGUCCNN
2503
GGACUCAGCAGACCCGGCCNN
35





 523-541
GCCGGGUCUGCUGAGUCCGNN
2504
CGGACUCAGCAGACCCGGCNN
35





 524-542
CCGGGUCUGCUGAGUCCGCNN
2505
GCGGACUCAGCAGACCCGGNN
35





 525-543
CGGGUCUGCUGAGUCCGCANN
2506
UGCGGACUCAGCAGACCCGNN
35





 526-544
GGGUCUGCUGAGUCCGCAGNN
2507
CUGCGGACUCAGCAGACCCNN
35





 574-592
GUGCAGGCCCAGUUGUCACNN
2508
GUGACAACUGGGCCUGCACNN
35





 575-593
UGCAGGCCCAGUUGUCACCNN
2509
GGUGACAACUGGGCCUGCANN
35





 576-594
GCAGGCCCAGUUGUCACCCNN
2510
GGGUGACAACUGGGCCUGCNN
35





 577-595
CAGGCCCAGUUGUCACCCCNN
2511
GGGGUGACAACUGGGCCUGNN
35





 578-596
AGGCCCAGUUGUCACCCCUNN
2512
AGGGGUGACAACUGGGCCUNN
35





 579-597
GGCCCAGUUGUCACCCCUCNN
2513
GAGGGGUGACAACUGGGCCNN
35





 580-598
GCCCAGUUGUCACCCCUCCNN
2514
GGAGGGGUGACAACUGGGCNN
35





 581-599
CCCAGUUGUCACCCCUCCANN
2515
UGGAGGGGUGACAACUGGGNN
35





 582-600
CCAGUUGUCACCCCUCCAGNN
2516
CUGGAGGGGUGACAACUGGNN
35





 583-601
CAGUUGUCACCCCUCCAGANN
2517
UCUGGAGGGGUGACAACUGNN
35





 584-602
AGUUGUCACCCCUCCAGAANN
2518
UUCUGGAGGGGUGACAACUNN
35





 585-603
GUUGUCACCCCUCCAGAACNN
2519
GUUCUGGAGGGGUGACAACNN
35





 586-604
UUGUCACCCCUCCAGAACANN
2520
UGUUCUGGAGGGGUGACAANN
35





 587-605
UGUCACCCCUCCAGAACAUNN
2521
AUGUUCUGGAGGGGUGACANN
35





 588-606
GUCACCCCUCCAGAACAUCNN
2522
GAUGUUCUGGAGGGGUGACNN
35





 589-607
UCACCCCUCCAGAACAUCUNN
2523
AGAUGUUCUGGAGGGGUGANN
35





 590-608
CACCCCUCCAGAACAUCUCNN
2524
GAGAUGUUCUGGAGGGGUGNN
35





 591-609
ACCCCUCCAGAACAUCUCCNN
2525
GGAGAUGUUCUGGAGGGGUNN
35





 592-610
CCCCUCCAGAACAUCUCCCNN
2526
GGGAGAUGUUCUGGAGGGGNN
35





 593-611
CCCUCCAGAACAUCUCCCCNN
2527
GGGGAGAUGUUCUGGAGGGNN
35





 594-612
CCUCCAGAACAUCUCCCCANN
2528
UGGGGAGAUGUUCUGGAGGNN
35





 595-613
CUCCAGAACAUCUCCCCAUNN
2529
AUGGGGAGAUGUUCUGGAGNN
35





 596-614
UCCAGAACAUCUCCCCAUGNN
2530
CAUGGGGAGAUGUUCUGGANN
35





 597-615
CCAGAACAUCUCCCCAUGGNN
2531
CCAUGGGGAGAUGUUCUGGNN
35





 598-616
CAGAACAUCUCCCCAUGGANN
2532
UCCAUGGGGAGAUGUUCUGNN
35





 599-617
AGAACAUCUCCCCAUGGAUNN
2533
AUCCAUGGGGAGAUGUUCUNN
35





 600-618
GAACAUCUCCCCAUGGAUUNN
2534
AAUCCAUGGGGAGAUGUUCNN
35





 601-619
AACAUCUCCCCAUGGAUUCNN
2535
GAAUCCAUGGGGAGAUGUUNN
35





 602-620
ACAUCUCCCCAUGGAUUCUNN
2536
AGAAUCCAUGGGGAGAUGUNN
35





 603-621
CAUCUCCCCAUGGAUUCUGNN
2537
CAGAAUCCAUGGGGAGAUGNN
35





 604-622
AUCUCCCCAUGGAUUCUGGNN
2538
CCAGAAUCCAUGGGGAGAUNN
35





 605-623
UCUCCCCAUGGAUUCUGGCNN
2539
GCCAGAAUCCAUGGGGAGANN
35





 606-624
CUCCCCAUGGAUUCUGGCGNN
2540
CGCCAGAAUCCAUGGGGAGNN
35





 607-625
UCCCCAUGGAUUCUGGCGGNN
2541
CCGCCAGAAUCCAUGGGGANN
36





 608-626
CCCCAUGGAUUCUGGCGGUNN
2542
ACCGCCAGAAUCCAUGGGGNN
36





 609-627
CCCAUGGAUUCUGGCGGUANN
2543
UACCGCCAGAAUCCAUGGGNN
36





 610-628
CCAUGGAUUCUGGCGGUAUNN
2544
AUACCGCCAGAAUCCAUGGNN
36





 611-629
CAUGGAUUCUGGCGGUAUUNN
2545
AAUACCGCCAGAAUCCAUGNN
36





 612-630
AUGGAUUCUGGCGGUAUUGNN
2546
CAAUACCGCCAGAAUCCAUNN
36





 613-631
UGGAUUCUGGCGGUAUUGANN
2547
UCAAUACCGCCAGAAUCCANN
36





 614-632
GGAUUCUGGCGGUAUUGACNN
2548
GUCAAUACCGCCAGAAUCCNN
36





 615-633
GAUUCUGGCGGUAUUGACUNN
2549
AGUCAAUACCGCCAGAAUCNN
36





 616-634
AUUCUGGCGGUAUUGACUCNN
2550
GAGUCAAUACCGCCAGAAUNN
36





 617-635
UUCUGGCGGUAUUGACUCUNN
2551
AGAGUCAAUACCGCCAGAANN
36





 618-636
UCUGGCGGUAUUGACUCUUNN
2552
AAGAGUCAAUACCGCCAGANN
36





 619-637
CUGGCGGUAUUGACUCUUCNN
2553
GAAGAGUCAAUACCGCCAGNN
36





 620-638
UGGCGGUAUUGACUCUUCANN
2554
UGAAGAGUCAAUACCGCCANN
36





 621-639
GGCGGUAUUGACUCUUCAGNN
2555
CUGAAGAGUCAAUACCGCCNN
36





 622-640
GCGGUAUUGACUCUUCAGANN
2556
UCUGAAGAGUCAAUACCGCNN
36





 623-641
CGGUAUUGACUCUUCAGAUNN
2557
AUCUGAAGAGUCAAUACCGNN
36





 624-642
GGUAUUGACUCUUCAGAUUNN
2558
AAUCUGAAGAGUCAAUACCNN
36





 625-643
GUAUUGACUCUUCAGAUUCNN
2559
GAAUCUGAAGAGUCAAUACNN
36





 626-644
UAUUGACUCUUCAGAUUCANN
2560
UGAAUCUGAAGAGUCAAUANN
36





 627-645
AUUGACUCUUCAGAUUCAGNN
2561
CUGAAUCUGAAGAGUCAAUNN
36





 628-646
UUGACUCUUCAGAUUCAGANN
2562
UCUGAAUCUGAAGAGUCAANN
36





 629-647
UGACUCUUCAGAUUCAGAGNN
2563
CUCUGAAUCUGAAGAGUCANN
36





 630-648
GACUCUUCAGAUUCAGAGUNN
2564
ACUCUGAAUCUGAAGAGUCNN
36





 631-649
ACUCUUCAGAUUCAGAGUCNN
2565
GACUCUGAAUCUGAAGAGUNN
36





 632-650
CUCUUCAGAUUCAGAGUCUNN
2566
AGACUCUGAAUCUGAAGAGNN
36





 633-651
UCUUCAGAUUCAGAGUCUGNN
