Modified RNAi agents

FIELD OF THE INVENTION

The invention relates to RNAi duplex agents having particular motifs that are advantageous for inhibition of target gene expression, as well as RNAi compositions suitable for therapeutic use. Additionally, the invention provides methods of inhibiting the expression of a target gene by administering these RNAi duplex agents, e.g., for the treatment of various diseases.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al. (1998) Nature 391, 806-811; Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multi-component nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity remained unknown.

Double-stranded RNA (dsRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA induced silencing complex (RISC), which requires degradation of the sense strand of the dsRNA duplex. Sense strand was known to act as the first RISC substrate that is cleaved by Argonaute 2 in the middle of the duplex region. Immediately after the cleaved 5′-end and 3′-end fragments of the sense strand are removed from the endonuclease Ago2, the RISC becomes activated by the antisense strand (Rand et al. (2005) Cell 123, 621).

It was believed that when the cleavage of the sense strand is inhibited, the endonucleolytic cleavage of target mRNA is impaired (Leuschner et al. (2006) EMBO Rep., 7, 314; Rand et al. (2005) Cell 123, 621; Schwarz et al. (2004) Curr. Biol. 14, 787). Leuschner et al. showed that incorporation of a 2′-O-Me ribose to the Ago2 cleavage site in the sense strand inhibits RNAi in HeLa cells (Leuschner et al. (2006) EMBO Rep., 7, 314). A similar effect was observed with phosphorothioate modifications, showing that cleavage of the sense strand was required for efficient RNAi also in mammals.

Morrissey et al. used a siRNA duplex containing 2′-F modified residues, among other sites and modifications, also at the Ago2 cleavage site, and obtained compatible silencing compared to the unmodified siRNAs (Morrissey et al. (2005) Hepatology 41, 1349). However, Morrissey's modification is not motif specific, e.g., one modification includes 2′-F modifications on all pyrimidines on both sense and antisense strands as long as pyrimidine residue is present, without any selectivity; and hence it is uncertain, based on these teachings, if specific motif modification at the cleavage site of sense strand can have any actual effect on gene silencing activity.

Muhonen et al. used a siRNA duplex containing two 2′-F modified residues at the Ago2 cleavage site on the sense or antisense strand and found it was tolerated (Muhonen et al. (2007) Chemistry & Biodiversity 4, 858-873). However, Muhonen's modification is also sequence specific, e.g., for each particular strand, Muhonen only modifies either all pyrimidines or all purines, without any selectivity.

Choung et al. used a siRNA duplex containing alternative modifications by 2′-OMe or various combinations of 2′-F, 2′-OMe and phosphorothioate modifications to stabilize siRNA in serum to Sur10058 (Choung et al. (2006) Biochemical and Biophysical Research Communications 342, 919-927). Choung suggested that the residues at the cleavage site of the antisense strand should not be modified with 2′-OMe in order to increase the stability of the siRNA.

There is thus an ongoing need for iRNA duplex agents to improve the gene silencing efficacy of siRNA gene therapeutics. This invention is directed to that need.

SUMMARY

This invention provides effective nucleotide or chemical motifs for dsRNA agents optionally conjugated to at least one ligand, which are advantageous for inhibition of target gene expression, as well as RNAi compositions suitable for therapeutic use.

The inventors surprisingly discovered that introducing one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of a dsRNA agent that is comprised of modified sense and antisense strands enhances the gene silencing activity of the dsRNA agent.

In one aspect, the invention relates to a double-stranded RNAi (dsRNA) agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The dsRNA duplex is represented by formula (III):

(III)

sense: 5′ n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

antisense: 3′ n_p′-N_a′-(X′ X′ X′)_k-N_b′-Y′Y′Y′-N_b′-

(Z′Z′Z′)₁-N_a′-n_q′ 5′,

In formula (III), i, j, k, and l are each independently 0 or 1; p and q are each independently 0-6; n represents a nucleotide; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; each n_pand n_qindependently represents an overhang nucleotide sequence comprising 0-6 nucleotides; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; wherein the modifications on Nb is different than the modification on Y and the modifications on Nb′ is different than the modification on Y′. At least one of the Y nucleotides forms a base pair with its complementary Y′ nucleotides, and wherein the modification on the Y nucleotide is different than the modification on the Y′ nucleotide.

Each n_pand n_qindependently represents an overhang nucleotide sequence comprising 0-6 nucleotides; each n and n′ represents an overhang nucleotide; and p and q are each independently 0-6.

In another aspect, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one nucleotide. The antisense strand contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at or near cleavage site by at least one nucleotide. The modification in the motif occurring at or near the cleavage site in the sense strand is different than the modification in the motif occurring at or near the cleavage site in the antisense strand.

In another aspect, the invention further provides a method for delivering the dsRNA to a specific target in a subject by subcutaneous or intravenenuous administration.

DETAILED DESCRIPTION

A superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand and/or antisense strand of a dsRNA agent, particularly at or near the cleavage site. The sense strand and antisense strand of the dsRNA agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense and/or antisense strand. The dsRNA agent optionally conjugates with a GalNAc derivative ligand, for instance on the sense strand. The resulting dsRNA agents present superior gene silencing activity.

The inventors surprisingly discovered that having one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of a dsRNA agent superiorly enhanced the gene silencing activity of the dsRNA agent.

Accordingly, the invention provides a double-stranded RNAi (dsRNA) agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand. Each strand of the dsRNA agent can range from 12-30 nucleotides in length. For example, each strand can be between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex dsRNA. The duplex region of a dsRNA agent may be 12-30 nucleotide pairs in length. For example, the duplex region can be between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-30 nucleotides in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27.

In one embodiment, the dsRNA agent of the invention comprises may contain one or more overhang regions and/or capping groups of dsRNA agent at the 3′-end, or 5′-end or both ends of a strand. The overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the dsRNA agent of the invention can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2-F 2′-Omethyl, thymidine (T), 2′-β-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the dsRNA agent of the invention may be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.

The dsRNA agent of the invention comprises only single overhang, which can strengthen the interference activity of the dsRNA, without affecting its overall stability. For example, the single-stranded overhang is located at the 3′-terminal end of the sense strand or, alternatively, at the 3′-terminal end of the antisense strand. The dsRNA may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In one embodiment, the dsRNA agent of the invention may also have two blunt ends, at both ends of the dsRNA duplex.

In one embodiment, the dsRNA agent of the invention is a double ended bluntmer of 19 nt in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7,8,9 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention is a double ended bluntmer of 20 nt in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8,9,10 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention is a double ended bluntmer of 21 nt in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5′ end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5′ end, wherein one end of the dsRNA is blunt, while the other end is comprises a 2 nt overhang. Preferably, the 2 nt overhang is at the 3′-end of the antisense. Optionally, the dsRNA further comprises a ligand (preferably GalNAc₃).

In one embodiment, the dsRNA agent of the invention comprising a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of said first strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3′ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In one embodiment, the dsRNA agent of the invention comprising a sense and antisense strands, wherein said dsRNA agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11,12,13 from the 5′ end; wherein said 3′ end of said first strand and said 5′ end of said second strand form a blunt end and said second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region which is at least 25 nucleotides in length, and said second strand is sufficiently complementary to a target mRNA along at least 19 nt of said second strand length to reduce target gene expression when said dsRNA agent is introduced into a mammalian cell, and wherein dicer cleavage of said dsRNA preferentially results in an siRNA comprising said 3′ end of said second strand, thereby reducing expression of the target gene in the mammal. Optionally, the dsRNA agent further comprises a ligand.

In one embodiment, the sense strand of the dsRNA agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In one embodiment, the antisense strand of the dsRNA agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For dsRNA agent having a duplex region of 17-23 nt in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1^stnucleotide from the 5′-end of the antisense strand, or, the count starting from the 1^stpaired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the dsRNA from the 5′-end.

The sense strand of the dsRNA agent comprises at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides of the motifs from both strands may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the dsRNA agent comprises more than one motif of three identical modifications on three consecutive nucleotides. The first motif should occur at or near the cleavage site of the strand and the other motifs may be a wing modifications. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other the chemistry of the motifs are distinct from each other and when the motifs are separated by one or more nucleotide the chemistries of the motifs can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, the wing modifications may both occur at one end of the duplex region relative to the first motif which is at or near the cleavage site or each of the wing modifications may occur on either side of the first motif.

Like the sense strand, the antisense strand of the dsRNA agent comprises at least two motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that is present on the sense strand.

In one embodiment, the wing modification on the sense strand, antisense strand, or both strands of the dsRNA agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end or both ends of the strand.

In another embodiment, the wing modification on the sense strand, antisense strand, or both strands of the dsRNA agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end or both ends of the strand.

When the sense strand and the antisense strand of the dsRNA agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.

When the sense strand and the antisense strand of the dsRNA agent each contain at least two wing modifications, the sense strand and the antisense strand can be aligned so that two wing modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides.

In one embodiment, every nucleotide in the sense strand and antisense strand of the dsRNA agent, including the nucleotides that are part of the motifs, may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking 0 of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a RNA or may only occur in a single strand region of a RNA. E.g., a phosphorothioate modification at a non-linking 0 position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In one embodiment, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-β-allyl, 2′-C— allyl, 2′-deoxy, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the sense strand and antisense strand each contains two differently modified nucleotides selected from 2′-O-methyl or 2′-fluoro.

In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl nucleotide, 2′-deoxyfluoro nucleotide, 2′-O—N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-β-DMAEOE) nucleotide, 2′-O-aminopropyl (2′-O-AP) nucleotide, or 2′-ara-F nucleotide.

In one embodiment, the N_aand/or N_bcomprise modifications of an alternating pattern. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

In one embodiment, the N_a′ and/or N_b′ comprise modifications of an alternating pattern. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In one embodiment, the dsRNA agent of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3′-5′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisenese strand may start with “BBAABBAA” from 3′-5′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

In one embodiment, the dsRNA agent of the invention comprises the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the antisense strand initially, i.e., the 2′-O-methyl modified nucleotide on the sense strand base pairs with a 2′-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2′-F modification, and the 1 position of the antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand and/or antisense strand interrupts the initial modification pattern present in the sense strand and/or antisense strand. This interruption of the modification pattern of the sense and/or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense and/or antisense strand surprisingly enhances the gene silencing activity to the target gene.

In one embodiment, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif is “ . . . N_aYYYN_b. . . ,” where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotide, and “N_a” and “N_b” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of Y, and where N_aand N_bcan be the same or different modifications. Alternatively, N_aand/or N_bmay be present or absent when there is a wing modification present.

The dsRNA agent of the invention may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.

In one embodiment, the dsRNA comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3′-end of the antisense strand.

In one embodiment the sense strand of the dsRNA comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7 or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5 or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3 or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment, the dsRNA of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s) of the sense and/or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage at one end or both ends of the sense and/or antisense strand.

In one embodiment, the dsRNA of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the internal region of the duplex of each of the sense and/or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleotide linkage at position 8-16 of the duplex region counting from the 5′-end of the sense strand; the dsRNA can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s).

In one embodiment, the dsRNA of the invention further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5′-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5′-end), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end), and one phosphorothioateinternucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 20 and 21 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 21 and 22 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 22 and 23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In one embodiment, the dsRNA agent of the invention comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In one embodiment, the sense strand sequence may be represented by formula (I):

(I)

5′ n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_aindependently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_bindependently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_pand n_qindependently represent an overhang nucleotide;

wherein N_band Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.

In one embodiment, the N_aand/or N_bcomprise modifications of alternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the dsRNA agent has a duplex region of 17-23 nucleotide pairs in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sense strand, the count starting from the 1^stnucleotide, from the 5′-end; or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

(Ia)

5′ n_p-N_a-YYY-N_b-ZZZ-N_a-n_q 3′;

(Ib)

5′ n_p-N_a-XXX-N_b-YYY-N_a-n_q 3′;

or

(Ic)

5′ n_p-N_a-XXX-N_b-YYY-N_b-ZZZ-N_a-n_q 3′.

When the sense strand is represented by formula (Ia), N_brepresents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_aindependently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ib), N_brepresents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_acan independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), each N_bindependently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_bis 0, 1, 2, 3, 4, 5 or 6 Each N_acan independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In one embodiment, the antisense strand sequence of the dsRNA may be represented by formula (II):

(II)

5′ n_q′-N_a′-(Z′Z′Z′)_k-N_b′-Y′Y′Y′-N_b′-

(X′X′X′)₁-N′_a-n_p′ 3′

wherein:

k and l are each independently 0 or 1;

p and q are each independently 0-6; each N_a′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_b′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_p′ and n_q′ independently represent an overhang nucleotide comprising 0-6 nucleotides;

wherein N_b′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, the N_a′ and/or N_b′ comprise modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the dsRNA agent has a duplex region of 17-23 nt in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1^stnucleotide, from the 5′-end; or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas:

(IIa)

5′ n_q′-N_a′-Z′Z′Z′-N_b′-Y′Y′Y′-N_a′-n_p′ 3′;

(IIb)

5′ n_q′-N_a′-Y′Y′Y′-N_b′-X′X′X′-n_p′ 3′;

or

(IIc)

5′ n_q′-N_a′-Z′Z′Z′-N_b′-Y′Y′Y′-N_b′-X′X′X′-N_a′-n_p′ 3′.

When the antisense strand is represented by formula (IIa), N_b′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIb), N_b′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), each N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_bis 0, 1, 2, 3, 4, 5 or 6.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C— allyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the dsRNA agent comprises YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1^stnucleotide from the 5′-end, or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1^stnucleotide from the 5′-end, or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib) and (Ic) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb) and (IIc), respectively.

Accordingly, the dsRNA agent may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the dsRNA duplex represented by formula (III):

(III)

sense:

5′ n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)₁-

N_a′-n_q′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p and q are each independently 0-6;

each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein

each n_p′, n_p, n_q′, and n_qindependently represents an overhang nucleotide sequence; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 1. In another embodiment, k is 1 and l is 0; k is 0 and l is 1; or both k and l are 1.

In one embodiment, the dsRNA agent of the invention comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the dsRNA duplex represented by formula (V):

(V)

sense:

5′ N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)₁-N_a′ 5′

wherein:

j, k, and 1 are each independently 0 or 1;

p and q are each independently 2;

each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein

each n_p′, and n_qindependently represents an overhang nucleotide sequence; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 1. In another embodiment, k is 1 and l is 0; k is 0 and l is 1; or both k and l are 1.