2567
CAGACUCUGAAUCUGAAGANN
36





 634-652
CUUCAGAUUCAGAGUCUGANN
2568
UCAGACUCUGAAUCUGAAGNN
36





 635-653
UUCAGAUUCAGAGUCUGAUNN
2569
AUCAGACUCUGAAUCUGAANN
36





 636-654
UCAGAUUCAGAGUCUGAUANN
2570
UAUCAGACUCUGAAUCUGANN
36





 637-655
CAGAUUCAGAGUCUGAUAUNN
2571
AUAUCAGACUCUGAAUCUGNN
36





 638-656
AGAUUCAGAGUCUGAUAUCNN
2572
GAUAUCAGACUCUGAAUCUNN
36





 639-657
GAUUCAGAGUCUGAUAUCCNN
2573
GGAUAUCAGACUCUGAAUCNN
36





 640-658
AUUCAGAGUCUGAUAUCCUNN
2574
AGGAUAUCAGACUCUGAAUNN
36





 641-659
UUCAGAGUCUGAUAUCCUGNN
2575
CAGGAUAUCAGACUCUGAANN
36





 642-660
UCAGAGUCUGAUAUCCUGUNN
2576
ACAGGAUAUCAGACUCUGANN
36





 643-661
CAGAGUCUGAUAUCCUGUUNN
2577
AACAGGAUAUCAGACUCUGNN
36





 644-662
AGAGUCUGAUAUCCUGUUGNN
2578
CAACAGGAUAUCAGACUCUNN
36





 645-663
GAGUCUGAUAUCCUGUUGGNN
2579
CCAACAGGAUAUCAGACUCNN
36





 646-664
AGUCUGAUAUCCUGUUGGGNN
2580
CCCAACAGGAUAUCAGACUNN
36





 647-665
GUCUGAUAUCCUGUUGGGCNN
2581
GCCCAACAGGAUAUCAGACNN
36





 648-666
UCUGAUAUCCUGUUGGGCANN
2582
UGCCCAACAGGAUAUCAGANN
36





 649-667
CUGAUAUCCUGUUGGGCAUNN
2583
AUGCCCAACAGGAUAUCAGNN
36





 650-668
UGAUAUCCUGUUGGGCAUUNN
2584
AAUGCCCAACAGGAUAUCANN
36





 651-669
GAUAUCCUGUUGGGCAUUCNN
2585
GAAUGCCCAACAGGAUAUCNN
36





 652-670
AUAUCCUGUUGGGCAUUCUNN
2586
AGAAUGCCCAACAGGAUAUNN
36





 653-671
UAUCCUGUUGGGCAUUCUGNN
2587
CAGAAUGCCCAACAGGAUANN
36





 654-672
AUCCUGUUGGGCAUUCUGGNN
2588
CCAGAAUGCCCAACAGGAUNN
36





 655-673
UCCUGUUGGGCAUUCUGGANN
2589
UCCAGAAUGCCCAACAGGANN
36





 656-674
CCUGUUGGGCAUUCUGGACNN
2590
GUCCAGAAUGCCCAACAGGNN
36





 657-675
CUGUUGGGCAUUCUGGACANN
2591
UGUCCAGAAUGCCCAACAGNN
36





 658-676
UGUUGGGCAUUCUGGACAANN
2592
UUGUCCAGAAUGCCCAACANN
36





 659-677
GUUGGGCAUUCUGGACAACNN
2593
GUUGUCCAGAAUGCCCAACNN
36





 660-678
UUGGGCAUUCUGGACAACUNN
2594
AGUUGUCCAGAAUGCCCAANN
36





 661-679
UGGGCAUUCUGGACAACUUNN
2595
AAGUUGUCCAGAAUGCCCANN
36





 662-680
GGGCAUUCUGGACAACUUGNN
2596
CAAGUUGUCCAGAAUGCCCNN
36





 663-681
GGCAUUCUGGACAACUUGGNN
2597
CCAAGUUGUCCAGAAUGCCNN
36





 664-682
GCAUUCUGGACAACUUGGANN
2598
UCCAAGUUGUCCAGAAUGCNN
36





 665-683
CAUUCUGGACAACUUGGACNN
2599
GUCCAAGUUGUCCAGAAUGNN
36





 666-684
AUUCUGGACAACUUGGACCNN
2600
GGUCCAAGUUGUCCAGAAUNN
36





 667-685
UUCUGGACAACUUGGACCCNN
2601
GGGUCCAAGUUGUCCAGAANN
36





 668-686
UCUGGACAACUUGGACCCANN
2602
UGGGUCCAAGUUGUCCAGANN
36





 669-687
CUGGACAACUUGGACCCAGNN
2603
CUGGGUCCAAGUUGUCCAGNN
36





 670-688
UGGACAACUUGGACCCAGUNN
2604
ACUGGGUCCAAGUUGUCCANN
36





 671-689
GGACAACUUGGACCCAGUCNN
2605
GACUGGGUCCAAGUUGUCCNN
36





 672-690
GACAACUUGGACCCAGUCANN
2606
UGACUGGGUCCAAGUUGUCNN
36





 673-691
ACAACUUGGACCCAGUCAUNN
2607
AUGACUGGGUCCAAGUUGUNN
36





 674-692
CAACUUGGACCCAGUCAUGNN
2608
CAUGACUGGGUCCAAGUUGNN
36





 675-693
AACUUGGACCCAGUCAUGUNN
2609
ACAUGACUGGGUCCAAGUUNN
36





 676-694
ACUUGGACCCAGUCAUGUUNN
2610
AACAUGACUGGGUCCAAGUNN
36





 677-695
CUUGGACCCAGUCAUGUUCNN
2611
GAACAUGACUGGGUCCAAGNN
36





 678-696
UUGGACCCAGUCAUGUUCUNN
2612
AGAACAUGACUGGGUCCAANN
36





 679-697
UGGACCCAGUCAUGUUCUUNN
2613
AAGAACAUGACUGGGUCCANN
36





 680-698
GGACCCAGUCAUGUUCUUCNN
2614
GAAGAACAUGACUGGGUCCNN
36





 681-699
GACCCAGUCAUGUUCUUCANN
2615
UGAAGAACAUGACUGGGUCNN
36





 682-700
ACCCAGUCAUGUUCUUCAANN
2616
UUGAAGAACAUGACUGGGUNN
36





 683-701
CCCAGUCAUGUUCUUCAAANN
2617
UUUGAAGAACAUGACUGGGNN
36





 684-702
CCAGUCAUGUUCUUCAAAUNN
2618
AUUUGAAGAACAUGACUGGNN
36





 685-703
CAGUCAUGUUCUUCAAAUGNN
2619
CAUUUGAAGAACAUGACUGNN
36





 686-704
AGUCAUGUUCUUCAAAUGCNN
2620
GCAUUUGAAGAACAUGACUNN
36





 687-705
GUCAUGUUCUUCAAAUGCCNN
2621
GGCAUUUGAAGAACAUGACNN
36





 688-706
UCAUGUUCUUCAAAUGCCCNN
2622
GGGCAUUUGAAGAACAUGANN
36





 689-707
CAUGUUCUUCAAAUGCCCUNN
2623
AGGGCAUUUGAAGAACAUGNN
36





 690-708
AUGUUCUUCAAAUGCCCUUNN
2624
AAGGGCAUUUGAAGAACAUNN
36





 691-709
UGUUCUUCAAAUGCCCUUCNN
2625
GAAGGGCAUUUGAAGAACANN
36





 692-710
GUUCUUCAAAUGCCCUUCCNN
2626
GGAAGGGCAUUUGAAGAACNN
36





 693-711
UUCUUCAAAUGCCCUUCCCNN
2627
GGGAAGGGCAUUUGAAGAANN
36





 694-712
UCUUCAAAUGCCCUUCCCCNN
2628
GGGGAAGGGCAUUUGAAGANN
36





 695-713
CUUCAAAUGCCCUUCCCCANN
2629
UGGGGAAGGGCAUUUGAAGNN
36





 696-714
UUCAAAUGCCCUUCCCCAGNN
2630
CUGGGGAAGGGCAUUUGAANN
36





 697-715
UCAAAUGCCCUUCCCCAGANN
2631
UCUGGGGAAGGGCAUUUGANN
36





 698-716
CAAAUGCCCUUCCCCAGAGNN
2632
CUCUGGGGAAGGGCAUUUGNN
36





 718-736
CUGCCAGCCUGGAGGAGCUNN
2633
AGCUCCUCCAGGCUGGCAGNN
36





 719-737
UGCCAGCCUGGAGGAGCUCNN
2634
GAGCUCCUCCAGGCUGGCANN
36





 720-738
GCCAGCCUGGAGGAGCUCCNN
2635
GGAGCUCCUCCAGGCUGGCNN
36





 721-739
CCAGCCUGGAGGAGCUCCCNN
2636
GGGAGCUCCUCCAGGCUGGNN
36





 722-740
CAGCCUGGAGGAGCUCCCANN
2637
UGGGAGCUCCUCCAGGCUGNN
36





 723-741
AGCCUGGAGGAGCUCCCAGNN
2638
CUGGGAGCUCCUCCAGGCUNN
36





 724-742
GCCUGGAGGAGCUCCCAGANN
2639
UCUGGGAGCUCCUCCAGGCNN
36





 725-743
CCUGGAGGAGCUCCCAGAGNN
2640
CUCUGGGAGCUCCUCCAGGNN
36





 726-744
CUGGAGGAGCUCCCAGAGGNN
2641
CCUCUGGGAGCUCCUCCAGNN
37





 727-745
UGGAGGAGCUCCCAGAGGUNN
2642
ACCUCUGGGAGCUCCUCCANN
37





 728-746
GGAGGAGCUCCCAGAGGUCNN
2643
GACCUCUGGGAGCUCCUCCNN
37





 729-747
GAGGAGCUCCCAGAGGUCUNN
2644
AGACCUCUGGGAGCUCCUCNN
37





 730-748
AGGAGCUCCCAGAGGUCUANN
2645
UAGACCUCUGGGAGCUCCUNN
37





 731-749
GGAGCUCCCAGAGGUCUACNN
2646
GUAGACCUCUGGGAGCUCCNN
37





 732-750
GAGCUCCCAGAGGUCUACCNN
2647
GGUAGACCUCUGGGAGCUCNN
37





 733-751
AGCUCCCAGAGGUCUACCCNN
2648
GGGUAGACCUCUGGGAGCUNN
37





 734-752
GCUCCCAGAGGUCUACCCANN
2649
UGGGUAGACCUCUGGGAGCNN
37





 735-753
CUCCCAGAGGUCUACCCAGNN
2650
CUGGGUAGACCUCUGGGAGNN
37





 736-754
UCCCAGAGGUCUACCCAGANN
2651
UCUGGGUAGACCUCUGGGANN
37





 737-755
CCCAGAGGUCUACCCAGAANN
2652
UUCUGGGUAGACCUCUGGGNN
37





 738-756
CCAGAGGUCUACCCAGAAGNN
2653
CUUCUGGGUAGACCUCUGGNN
37





 739-757
CAGAGGUCUACCCAGAAGGNN
2654
CCUUCUGGGUAGACCUCUGNN
37





 740-758
AGAGGUCUACCCAGAAGGANN
2655
UCCUUCUGGGUAGACCUCUNN
37





 741-759
GAGGUCUACCCAGAAGGACNN
2656
GUCCUUCUGGGUAGACCUCNN
37





 742-760
AGGUCUACCCAGAAGGACCNN
2657
GGUCCUUCUGGGUAGACCUNN
37





 743-761
GGUCUACCCAGAAGGACCCNN
2658
GGGUCCUUCUGGGUAGACCNN
37





 744-762
GUCUACCCAGAAGGACCCANN
2659
UGGGUCCUUCUGGGUAGACNN
37





 745-763
UCUACCCAGAAGGACCCAGNN
2660
CUGGGUCCUUCUGGGUAGANN
37





 746-764
CUACCCAGAAGGACCCAGUNN
2661
ACUGGGUCCUUCUGGGUAGNN
37





 747-765
UACCCAGAAGGACCCAGUUNN
2662
AACUGGGUCCUUCUGGGUANN
37





 748-766
ACCCAGAAGGACCCAGUUCNN
2663
GAACUGGGUCCUUCUGGGUNN
37





 749-767
CCCAGAAGGACCCAGUUCCNN
2664
GGAACUGGGUCCUUCUGGGNN
37





 750-768
CCAGAAGGACCCAGUUCCUNN
2665
AGGAACUGGGUCCUUCUGGNN
37





 751-769
CAGAAGGACCCAGUUCCUUNN
2666
AAGGAACUGGGUCCUUCUGNN
37





 752-770
AGAAGGACCCAGUUCCUUANN
2667
UAAGGAACUGGGUCCUUCUNN
37





 753-771
GAAGGACCCAGUUCCUUACNN
2668
GUAAGGAACUGGGUCCUUCNN
37





 754-772
AAGGACCCAGUUCCUUACCNN
2669
GGUAAGGAACUGGGUCCUUNN
37





 755-773
AGGACCCAGUUCCUUACCANN
2670
UGGUAAGGAACUGGGUCCUNN
37





 756-774
GGACCCAGUUCCUUACCAGNN
2671
CUGGUAAGGAACUGGGUCCNN
37





 757-775
GACCCAGUUCCUUACCAGCNN
2672
GCUGGUAAGGAACUGGGUCNN
37





 758-776
ACCCAGUUCCUUACCAGCCNN
2673
GGCUGGUAAGGAACUGGGUNN
37





 759-777
CCCAGUUCCUUACCAGCCUNN
2674
AGGCUGGUAAGGAACUGGGNN
37





 760-778
CCAGUUCCUUACCAGCCUCNN
2675
GAGGCUGGUAAGGAACUGGNN
37





 761-779
CAGUUCCUUACCAGCCUCCNN
2676
GGAGGCUGGUAAGGAACUGNN
37





 762-780
AGUUCCUUACCAGCCUCCCNN
2677
GGGAGGCUGGUAAGGAACUNN
37





 763-781
GUUCCUUACCAGCCUCCCUNN
2678
AGGGAGGCUGGUAAGGAACNN
37





 764-782
UUCCUUACCAGCCUCCCUUNN
2679
AAGGGAGGCUGGUAAGGAANN
37





 765-783
UCCUUACCAGCCUCCCUUUNN
2680
AAAGGGAGGCUGGUAAGGANN
37





 766-784
CCUUACCAGCCUCCCUUUCNN
2681
GAAAGGGAGGCUGGUAAGGNN
37





 767-785
CUUACCAGCCUCCCUUUCUNN
2682
AGAAAGGGAGGCUGGUAAGNN
37





 768-786
UUACCAGCCUCCCUUUCUCNN
2683
GAGAAAGGGAGGCUGGUAANN
37





 769-787
UACCAGCCUCCCUUUCUCUNN
2684
AGAGAAAGGGAGGCUGGUANN
37





 770-788
ACCAGCCUCCCUUUCUCUGNN
2685
CAGAGAAAGGGAGGCUGGUNN
37





 771-789
CCAGCCUCCCUUUCUCUGUNN
2686
ACAGAGAAAGGGAGGCUGGNN
37





 772-790
CAGCCUCCCUUUCUCUGUCNN
2687
GACAGAGAAAGGGAGGCUGNN
37





 773-791
AGCCUCCCUUUCUCUGUCANN
2688
UGACAGAGAAAGGGAGGCUNN
37





 774-792
GCCUCCCUUUCUCUGUCAGNN
2689
CUGACAGAGAAAGGGAGGCNN
37





 775-793
CCUCCCUUUCUCUGUCAGUNN
2690
ACUGACAGAGAAAGGGAGGNN
37





 776-794
CUCCCUUUCUCUGUCAGUGNN
2691
CACUGACAGAGAAAGGGAGNN
37





 777-795
UCCCUUUCUCUGUCAGUGGNN
2692
CCACUGACAGAGAAAGGGANN
37





 778-796
CCCUUUCUCUGUCAGUGGGNN
2693
CCCACUGACAGAGAAAGGGNN
37





 779-797
CCUUUCUCUGUCAGUGGGGNN
2694
CCCCACUGACAGAGAAAGGNN
37





 780-798
CUUUCUCUGUCAGUGGGGANN