In one embodiment, the dsRNA agent of the invention comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the dsRNA duplex represented by formula (Va):

(Va)

sense:

5′ N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a 3′

antisense:

3′ n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)₁-N_a′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p and q are each independently 2;

each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein

n_p′ represents an overhang nucleotide sequence; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

Exemplary combinations of the sense strand and antisense strand forming a dsRNA duplex include the formulas below:

(IIIa)

5′ n_p-N_a-Y Y Y-N_b-Z Z Z-N_a-n_q 3′

3′ n_p′-N_a′-Y′Y′Y′-N_b′-Z′Z′Z′-N_a′n_q′ 5′

(IIIb)

5′ n_p-N_a-X X X-N_b-Y Y Y-N_a-n_q 3′

3′ n_p′-N_a′-X′X′X′-N_b′-Y′Y′Y′-N_a′-n_q′ 5′

(IIIc)

5′ n_p-N_a-X X X-N_b-Y Y Y-N_b-Z Z Z-N_a-nq 3′

3′ n_p′-N_a′-X′X′X′-N_b′-Y′Y′Y′-N_b′-Z′Z′Z′-N_a-n_q′ 5′

When the dsRNA agent is represented by formula (IIIa), each N_band N_b′ independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N_aand N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNA agent is represented as formula (IIIb), each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_aand N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNA agent is represented as formula (IIIc), each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_aand N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_a, N_a′, N_band N_b′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb) and (IIc) may be the same or different from each other.

When the dsRNA agent is represented by formula (III), (IIIa), (IIIb) or (IIc), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

It is understood that N_anucleotides from base pair with N_a′, N_bnucleotides from base pair with N_b′, X nucleotides from base pair with X′, Y nucleotides from base pair with Y′, and Z nucleotides from base pair with Z′.

When the dsRNA agent is represented by formula (IIIa) or (IIIc), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNA agent is represented as formula (IIIb) or (IIIc), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In one embodiment, the dsRNA agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb) or (IIIc), wherein said duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, said multimer further comprise a ligand. Each of the dsRNA can target the same gene or two different genes; or each of the dsRNA can target same gene at two different target sites.

In one embodiment, the dsRNA agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb) or (IIIc), wherein said duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, said multimer further comprises a ligand. Each of the dsRNA can target the same gene or two different genes; or each of the dsRNA can target same gene at two different target sites.

In one embodiment, two dsRNA agent represented by formula (III), (IIIa), (IIIb) or (IIIc) are linked to each other at the 5′ end, and one or both of the 3′ ends of the are optionally conjugated to a ligand. Each of the dsRNA can target the same gene or two different genes; or each of the dsRNA can target same gene at two different target sites.

Various publications described multimeric siRNA and can all be used with the dsRNA of the invention. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520 which are hereby incorporated by their entirely.

The dsRNA agent that contains conjugations of one or more carbohydrate moieties to a dsRNA agent can optimize one or more properties of the dsRNA agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the dsRNA agent. E.g., the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

In embodiment the dsRNA of the invention is conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

The double-stranded RNA (dsRNA) agent of the invention may optionally be conjugated to one or more ligands. The ligand can be attached to the sense strand, antisense strand or both strands, at the 3′-end, 5′-end or both ends. For instance, the ligand may be conjugated to the sense strand, in particular, the 3′-end of the sense strand.

Ligands

A wide variety of entities can be coupled to the oligonucleotides of the present invention. Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether.

In preferred embodiments, a ligand alters the distribution, targeting or lifetime of the molecule 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, receptor 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. Ligands providing enhanced affinity for a selected target are also termed targeting ligands.

Some ligands can have endosomolytic properties. The endosomolytic ligands promote the lysis of the endosome and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell. The endosomolytic ligand may be a polyanionic peptide or peptidomimetic which shows pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand assumes its active conformation at endosomal pH. The “active” conformation is that conformation in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

Ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); a 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, an oligonucleotide (e.g. an aptamer). 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, an RGD peptide, an RGD peptide mimetic or an aptamer. Table 2 shows some examples of targeting ligands and their associated receptors.

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 or a chelator (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, multivalent fucose, or aptamers. 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.

The ligand can increase the uptake of the oligonucleotide into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, 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, naproxen 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 B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HAS, low density lipoprotein (LDL) and high-density lipoprotein (HDL).

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 alphα-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 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: 1). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 2)) 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: 3) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) (SEQ ID NO: 4) 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 an iRNA 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 iRNA 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 an iRNA agent to a tumor cell expressing α_Vβ₃(Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type lignads that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.

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).

In one embodiment, a targeting peptide can be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i±3, or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting a specific receptor. Examples are: folate, GalNAc, galactose, mannose, mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster, galactose cluster, or an apatamer. A cluster is a combination of two or more sugar units. The targeting ligands also include integrin receptor ligands, Chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. The ligands can also be based on nucleic acid, e.g., an aptamer. The aptamer can be unmodified or have any combination of modifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles, PEIs, peptides, fusogenic peptides, polycarboxylates, polyacations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketyals, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulator include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g. oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbaone are also amenable to the present invention as ligands (e.g. as PK modulating ligands).

In addition, aptamers that bind serum components (e.g. serum proteins) are also amenable to the present invention as PK modulating ligands.

Other ligand conjugates amenable to the invention are described in U.S. patent applications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser. No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filed Aug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser. No. 11/944,227 filed Nov. 21, 2007, which are incorporated by reference in their entireties for all purposes.

When two or more ligands are present, the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties. For example, a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties. In a preferred embodiment, all the ligands have different properties.

Ligands can be coupled to the oligonucleotides at various places, for example, 3′-end, 5′-end, and/or at an internal position. In preferred embodiments, the ligand is attached to the oligonucleotides via an intervening tether, e.g. a carrier described herein. The ligand or tethered ligand may be present on a monomer when said monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated via coupling to a “precursor” monomer after said “precursor” monomer has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH₂)_nNH₂may be incorporated into a growing oligonucleotide strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer's tether.

In another example, a monomer having a chemical group suitable for taking part in Click Chemistry reaction may be incorporated e.g., an azide or alkyne terminated tether/linker. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having complementary chemical group, e.g. an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.

For double-stranded oligonucleotides, ligands can be attached to one or both strands. In some embodiments, a double-stranded iRNA agent contains a ligand conjugated to the sense strand. In other embodiments, a double-stranded iRNA agent contains a ligand conjugated to the antisense strand.

In some embodiments, ligand can be conjugated to nucleobases, sugar moieties, or internucleosidic linkages of nucleic acid molecules. Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a conjugate moiety. Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to sugar moieties of nucleosides can occur at any carbon atom. Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2′, 3′, and 5′ carbon atoms. The 1′ position can also be attached to a conjugate moiety, such as in an abasic residue. Internucleosidic linkages can also bear conjugate moieties. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleosidic linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

Any suitable ligand in the field of RNA interference may be used, although the ligand is typically a carbohydrate e.g. monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, polysaccharide.

Linkers that conjugate the ligand to the nucleic acid include those discussed above. For example, the ligand can be one or more GalNAc (N-acetylglucosamine) derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, the dsRNA of the invention is conjugated to a bivalent and trivalent branched linkers include the structures shown in any of formula (IV)-(VII):

embedded image

wherein:

q^2A, q^2B, q^3A, q^3B, q^4A, q^4B, q^5Band q^5Cfor each represent independently occurrence 0-20 and wherein the repeating unit can be the same or different;

P^2A, P^2B, P^3A, P^3B, P^4A, P^4B, P^5A, P^5B, P^5C, T^2A, T^2B, T^3A, T^3B, T^4A, T^4B, T^4A, T^5B, T^5Care each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^2A, Q^2B, Q^3A, Q^3B, Q^4A, Q^4B, Q^5A, Q^5B, Q^5Care independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^N), C(R′)═C(R″), CC or C(O);

R^2A, R^2B, R^3A, R^3B, R^4A, R^4B, R^5A, R^5B, R^5Care each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^a)C(O), —C(O)—CH(R^a)—NH—, CO, CH═N—O,

embedded image

or heterocyclyl;

L^2A, L^2B, L^3A, L^3B, L^4A, L^4B, L^5A, L^5Band L^5Crepresent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and

R^ais H or amino acid side chain.

Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (VII):

embedded image

wherein L^5A, L^5Band L^5Crepresent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the following compounds:

embedded image

Definitions

As used herein, the terms “dsRNA”, “siRNA”, and “iRNA agent” are used interchangeably to agents that can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., an siRNA agent of 21 to 23 nucleotides.

As used herein, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, a dsRNA agent of the invention is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the dsRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the dsRNA agent of the invention is “exactly complementary” to a target RNA, e.g., the target RNA and the dsRNA duplex agent anneal, for example to form a hybrid made exclusively of Watson-Crick base pairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the dsRNA agent of the invention specifically discriminates a single-nucleotide difference. In this case, the dsRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) for example of length less than 100, 200, 300, or 400 nucleotides.

The term “halo” refers to any radical of fluorine, chlorine, bromine or iodine. The term “alkyl” refers to saturated and unsaturated non-aromatic hydrocarbon chains that may be a straight chain or branched chain, containing the indicated number of carbon atoms (these include without limitation propyl, allyl, or propargyl), which may be optionally inserted with N, O, or S. For example, C₁-C₁₀indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it. The term “alkoxy” refers to an —O-alkyl radical. The term “alkylene” refers to a divalent alkyl (i.e., —R—). The term “alkylenedioxo” refers to a divalent species of the structure —O—R—O—, in which R represents an alkylene. The term “aminoalkyl” refers to an alkyl substituted with an amino The term “mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an —S-alkyl radical.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refers to alkyl substituted with an aryl. The term “arylalkoxy” refers to an alkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. The term “heteroarylalkyl” or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.

The term “substituted” refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylamino carbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent can be further substituted.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, liver targeting ligands can be linked to the cationic lipids through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groups that are cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In a preferred embodiment, candidate compounds are cleaved by at most 10% in the blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHR^AC(O)NHCHR^BC(O)—, where R^Aand R^Bare the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4-9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C₅and above (preferably C₅-C₈) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (preferably C₅-C₈).

Alternative Embodiments

In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at the cleavage site in the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one nucleotide. The antisense strand contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at or near cleavage site by at least one nucleotide. The modification in the motif occurring at the cleavage site in the sense strand is different than the modification in the motif occurring at or near the cleavage site in the antisense strand. In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 12 to 30 nucleotides. The sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at the cleavage site in the strand. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides.

The sense strand may further comprises one or more motifs of three identical modifications on three consecutive nucleotides, where the one or more additional motifs occur at another portion of the strand that is separated from the three 2′-F modifications at the cleavage site by at least one nucleotide. The antisense strand may further comprises one or more motifs of three identical modifications on three consecutive nucleotides, where the one or more additional motifs occur at another portion of the strand that is separated from the three 2′-O-methyl modifications by at least one nucleotide. At least one of the nucleotides having a 2′-F modification may form a base pair with one of the nucleotides having a 2′-O-methyl modification.

In one embodiment, the dsRNA of the invention is administered in buffer.

In one embodiment, siRNA compounds described herein can be formulated for administration to a subject. A formulated siRNA composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the siRNA is in an aqueous phase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the siRNA composition is formulated in a manner that is compatible with the intended method of administration, as described herein. For example, in particular embodiments the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.

A siRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a siRNA, e.g., a protein that complexes with siRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the siRNA preparation includes another siNA compound, e.g., a second siRNA that can mediate RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different siRNA species. Such siRNAs can mediate RNAi with respect to a similar number of different genes.

In one embodiment, the siRNA preparation includes at least a second therapeutic agent (e.g., an agent other than a RNA or a DNA). For example, a siRNA composition for the treatment of a viral disease, e.g., HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a siRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.

Exemplary formulations are discussed below.

Liposomes.

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified siRNA compounds. It may be understood, however, that these formulations, compositions and methods can be practiced with other siRNA compounds, e.g., modified siRNAs, and such practice is within the invention. An siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof) preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the siRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the siRNA composition, although in some examples, it may. 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 liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the siRNA are delivered into the cell where the siRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the siRNA to particular cell types.

A liposome containing a siRNA can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The siRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the siRNA and condense around the siRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of siRNA.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging siRNA preparations into liposomes.

Liposomes that are pH-sensitive or negatively-charged entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are 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, 19, (1992) 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.

Examples of other methods to introduce liposomes into cells in vitro and include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver siRNAs to macrophages.

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 siRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, 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.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of siRNA (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Choi”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited 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 siRNA, into the skin. In some implementations, liposomes are used for delivering siRNA to epidermal cells and also to enhance the penetration of siRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

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 a drug into the dermis of mouse skin. Such formulations with siRNA are useful for treating a dermatological disorder.

Liposomes that include siRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include siRNA can be delivered, for example, subcutaneously by infection in order to deliver siRNA to keratinocytes in the skin. In order to cross 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. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described in U.S. provisional application Ser. Nos. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.

Surfactants.

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical 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).

Micelles and Other Membranous Formulations.

For ease of exposition the micelles and other formulations, compositions and methods in this section are discussed largely with regard to unmodified siRNA compounds. It may be understood, however, that these micelles and other formulations, compositions and methods can be practiced with other siRNA compounds, e.g., modified siRNA compounds, and such practice is within the invention. The siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof)) composition can be provided as a micellar formulation. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C₈to C₂₂alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, mono laurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, late, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

Particles.

For ease of exposition the particles, formulations, compositions and methods in this section are discussed largely with regard to modified siRNA compounds. It may be understood, however, that these particles, formulations, compositions and methods can be practiced with other siRNA compounds, e.g., unmodified siRNA compounds, and such practice is within the invention. In another embodiment, an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof) preparations may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

Pharmaceutical Compositions

The iRNA agents of the invention may be formulated for pharmaceutical use. Pharmaceutically acceptable compositions comprise a therapeutically-effective amount of one or more of the dsRNA agents in any of the preceding embodiments, taken alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents.

The pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous.

The phrase “therapeutically-effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention. In certain embodiments, an aforementioned formulation renders orally bio available a compound of the present invention.

iRNA agent preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a iRNA, e.g., a protein that complexes with iRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

The term “treatment” is intended to encompass also prophylaxis, therapy and cure. The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

Double-stranded RNAi agents are produced in a cell in vivo, e.g., from exogenous DNA templates that are delivered into the cell. For example, the DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. The DNA templates, for example, can include two transcription units, one that produces a transcript that includes the top strand of a dsRNA agent and one that produces a transcript that includes the bottom strand of a dsRNA agent. When the templates are transcribed, the dsRNA agent is produced, and processed into siRNA agent fragments that mediate gene silencing.

Routes of Delivery

A composition that includes an iRNA can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of iRNA and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The 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 (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iRNA in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.

Dosage

In one aspect, the invention features a method of administering a dsRNA agent, e.g., a siRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the dsRNA agent, e.g., a siRNA agent, e.g., double stranded siRNA agent that (a) the double-stranded part is 14-30 nucleotides (nt) long, for example, 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 10 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g., about 4.4×10¹⁶copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA. The unit dose, for example, can be administered by injection (e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, or a topical application. In some embodiments dosages may be less than 10, 5, 2, 1, or 0.1 mg/kg of body weight.

In some embodiments, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.

In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than a dsRNA agent, e.g., other than a siRNA agent. In another embodiment, the subject has atherosclerosis and the effective dose of a dsRNA agent, e.g., a siRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one or more maintenance doses of a dsRNA agent, e.g., a siRNA agent, (e.g., a precursor, e.g., a larger dsRNA agent which can be processed into a siRNA agent, or a DNA which encodes a dsRNA agent, e.g., a siRNA agent, or precursor thereof). The maintenance dose or doses can be the same or lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 15 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are, for example, administered no more than once every 2, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In certain embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.

In one embodiment, the composition includes a plurality of dsRNA agent species. In another embodiment, the dsRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of dsRNA agent species is specific for different naturally occurring target genes. In another embodiment, the dsRNA agent is allele specific.

The dsRNA agents of the invention described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

In one embodiment, the administration of the dsRNA agent, e.g., a siRNA agent, composition is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

The invention provides methods, compositions, and kits, for rectal administration or delivery of dsRNA agents described herein

Methods of Inhibiting Expression of the Target Gene

Embodiments of the invention also relate to methods for inhibiting the expression of a target gene. The method comprises the step of administering the dsRNA agents in any of the preceding embodiments, in an amount sufficient to inhibit expression of the target gene.

Another aspect the invention relates to a method of modulating the expression of a target gene in a cell, comprising providing to said cell a dsRNA agent of this invention. In one embodiment, the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepciden, Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73 gene, mutations in the p21(WAF1/CIP1) gene, mutations in the p27(KIP1) gene, mutations in the PPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor suppressor genes, and mutations in the p53 tumor suppressor gene.

The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

EXAMPLES
Example 1. In Vitro Screening of siRNA Duplexes

Cell Culture and Transfections:

Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×10⁴Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done using 8, 4 fold serial dilutions with a maximum dose of 10 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 1.6 μl of H₂O per reaction were added into 5 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E (human) Cat #4308313 (rodent)), 0.5 μl TTR TaqMan probe (Applied Biosystems cat #HS00174914_ml (human) cat #Rn00562124_ml (rat)) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plate (Roche cat #04887301001). Real time PCR was done in a Roche LC 480 Real Time PCR machine (Roche). Each duplex was tested in at least two independent transfections and each transfection was assayed in duplicate, unless otherwise noted.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose. IC₅₀s were calculated for each individual transfection as well as in combination, where a single IC₅₀was fit to the data from both transfections.

The results of gene silencing of the exemplary siRNA duplex with various motif modifications of the invention are shown in the table below.

Example 2. RNA Synthesis and Duplex Annealing

1. Oligonucleotide Synthesis:

All oligonucleotides were synthesized on an AKTAoligopilot synthesizer or an ABI 394 synthsizer. 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 unless otherwise specified. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite were purchased from (Promega). All phosphoramidites were used at a concentration of 0.2M in acetonitrile (CH₃CN) except for guanosine which was used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes was used. The activator was 5-ethyl thiotetrazole (0.75M, American International Chemicals), for the PO-oxidation Iodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) was used.

Ligand conjugated strands were synthesized using solid support containing the corresponding ligand. For example, the introduction of carbohydrate moiety/ligand (for e.g., GalNAc) at the 3′-end of a sequence was achieved by starting the synthesis with the corresponding carbohydrate solid support. Similarly a cholesterol moiety at the 3′-end was introduced by starting the synthesis on the cholesterol support. In general, the ligand moiety was tethered to trans-4-hydroxyprolinol via a tether of choice as described in the previous examples to obtain a hydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety was then coupled to a solid support via a succinate linker or was converted to phosphoramidite via standard phosphitylation conditions to obtain the desired carbohydrate conjugate building blocks. Fluorophore labeled siRNAs were synthesized from the corresponding phosphoramidite or solid support, purchased from Biosearch Technologies. The oleyl lithocholic (GalNAc)₃polymer support made in house at a loading of 38.6 mmol/gram. The Mannose (Man)₃polymer support was also made in house at a loading of 42.0 mmol/gram.

Conjugation of the ligand of choice at desired position, for example at the 5′-end of the sequence, was achieved by coupling of the corresponding phosphoramidite to the growing chain under standard phosphoramidite coupling conditions unless otherwise specified. An extended 15 min coupling of 0.1M solution of phosphoramidite in anhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate was 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 was 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 was synthesized in house, and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite was 16 minutes.

2. Deprotection-I (Nucleobase Deprotection)

After completion of synthesis, the support was transferred to a 100 ml glass bottle (VWR). The oligonucleotide was 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 was cooled briefly on ice and then the ethanolic ammonia mixture was filtered into a new 250 ml bottle. The CPG was washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture was then reduced to ˜30 ml by roto-vap. The mixture was then frozen on dry ice and dried under vacuum on a speed vac.

3. Deprotection-II (Removal of 2′ TBDMS Group)

The dried residue was 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 was then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to 6.5, and stored in freezer until purification.

4. Analysis

The oligoncuelotides were 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.

5. HPLC Purification

The ligand conjugated oligonucleotides were purified reverse phase preparative HPLC. The unconjugated oligonucleotides were purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers were 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 were pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotidess were diluted in water to 150 μl and then pipetted in special vials for CGE and LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

6. siRNA Preparation

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

TABLE 2

ANGPTL3 modified duplex

% of mRNA

Antisense strand (AS)
remained conc.

Du-

Sense strand (S) (SEQ ID NOS 5-

(SEQ ID NOS 425-844,
of siRNA

plex

424, respectively, in order of

respectively, in
1
0.1
0.01
IC50

ID
S ID
appearance)
AS ID
order of appearance)
nM
nM
nM
(nM)

D1000
51000
AfuGfuAfaCfcAfAfGfaGfuAfuUfcCfasu
A51000
AfUfgGfaAfuAfcUfcu
0.03
0.1
0.47
0.006

uGfgUfuAfcAfusGfsa

D1001
51001
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1001
aUfsgGfAfAfuAfcUfc
0.03
0.10
0.49
0.0065

uuGfgUfuAfcAfusGfsa

D1002
51002
AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
A51002
aUfgGfAfAfuAfcUfc
0.04
0.10
0.46
0.0068

uuGfgsUfuAfcAfusGfsa

D1003
51003
AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
A51003
aUfgGfAfAfuAfcUfcu
0.05
0.12
0.56
0.0073

uGfgUfsuAfcAfusGfsa

D1004
51004
aUGuaACccAGagUAuuCCasu
A51004
AUggAAuaCUcuUGguUA
0.07
0.13
0.44
0.008

caUsGsa

D1005
51005
AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
A51005
aUfgGfAfAfuAfcUfcuu
0.06
0.11
0.53
0.0093

GfgsUfsuAfcAfusGfsa

D1006
51006
AfuGfuAfAfccAfAfGfaGfuAfuUfcCfasUf
A51006
aUfgGfaAfuAfcUfcuu
0.05
0.16
0.55
0.0095

GfGfuuAfcAfusGfsa

D1007
51007
AfuGfuAfAfCfcAfAfGfaGfuAfuUfc
A51007
aUfgGfaAfuAfcUfcuu
0.05
0.14
0.48
0.0098

CfasUf

GfguuAfcAfusGfsa

D1008
51008
auguaaccaadGadGudAudAcdGasu
A51008
aUfgGfaAfuAfcUfca
0.07
0.11
0.33
0.010

UfuGfgUfuAfcAfusGfs

D1009
51009
UfgGfGfAfuUfuCfAfUfgUfa
A51009
uCfuugGfuUfaCfaugA
0.03
0.14
0.56
0.0101

AfcCfAfAfgsAf

faAfuccCfasUfsc

D1010
51010
UfgGfgauUfuCfAfUfgUfaAfcCfaAfgsAf
AS1010
uCfuUfgGfuUfaCfau
0.03
0.14
0.65
0.0101

gAfaAfUfCfcCfasUfsc

D1011
51011
aUfGfuAfAfccAfAfGfaGfuAfuUfcCfasUf
AS1011
aUfgGfaAfuAfcUfcuuG
0.06
0.10
0.55
0.011

fGfuuAfcaUfsgsa

D1012
51012
UfgGfgAfuUfuCfAfUfgUfaacCfaAfgsAf
A51012
uCfuUfgGfUfUfaCfa
0.04
0.13
0.54
0.0114

ugAfaAfuCfcCfasUfsc

D1013
51013
auguaaccaadGadGudAudAcdGasu
A51013
aUfgGfaAfuAfcUfcUf
0.11
0.19
0.49
0.011

ugdGudTadCadTsgsa

D1014
51014
AfuGfuaaCfcAfAfGfaGfuAfuUfcCfasUf
A51014
aUfgGfaAfuAfcUfcuu
0.04
0.16
0.59
0.013

GfgUfUfAfcAfusGfsa

D1015
51015
AfuguAfaccAfaGfdAGfdTAdTudCcdAsu
A51015
dAUdGgdAadTAfdCUfcU
0.07
0.15
0.51
0.013

fuGfgUfuAfcAfusGfsa

D1016
51016
auGfuAfaCfcAfAfGfaGfuAfuUfcCfasUf
A51016
aUfgGfaAfuAfcUfcuu
0.05
0.14
0.64
0.013

GfgUfuAfcAfUfsGfsa

D1017
51017
UfGfggAfuUfuCfAfUfgUfAfA
A51017
uCfuUfgGfuuaCfaug
0.09
0.41
0.74
0.0133

fcCfaAfgsAf

AfaAfuCfCfcasUfsc

D1018
51018
AfuguAfaCfcAfAfGfaGfuAfuUfcCfasUf
A51018
aUfgGfaAfuAfcUfcuu
0.03
0.14
0.61
0.014

GfgUfuAfCfAfusGfsa

D1019
51019
AfuGfuAfaccAfAfGfaGfuAfuUfcCfasUf
A51019
aUfgGfaAfuAfcUfcuuG
0.02
0.2
0.7
0.014

fGfUfuAfcAfusGfsa

D1020
51020
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
A51020
asUfsgGfAfAfuAfcUfc
0.04
0.16
0.67
0.0156

uuGfgUfuAfcAfusGfsa

D1021
51021
aUfguAfAfccAfAfgagUfaUfuCfcasUf
A51021
aUfGfgAfaUfaCfUfCf
0.11
0.24
0.64
0.016

uuGfGfuuAfCfaUfsgsa

D1022
51022
dTdGggdAdTuudCdAugdTdAacdCdAagsdA
AS1022
udCdTugdGdTuadCdAug
0.08
0.27
0.64
0.0161

dAdAaudCdCcasdTsc

D1023
51023
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1023
aUfgsGfAfAfuAfcUfc
0.03
0.19
0.63
0.0163

uuGfgUfuAfcAfusGfsa

D1024
51024
UfgGfgAfu UfuCfAfUfguaAfcCfaAfgsAf
AS1024
uCfuUfgGfuUfAfCfaug
0.05
0.25
0.69
0.0164

AfaAfuCfcCfasUfsc

D1025
51025
UfgGfgAfuUfuCfAfUfgUfAfA
AS1025
uCfuUfgGfuuaCfaug
0.04
0.18
0.75
0.0166

fcCfaAfgsAf

AfaAfuCfcCfasUfsc

D1026
51026
UfgGfgAfuUfuCfAfUfgUfaAfcCfaAfgsAf
AS1026
uCfuUfgGfuUfaCfau
0.04
0.19
0.66
0.0178

gAfaAfuCfcCfasUfsc

D1027
51027
UfgGfgAfuUfuCfAfUfgUfaAfccaAfgsAf
AS1027
uCfuUfGfGfuUfaCfaug
0.04
0.19
0.69
0.018

AfaAfuCfcCfasUfsc

D1028
51028
dAdTgudAdAccdAdAgadGdTaudTdCcasdT
AS1028
adTdGgadAdTacdTdCu
0.15
0.29
0.72
0.018

udGdGuudAdCausdGsa

D1029
51029
AdTGdTAdACdCAdAGdAGdTAdTUdCCdAsU
AS1029
dAUdGGdAAdTAdCUdCUd
0.1
0.27
0.61
0.018

TGdGUdTAdCAdTsGsdA

D1030
51030
UfgGfGfAfuuuCfAfUfgUfaAfcCfaAfgsAf
AS1030
uCfuUfgGfuUfaCfaug
0.04
0.21
0.64
0.0187

AfAfAfuccCfasUfsc

D1031
51031
AfuGfuAfAfccAfAfGfAfGfuAfuuccAfsu
AS1031
AfUfGfGfAfAfuAf
0.06
0.15
0.62
0.019

CfUfCfUfuGfGf

uuAfcAfusGfsa

D1032
51032
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1032
asUfgGfAfAfuAfcUfcuu
0.09
0.34
0.78
0.021

GfgUfsuAfcAfusGfsa

D1033
51033
UfgGfgAfuUfuCfaUfGfUfaacCfaAfgsAf
AS1033
uCfuUfgGfUfUfacaUfg
0.06
0.26
0.57
0.0212

AfaAfuCfcCfasUfsc

D1034
51034
AfuGfuAfAfccAfaGfaGfuAfuUfcCfasUf
AS1034
aUfgGfaAfuAfcUfcUfu
0.11
0.39
0.82
0.0216

GfGfuuAfcAfusGfsa

D1035
51035
UfgGfgAfuuuCfAfUfgUfaAfcCfaAfgsAf
AS1035
uCfuUfgGfuUfaCfaug
0.04
0.16
0.56
0.0222

AfAfAfuCfcCfasUfsc

D1036
51036
UfgGfGfAfuUfuCfaUfgUfaAfcCfAfAfgsAf
AS1036
uCfuugGfuUfaCfaUfgA
0.06
0.31
0.78
0.0234

faAfuccCfasUfsc

D1037
51037
UfgGfGfAfuUfuCfAfUfgUfaAfcCfaAfgsAf
AS1037
uCfuUfgGfuUfaCfaugA
0.03
0.14
0.62
0.0235

faAfuccCfasUfsc

D1038
51038
UfGfggAfUfuuCfAfugUfAfacCfAfagsAf
AS1038
uCfUfugGfUfuaCfAfug
0.09
0.39
0.78
0.0239

AfAfauCfCfcasUfsc

D1039
51039
AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1039
aUfgGfAfAfuAfcUfcu
0.03
0.14
0.59
0.025

uGfgUfuAfcAfusGfsa

D1040
51040
AfuGfuAfaCfcAfAfGfaGfuAfuUfccasUf
AS1040
aUfGfGfaAfuAfcUfcu
0.03
0.13
0.56
0.025

uGfgUfuAfcAfusGfsa

D1041
51041
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1041
asUfgGfAfAfuAfcUfc
0.06
0.27
0.79
0.0252

uuGfgUfuAfcAfusGfsa

D1042
51042
UfgGfgAfuuuCfAfUfgUfAfAfcCfaAfgsAf
AS1042
uCfuUfgGfuuaCfaugAf
0.05
0.27
0.67
0.0259

AfAfuCfcCfasUfsc

D1043
51043
AfuGfuAfaCfcAfAfGfaGfuauUfcCfasUf
AS1043
aUfgGfaAfUfAfcUfcuu
0.02
0.16
0.63
0.027