2695
UCCCCACUGACAGAGAAAGNN
37





 781-799
UUUCUCUGUCAGUGGGGACNN
2696
GUCCCCACUGACAGAGAAANN
37





 782-800
UUCUCUGUCAGUGGGGACGNN
2697
CGUCCCCACUGACAGAGAANN
37





 783-801
UCUCUGUCAGUGGGGACGUNN
2698
ACGUCCCCACUGACAGAGANN
37





 784-802
CUCUGUCAGUGGGGACGUCNN
2699
GACGUCCCCACUGACAGAGNN
37





 785-803
UCUGUCAGUGGGGACGUCANN
2700
UGACGUCCCCACUGACAGANN
37





 786-804
CUGUCAGUGGGGACGUCAUNN
2701
AUGACGUCCCCACUGACAGNN
37





 787-805
UGUCAGUGGGGACGUCAUCNN
2702
GAUGACGUCCCCACUGACANN
37





 788-806
GUCAGUGGGGACGUCAUCANN
2703
UGAUGACGUCCCCACUGACNN
37





 789-807
UCAGUGGGGACGUCAUCAGNN
2704
CUGAUGACGUCCCCACUGANN
37





 790-808
CAGUGGGGACGUCAUCAGCNN
2705
GCUGAUGACGUCCCCACUGNN
37





 791-809
AGUGGGGACGUCAUCAGCCNN
2706
GGCUGAUGACGUCCCCACUNN
37





 792-810
GUGGGGACGUCAUCAGCCANN
2707
UGGCUGAUGACGUCCCCACNN
37





 793-811
UGGGGACGUCAUCAGCCAANN
2708
UUGGCUGAUGACGUCCCCANN
37





 794-812
GGGGACGUCAUCAGCCAAGNN
2709
CUUGGCUGAUGACGUCCCCNN
37





 795-813
GGGACGUCAUCAGCCAAGCNN
2710
GCUUGGCUGAUGACGUCCCNN
37





 796-814
GGACGUCAUCAGCCAAGCUNN
2711
AGCUUGGCUGAUGACGUCCNN
37





 797-815
GACGUCAUCAGCCAAGCUGNN
2712
CAGCUUGGCUGAUGACGUCNN
37





 798-816
ACGUCAUCAGCCAAGCUGGNN
2713
CCAGCUUGGCUGAUGACGUNN
37





 799-817
CGUCAUCAGCCAAGCUGGANN
2714
UCCAGCUUGGCUGAUGACGNN
37





 800-818
GUCAUCAGCCAAGCUGGAANN
2715
UUCCAGCUUGGCUGAUGACNN
37





 801-819
UCAUCAGCCAAGCUGGAAGNN
2716
CUUCCAGCUUGGCUGAUGANN
37





 802-820
CAUCAGCCAAGCUGGAAGCNN
2717
GCUUCCAGCUUGGCUGAUGNN
37





 803-821
AUCAGCCAAGCUGGAAGCCNN
2718
GGCUUCCAGCUUGGCUGAUNN
37





 804-822
UCAGCCAAGCUGGAAGCCANN
2719
UGGCUUCCAGCUUGGCUGANN
37





 805-823
CAGCCAAGCUGGAAGCCAUNN
2720
AUGGCUUCCAGCUUGGCUGNN
37





 806-824
AGCCAAGCUGGAAGCCAUUNN
2721
AAUGGCUUCCAGCUUGGCUNN
37





 807-825
GCCAAGCUGGAAGCCAUUANN
2722
UAAUGGCUUCCAGCUUGGCNN
37





 808-826
CCAAGCUGGAAGCCAUUAANN
2723
UUAAUGGCUUCCAGCUUGGNN
37





 809-827
CAAGCUGGAAGCCAUUAAUNN
2724
AUUAAUGGCUUCCAGCUUGNN
37





 810-828
AAGCUGGAAGCCAUUAAUGNN
2725
CAUUAAUGGCUUCCAGCUUNN
37





 811-829
AGCUGGAAGCCAUUAAUGANN
2726
UCAUUAAUGGCUUCCAGCUNN
37





 812-830
GCUGGAAGCCAUUAAUGAANN
2727
UUCAUUAAUGGCUUCCAGCNN
37





 813-831
CUGGAAGCCAUUAAUGAACNN
2728
GUUCAUUAAUGGCUUCCAGNN
37





 814-832
UGGAAGCCAUUAAUGAACUNN
2729
AGUUCAUUAAUGGCUUCCANN
37





 815-833
GGAAGCCAUUAAUGAACUANN
2730
UAGUUCAUUAAUGGCUUCCNN
37





 816-834
GAAGCCAUUAAUGAACUAANN
2731
UUAGUUCAUUAAUGGCUUCNN
37





 817-835
AAGCCAUUAAUGAACUAAUNN
2732
AUUAGUUCAUUAAUGGCUUNN
37





 818-836
AGCCAUUAAUGAACUAAUUNN
2733
AAUUAGUUCAUUAAUGGCUNN
37





 819-837
GCCAUUAAUGAACUAAUUCNN
2734
GAAUUAGUUCAUUAAUGGCNN
37





 820-838
CCAUUAAUGAACUAAUUCGNN
2735
CGAAUUAGUUCAUUAAUGGNN
37





 821-839
CAUUAAUGAACUAAUUCGUNN
2736
ACGAAUUAGUUCAUUAAUGNN
37





 822-840
AUUAAUGAACUAAUUCGUUNN
2737
AACGAAUUAGUUCAUUAAUNN
37





 823-841
UUAAUGAACUAAUUCGUUUNN
2738
AAACGAAUUAGUUCAUUAANN
37





 824-842
UAAUGAACUAAUUCGUUUUNN
2739
AAAACGAAUUAGUUCAUUANN
37





 825-843
AAUGAACUAAUUCGUUUUGNN
2740
CAAAACGAAUUAGUUCAUUNN
37





 826-844
AUGAACUAAUUCGUUUUGANN
2741
UCAAAACGAAUUAGUUCAUNN
38





 827-845
UGAACUAAUUCGUUUUGACNN
2742
GUCAAAACGAAUUAGUUCANN
38





 828-846
GAACUAAUUCGUUUUGACCNN
2743
GGUCAAAACGAAUUAGUUCNN
38





 829-847
AACUAAUUCGUUUUGACCANN
2744
UGGUCAAAACGAAUUAGUUNN
38





 830-848
ACUAAUUCGUUUUGACCACNN
2745
GUGGUCAAAACGAAUUAGUNN
38





 831-849
CUAAUUCGUUUUGACCACANN
2746
UGUGGUCAAAACGAAUUAGNN
38





 832-850
UAAUUCGUUUUGACCACAUNN
2747
AUGUGGUCAAAACGAAUUANN
38





 833-851
AAUUCGUUUUGACCACAUANN
2748
UAUGUGGUCAAAACGAAUUNN
38





 834-852
AUUCGUUUUGACCACAUAUNN
2749
AUAUGUGGUCAAAACGAAUNN
38





 835-853
UUCGUUUUGACCACAUAUANN
2750
UAUAUGUGGUCAAAACGAANN
38





 836-854
UCGUUUUGACCACAUAUAUNN
2751
AUAUAUGUGGUCAAAACGANN
38





 837-855
CGUUUUGACCACAUAUAUANN
2752
UAUAUAUGUGGUCAAAACGNN
38





 838-856
GUUUUGACCACAUAUAUACNN
2753
GUAUAUAUGUGGUCAAAACNN
38





 839-857
UUUUGACCACAUAUAUACCNN
2754
GGUAUAUAUGUGGUCAAAANN
38





 840-858
UUUGACCACAUAUAUACCANN
2755
UGGUAUAUAUGUGGUCAAANN
38





 841-859
UUGACCACAUAUAUACCAANN
2756
UUGGUAUAUAUGUGGUCAANN
38





 842-860
UGACCACAUAUAUACCAAGNN
2757
CUUGGUAUAUAUGUGGUCANN
38





 843-861
GACCACAUAUAUACCAAGCNN
2758
GCUUGGUAUAUAUGUGGUCNN
38





 844-862