GfgUfuAfcAfusGfsa

D1044
51044
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1044
asUfgGfAfAfuAfcUfcu
0.06
0.30
0.81
0.0271

uGfgsUfsuAfcAfusGfsa

D1045
51045
aUfguAfAfccAfAfgaGfGfauUfCfcasUf
AS1045
aUfGfgaAfUfacUfCfuu
0.12
0.29
0.8
0.028

GfGfuuAfCfaUfsgsa

D1046
51046
AfuGfuAfaCfcAfAfGfaguAfuUfcCfasUf
AS1046
aUfgGfaAfuAfCfUfcuu
0.03
0.15
0.59
0.030

GfgUfuAfcAfusGfsa

D1047
51047
UfgGfGfAfuUfuCfaUfgUfAfAfcCfaAfgsAf
AS1047
uCfuUfgGfuuaCfaUfg
0.08
0.44
0.83
0.0324

AfaAfuccCfasUfsc

D1048
51048
AfuGfuAfaCfcAfAfGfaGfuAfuUfcCfasUf
AS1048
aUfgGfaAfuAfcUfcu
0.07
0.23
0.67
0.036

uGfgUfuAfcAfusGfsa

D1049
51049
AfuGfuAfAfccAfAfGfAfGfuAfuuccAfsu
AS1049
AfUfGfGfAfAfuAf
0.08
0.23
0.73
0.037

CfUfCfUfUfGfGfU

fuAfCfAfusGfsa

D1050
51050
UfgGfgAfuuuCfaUfgUfaAfcCfAfAfgsAf
AS1050
uCfuugGfuUfaCfaUfg
0.06
0.29
0.78
0.0372

AfAfAfuCfcCfasUfsc

D1051
51051
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1051
aUfgGfaAfudAcdTcdT
0.12
0.41
0.86
0.040

udGgdTuAfcAfusgsa

D1052
51052
AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu
AS1052
aUfgGfaAfuAfcUfcUf
0.1
0.22
0.72
0.042

uGfgUfuAfcAfusGfsa

D1053
51053
AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu
AS1053
dAUdGGdAAfuAfcUfcUfu
0.09
0.31
0.69
0.044

GfGfUfuAfCfAfusGfsa

D1054
51054
AfuGfuAfaCfcAfaGfadGdTAfuUfcdCdAsUf
AS1054
adTdGGfaAfudAdCUfcU
0.1
0.45
0.75
0.047

fuGfgUfuAfcAfusGfsa

D1055
51055
AfuguAfaccAfaGfaGfdTAdTUdCcdAsu
AS1055
dAUdGGdAadTAfcUfcUf
0.12
0.26
0.7
0.049

uGfgUfuAfcAfusGfsa

D1056
51056
AuGuAaCcAaGaGuAuUcCasU
AS1056
aUgGaAuAcUcUuGgU
0.08
0.24
0.65
0.050

uAcAusGsa

D1057
51057
AfuguAfaccAfagaGfuauUfccasUf
AS1057
aUfGfGfaAfUfAfcUfCfUf
0.14
0.42
0.62
0.051

uGfGfUfuAfCfAfusGfsa

D1058
51058
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1058
aUfgGfaAfudAcdTcdTu
0.12
0.36
0.86
0.053

dGgdTuAfcAfusGfsa

D1059
51059
AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu
AS1059
dAUdGGdAadTAfdCUfcUfu
0.09
0.27
0.7
0.054

GfgUfuAfcAfusGfsa

D1060
51060
adTgudAdAccdAdAgagdTadTudCcasdT
AS1060
adTdGgdAadTadCdTdC
0.11
0.37
0.66
0.056

uudGdGuudAdCadTsgsa

D1061
51061
AfuGfuAfaCfcAfaGfdAdG
AS1061
adTdGGfaAfuAfdCdTcU
0.1
0.31
0.77
0.059

uAfuUfcdCdAsUf

fuGfgUfuAfcAfusGfsa

D1062
51062
AfuguAfaccAfaGfdAGfdTAdTudCcdAsu
AS1062
aUfgGfaAfuAfcUfcUf
0.1
0.27
0.65
0.059

uGfgUfuAfcAfusGfsa

D1063
51063
adTdGuadAdCccdAdGagdTdAuudCdCasu
AS1063
dAdTggdAdAuadCdTcu
0.12
0.44
0.82
0.064

dTdGgudTdAcadTsdGsa

D1064
51064
AfuGfuAfaCfcAfaGfaGfdTdAuUfcdC
AS1064
adTdGGfaAfdTdAcUfcU
0.12
0.32
0.83
0.064

dAsUf

fuGfgUfuAfcAfusGfsa

D1065
51065
AfuguAfaccAfaGfaGfdTAdTudCcdAsu
AS1065
dAUdGgdAadTAfcUfcU
0.13
0.34
0.72
0.066

fuGfgUfuAfcAfusGfsa

D1066
51066
AfuGfuAfaCfcAfaGfaGfudAdTUf
AS1066
adTdGGfadAdTAfcUfcU
0.11
0.33
0.72
0.067

cdCdAsUf

fuGfgUfuAfcAfusGfsa

D1067
51067
AfuguAfaccAfaGfaGfdTAdTUdCcdAsu
AS1067
aUfgGfaAfuAfcUfcUf
0.11
0.37
0.62
0.070

uGfgUfuAfcAfusGfsa

D1068
51068
AfuguAfaccAfaGfaGfdTAdTUdCcdAsu
AS1068
dAUdGGdAAuAfcUfcUfu
0.16
0.33
0.64
0.072

GfGfUfuAfCfAfusGfsa

D1069
51069
aUfGfuaAfCfccAfGfagUfAfuuCfCfasu
AS1069
AfUfggAfAfuaCfUfcuU
0.14
0.43
0.73
0.074

fGfguUfAfcaUfsGfsa

D1070
51070
AfuGfuAfaCfCfAfaGfaguAfuUfcCfasUf
AS1070
aUfgGfaAfuAfCfUfcU
0.08
0.42
0.94
0.075

fuggUfuAfcAfusGfsa

D1071
51071
UfgGfgAfuuuCfaUfgUfaAfcCfaAfgsAf
AS1071
uCfuUfgGfuUfaCfaUf
0.14
0.28
0.83
0.0797

gAfAfAfuCfcCfasUfsc

D1072
51072
AfuGfuAfaCfcAfaGfAfGfuauUfcCfasUf
AS1072
aUfgGfaAfUfAfcucUf
0.05
0.26
0.8
0.082

uGfgUfuAfcAfusGfsa

D1073
51073
AfuGfuAfaCfcAfaGfadGdT
AS1073
aUfgGfadAdTdAdCUfc
0.12
0.41
0.73
0.083

dAdTUfcCfasUf

UfuGfgUfuAfcAfusGfsa

D1074
51074
AfUfguAfAfccAfAfgaGfUfauUfCfcasUf
AS1074
aUfGfgaAfUfacUfCf
0.14
0.44
0.75
0.086

uuGfGfuuAfCfausGfsa

D1075
51075
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1075
aUfgGfdAdAdTdAcUfcU
0.1
0.41
0.72
0.088

fuGfgUfuAfcAfusGfsa

D1076
51076
AfuGfuAfaCfcAfaGfaGfudAdT
AS1076
aUfgdGdAdAdTAfcUfcU
0.15
0.45
0.86
0.088

dTdCCfasUf

fuGfgUfuAfcAfusGfsa

D1077
51077
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasu
AS1077
AfUfgGfaAfuAfcUfcU
0.08
0.46
0.95
0.092

fuGfgUfuAfcAfusGfsa

D1078
51078
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1078
dAUdGGdAadTAfcUfcU
0.09
0.32
0.76
0.093

fuGfgUfuAfcAfusGfsa

D1079
51079
AfuguAfaccAfaGfaGfdTadTudCcdAsu
AS1079
dAudGgdAadTAfcUfcUf
0.14
0.38
0.76
0.095

uGfgUfuAfcAfusGfsa

D1080
51080
AfuGfuAfaCfcAfaGfAfGfuAfuucCfasUf
AS1080
aUfgGfAfAfuAfcucU
0.05
0.42
0.86
0.099

fuGfgUfuAfcAfusGfsa

D1081
51081
AfuGfuAfaCfcAfaGfaGfu
AS1081
dAdTdGdGaAfuAfcUfc
0.17
0.47
0.9
0.105

AfuUfdCdCdAsdT

UfuGfgUfuAfcAfusGfsa

D1082
51082
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1082
aUfgGfaAfudACfudCU
0.12
0.44
0.83
0.106

fudGGfudTAfcAfusgsa

D1083
51083
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1083
adTdGGfaAfdTdAcUfcU
0.11
0.34
0.74
0.109

fuGfgUfuAfcAfusGfsa

D1084
51084
AfuGfuAfAfCfcAfaGfaGfuauUfcCfasUf
AS1084
aUfgGfaAfUfAfcUfcUf
0.1
0.45
0.93
0.117

uGfguuAfcAfusGfsa

D1085
51085
AfuGfUfAfaCfcAfaGfaGfuauUfcCfasUf
AS1085
aUfgGfaAfUfAfcUfcUf
0.07
0.42
0.78
0.120

uGfgUfuacAfusGfsa

D1086
51086
aUfguAfAfccAfAfgaGfuAfuUfcCfasUf
AS1086
aUfgGfaAfuAfcUfCfuu
0.17
0.45
0.83
0.1197

GfGfuuAfCfaUfsgsa

D1087
51087
AfuGfuAfaCfcAfaGfaGfUfAfuUfcCfasu
AS1087
AfUfgGfaAfuacUfcUfu
0.05
0.3
0.7
0.120

GfgUfuAfcAfusGfsa

D1088
51088
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1088
aUfgGfaAfuAfcUfcUf
0.11
0.46
0.8
0.120

uGfgUfuAfcAfusgsa

D1089
51089
AfuGfuAfaCfcAfaGfaGfUfAfuUfcCfasUf
AS1089
aUfgGfaAfuacUfcUfu
0.14
0.49
0.85
0.122

GfgUfuAfcAfusGfsa

D1090
51090
AfuGfuAfaCfcAfaGfaGfuauUfcCfasUf
AS1090
aUfgGfaAfUfAfcUfcU
0.1
0.41
0.85
0.125

fuGfgUfuAfcAfusGfsa

D1091
51091
AfuguAfaccAfaGfaGfdTAdTudCcdAsu
AS1091
aUfgGfaAfuAfcUfcUf
0.16
0.38
0.77
0.125

uGfgUfuAfcAfusGfsa

D1092
51092
AfuGfuAfaCfcAfaGfAfGfuAfuUfcCfasu
AS1092
AfUfgGfaAfuAfcucUf
0.05
0.31
0.93
0.126

uGfgUfuAfcAfusGfsa

D1093
51093
auGfuAfaCfcAfaGfAfGfuAfuUfcCfasUf
AS1093
aUfgGfaAfuAfcucUfu
0.06
0.33
0.9
0.135

GfgUfuAfcAfUfsGfsa

D1094
51094
AfuGfuAfaCfcAfaGfaGfUfAfuUfccasUf
AS1094
aUfGfGfaAfuacUfcUf
0.07
0.39
0.85
0.142

uGfgUfuAfcAfusGfsa

D1095
51095
AfuGfuAfaCfcAfaGfAfGfuAfuUfcCfasUf
AS1095
aUfgGfaAfuAfcucUfu
0.09
0.39
0.76
0.146

GfgUfuAfcAfusGfsa

D1096
51096
AfuGfuAfaCfcAfaGfaGfUfAfuucCfasUf
AS1096
aUfgGfAfAfuacUfcUf
0.06
0.38
0.85
0.147

uGfgUfuAfcAfusGfsa

D1097
51097
AfuGfUfAfaCfcAfaGfaGfuAfuucCfasUf
AS1097
aUfgGfAfAfuAfcUfcU
0.12
0.47
0.87
0.147

fuGfgUfuacAfusGfsa

D1098
51098
AfuGfuAfaCfcAfaGfaGfuAfUfUfccasUf
AS1098
aUfGfGfaauAfcUfcUf
0.06
0.42
0.85
0.151

uGfgUfuAfcAfusGfsa

D1099
51099
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1099
dAUdGGdAadTAfdCUfcU
0.16
0.41
0.85
0.152

fuGfgUfuAfcAfusGfsa

D1100
51100
AfuguAfaccAfaGfaGfuAfuUfcCfasUf
AS1100
aUfgGfaAfuAfcUfcUf
0.15
0.48
0.72
0.152

uGfgUfuAfcAfusGfsa

D1101
51101
AfuGfuAfaCfcAfaGfAfGfuAfuUfccasUf
AS1101
aUfGfGfaAfuAfcucUf
0.06
0.38
0.94
0.158

uGfgUfuAfcAfusGfsa

D1102
51102
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1102
aUfgGfaAfuAfdCuCfd
0.21
0.45
0.89
0.162

TuGfdGuUfacAfusGfsa

D1103
51103
AfuGfuaaCfCfAfaGfaGfuAfuUfcCfasUf
AS1103
aUfgGfaAfuAfcUfcUf
0.14
0.49
0.95
0.163

uggUfUfAfcAfusGfsa

D1104
51104
AfuGfuAfaccAfaGfaGfUfAfuUfcCfasUf
AS1104
aUfgGfaAfuacUfcUfu
0.06
0.36
0.92
0.163

GfGfUfuAfcAfusGfsa

D1105
51105
AfuGfuAfaCfcAfaGfaGfuAfuucCfasUf
AS1105
aUfgGfAfAfuAfcUfcU
0.1
0.45
0.84
0.167

fuGfgUfuAfcAfusGfsa

D1106
51106
AfuGfuaaCfcAfaGfAfGfuAfuUfcCfasUf
AS1106
aUfgGfaAfuAfcucUf
0.09
0.43
0.91
0.170

uGfgUfUfAfcAfusGfsa

D1107
51107
AfuGfuAfaccAfaGfAfGfuAfuUfcCfasUf
AS1107
aUfgGfaAfuAfcucUfuG
0.09
0.46
1
0.171

fGfUfuAfcAfusGfsa

D1108
51108
AfuguAfaccAfaGfaGfdTadTudCcdAsu
AS1108
aUfgGfaAfuAfcUfcUf
0.11
0.39
0.71
0.176

uGfgUfuAfcAfusGfsa

D1109
51109
AfuGfUfAfaCfcAfaGfaGfuAfuUfccasUf
AS1109
aUfGfGfaAfuAfcUfcU
0.1
0.43
0.9
0.180

fuGfgUfuacAfusGfsa

D1110
51110
AfuGfuAfaCfcAfaGfaguAfUfUfcCfasUf
AS1110
aUfgGfaauAfCfUfcUf
0.06
0.42
0.88
0.182

uGfgUfuAfcAfusGfsa

D1111
51111
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1111
dAUdGGdAAuAfcUfcUf
0.18
0.49
0.79
0.183

uGfGfUfuAfCfAfusGfs

D1112
51112
AfuGfUfAfaccAfaGfaGfuAfuUfcCfasUf
AS1112
aaUfgGfaAfuAfcUfcUf
0.14
0.48
0.85
0.195

uGfGfUfuacAfusGfsa

D1113
51113
AfuGfuAfaCfcAfaGfaguAfuUfcCfasUf
AS1113
aUfgGfaAfuAfCfUfcUf
0.09
0.41
0.85
0.201

uGfgUfuAfcAfusGfsa

D1114
51114
auGfuAfaCfcAfaGfaGfUfAfuUfcCfasUf
AS1114
aUfgGfaAfuacUfcUfu
0.05
0.44
0.94
0.201

GfgUfuAfcAfUfsGfsa

D1115
51115
AfuguAfaCfcAfaGfaGfUfAfuUfcCfasUf
AS1115
aUfgGfaAfuacUfcUfu
0.08
0.41
0.96
0.204

GfgUfuAfCfAfusGfsa

D1116
51116
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1116
adTdGGfadAdTAfcUfc
0.15
0.47
0.79
0.208

UfuGfgUfuAfcAfusGfsa

D1117
51117
AfuGfuaaCfcAfaGfaGfUfAfuUfcCfasUf
AS1117
aUfgGfaAfuacUfcUfu
0.08
0.42
0.92
0.224

GfgUfUfAfcAfusGfsa

D1118
51118
auguaaccaagaguauuccasu
AS1118
AfUfGfGfAfAfUfAfCf
0.19
0.5
0.87
0.303

UfCfUfUfGfGfUf

UfAfCfAfUfsgsa

D1119
51119
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1119
aUfgGfaAfuAfcUfcUf
0.14
0.55
0.89

uGfgUfuAfcAfusGfsa

D1120
51120
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1120
aUfgGfaAfuAfcUfcUfu
0.19
0.63
0.72

GfgUfuAfcAfusGfsa

D1121
51121
AfuGfuAfaccAfaGfaGfuAfuUfcCfasUf
AS1121
aUfgGfaAfuAfcUfcUfu
0.14
0.61
0.91

GfGfUfuAfcAfusGfsa

D1122
51122
AfUfGfuAfaCfcAfaGfaGfuAfuUfccasUf
AS1122
aUfGfGfaAfuAfcUfcU
0.14
0.54
0.95

fuGfgUfuAfcausGfsa

D1123
51123
auGfuAfAfCfcAfaGfaGfuAfuUfcCfasUf
AS1123
aUfgGfaAfuAfcUfcU
0.13
0.61
0.97

fuGfguuAfcAfUfsGfsa

D1124
51124
AfuGfuAfaCfcAfaGfaGfuAfUfUfcCfasUf
AS1124
aUfgGfaauAfcUfcUf
0.14
0.56
0.94

uGfgUfuAfcAfusGfsa

D1125
51125
AfuGfuAfaCfcaaGfaGfuAfuUfcCfasUf
AS1125
aUfgGfaAfuAfcUfcU
0.21
0.74
0.95

fUfGfgUfuAfcAfusGfsa

D1126
51126
AfUfGfuAfaCfcAfaGfaGfuAfuucCfasUf
AS1126
aUfgGfAfAfuAfcUfcU
0.2
0.69
0.91

fuGfgUfuAfcausGfsa

D1127
51127
AfuguAfAfCfcAfaGfaGfuAfuUfcCfasUf
AS1127
aUfgGfaAfuAfcUfcUf
0.17
0.7
0.96

uGfguuAfCfAfusGfsa

D1128
51128
AfUfGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1128
aUfgGfaAfuAfcUfcUf
0.19
0.62
0.85

uGfgUfuAfcausGfsa

D1129
51129
AfuGfuAfaCfcAfaGfaGfuAfuUfCfCfasUf
AS1129
aUfggaAfuAfcUfcUf
0.23
0.76
0.98

uGfgUfuAfcAfusGfsa

D1130
51130
AfuGfuAfaCfcAfagaGfuAfuUfcCfasUf
AS1130
aUfgGfaAfuAfcUfCfU
0.21
0.64
0.9

fuGfgUfuAfcAfusGfsa

D1131
51131
AfuGfuAfAfCfcaaGfaGfuAfuUfcCfasUf
AS1131
aUfgGfaAfuAfcUfcUfU
0.17
0.7
1.01

fGfguuAfcAfusGfsa

D1132
51132
AfuGfUfAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1132
aUfgGfaAfuAfcUfcUf
0.17
0.58
0.87

uGfgUfuacAfusGfsa

D1133
51133
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfAfsUf
AS1133
augGfaAfuAfcUfcUfu
0.33
0.89
1.05

GfgUfuAfcAfusGfsa

D1134
51134
AfUfGfuAfaCfcAfaGfaguAfuUfcCfasUf
AS1134
aUfgGfaAfuAfCfUfc
0.16
0.64
0.96

UfuGfgUfuAfcausGfsa

D1135
51135
AfuGfUfAfaCfcAfaGfaguAfuUfcCfasUf
AS1135
aUfgGfaAfuAfCfUfcU
0.12
0.53
0.96

fuGfgUfuacAfusGfsa

D1136
51136
AfuGfuAfAfCfcAfagaGfuAfuUfcCfasUf
AS1136
aUfgGfaAfuAfcUfCfU
0.16
0.58
0.98

fuGfguuAfcAfusGfsa

D1137
51137
AfuGfuAfAfCfcAfaGfaGfuAfuUfcCfasUf
AS1137
aUfgGfaAfuAfcUfcUf
0.16
0.6
0.91

uGfguuAfcAfusGfsa

D1138
51138
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1138
aUfgGfaAfuAfcUfcUfu
0.1
0.54
0.91

GfgUfuAfcAfusGfsAf

D1139
51139
AfUfGfuAfaCfcAfagaGfuAfuUfcCfasUf
AS1139
aUfgGfaAfuAfcUfCfU
0.24
0.68
0.98

fuGfgUfuAfcausGfsa

D1140
51140
AfuGfUfAfaCfcAfagaGfuAfuUfcCfasUf
AS1140
aUfgGfaAfuAfcUfCfU
0.13
0.75
0.9

fuGfgUfuacAfusGfsa

D1141
51141
AfuGfuAfAfCfcAfaGfaguAfuUfcCfasUf
AS1141
aUfgGfaAfuAfCfUfcU
0.15
0.52
1.05

fuGfguuAfcAfusGfsa

D1142
51142
AfuGfuAfaCfCfAfaGfaGfuAfuUfcCfasUf
AS1142
aUfgGfaAfuAfcUfcUf
0.16
0.66
0.89

uggUfuAfcAfusGfsa

D1143
51143
auGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1143
aUfgGfaAfuAfcUfcUfu
0.12
0.51
0.89

GfgUfuAfcAfUfsGfsa

D1144
51144
AfUfGfuAfaCfcaaGfaGfuAfuUfcCfasUf
AS1144
aUfgGfaAfuAfcUfcUf
0.25
0.71
0.95

UfGfgUfuAfcausGfsa

D1145
51145
AfuGfUfAfaCfcaaGfaGfuAfuUfcCfasUf
AS1145
aUfgGfaAfuAfcUfcUf
0.17
0.74
0.98

UfGfgUfuacAfusGfsa

D1146
51146
AfuguAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1146
aUfgGfaAfuAfcUfcUf
0.11
0.51
0.86

uGfgUfuAfCfAfusGfsa

D1147
51147
AfuGfuAfaCfcAfaGfaGfuAfuUfccasUf
AS1147
aUfGfGfaAfuAfcUfcU
0.1
0.52
0.83

fuGfgUfuAfcAfusGfsa

D1148
51148
AfUfGfuAfaccAfaGfaGfuAfuUfcCfasUf
AS1148
aUfgGfaAfuAfcUfcUfu
0.14
0.63
0.98

GfGfUfuAfcausGfsa

D1149
51149
AfuGfuAfAfCfcAfaGfaGfuAfuucCfasUf
AS1149
aUfgGfAfAfuAfcUfcUf
0.13
0.58
0.88

uGfguuAfcAfusGfsa

D1150
51150
AfuGfuaaCfcAfaGfaGfuAfuUfcCfasUf
AS1150
aUfgGfaAfuAfcUfcUfu
0.15
0.62
0.94

GfgUfUfAfcAfusGfsa

D1151
51151
AfUfGfuaaCfcAfaGfaGfuAfuUfcCfasUf
AS1151
aUfgGfaAfuAfcUfcUf
0.18
0.73
0.94

uGfgUfUfAfcausGfsa

D1152
51152
auGfUfAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1152
aUfgGfaAfuAfcUfcUf
0.13
0.53
0.97

uGfgUfuacAfUfsGfsa

D1153
51153
AfuGfuAfAfCfcAfaGfaGfuAfuUfccasUf
AS1153
aUfGfGfaAfuAfcUfcU
0.13
0.53
0.98

fuGfguuAfcAfusGfsa

D1154
51154
UfgGfgAfuUfuCfaUfgUfaAfcCfaAfgsAf
AS1154
uCfuUfgGfuUfaCfaUf
0.09
0.5
0.78

gAfaAfuCfcCfasUfsc

D1155
51155
UfgGfGfAfuuuCfaUfgUfAfAfcCfaAfgsAf
AS1155
uCfuUfgGfuuaCfaUfg
0.13
0.62
0.89

AfAfAfuccCfasUfsc

D1156
51156
UfgGfgAfuuuCfaUfGfUfaAfcCfaAfgsAf
AS1156
uCfuUfgGfuUfacaUfg
0.12
0.65
0.85

AfAfAfuCfcCfasUfsc

D1157
51157
UfgGfgAfuUfuCfaUfgUfAfAfcCfaAfgsAf
AS1157
uCfuUfgGfuuaCfaUfg
0.11
0.54
0.85

AfaAfuCfcCfasUfsc

D1158
51158
UfgGfgAfuuuCfaUfgUfAfAfcCfaAfgsAf
AS1158
uCfuUfgGfuuaCfaUfg
0.13
0.53
0.8

AfAfAfuCfcCfasUfsc

D1159
51159
UfGfggAfUfuUfcAfuGfuAfAfccAfAfgsAf
AS1159
uCfuuGfGfuuAfcAfu
0.59
0.89
0.81

GaAfauCfCfcasUfsc

D1160
51160
UfGfggAfUfuuCfaUfgUfAfAfcCfaAfgsAf
AS1160
uCfuUfgGfuuaCfaUf
0.16
0.72
0.9

gAfAfauCfCfcasUfsc

D1161
51161
UfgGfgAfuUfucaUfGfUfaAfcCfaAfgsAf
AS1161
uCfuUfgGfuUfacaUf
0.27
0.69
0.86

GfAfaAfuCfcCfasUfsc

D1162
51162
AfuGfuAfaCfcaaGfaGfUfAfuUfcCfasUf
AS1162
aUfgGfaAfuacUfcUf
0.12
0.6
0.95

UfGfgUfuAfcAfusGfsa

D1163
51163
AfuGfuAfaccAfaGfaGfuAfUfUfcCfasUf
AS1163
aUfgGfaauAfcUfcUf
0.05
0.56
1.02

uGfGfUfuAfcAfusGfsa

D1164
51164
AfuGfuAfaCfcAfagaGfUfAfuUfcCfasUf
AS1164
aUfgGfaAfuacUfCfU
0.13
0.55
1

fuGfgUfuAfcAfusGfsa

D1165
51165
AfuGfuAfaCfcaaGfaGfuAfUfUfcCfasUf
AS1165
aUfgGfaauAfcUfcUf
0.09
0.6
0.97

UfGfgUfuAfcAfusGfsa

D1166
51166
AfuguAfaCfCfAfaGfaGfuAfuUfcCfasUf
AS1166
aUfgGfaAfuAfcUfcU
0.15
0.59
0.91

fuggUfuAfCfAfusGfsa

D1167
51167
AfuGfuAfaCfcAfagaGfuAfUfUfcCfasUf
AS1167
aUfgGfaauAfcUfCfUf
0.11
0.59
1

uGfgUfuAfcAfusGfsa

D1168
51168
AfuGfuAfaCfCfAfagaGfuAfuUfcCfasUf
AS1168
aUfgGfaAfuAfcUfCfU
0.13
0.57
0.94

fuggUfuAfcAfusGfsa

D1169
51169
auGfuAfaCfcAfaGfaGfuAfUfUfcCfasUf
AS1169
aUfgGfaauAfcUfcUfu
0.08
0.5
0.9

GfgUfuAfcAfUfsGfsa

D1170
51170
AfuguAfaCfcAfaGfaGfuAfUfUfcCfasUf
AS1170
aUfgGfaauAfcUfcUfu
0.06
0.53
0.91

GfgUfuAfCfAfusGfsa

D1171
51171
auGfuAfaCfcAfaGfaGfuAfuUfCfCfasUf
AS1171
aUfggaAfuAfcUfcUf
0.07
0.56
0.89

uGfgUfuAfcAfUfsGfsa

D1172
51172
AfuGfuAfaCfCfAfaGfaGfuAfuucCfasUf
AS1172
aUfgGfAfAfuAfcUfcU
0.13
0.59
0.98

fuggUfuAfcAfusGfsa

D1173
51173
AfuGfuAfaCfcaaGfAfGfuAfuUfcCfasUf
AS1173
aUfgGfaAfuAfcucUfU
0.2
0.65
1.03

fGfgUfuAfcAfusGfsa

D1174
51174
AfuGfuaaCfcAfaGfaGfuAfUfUfcCfasUf
AS1174
aUfgGfaauAfcUfcUfu
0.07
0.51
0.95

GfgUfUfAfcAfusGfsa

D1175
51175
AfuguAfaCfcAfaGfaGfuAfuUfCfCfasUf
AS1175
aUfggaAfuAfcUfcUfu
0.2
0.53
0.76

GfgUfuAfCfAfusGfsa

D1176
51176
auGfuAfaCfcAfaGfaGfuAfuUfcCfAfsUf
AS1176
augGfaAfuAfcUfcUfu
0.74
0.98
0.81

GfgUfuAfcAfusGfsa

D1177
51177
AfuGfuAfaCfcAfaGfaGfuAfuucCfAfsUf
AS1177
augGfAfAfuAfcUfcU
0.43
0.64
0.88

fuGfgUfuAfcAfusGfsa

D1178
51178
auguaaccAfaGfaGfuAfuUfcCfasUf
AS1178
aUfgGfaAfuAfcUfcUfu
0.17
0.49
0.81

GfgUfuAfcAfusGfsa

D1179
51179
AfuGfuaaCfcAfaGfaGfuAfuUfCfCfasUf
AS1179
aUfggaAfuAfcUfcUfu
0.22
0.65
0.73

GfgUfUfAfcAfusGfsa

D1180
51180
AfuguAfaCfcAfaGfaGfuAfuUfcCfAfsUf
AS1180
augGfaAfuAfcUfcUfuG
0.6
1.09
0.8

fgUfuAfcAfUfsGfsa

D1181
51181
auGfuAfaCfcAfaGfaGfuAfuUfccasu
AS1181
aUfgGfaAfuAfcUfcUfu
0.3
0.78
0.78