ACCACAUAUAUACCAAGCCNN
2759
GGCUUGGUAUAUAUGUGGUNN
38





 845-863
CCACAUAUAUACCAAGCCCNN
2760
GGGCUUGGUAUAUAUGUGGNN
38





 846-864
CACAUAUAUACCAAGCCCCNN
2761
GGGGCUUGGUAUAUAUGUGNN
38





 847-865
ACAUAUAUACCAAGCCCCUNN
2762
AGGGGCUUGGUAUAUAUGUNN
38





 867-885
GUCUUAGAGAUACCCUCUGNN
2763
CAGAGGGUAUCUCUAAGACNN
38





 868-886
UCUUAGAGAUACCCUCUGANN
2764
UCAGAGGGUAUCUCUAAGANN
38





 869-887
CUUAGAGAUACCCUCUGAGNN
2765
CUCAGAGGGUAUCUCUAAGNN
38





 870-888
UUAGAGAUACCCUCUGAGANN
2766
UCUCAGAGGGUAUCUCUAANN
38





 871-889
UAGAGAUACCCUCUGAGACNN
2767
GUCUCAGAGGGUAUCUCUANN
38





 872-890
AGAGAUACCCUCUGAGACANN
2768
UGUCUCAGAGGGUAUCUCUNN
38





 873-891
GAGAUACCCUCUGAGACAGNN
2769
CUGUCUCAGAGGGUAUCUCNN
38





 874-892
AGAUACCCUCUGAGACAGANN
2770
UCUGUCUCAGAGGGUAUCUNN
38





 875-893
GAUACCCUCUGAGACAGAGNN
2771
CUCUGUCUCAGAGGGUAUCNN
38





 876-894
AUACCCUCUGAGACAGAGANN
2772
UCUCUGUCUCAGAGGGUAUNN
38





 877-895
UACCCUCUGAGACAGAGAGNN
2773
CUCUCUGUCUCAGAGGGUANN
38





 878-896
ACCCUCUGAGACAGAGAGCNN
2774
GCUCUCUGUCUCAGAGGGUNN
38





 879-897
CCCUCUGAGACAGAGAGCCNN
2775
GGCUCUCUGUCUCAGAGGGNN
38





 880-898
CCUCUGAGACAGAGAGCCANN
2776
UGGCUCUCUGUCUCAGAGGNN
38





 881-899
CUCUGAGACAGAGAGCCAANN
2777
UUGGCUCUCUGUCUCAGAGNN
38





 882-900
UCUGAGACAGAGAGCCAAGNN
2778
CUUGGCUCUCUGUCUCAGANN
38





 883-901
CUGAGACAGAGAGCCAAGCNN
2779
GCUUGGCUCUCUGUCUCAGNN
38





 884-902
UGAGACAGAGAGCCAAGCUNN
2780
AGCUUGGCUCUCUGUCUCANN
38





 885-903
GAGACAGAGAGCCAAGCUANN
2781
UAGCUUGGCUCUCUGUCUCNN
38





 886-904
AGACAGAGAGCCAAGCUAANN
2782
UUAGCUUGGCUCUCUGUCUNN
38





 887-905
GACAGAGAGCCAAGCUAAUNN
2783
AUUAGCUUGGCUCUCUGUCNN
38





 888-906
ACAGAGAGCCAAGCUAAUGNN
2784
CAUUAGCUUGGCUCUCUGUNN
38





 889-907
CAGAGAGCCAAGCUAAUGUNN
2785
ACAUUAGCUUGGCUCUCUGNN
38





 890-908
AGAGAGCCAAGCUAAUGUGNN
2786
CACAUUAGCUUGGCUCUCUNN
38





 891-909
GAGAGCCAAGCUAAUGUGGNN
2787
CCACAUUAGCUUGGCUCUCNN
38





 892-910
AGAGCCAAGCUAAUGUGGUNN
2788
ACCACAUUAGCUUGGCUCUNN
38





 893-911
GAGCCAAGCUAAUGUGGUANN
2789
UACCACAUUAGCUUGGCUCNN
38





 894-912
AGCCAAGCUAAUGUGGUAGNN
2790
CUACCACAUUAGCUUGGCUNN
38





 895-913
GCCAAGCUAAUGUGGUAGUNN
2791
ACUACCACAUUAGCUUGGCNN
38





 896-914
CCAAGCUAAUGUGGUAGUGNN
2792
CACUACCACAUUAGCUUGGNN
38





 897-915
CAAGCUAAUGUGGUAGUGANN
2793
UCACUACCACAUUAGCUUGNN
38





 898-916
AAGCUAAUGUGGUAGUGAANN
2794
UUCACUACCACAUUAGCUUNN
38





 899-917
AGCUAAUGUGGUAGUGAAANN
2795
UUUCACUACCACAUUAGCUNN
38





 900-918
GCUAAUGUGGUAGUGAAAANN
2796
UUUUCACUACCACAUUAGCNN
38





 901-919
CUAAUGUGGUAGUGAAAAUNN
2797
AUUUUCACUACCACAUUAGNN
38





 902-920
UAAUGUGGUAGUGAAAAUCNN
2798
GAUUUUCACUACCACAUUANN
38





 903-921
AAUGUGGUAGUGAAAAUCGNN
2799
CGAUUUUCACUACCACAUUNN
38





 904-922
AUGUGGUAGUGAAAAUCGANN
2800
UCGAUUUUCACUACCACAUNN
38





 905-923
UGUGGUAGUGAAAAUCGAGNN
2801
CUCGAUUUUCACUACCACANN
38





 906-924
GUGGUAGUGAAAAUCGAGGNN
2802
CCUCGAUUUUCACUACCACNN
38





 907-925
UGGUAGUGAAAAUCGAGGANN
2803
UCCUCGAUUUUCACUACCANN
38





 908-926
GGUAGUGAAAAUCGAGGAANN
2804
UUCCUCGAUUUUCACUACCNN
38





 909-927
GUAGUGAAAAUCGAGGAAGNN
2805
CUUCCUCGAUUUUCACUACNN
38





 910-928
UAGUGAAAAUCGAGGAAGCNN
2806
GCUUCCUCGAUUUUCACUANN
38





 911-929
AGUGAAAAUCGAGGAAGCANN
2807
UGCUUCCUCGAUUUUCACUNN
38





 912-930
GUGAAAAUCGAGGAAGCACNN
2808
GUGCUUCCUCGAUUUUCACNN
38





 913-931
UGAAAAUCGAGGAAGCACCNN
2809
GGUGCUUCCUCGAUUUUCANN
38





 914-932
GAAAAUCGAGGAAGCACCUNN
2810
AGGUGCUUCCUCGAUUUUCNN
38





 915-933
AAAAUCGAGGAAGCACCUCNN
2811
GAGGUGCUUCCUCGAUUUUNN
38





 916-934
AAAUCGAGGAAGCACCUCUNN
2812
AGAGGUGCUUCCUCGAUUUNN
38





 917-935
AAUCGAGGAAGCACCUCUCNN
2813
GAGAGGUGCUUCCUCGAUUNN
38





 918-936
AUCGAGGAAGCACCUCUCANN
2814
UGAGAGGUGCUUCCUCGAUNN
38





 919-937
UCGAGGAAGCACCUCUCAGNN
2815
CUGAGAGGUGCUUCCUCGANN
38





 920-938
CGAGGAAGCACCUCUCAGCNN
2816
GCUGAGAGGUGCUUCCUCGNN
38





 921-939
GAGGAAGCACCUCUCAGCCNN
2817
GGCUGAGAGGUGCUUCCUCNN
38





 922-940
AGGAAGCACCUCUCAGCCCNN
2818
GGGCUGAGAGGUGCUUCCUNN
38





 923-941
GGAAGCACCUCUCAGCCCCNN
2819
GGGGCUGAGAGGUGCUUCCNN
38





 924-942
GAAGCACCUCUCAGCCCCUNN
2820
AGGGGCUGAGAGGUGCUUCNN
38





 925-943
AAGCACCUCUCAGCCCCUCNN
2821
GAGGGGCUGAGAGGUGCUUNN
38





 926-944
AGCACCUCUCAGCCCCUCANN
2822
UGAGGGGCUGAGAGGUGCUNN
38





 927-945