GfgUfuAfcAfusGfsa

D1182
51182
auguaaccaaGfaGfuAfuUfcCfasUf
AS1182
aUfgGfaAfuAfcUfcU
0.35
0.73
0.84

fuGfgUfuAfcAfusGfsa

D1183
51183
AfuGfuAfaccAfaGfaGfuAfuUfCfCfasUf
AS1183
aUfggaAfuAfcUfcUf
0.19
0.6
0.94

uGfGfUfuAfcAfusGfsa

D1184
51184
AfuGfuaaCfcAfaGfaGfuAfuUfcCfAfsUf
AS1184
augGfaAfuAfcUfcUfuG
0.61
1.08
0.8

fgUfuAfCfAfusGfsa

D1185
51185
auGfuAfaCfcAfaGfaGfuAfuuccasu
AS1185
aUfgGfaAfuAfcUfcU
0.16
0.52
0.72

fuGfgUfuAfcAfusGfsa

D1186
51186
auguaaccaagaGfuAfuUfcCfasUf
AS1186
aUfgGfaAfuAfcUfcUf
0.2
0.53
0.74

uGfgUfuAfcAfusGfsa

D1187
51187
AfuGfuAfaCfcaaGfaGfuAfuUfCfCfasUf
AS1187
aUfggaAfuAfcUfcUfU
0.34
0.66
0.85

fGfgUfuAfcAfusGfsa

D1188
51188
AfuGfuAfaccAfaGfaGfuAfuUfcCfAfsUf
AS1188
augGfaAfuAfcUfcUfu
0.61
0.98
1.02

GfgUfUfAfcAfusGfsa

D1189
51189
AfuGfuAfaCfcAfaGfaGfuAfuuccasu
AS1189
aUfgGfaAfuAfcUfcU
0.3
0.73
0.85

fuGfgUfuAfcAfusGfsa

D1190
51190
auguaaccaagaguauuccasu
AS1190
aUfgGfaAfuAfcUfcU
0.28
0.69
0.78

fuGfgUfuAfcAfusGfsa

D1191
51191
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1191
aUfgGfaAfuAfcUfcU
0.33
0.88
0.64

fugdGudTadCadTsgsa

D1192
51192
AfuGfuAfaCfcAfagaGfuAfuUfCfCfasUf
AS1192
aUfggaAfuAfcUfCfUf
0.31
0.64
0.83

uGfgUfuAfcAfusGfsa

D1193
51193
AfuGfuAfaCfcaaGfaGfuAfuUfcCfAfsUf
AS1193
augGfaAfuAfcUfcUfu
0.64
0.82
0.92

GfGfUfuAfcAfusGfsa

D1194
51194
AfuGfuAfaCfcAfaGfaGfuauuccasu
AS1194
aUfgGfaAfuAfcUfcUf
0.21
0.62
0.77

uGfgUfuAfcAfusGfsa

D1195
51195
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1195
aUfgGfaAfuAfcUfcUf
0.17
0.7
0.95

uGfGfUfuAfCfAfusGfsa

D1196
51196
AfuGfuAfaCfcAfaGfaguAfuUfCfCfasUf
AS1196
aUfggaAfuAfCfUfcUf
0.19
0.71
0.65

uGfgUfuAfcAfusGfsa

D1197
51197
AfuGfuAfaCfcAfagaGfuAfuUfcCfAfsUf
AS1197
augGfaAfuAfcUfcUf
0.64
0.82
0.93

UfGfgUfuAfcAfusGfsa

D1198
51198
auguAfaCfcAfaGfaGfuAfuUfccasu
AS1198
aUfgGfaAfuAfcUfcU
0.19
0.65
0.72

fuGfgUfuAfcAfusGfsa

D1199
51199
AfuGfuAfaCfcAfaGfaGfuauUfCfCfasUf
AS1199
aUfggaAfUfAfcUfcUf
0.15
0.52
0.64

uGfgUfuAfcAfusGfsa

D1200
51200
AfuGfuAfaCfcAfaGfaguAfuUfcCfAfsUf
AS1200
augGfaAfuAfcUfCfUf
0.48
0.74
0.92

uGfgUfuAfcAfusGfsa

D1201
51201
auguAfaCfcAfaGfaGfuAfuUfcCfasu
AS1201
aUfgGfaAfuAfcUfcUf
0.17
0.71
0.77

uGfgUfuAfcAfusGfsa

D1202
51202
AfuGfuAfaCfcAfaGfaGfuauUfcCfAfsUf
AS1202
augGfaAfuAfCfUfcUf
0.43
0.69
0.85

uGfgUfuAfcAfusGfsa

D1203
51203
auguaaCfcAfaGfaGfuAfuUfcCfasUf
AS1203
aUfgGfaAfuAfcUfcUf
0.14
0.61
0.76

uGfgUfuAfcAfusGfsa

D1204
51204
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1204
adTdGGfaAfudAdCUfc
0.16
0.56
0.89

UfuGfgUfuAfcAfusGfsa

D1205
51205
AfuGfuAfaCfcAfaGfaGfdTdAdTdTcCfasUf
AS1205
aUfgGfdAdAdTdAcUfc
0.13
0.57
0.9

UfuGfgUfuAfcAfusGfsa

D1206
51206
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1206
adTdGdGdAAfuAfcUfc
0.29
0.73
0.89

UfuGfgUfuAfcAfusGfsa

D1207
51207
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1207
adTdGGfaAfuAfdCdTcUf
0.16
0.56
0.78

uGfgUfuAfcAfusGfsa

D1208
51208
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1208
aUfdGdGdAdAuAfcUfcU
0.22
0.67
0.89

fuGfgUfuAfcAfusGfsa

D1209
51209
AfuguAfaccAfaGfaGfuAfuUfcCfasUf
AS1209
aUfgGfaAfuAfcUfcUf
0.14
0.55
0.78

uGfGfUfuAfCfAfusGfsa

D1210
51210
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1210
aUfgdGdAdAdTAfcUfcU
0.14
0.5
0.84

fuGfgUfuAfcAfusGfsa

D1211
51211
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1211
aUfgGfadAdTdAdCUfcU
0.14
0.59
0.72

fuGfgUfuAfcAfusGfsa

D1212
51212
auguaaccaaGfaGfuAfuUfcCfasUf
AS1212
aUfgGfaAfuAfcUfcUf
0.21
0.74
0.77

ugdGudTadCadTsgsa

D1213
51213
AfuGfuAfaCfcAfaGfaGfuAfu
AS1213
adTdGdGdAAfuAfcUfcU
0.15
0.53
0.91

dTdCdCdAsUf

fuGfgUfuAfcAfusGfsa

D1214
51214
aUfgUfaAfcCfaAfgAfgUfaUfuCfcAfsu
AS1214
aUfgGfaAfuAfcUfcUf
0.12
0.71
0.87

uGfgUfuAfcAfusGfsa

D1215
51215
AfuGfuAfaCfcAfaGfaGfuAfdTdT
AS1215
aUfdGdGdAdAuAfcUfcU
0.18
0.67
0.97

dCdCasUf

fuGfgUfuAfcAfusGfsa

D1216
51216
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1216
aUfgGfaAfuacucuug
0.36
0.87
1.07

gUfuAfcAfusgsa

D1217
51217
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1217
aUfgGfaAfuAfCfUfC
0.37
0.73
1.03

fUfuGfGfuuAfcAfusgsa

D1218
51218
AfUfguAfAfccAfAfgaGfUfauUfCfcasUf
AS1218
aUfGfgaAfUfacUfCfuu
0.23
0.42
0.84

GfGfuuAfCfausGfsa

D1219
51219
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1219
aUfgGfaAfuaCfUfc
0.43
0.71
1.03

UfUfgGfuuAfcAfusgsa

D1220
51220
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1220
aUfgGfaAfuAfcUfCfUf
0.37
0.63
0.99

uGfGfuuAfcAfusgsa

D1221
51221
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1221
aUfgGfaAfuAfcUfCfU
0.29
0.84
0.88

fuGfGfuUfacAfusgsa

D1222
51222
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1222
aUfgGfaAfuaCfuCfuU
0.31
0.8
0.99

fgGfuuAfcAfusgsa

D1223
51223
auGfuAfAfccAfaGfagUfaUfUfcCfasUf
AS1223
aUfgGfaaUfaCfUfcUf
0.09
0.52
0.82

uGfGfuuAfcAfAfsgsa

D1224
51224
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1224
aUfgGfaAfuadCudCud
0.22
0.79
1

TgdGuuAfcAfusgsa

D1225
51225
auGfuaAfccAfagAfguAfuuCfcasUf
AS1225
aUfGfgAfAfuAfCfuCfUf
0.31
0.76
0.84

uGfGfuUfAfcAfUfsGfsa

D1226
51226
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1226
aUfgGfaAfuadCUfcd
0.26
0.64
0.87

TUfgdGuuAfcAfusgsa

D1227
51227
augUfaacCfaagAfguaUfuccAfsu
AS1227
aUfgGfAfaUfAfCfuCfUf
0.33
0.79
0.81

UfgGfUfUfaCfAfUfsGfsa

D1228
51228
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1228
aUfgGfaAfuAfcUfcUfu
0.464
0.932
0.978

GfgUfuAfcAfusGfsa

D1229
51229
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1229
aUfgGfaAfuAfcUfcUf
0.453
1.047
1.178

uGfgUfuAfcAfusGfsa

D1230
51230
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1230
aUfgGfaAfuAfcUfcUf
0.831
0.967
1.151

uGfgUfuAfcAfusGfsa

D1231
51231
auGfuAfAfCfcAfaGfaGfuAfuUfcCfasu
AS1231
AfUfgGfaAfuAfcUfcU
0.09
0.5
1.07

fuGfguuAfcAfUfsGfsa

D1232
51232
AfuGfuAfaCfCfAfaGfaGfuAfuUfcCfasu
AS1232
AfUfgGfaAfuAfcUfcU
0.11
0.54
1.1

fuggUfuAfcAfusGfsa

D1233
51233
AfuGfuAfaCfcAfaGfaGfuAfuUfCfCfasu
AS1233
AfUfggaAfuAfcUfcU
0.19
0.61
0.74

fuGfgUfuAfcAfusGfsa

D1234
51234
aUfgUfaAfcCfaAfgAfgUfaUfuCfcAfsu
AS1234
AfuGfgAfaUfaCfuCf
0.22
0.61
0.98

uUfgGfuUfaCfaUfsgsAf

D1235
51235
aUfgUfaAfcCfaAfgAfgUfaUfuCfcAfsu
AS1235
AfuGfgAfaUfaCfuCfuUf
0.27
0.69
0.92

gGfuUfaCfaUfsgsAf

D1236
51236
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1236
AfuGfgAfaUfaCfuC
0.54
1.08
0.8

fuUfgGfuUfaCfaUfsgsAf

D1237
51237
augUfaAfccaAfgAfguaUfuCfcasu
AS1237
AfUfGfgAfaUfAfCfuCf
0.29
0.61
0.79

uUfGfGfuUfaCfAfUfsgsa

D1238
51238
AfugUfaAfccaAfgAfguaUfuCfcasu
AS1238
AfUfGfgAfaUfAfCfuCfu
0.31
0.6
0.88

UfGfGfuUfaCfAfusgsa

D1239
51239
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1239
dAUdGGdAauAfcUfcUf
0.2
0.67
0.85

uGfgUfuAfcAfusGfsa

D1240
51240
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1240
dAUdGgdAauAfcUfcUf
0.23
0.58
0.68

uGfgUfuAfcAfusGfsa

D1241
51241
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1241
dAudGgdAauAfcUfcUf
0.25
0.65
0.78

uGfgUfuAfcAfusGfsa

D1242
51242
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1242
dAUdGgdAadTAfcUfc
0.18
0.64
0.84

UfuGfgUfuAfcAfusGfsa

D1243
51243
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1243
dAUdGGdAAfuAfcUfcUf
0.19
0.72
0.87

uGfGfUfuAfCfAfusGfsa

D1244
51244
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1244
dAUdGgdAadTAfdCUfcUf
0.16
0.55
0.8

uGfgUfuAfcAfusGfsa

D1245
51245
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1245
dAUdGGdAAuAfcUfcUfu
0.22
0.51
0.9

GfgUfuAfcAfusGfsa

D1246
51246
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1246
dAudGgdAadTAfcUf
0.27
0.78
0.66

cUfuGfgUfuAfcAfusGfsa

D1247
51247
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1247
dAdTdGdGaAfuAfcUfcU
0.16
0.57
0.97

fuGfgUfuAfcAfusGfsa

D1248
51248
AfacaAfuguUfcUfuGfdCUdCudAudAsa
AS1248
dTUdAudAgdAGfcAfaGf
0.06
0.09
0.36
0.0047

aAfcAfcUfgUfusUfsu

D1249
51249
AfaCfaGfuGfuUfcUfuGfCfUfcUfaUfasa
AS1249
UfUfaUfaGfagcAfaGfa
0.06
0.10
0.47
0.005

AfcAfcUfgUfusUfsu

D1250
51250
AfaCfaGfuGfuUfcUfugcUfcUfAfUfasAf
AS1250
uUfauaGfaGfCfAfaGf
0.07
0.14
0.55
0.005

aAfcAfcUfgUfusUfsu

D1251
51251
AfaCfaGfuGfuUfcUfuGfcucUfAfUfasAf
AS1251
uUfauaGfAfGfcAfaGfa
0.07
0.14
0.49
0.006

AfcAfcUfgUfusUfsu

D1252
51252
cAGuGuucuuGcucuAuAAdTdT
AS1252
UuAuAGAGcAAGAAcAC

UGdTdT

D1253
51253
AfaCfaGfuGfuUfcUfugcUfCfUfaUfasAf
AS1253
uUfaUfagaGfCfAfaGf
0.05
0.12
0.43
0.006

aAfcAfcUfgUfusUfsu

D1254
51254
AfaCfaGfuGfuUfCfUfuGfcUfcUfaUfasa
AS1254
UfUfaUfaGfaGfcAfa
0.06
0.13
0.39
0.006

gaAfcAfcUfgUfusUfsu

D1255
51255
AfaCfaGfuGfuUfcUfuGfcUfCfUfaUfasa
AS1255
UfUfaUfagaGfcAfaG
0.08
0.17
0.48
0.007

faAfcAfcUfgUfusUfsu

D1256
51256
AfaCfaGfuGfuUfcUfUfGfcUfcUfaUfasa
AS1256
UfUfaUfaGfaGfcaa
0.08
0.14
0.40
0.007

GfaAfcAfcUfgUfusUfsu

D1257
51257
AfaCfaGfuGfuUfcUfuGfcUfCfUfaUfasAf
AS1257
uUfaUfagaGfcAfaGfaA
0.07
0.12
0.40
0.007

fcAfcUfgUfusUfsUf

D1258
51258
AfaCfaguGfuUfCfUfuGfcUfcUfaUfasAf
AS1258
uUfaUfaGfaGfcAfaga
0.08
0.13
0.41
0.007

AfcAfCfUfgUfusUfsu

D1259
51259
AfaCfAfGfuGfuUfcUfuGfcucUfaUfasAf
AS1259
uUfaUfaGfAfGfcAfaG
0.05
0.11
0.35
0.008

faAfcAfcugUfusUfsu

D1260
51260
AfacaGfuGfuUfCfUfuGfcUfcUfaUfasAf
AS1260
uUfaUfaGfaGfcAfaga
0.06
0.12
0.40
0.008

AfcAfcUfGfUfusUfsu

D1261
51261
AfacaGfuGfuUfcUfuGfcUfCfUfaUfasAf
AS1261
uUfaUfagaGfcAfa
0.06
0.13
0.42
0.008

GfaAfcAfcUfGfUfusUfsu

D1262
51262
AfaCfaGfuGfuUfcUfuGfcucUfaUfasAf
AS1262
uUfaUfaGfAfGfcAfa
0.06
0.13
0.37
0.008

GfaAfcAfcUfgUfusUfsu

D1263
51263
cAGuGuucuuGcucuAuAAdTdT
AS1263
UuAuAGAGcAAGAAcAC

0.008

UGdTdT

D1264
51264
AfaCfaGfuGfuUfcUfuGfCfUfcUfauasAf
AS1264
uUfAfUfaGfagcAfaGfaA
0.07
0.12
0.50
0.008

fcAfcUfgUfusUfsu

D1265
51265
AfaCfaGfuguUfCfUfuGfcUfcUfaUfasAf
AS1265
uUfaUfaGfaGfcAfaga
0.12
0.13
0.48
0.009

AfCfAfcUfgUfusUfsu

D1266
51266
AfacaGfuGfuUfcUfuGfcUfcUfAfUfasAf
AS1266
uUfauaGfaGfcAfaGfaAf
0.07
0.15
0.51
0.009

cAfcUfGfUfusUfsu

D1267
51267
AfacaAfuguUfcUfuGfdCudCudAudAsa
AS1267
dTudAudAgdAGfcAfaGf
0.06
0.14
0.48
0.0088

aAfcAfcAfgUfusUfsu

D1268
51268
AfaCfaGfuGfuUfCfUfuGfcucUfaUfasAf
AS1268
uUfaUfaGfAfGfcAfagaA
0.05
0.09
0.35
0.009

fcAfcUfgUfusUfsu

D1269
51269
cAGuGuucuuGcucuAuAAdTdT
AS1269
UuAuAGAGcAAGAAc

0.009

ACUGdTdT

D1270
51270
aaCfaGfuGfuUfcUfuGfcUfCfUfaUfasAf
AS1270
uUfaUfagaGfcAfaGfaA
0.07
0.14
0.49
0.009

fcAfcUfgUfUfsUfsu

D1271
51271
AfaCfaGfUfGfuUfcUfuGfcucUfaUfasAf
AS1271
uUfaUfaGfAfGfcAfaGfaA
0.06
0.10
0.36
0.009

fcacUfgUfusUfsu

D1272
51272
cAGuGuucuuGcucuAuAAdTdT
AS1272
UuAuAGAGcAAGAAcACU

0.009

GdTdT

D1273
51273
AfaCfaGfUfGfuUfcUfuGfcUfcUfaUfasAf
AS1273
uUfaUfaGfaGfcAfaGf
0.06
0.13
0.51
0.009

aAfcacUfgUfusUfsUf

D1274
51274
AfaCfaGfuGfuUfCfUfuGfcUfcuaUfasAf
AS1274
uUfaUfAfGfaGfcAfag
0.06
0.12
0.46
0.010

aAfcAfcUfgUfusUfsu

D1275
51275
cAGuGuucuuGcucuAuAAdTdT
AS1275
UuAuAGAGcAAGAAcA

0.010

CUGdTdT

D1276
51276
AfaCfaGfuGfuUfCfUfuGfcUfcUfauasAf
AS1276
uUfAfUfaGfaGfcAfa
0.06
0.14
0.47
0.010

gaAfcAfcUfgUfusUfsu

D1277
51277
AfaCfaguGfuUfcUfuGfcUfCfUfaUfasAf
AS1277
uUfaUfagaGfcAfaGfaA
0.07
0.15
0.50
0.010

fcAfCfUfgUfusUfsu

D1278
51278
AfaCfaGfuGfuUfCfUfugcUfcUfaUfasAf
AS1278
uUfaUfaGfaGfCfAfaga
0.06
0.13
0.43
0.010

AfcAfcUfgUfusUfsu

D1279
51279
cAGuGuucuuGcucuAuAAdTdT
AS1279
UuAuAGAGcAAGAAcAC

0.010

UGdTdT

D1280
51280
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasa
AS1280
UfUfaUfaGfaGfcAfaGfa
0.06
0.14
0.45
0.010

AfcAfcUfgUfususu

D1281
51281
AfaCfAfGfuGfuUfcUfuGfcUfcUfaUfasa
AS1281
UfUfaUfaGfaGfcAfaGf
0.07
0.18
0.46
0.011

aAfcAfcugUfusUfsu

D1282
51282
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1282
uUfaUfaGfaGfcAfaGfa
0.07
0.15
0.55
0.011

AfcAfcUfgUfusUfsu

D1283
51283
AfaCfaGfuGfuUfcUfuGfcucUfaUfasAf
AS1283
uUfaUfaGfAfGfcAfaGf
0.07
0.12
0.45
0.011

aAfcAfcUfgUfususu

D1284
51284
AfacaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1284
uUfaUfaGfaGfcAfaGfa
0.06
0.13
0.48
0.011

AfcAfcUfGfUfusUfsu

D1285
51285
AfAfCfaGfuGfuUfcUfuGfcucUfaUfasAf
AS1285
uUfaUfaGfAfGfcAfaG
0.06
0.11
0.40
0.011

faAfcAfcUfguusUfsu

D1286
51286
AfaCfAfGfuGfuUfcUfuGfcUfcUfauasAf
AS1286
uUfAfUfaGfaGfcAfaGf
0.06
0.16
0.47
0.011

aAfcAfcugUfusUfsu

D1287
51287
AfaCfaGfuGfuUfcUfugcUfcUfaUfasAf
AS1287
uUfaUfaGfaGfCfAfaGf
0.07
0.19
0.46
0.012

aAfcAfcUfgUfususu

D1288
51288
AfaCfaGfuGfuUfcUfugcUfcUfaUfasAf
AS1288
uUfaUfaGfaGfCfAfaGf
0.06
0.17
0.46
0.012

aAfcAfcUfgUfusUfsu

D1289
51289
AfaCfaGfuGfuUfcUfUfGfcucUfaUfasAf
AS1289
uUfaUfaGfAfGfcaaGf
0.05
0.09
0.31
0.012

aAfcAfcUfgUfusUfsu

D1290
51290
AfAfCfaGfuGfuUfcUfuGfcUfcUfaUfasa
AS1290
UfUfaUfaGfaGfcAfaG
0.06
0.16
0.49
0.013

faAfcAfcUfguusUfsu

D1291
51291
AfaCfaGfuGfuUfCfUfuGfcUfcUfaUfasAf
AS1291
uUfaUfaGfaGfcAfaga
0.06
0.11
0.32
0.013

AfcAfcUfgUfusUfsUf

D1292
51292
AfaCfAfGfuGfuUfcUfugcUfcUfaUfasAf
AS1292
uUfaUfaGfaGfCfAfaG
0.06
0.14
0.44
0.013

faAfcAfcugUfusUfsu

D1293
51293
AfaCfaGfUfGfuUfcUfuGfcUfcUfaUfasa
AS1293
UfUfaUfaGfaGfcAfaG
0.07
0.16
0.39
0.013

faAfcacUfgUfusUfsu

D1294
51294
AfaCfAfGfuGfuUfcUfuGfcUfcuaUfasAf
AS1294
uUfaUfAfGfaGfcAfaGf
0.07
0.18
0.41
0.014

aAfcAfcugUfusUfsu

D1295
51295
AfaCfaGfUfGfuUfcUfuGfcUfcuaUfasAf
AS1295
uUfaUfAfGfaGfcAfaG
0.07
0.18
0.47
0.014

faAfcacUfgUfusUfsu

D1296
51296
adAdCagdTdGuudCdTugdCdTcudAdTasa
AS1296
dTdTaudAdGagdCdAagd
0.12
0.21
0.68
0.0146

AdAcadCdTgudTsdTsu

D1297
51297
AfacaGfUfGfuUfcUfuGfcUfcUfaUfasAf
AS1297
uUfaUfaGfaGfcAfaGf
0.06
0.15
0.50
0.016

aAfcacUfGfUfusUfsu

D1298
51298
AfaCfaGfUfGfuUfcUfuGfcUfcUfauasAf
AS1298
uUfAfUfaGfaGfcAfaG
0.08
0.17
0.50
0.016

faAfcacUfgUfusUfsu

D1299
51299
AfaCfaguGfuUfcUfuGfcUfcUfaUfasAf
AS1299
uUfaUfaGfaGfcAfaGf
0.07
0.16
0.50
0.018

aAfcAfCfUfgUfususu

D1300
51300
AfaCfaGfuGfuUfcUfUfGfcUfcUfauasAf
AS1300
uUfAfUfaGfaGfcaaGf
0.06
0.12
0.43
0.020

aAfcAfcUfgUfusUfsu

D1301
51301
AfaCfaGfUfGfuUfcUfugcUfcUfaUfasAf
AS1301
uUfaUfaGfaGfCfAfaG
0.07
0.17
0.45
0.021

faAfcacUfgUfusUfsu

D1302
51302
AfaCfaGfuguUfcUfUfGfcUfcUfaUfasAf
AS1302
uUfaUfaGfaGfcaaGf
0.06
0.14
0.49
0.021

aAfCfAfcUfgUfusUfsu

D1303
51303
AfAfCfaguGfuUfcUfuGfcUfcUfaUfasAf
AS1303
uUfaUfaGfaGfcAfaG
0.07
0.24
0.51
0.022

faAfcAfCfUfguusUfsu

D1304
51304
AfaCfaGfuGfuucUfuGfcUfcUfaUfasAf
AS1304
uUfaUfaGfaGfcAfaGfA
0.09
0.27
0.47
0.033

fAfcAfcUfgUfususu

D1305
51305
aadCdAgudGdTucdTdTgcdTdCuadTdAsa
AS1305
udTadTdAgadGdCaadG
0.19
0.36
0.86
0.045

dAacdAdCugdTdTsusu

D1306
51306
AfacaGfuguUfcUfuGfdCUdCUdAudAsa
AS1306
dTUdAUdAGfaGfcAfaGf
0.08
0.22
0.61

aAfCfAfcUfGfUfusUfsu

D1307
51307
AfacaGfuguUfcUfdTGfdCUdCUdAudAsa
AS1307
dTUdAUdAGfaGfcAfaGf
0.13
0.39
0.84

aAfCfAfcUfGfUfusUfsu

D1308
51308
AfacaGfuguUfcUfuGfdCUdCUdAudAsa
AS1308
dTUdAUdAgdAGfcAfaGfa
0.09
0.13
0.48

AfcAfcUfgUfusUfsu

D1309
51309
AfacaGfuguUfcUfdTGfdCUdCUdAudAsa
AS1309
dTUdAUdAgdAGfdCAfaG
0.07
0.13
0.58

faAfcAfcUfgUfusUfsu

D1310
51310
AfacaAfuguUfcUfdTGfdCUdCudAudAsa
AS1310
dTUdAudAgdAGfdCAfaG
0.07
0.14
0.55

faAfcAfcAfgUfusUfsu

D1311
51311
AfaCfaAfuGfuUfcUfuGfcUfcUfd
AS1311
dTdTdAdTaGfaGfcAfaGf
0.10
0.30
0.66

AdTdAsdA

aAfcAfcAfgUfusUfsu

D1312
51312
AfacaGfuguUfcUfuGfdCUdCUdAudAsa
AS1312
dTUdAUdAgdAGfcAfaGf
0.09
0.13
0.48

aAfcAfcUfgUfusUfsu

D1313
51313
AfAfCfaGfuGfuucUfuGfcUfcUfaUfasAf
AS1313
uUfaUfaGfaGfcAfaGf
0.14
0.38
0.74

AfAfcAfcUfguusUfsu

D1314
51314
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1314
uUfaUfaGfaGfcAfaG
0.07
0.19
0.54

faAfcAfcUfgUfusUfsu

D1315
51315
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1315
uUfaUfaGfaGfcAfaGfa
0.07
0.15
0.55

AfcAfcUfgUfusUfsu

D1316
51316
AfaCfaGfuGfuUfcUfuGfcUfcUfauasAf
AS1316
uUfAfUfaGfaGfcAfaG
0.07
0.16
0.53

faAfcAfcUfgUfususu

D1317
51317
AfacaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1317
uUfaUfaGfaGfcAfaGf
0.07
0.16
0.55

aAfcAfcUfGfUfususu

D1318
51318
AfAfCfaGfuguUfcUfuGfcUfcUfaUfasAf
AS1318
uUfaUfaGfaGfcAfaGf
0.10
0.32
0.61

aAfCfAfcUfguusUfsu

D1319
51319
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1319
uUfaUfaGfaGfcAfaG
0.08
0.16
0.53

faAfcAfcUfgUfususu

D1320
51320
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1320
uUfaUfaGfaGfcAfaGf
0.08
0.16
0.61

aAfcAfcUfgUfususu

D1321
51321
AfaCfaGfuGfuUfcUfuGfcUfCfUfaUfasAf
AS1321
uUfaUfagaGfcAfaGfa
0.06
0.14
0.58

AfcAfcUfgUfusUfsu

D1322
51322
AfaCfaGfuGfuUfcuuGfcUfcUfaUfasAf
AS1322
uUfaUfaGfaGfcAfAfG
0.15
0.49
0.84

faAfcAfcUfgUfusUfsu

D1323
51323
AfaCfaGfuGfuUfcUfuGfcUfcuaUfasAf
AS1323
uUfaUfAfGfaGfcAfa
0.07
0.20
0.62

GfaAfcAfcUfgUfususu

D1324
51324
AfAfCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1324
uUfaUfaGfaGfcAfaG
0.08
0.25
0.78

faAfcAfcUfguusUfsu

D1325
51325
AfAfCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1325
uUfaUfaGfaGfcAfaGf
0.08
0.18
0.80

aAfcAfcUfguusUfsu

D1326
51326
AfaCfaGfuGfuUfcUfuGfcUfcUfAfUfasAf
AS1326
uUfauaGfaGfcAfaGf
0.07
0.21
0.66

aAfcAfcUfgUfusUfsu

D1327
51327
AfaCfaGfuGfuucUfuGfcUfcUfaUfasAf
AS1327
uUfaUfaGfaGfcAfaG
0.10
0.31
0.70

fAfAfcAfcUfgUfusUfsu

D1328
51328
AfAfCfaGfuGfuUfcUfuGfcUfcUfauasAf
AS1328
uUfAfUfaGfaGfcAfaGf
0.07
0.15
0.55

aAfcAfcUfguusUfsu

D1329
51329
AfaCfAfGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1329
uUfaUfaGfaGfcAfaGf
0.08
0.19
0.71

aAfcAfcugUfusUfsu

D1330
51330
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfAfsAf
AS1330
uuaUfaGfaGfcAfaGfa
0.09
0.27
0.76