GCACCUCUCAGCCCCUCAGNN
2823
CUGAGGGGCUGAGAGGUGCNN
38





 928-946
CACCUCUCAGCCCCUCAGANN
2824
UCUGAGGGGCUGAGAGGUGNN
38





 929-947
ACCUCUCAGCCCCUCAGAGNN
2825
CUCUGAGGGGCUGAGAGGUNN
38





 930-948
CCUCUCAGCCCCUCAGAGANN
2826
UCUCUGAGGGGCUGAGAGGNN
38





 931-949
CUCUCAGCCCCUCAGAGAANN
2827
UUCUCUGAGGGGCUGAGAGNN
38





 932-950
UCUCAGCCCCUCAGAGAAUNN
2828
AUUCUCUGAGGGGCUGAGANN
38





 933-951
CUCAGCCCCUCAGAGAAUGNN
2829
CAUUCUCUGAGGGGCUGAGNN
38





 934-952
UCAGCCCCUCAGAGAAUGANN
2830
UCAUUCUCUGAGGGGCUGANN
38





 935-953
CAGCCCCUCAGAGAAUGAUNN
2831
AUCAUUCUCUGAGGGGCUGNN
38





 936-954
AGCCCCUCAGAGAAUGAUCNN
2832
GAUCAUUCUCUGAGGGGCUNN
38





 937-955
GCCCCUCAGAGAAUGAUCANN
2833
UGAUCAUUCUCUGAGGGGCNN
38





 938-956
CCCCUCAGAGAAUGAUCACNN
2834
GUGAUCAUUCUCUGAGGGGNN
38





 939-957
CCCUCAGAGAAUGAUCACCNN
2835
GGUGAUCAUUCUCUGAGGGNN
38





 940-958
CCUCAGAGAAUGAUCACCCNN
2836
GGGUGAUCAUUCUCUGAGGNN
38





 941-959
CUCAGAGAAUGAUCACCCUNN
2837
AGGGUGAUCAUUCUCUGAGNN
38





 942-960
UCAGAGAAUGAUCACCCUGNN
2838
CAGGGUGAUCAUUCUCUGANN
38





 943-961
CAGAGAAUGAUCACCCUGANN
2839
UCAGGGUGAUCAUUCUCUGNN
38





 944-962
AGAGAAUGAUCACCCUGAANN
2840
UUCAGGGUGAUCAUUCUCUNN
38





 945-963
GAGAAUGAUCACCCUGAAUNN
2841
AUUCAGGGUGAUCAUUCUCNN
39





 946-964
AGAAUGAUCACCCUGAAUUNN
2842
AAUUCAGGGUGAUCAUUCUNN
39





 947-965
GAAUGAUCACCCUGAAUUCNN
2843
GAAUUCAGGGUGAUCAUUCNN
39





 948-966
AAUGAUCACCCUGAAUUCANN
2844
UGAAUUCAGGGUGAUCAUUNN
39





 949-967
AUGAUCACCCUGAAUUCAUNN
2845
AUGAAUUCAGGGUGAUCAUNN
39





 950-968
UGAUCACCCUGAAUUCAUUNN
2846
AAUGAAUUCAGGGUGAUCANN
39





 951-969
GAUCACCCUGAAUUCAUUGNN
2847
CAAUGAAUUCAGGGUGAUCNN
39





 952-970
AUCACCCUGAAUUCAUUGUNN
2848
ACAAUGAAUUCAGGGUGAUNN
39





 953-971
UCACCCUGAAUUCAUUGUCNN
2849
GACAAUGAAUUCAGGGUGANN
39





 954-972
CACCCUGAAUUCAUUGUCUNN
2850
AGACAAUGAAUUCAGGGUGNN
39





 955-973
ACCCUGAAUUCAUUGUCUCNN
2851
GAGACAAUGAAUUCAGGGUNN
39





 956-974
CCCUGAAUUCAUUGUCUCANN
2852
UGAGACAAUGAAUUCAGGGNN
39





 957-975
CCUGAAUUCAUUGUCUCAGNN
2853
CUGAGACAAUGAAUUCAGGNN
39





 958-976
CUGAAUUCAUUGUCUCAGUNN
2854
ACUGAGACAAUGAAUUCAGNN
39





 959-977
UGAAUUCAUUGUCUCAGUGNN
2855
CACUGAGACAAUGAAUUCANN
39





 960-978
GAAUUCAUUGUCUCAGUGANN
2856
UCACUGAGACAAUGAAUUCNN
39





 961-979
AAUUCAUUGUCUCAGUGAANN
2857
UUCACUGAGACAAUGAAUUNN
39





 962-980
AUUCAUUGUCUCAGUGAAGNN
2858
CUUCACUGAGACAAUGAAUNN
39





 963-981
UUCAUUGUCUCAGUGAAGGNN
2859
CCUUCACUGAGACAAUGAANN
39





 964-982
UCAUUGUCUCAGUGAAGGANN
2860
UCCUUCACUGAGACAAUGANN
39





 965-983
CAUUGUCUCAGUGAAGGAANN
2861
UUCCUUCACUGAGACAAUGNN
39





 966-984
AUUGUCUCAGUGAAGGAAGNN
2862
CUUCCUUCACUGAGACAAUNN
39





 967-985
UUGUCUCAGUGAAGGAAGANN
2863
UCUUCCUUCACUGAGACAANN
39





 968-986
UGUCUCAGUGAAGGAAGAANN
2864
UUCUUCCUUCACUGAGACANN
39





 969-987
GUCUCAGUGAAGGAAGAACNN
2865
GUUCUUCCUUCACUGAGACNN
39





 970-988
UCUCAGUGAAGGAAGAACCNN
2866
GGUUCUUCCUUCACUGAGANN
39





 971-989
CUCAGUGAAGGAAGAACCUNN
2867
AGGUUCUUCCUUCACUGAGNN
39





 972-990
UCAGUGAAGGAAGAACCUGNN
2868
CAGGUUCUUCCUUCACUGANN
39





 973-991
CAGUGAAGGAAGAACCUGUNN
2869
ACAGGUUCUUCCUUCACUGNN
39





 974-992
AGUGAAGGAAGAACCUGUANN
2870
UACAGGUUCUUCCUUCACUNN
39





 975-993
GUGAAGGAAGAACCUGUAGNN
2871
CUACAGGUUCUUCCUUCACNN
39





 976-994
UGAAGGAAGAACCUGUAGANN
2872
UCUACAGGUUCUUCCUUCANN
39





 977-995
GAAGGAAGAACCUGUAGAANN
2873
UUCUACAGGUUCUUCCUUCNN
39





 978-996
AAGGAAGAACCUGUAGAAGNN
2874
CUUCUACAGGUUCUUCCUUNN
39





 979-997
AGGAAGAACCUGUAGAAGANN
2875
UCUUCUACAGGUUCUUCCUNN
39





 980-998
GGAAGAACCUGUAGAAGAUNN
2876
AUCUUCUACAGGUUCUUCCNN
39





 981-999
GAAGAACCUGUAGAAGAUGNN
2877
CAUCUUCUACAGGUUCUUCNN
39





 982-1000
AAGAACCUGUAGAAGAUGANN
2878
UCAUCUUCUACAGGUUCUUNN
39





 983-1001
AGAACCUGUAGAAGAUGACNN
2879
GUCAUCUUCUACAGGUUCUNN
39





 984-1002
GAACCUGUAGAAGAUGACCNN
2880
GGUCAUCUUCUACAGGUUCNN
39





 985-1003
AACCUGUAGAAGAUGACCUNN
2881
AGGUCAUCUUCUACAGGUUNN
39





 986-1004
ACCUGUAGAAGAUGACCUCNN
2882
GAGGUCAUCUUCUACAGGUNN
39
















TABLE 13







Sequences of dsRNA targeting both mouse and rhesus monkey XBP-1.


*Target refers location of target sequence in NM_013842 (Musmusculis XPB1 mRNA).


Sense and antisense sequences are described with optional dinucleotide (NN) overhangs.













SEQ ID

SEQ ID


*Target
sense (5′-3′)
NO
antisense (5′-3′)
NO





 369-387
AGAAAACUCACGGCCUUGUNN
3942
ACAAGGCCGUGAGUUUUCUNN
4042





 237-255
AACUGAAAAACAGAGUAGCNN
3943
GCUACUCUGUUUUUCAGUUNN
4043





 491-509
GGGUCUGCUGAGUCCGCAGNN
3944
CUGCGGACUCAGCAGACCCNN
4044





 917-935
AUCACCCUGAAUUCAUUGUNN
3945
ACAAUGAAUUCAGGGUGAUNN
4045





 923-941
CUGAAUUCAUUGUCUCAGUNN
3946
ACUGAGACAAUGAAUUCAGNN
4046





 702-720
CCCAGAGGUCUACCCAGAANN
3947
UUCUGGGUAGACCUCUGGGNN
4047





 926-944
AAUUCAUUGUCUCAGUGAANN
3948
UUCACUGAGACAAUGAAUUNN
4048





 391-409
UGAGAACCAGGAGUUAAGANN
3949
UCUUAACUCCUGGUUCUCANN
4049





 775-793
AAGCUGGAAGCCAUUAAUGNN
3950
CAUUAAUGGCUUCCAGCUUNN
4050





1150-1168
CCCCAGCUGAUUAGUGUCUNN
3951
AGACACUAAUCAGCUGGGGNN
4051





 776-794
AGCUGGAAGCCAUUAAUGANN
3952
UCAUUAAUGGCUUCCAGCUNN
4052





 921-939
CCCUGAAUUCAUUGUCUCANN
3953
UGAGACAAUGAAUUCAGGGNN
4053





 777-795
GCUGGAAGCCAUUAAUGAANN
3954
UUCAUUAAUGGCUUCCAGCNN
4054





 539-557
GUGCAGGCCCAGUUGUCACNN
3955
GUGACAACUGGGCCUGCACNN
4055





 731-749
CCUUACCAGCCUCCCUUUCNN
3956
GAAAGGGAGGCUGGUAAGGNN
4056





 924-942
UGAAUUCAUUGUCUCAGUGNN
3957
CACUGAGACAAUGAAUUCANN
4057





1151-1169
CCCAGCUGAUUAGUGUCUANN
3958
UAGACACUAAUCAGCUGGGNN
4058





1152-1170
CCAGCUGAUUAGUGUCUAANN
3959
UUAGACACUAAUCAGCUGGNN
4059





1718-1736
ACUAUGUAAAUGCUUGAUGNN
3960
CAUCAAGCAUUUACAUAGUNN
4060





 368-386
GAGAAAACUCACGGCCUUGNN
3961
CAAGGCCGUGAGUUUUCUCNN
4061





 489-507
CCGGGUCUGCUGAGUCCGCNN
3962
GCGGACUCAGCAGACCCGGNN
4062





 238-256
ACUGAAAAACAGAGUAGCANN
3963
UGCUACUCUGUUUUUCAGUNN
4063





 240-258
UGAAAAACAGAGUAGCAGCNN
3964
GCUGCUACUCUGUUUUUCANN
4064





 390-408
UUGAGAACCAGGAGUUAAGNN
3965
CUUAACUCCUGGUUCUCAANN
4065





 487-505
GGCCGGGUCUGCUGAGUCCNN
3966
GGACUCAGCAGACCCGGCCNN
4066





 741-759
CUCCCUUUCUCUGUCAGUGNN
3967
CACUGACAGAGAAAGGGAGNN
4067





 918-936
UCACCCUGAAUUCAUUGUCNN
3968
GACAAUGAAUUCAGGGUGANN
4068





 919-937
CACCCUGAAUUCAUUGUCUNN
3969
AGACAAUGAAUUCAGGGUGNN
4069





1130-1148
CUUUUGCCAAUGAACUUUUNN
3970
AAAAGUUCAUUGGCAAAAGNN
4070





1712-1730
AAAUUUACUAUGUAAAUGCNN
3971
GCAUUUACAUAGUAAAUUUNN
4071





1714-1732
AUUUACUAUGUAAAUGCUUNN
3972
AAGCAUUUACAUAGUAAAUNN
4072





1717-1735
UACUAUGUAAAUGCUUGAUNN
3973
AUCAAGCAUUUACAUAGUANN
4073





1719-1737
CUAUGUAAAUGCUUGAUGGNN
3974
CCAUCAAGCAUUUACAUAGNN
4074





1775-1793
CCAUUUAUUUAAAACUACCNN
3975
GGUAGUUUUAAAUAAAUGGNN
4075





1776-1794
CAUUUAUUUAAAACUACCCNN
3976
GGGUAGUUUUAAAUAAAUGNN
4076





 239-257
CUGAAAAACAGAGUAGCAGNN
3977
CUGCUACUCUGUUUUUCAGNN
4077





 347-365
CUAGAAAAUCAGCUUUUACNN
3978
GUAAAAGCUGAUUUUCUAGNN
4078





 348-366
UAGAAAAUCAGCUUUUACGNN
3979
CGUAAAAGCUGAUUUUCUANN
4079





 485-503
GUGGCCGGGUCUGCUGAGUNN
3980
ACUCAGCAGACCCGGCCACNN
4080





 486-504
UGGCCGGGUCUGCUGAGUCNN
3981
GACUCAGCAGACCCGGCCANN
4081





 488-506
GCCGGGUCUGCUGAGUCCGNN
3982
CGGACUCAGCAGACCCGGCNN
4082





 540-558
UGCAGGCCCAGUUGUCACCNN
3983
GGUGACAACUGGGCCUGCANN
4083





 703-721
CCAGAGGUCUACCCAGAAGNN
3984
CUUCUGGGUAGACCUCUGGNN
4084





 705-723
AGAGGUCUACCCAGAAGGANN
3985
UCCUUCUGGGUAGACCUCUNN
4085





 730-748
UCCUUACCAGCCUCCCUUUNN
3986
AAAGGGAGGCUGGUAAGGANN
4086





 742-760
UCCCUUUCUCUGUCAGUGGNN
3987
CCACUGACAGAGAAAGGGANN
4087





 744-762
CCUUUCUCUGUCAGUGGGGNN
3988
CCCCACUGACAGAGAAAGGNN
4088





 767-785
CAUCAGCCAAGCUGGAAGCNN
3989
GCUUCCAGCUUGGCUGAUGNN
4089





 771-789
AGCCAAGCUGGAAGCCAUUNN
3990
AAUGGCUUCCAGCUUGGCUNN
4090





 916-934
GAUCACCCUGAAUUCAUUGNN
3991
CAAUGAAUUCAGGGUGAUCNN
4091





 920-938
ACCCUGAAUUCAUUGUCUCNN
3992
GAGACAAUGAAUUCAGGGUNN
4092





 922-940
CCUGAAUUCAUUGUCUCAGNN
3993
CUGAGACAAUGAAUUCAGGNN
4093





 925-943
GAAUUCAUUGUCUCAGUGANN
3994
UCACUGAGACAAUGAAUUCNN
4094





1720-1738
UAUGUAAAUGCUUGAUGGANN
3995
UCCAUCAAGCAUUUACAUANN
4095





 232-250
GAGGAAACUGAAAAACAGANN
3996
UCUGUUUUUCAGUUUCCUCNN
4096





 236-254
AAACUGAAAAACAGAGUAGNN
3997
CUACUCUGUUUUUCAGUUUNN
4097





 728-746
GUUCCUUACCAGCCUCCCUNN
3998
AGGGAGGCUGGUAAGGAACNN
4098





 729-747
UUCCUUACCAGCCUCCCUUNN
3999
AAGGGAGGCUGGUAAGGAANN
4099





 745-763
CUUUCUCUGUCAGUGGGGANN
4000
UCCCCACUGACAGAGAAAGNN
4100





 766-784
UCAUCAGCCAAGCUGGAAGNN
4001
CUUCCAGCUUGGCUGAUGANN
4101





 927-945
AUUCAUUGUCUCAGUGAAGNN
4002
CUUCACUGAGACAAUGAAUNN
4102





 234-252
GGAAACUGAAAAACAGAGUNN
4003
ACUCUGUUUUUCAGUUUCCNN
4103





 235-253
GAAACUGAAAAACAGAGUANN
4004
UACUCUGUUUUUCAGUUUCNN
4104





 346-364
GCUAGAAAAUCAGCUUUUANN
4005
UAAAAGCUGAUUUUCUAGCNN
4105





 490-508
CGGGUCUGCUGAGUCCGCANN
4006
UGCGGACUCAGCAGACCCGNN
4106





 700-718
CUCCCAGAGGUCUACCCAGNN
4007
CUGGGUAGACCUCUGGGAGNN
4107





1715-1733
UUUACUAUGUAAAUGCUUGNN
4008
CAAGCAUUUACAUAGUAAANN
4108





 734-752
UACCAGCCUCCCUUUCUCUNN
4009
AGAGAAAGGGAGGCUGGUANN
4109





 773-791
CCAAGCUGGAAGCCAUUAANN
4010
UUAAUGGCUUCCAGCUUGGNN
4110





 778-796
CUGGAAGCCAUUAAUGAACNN
4011
GUUCAUUAAUGGCUUCCAGNN
4111





 779-797
UGGAAGCCAUUAAUGAACUNN
4012
AGUUCAUUAAUGGCUUCCANN
4112





1774-1792
UCCAUUUAUUUAAAACUACNN
4013
GUAGUUUUAAAUAAAUGGANN
4113





 704-722
CAGAGGUCUACCCAGAAGGNN
4014
CCUUCUGGGUAGACCUCUGNN
4114





1716-1734
UUACUAUGUAAAUGCUUGANN
4015
UCAAGCAUUUACAUAGUAANN
4115





1713-1731
AAUUUACUAUGUAAAUGCUNN
4016
AGCAUUUACAUAGUAAAUUNN
4116





 768-786
AUCAGCCAAGCUGGAAGCCNN
4017
GGCUUCCAGCUUGGCUGAUNN
4117





1129-1147
ACUUUUGCCAAUGAACUUUNN
4018
AAAGUUCAUUGGCAAAAGUNN
4118





 389-407
GUUGAGAACCAGGAGUUAANN
4019
UUAACUCCUGGUUCUCAACNN
4119





 701-719
UCCCAGAGGUCUACCCAGANN
4020
UCUGGGUAGACCUCUGGGANN
4120





 706-724
GAGGUCUACCCAGAAGGACNN
4021
GUCCUUCUGGGUAGACCUCNN
4121





 707-725
AGGUCUACCCAGAAGGACCNN
4022
GGUCCUUCUGGGUAGACCUNN
4122





 727-745
AGUUCCUUACCAGCCUCCCNN
4023
GGGAGGCUGGUAAGGAACUNN
4123





 733-751
UUACCAGCCUCCCUUUCUCNN
4024
GAGAAAGGGAGGCUGGUAANN
4124





 736-754
CCAGCCUCCCUUUCUCUGUNN
4025
ACAGAGAAAGGGAGGCUGGNN
4125





 738-756
AGCCUCCCUUUCUCUGUCANN
4026
UGACAGAGAAAGGGAGGCUNN
4126





 743-761
CCCUUUCUCUGUCAGUGGGNN
4027
CCCACUGACAGAGAAAGGGNN
4127





 769-787
UCAGCCAAGCUGGAAGCCANN
4028
UGGCUUCCAGCUUGGCUGANN
4128





 772-790
GCCAAGCUGGAAGCCAUUANN
4029
UAAUGGCUUCCAGCUUGGCNN
4129





 774-792
CAAGCUGGAAGCCAUUAAUNN
4030
AUUAAUGGCUUCCAGCUUGNN
4130





 231-249
GGAGGAAACUGAAAAACAGNN
4031
CUGUUUUUCAGUUUCCUCCNN
4131





 233-251
AGGAAACUGAAAAACAGAGNN
4032
CUCUGUUUUUCAGUUUCCUNN
4132





 735-753
ACCAGCCUCCCUUUCUCUGNN
4033
CAGAGAAAGGGAGGCUGGUNN
4133





 737-755
CAGCCUCCCUUUCUCUGUCNN
4034
GACAGAGAAAGGGAGGCUGNN
4134





 739-757
GCCUCCCUUUCUCUGUCAGNN
4035
CUGACAGAGAAAGGGAGGCNN
4135





 740-758
CCUCCCUUUCUCUGUCAGUNN
4036
ACUGACAGAGAAAGGGAGGNN
4136





 746-764
UUUCUCUGUCAGUGGGGACNN
4037
GUCCCCACUGACAGAGAAANN
4137





 770-788
CAGCCAAGCUGGAAGCCAUNN
4038
AUGGCUUCCAGCUUGGCUGNN
4138





  26-44
GCUAUGGUGGUGGUGGCAGNN
4039
CUGCCACCACCACCAUAGCNN
4139





  27-45
CUAUGGUGGUGGUGGCAGCNN
4040
GCUGCCACCACCACCAUAGNN
4140





 732-750
CUUACCAGCCUCCCUUUCUNN
4041
AGAAAGGGAGGCUGGUAAGNN
4141








Claims
  • 1. A dual targeting siRNA agent comprising a first dsRNA targeting a PCSK9 gene and a second dsRNA targeting a second gene, wherein the first dsRNA and the second dsRNA are linked with a covalent linker.
  • 2. (canceled)
  • 3. The dual targeting siRNA agent of claim 1, wherein the second gene is selected from the group consisting of XBP-1, PCSK9, PCSK5, ApoC3, SCAP, and MIG12.
  • 4. (canceled)
  • 5. The dual targeting siRNA agent of claim 1, wherein the first dsRNA comprises at least 15 contiguous nucleotides of an antisense strand of one of Tables 1, 2, or 4-8, or comprises an antisense strand of one of Tables 1, 2, or 4-8, or comprises a sense strand and an antisense strand of one of Tables 1, 2, or 4-8.
  • 6. The dual targeting siRNA agent of claim 1, wherein the first dsRNA comprises AD-9680 or AD-10792.
  • 7. The dual targeting siRNA agent of claim 1, wherein the second dsRNA comprises at least 15 contiguous nucleotides of an antisense strand of one of Tables 3 or 9-13, or comprises an antisense strand of one of Tables 3 or 9-13, or comprises a sense strand and an antisense strand of one of Tables 3 or 9-13.
  • 8. The dual targeting siRNA agent of claim 1, wherein the second dsRNA comprises AD-18038.
  • 9. The dual targeting siRNA agent of claim 1, wherein the first and second dsRNA comprises at least one modified nucleotide.
  • 10. The dual targeting siRNA agent of claim 9, wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
  • 11. The dual targeting siRNA agent of claim 9, wherein the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
  • 12. The dual targeting siRNA agent of claim 1, wherein each strand of each dsRNA is 19-23 bases in length.
  • 13. The dual targeting siRNA agent of claim 1, wherein the first and second dsRNAs are linked with a disulfide linker.
  • 14. The dual targeting siRNA agent of claim 1, wherein the covalent linker links the sense strand of the first dsRNA to the sense strand of the second dsRNA.
  • 15. The dual targeting siRNA agent of claim 1, wherein the covalent linker links the antisense strand of the first dsRNA to the antisense strand of the second dsRNA.
  • 16. The dual targeting siRNA agent of claim 1, further comprising a ligand.
  • 17. (canceled)
  • 18. (canceled)
  • 19. A pharmaceutical composition comprising the dual targeting siRNA agent of claim 1 and a pharmaceutical carrier.
  • 20. The pharmaceutical composition of claim 19, wherein the pharmaceutical carrier is a lipid formulation.
  • 21.-23. (canceled)
  • 24. A method of inhibiting expression of the PCSK9 gene and a second gene in a cell, the method comprising (a) introducing into the cell the dual targeting siRNA agent of claim 1; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the PCSK9 gene and the second gene, thereby inhibiting expression of the PCSK9 gene and the second gene in the cell.
  • 25. A method of treating a disorder mediated by PCSK9 expression or of reducing total serum cholesterol in a subject comprising administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical composition of claim 19.
  • 26.-30. (canceled)
CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/521,422, filed Jul. 24, 2019, pending, which is a continuation of U.S. application Ser. No. 15/724,175, filed Oct. 3, 2017, pending, which is a continuation of U.S. application Ser. No. 14/885,342, filed Oct. 16, 2015, (abandoned) which is a continuation of U.S. application Ser. No. 13/497,226, with a 371(c) filing date of Oct. 10, 2012, now U.S. Pat. No. 9,187,746, issued Nov. 17, 2015, which is a National Stage of International Application No. PCT/US2010/049868, filed Sep. 22, 2010, which claims the benefit of U.S. Provisional Application No. 61/244,859, filed Sep. 22, 2009, and claims the benefit of U.S. Provisional Application No. 61/313,584, filed Mar. 12, 2010, all of which are hereby incorporated in their entirety by reference.

Provisional Applications (2)
Number Date Country
61313584 Mar 2010 US
61244859 Sep 2009 US
Continuations (4)
Number Date Country
Parent 16521422 Jul 2019 US
Child 16803738 US
Parent 15724175 Oct 2017 US
Child 16521422 US
Parent 14885342 Oct 2015 US
Child 15724175 US
Parent 13497226 Oct 2012 US
Child 14885342 US