AfcAfcUfgUfusUfsu

D1331
51331
AfaCfaGfuguUfcUfuGfcUfcUfaUfasAf
AS1331
uUfaUfaGfaGfcAfaG
0.07
0.21
0.65

faAfCfAfcUfgUfusUfsu

D1332
51332
AfAfCfaGfuGfuUfcUfuGfcUfcuaUfasAf
AS1332
uUfaUfAfGfaGfcAfaGf
0.07
0.17
0.53

aAfcAfcUfguusUfsu

D1333
51333
AfaCfaGfUfGfuUfcUfuGfcUfcUfaUfasAf
AS1333
uUfaUfaGfaGfcAfaGfa
0.08
0.25
0.73

AfcacUfgUfusUfsu

D1334
51334
AfaCfaguGfuUfcUfuGfcUfcUfaUfasAf
AS1334
uUfaUfaGfaGfcAfaGfa
0.07
0.18
0.54

AfcAfCfUfgUfusUfsu

D1335
51335
AfaCfaGfuGfuUfcuuGfcUfcUfaUfasAf
AS1335
uUfaUfaGfaGfcAfAfG
0.14
0.38
0.57

faAfcAfcUfgUfususu

D1336
51336
AfaCfaGfuGfUfUfcUfuGfcUfcUfaUfasAf
AS1336
uUfaUfaGfaGfcAfaGf
0.16
0.50
0.96

aacAfcUfgUfusUfsu

D1337
51337
AfaCfaGfuGfuUfcUfuGfcUfcUfauasAf
AS1337
uUfAfUfaGfaGfcAfaG
0.08
0.19
0.54

faAfcAfcUfgUfusUfsu

D1338
51338
AfAfCfaGfuGfuUfcUfugcUfcUfaUfasAf
AS1338
uUfaUfaGfaGfCfAfaGf
0.08
0.20
0.69

aAfcAfcUfguusUfsu

D1339
51339
AfaCfaGfuGfuUfCfUfuGfcUfcUfaUfasAf
AS1339
uUfaUfaGfaGfcAfagaA
0.07
0.16
0.55

fcAfcUfgUfusUfsu

D1340
51340
AfaCfaGfuGfuUfcUfuGfcUfcuaUfasAf
AS1340
uUfaUfAfGfaGfcAfaG
0.08
0.17
0.57

faAfcAfcUfgUfusUfsu

D1341
51341
AfaCfaGfuguUfcUfuGfcUfcUfaUfasAf
AS1341
uUfaUfaGfaGfcAfaG
0.08
0.22
0.63

faAfCfAfcUfgUfususu

D1342
51342
AfAfCfaGfuGfuUfcuuGfcUfcUfaUfasAf
AS1342
uUfaUfaGfaGfcAfAf
0.21
0.56
0.86

GfaAfcAfcUfguusUfsu

D1343
51343
AfacaGfuGfUfUfcUfuGfcUfcUfaUfasAf
AS1343
uUfaUfaGfaGfcAfaGf
0.14
0.37
0.73

aacAfcUfGfUfusUfsu

D1344
51344
AfaCfaGfuGfuucUfUfGfcUfcUfaUfasAf
AS1344
uUfaUfaGfaGfcaaGf
0.08
0.20
0.66

AfAfcAfcUfgUfusUfsu

D1345
51345
AfaCfAfGfuGfuUfcuuGfcUfcUfaUfasAf
AS1345
uUfaUfaGfaGfcAfAf
0.12
0.34
0.73

GfaAfcAfcugUfusUfsu

D1346
51346
AfaCfaGfuGfUfUfcUfuGfcUfcUfauasAf
AS1346
uUfAfUfaGfaGfcAfaG
0.16
0.42
0.90

faacAfcUfgUfusUfsu

D1347
51347
AfaCfaGfuGfUfUfcUfuGfcUfcUfaUfasAf
AS1347
uUfaUfaGfaGfcAfaGf
0.17
0.43
0.85

aacAfcUfgUfusUfsUf

D1348
51348
AfaCfAfGfuGfuucUfuGfcUfcUfaUfasAf
AS1348
uUfaUfaGfaGfcAfaG
0.08
0.21
0.58

fAfAfcAfcugUfusUfsu

D1349
51349
AfaCfaGfuGfUfUfcUfuGfcUfcuaUfasAf
AS1349
uUfaUfAfGfaGfcAfa
0.21
0.39
0.88

GfaacAfcUfgUfusUfsu

D1350
51350
AfaCfaguGfuUfcUfUfGfcUfcUfaUfasAf
AS1350
uUfaUfaGfaGfcaaGf
0.06
0.13
0.52

aAfcAfCfUfgUfusUfsu

D1351
51351
AfaCfAfGfuguUfcUfuGfcUfcUfaUfasAf
AS1351
uUfaUfaGfaGfcAfaG
0.08
0.21
0.58

faAfCfAfcugUfusUfsu

D1352
51352
AfaCfaGfUfGfuUfcuuGfcUfcUfaUfasAf
AS1352
uUfaUfaGfaGfcAfAf
0.18
0.49
0.84

GfaAfcacUfgUfusUfsu

D1353
51353
AfaCfaGfuGfUfUfcUfuGfcucUfaUfasAf
AS1353
uUfaUfaGfAfGfcAfa
0.11
0.25
0.68

GfaacAfcUfgUfusUfsu

D1354
51354
AfacaGfuGfuUfcUfUfGfcUfcUfaUfasAf
AS1354
uUfaUfaGfaGfcaaGfa
0.07
0.15
0.52

AfcAfcUfGfUfusUfsu

D1355
51355
AfaCfaGfUfGfuucUfuGfcUfcUfaUfasAf
AS1355
uUfaUfaGfaGfcAfaGf
0.10
0.26
0.63

AfAfcacUfgUfusUfsu

D1356
51356
AfaCfaGfuGfUfUfcUfugcUfcUfaUfasAf
AS1356
uUfaUfaGfaGfCfAfaG
0.16
0.33
0.79

faacAfcUfgUfusUfsu

D1357
51357
AfaCfAfGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1357
uUfaUfaGfaGfcAfaG
0.09
0.19
0.51

faAfcAfcugUfusUfsUf

D1358
51358
AfaCfaGfuGfUfUfcuuGfcUfcUfaUfasAf
AS1358
uUfaUfaGfaGfcAfAfG
0.22
0.48
0.71

faacAfcUfgUfusUfsu

D1359
51359
AfaCfaGfuGfuUfcUfUfGfcUfcUfaUfasAf
AS1359
uUfaUfaGfaGfcaaGfa
0.10
0.17
0.61

AfcAfcUfgUfusUfsUf

D1360
51360
AfaCfaguGfUfUfcUfuGfcUfcUfaUfasAf
AS1360
uUfaUfaGfaGfcAfaGf
0.14
0.40
0.87

aacAfCfUfgUfusUfsu

D1361
51361
AfaCfaGfuGfuUfcUfUfGfcUfcuaUfasAf
AS1361
uUfaUfAfGfaGfcaaGf
0.07
0.14
0.52

aAfcAfcUfgUfusUfsu

D1362
51362
aaCfaGfuGfuUfcUfuGfCfUfcUfaUfasAf
AS1362
uUfaUfaGfagcAfaGfa
0.10
0.28
0.81

AfcAfcUfgUfUfsUfsu

D1363
51363
AfaCfaGfuGfuucUfuGfcUfcUfAfUfasAf
AS1363
uUfauaGfaGfcAfaGfA
0.06
0.16
0.68

fAfcAfcUfgUfusUfsu

D1364
51364
AfaCfaGfuGfuUfcUfugcUfcUfaUfAfsAf
AS1364
uuaUfaGfaGfCfAfaGf
0.09
0.26
0.67

aAfcAfcUfgUfusUfsu

D1365
51365
aacaguguucuugcucuauasa
AS1365
uUfaUfaGfaGfcAfaGf
0.20
0.59
0.95

aAfcAfcUfgUfusUfsu

D1366
51366
AfaCfaGfuGfuUfcUfuGfCfUfcUfauasAf
AS1366
uUfAfUfaGfagcAfaG
0.06
0.13
0.53

faAfcAfcUfgUfusUfsu

D1367
51367
AfaCfaGfuGfuUfcUfuGfCfUfcUfaUfasAf
AS1367
uUfaUfaGfagcAfaGfa
0.08
0.16
0.53

AfcAfcUfgUfusUfsUf

D1368
51368
AfaCfaGfuguUfcUfuGfcUfcUfAfUfasAf
AS1368
uUfauaGfaGfcAfaGf
0.07
0.15
0.54

aAfCfAfcUfgUfusUfsu

D1369
51369
AfaCfaGfuGfuUfcuuGfcUfcUfaUfAfsAf
AS1369
uuaUfaGfaGfcAfAfGf
0.23
0.56
0.89

aAfcAfcUfgUfusUfsu

D1370
51370
AfaCfaGfuGfuUfcUfuGfCfUfcuaUfasAf
AS1370
uUfaUfAfGfagcAfaG
0.06
0.12
0.55

faAfcAfcUfgUfusUfsu

D1371
51371
AfaCfaGfuGfuUfcUfuGfCfUfcuaUfasAf
AS1371
uUfaUfAfGfagcAfaG
0.07
0.18
0.58

faAfcAfcUfgUfusUfsu

D1372
51372
AfaCfaguGfuUfcUfuGfcUfcUfAfUfasAf
AS1372
uUfauaGfaGfcAfaGfa
0.06
0.15
0.56

AfcAfCfUfgUfusUfsu

D1373
51373
AfaCfaGfuGfuucUfuGfcUfcUfaUfAfsAf
AS1373
uuaUfaGfaGfcAfaGf
0.21
0.51
0.89

AfAfcAfcUfgUfusUfsu

D1374
51374
AfacaGfuguUfcUfuGfcUfcUfaUfasAf
AS1374
uUfaUfaGfaGfcAfaGf
0.08
0.21
0.64

aAfCfAfcUfGfUfusUfsu

D1375
51375
AfaCfaGfuGfuUfcuuGfCfUfcUfaUfasAf
AS1375
uUfaUfaGfagcAfAfGf
0.15
0.40
0.94

aAfcAfcUfgUfusUfsu

D1376
51376
AfaCfaGfuGfuUfcuuGfCfUfcUfaUfasAf
AS1376
uUfaUfaGfagcAfAfGf
0.13
0.40
0.96

aAfcAfcUfgUfusUfsu

D1377
51377
AfaCfaGfuGfuUfcUfuGfcUfCfUfauasAf
AS1377
uUfAfUfagaGfcAfaG
0.08
0.17
0.64

faAfcAfcUfgUfusUfsu

D1378
51378
AfaCfaGfuguUfcUfuGfcUfcUfaUfAfsAf
AS1378
uuaUfaGfaGfcAfaGfa
0.18
0.50
0.97

AfCfAfcUfgUfusUfsu

D1379
51379
AfaCfaGfuGfuucUfuGfCfUfcUfaUfasAf
AS1379
uUfaUfaGfagcAfaGfA
0.08
0.24
0.79

fAfcAfcUfgUfusUfsu

D1380
51380
aaCfaGfuGfuUfcUfuGfcUfcUfAfUfasAf
AS1380
uUfauaGfaGfcAfaGfa
0.07
0.14
0.58

AfcAfcUfgUfUfsUfsu

D1381
51381
AfaCfaguGfuUfcUfuGfcUfcUfaUfAfsAf
AS1381
uuaUfaGfaGfcAfaGfa
0.11
0.34
0.96

AfcAfCfUfgUfusUfsu

D1382
51382
AfaCfaGfuguUfcUfuGfCfUfcUfaUfasAf
AS1382
uUfaUfaGfagcAfaGfa
0.08
0.18
0.69

AfCfAfcUfgUfusUfsu

D1383
51383
AfaCfaGfuGfuUfcuuGfcUfCfUfaUfasAf
AS1383
uUfaUfagaGfcAfAfGf
0.14
0.38
0.85

aAfcAfcUfgUfusUfsu

D1384
51384
AfaCfaGfuGfuUfcUfuGfcUfcUfAfUfasAf
AS1384
uUfauaGfaGfcAfaGf
0.07
0.16
0.54

aAfcAfcUfgUfusUfsUf

D1385
51385
AfacaGfuGfuUfcUfuGfcUfcUfaUfAfsAf
AS1385
uuaUfaGfaGfcAfaGf
0.08
0.20
0.75

aAfcAfcUfGfUfusUfsu

D1386
51386
aacaguguucUfuGfcUcUaudAsa
AS1386
uUfdAUdAGfaGfcAfaG
0.25
0.56
0.90

faadCadCudGdTdTsusu

D1387
51387
AfaCfaguGfuUfcUfuGfCfUfcUfaUfasAf
AS1387
uUfaUfaGfagcAfaGfa
0.08
0.19
0.70

AfcAfCfUfgUfusUfsu

D1388
51388
AfaCfaGfuGfuucUfuGfcUfCfUfaUfasAf
AS1388
uUfaUfagaGfcAfaGfA
0.08
0.14
0.60

fAfcAfcUfgUfusUfsu

D1389
51389
AfaCfaGfuGfuUfcUfuGfcUfcuaUfAfsAf
AS1389
uuaUfAfGfaGfcAfaGf
0.08
0.19
0.62

aAfcAfcUfgUfusUfsu

D1390
51390
aaCfaGfuGfuUfcUfuGfcUfcUfaUfAfsAf
AS1390
uuaUfaGfaGfcAfaGfa
0.08
0.27
0.76

AfcAfcUfgUfUfsUfsu

D1391
51391
aacaguguucdTudGcdTcdTadTasa
AS1391
uUfdAUdAGfaGfcAfa
0.18
0.36
0.81

GfaadCadCudGudTsusu

D1392
51392
AfacaGfuGfuUfcUfuGfCfUfcUfaUfasAf
AS1392
uUfaUfaGfagcAfaGfa
0.07
0.17
0.55

AfcAfcUfGfUfusUfsu

D1393
51393
AfaCfaGfuguUfcUfuGfcUfCfUfaUfasAf
AS1393
uUfaUfagaGfcAfaGfa
0.07
0.15
0.57

AfCfAfcUfgUfusUfsu

D1394
51394
AfaCfaGfuGfuUfcuuGfcUfcUfAfUfasAf
AS1394
uUfauaGfaGfcAfAfG
0.26
0.68
1.06

faAfcAfcUfgUfusUfsu

D1395
51395
AfaCfaGfuGfuUfcUfuGfcucUfaUfAfsAf
AS1395
uuaUfaGfAfGfcAfaG
0.06
0.18
0.58

faAfcAfcUfgUfusUfsu

D1396
51396
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfAfsAf
AS1396
uuaUfaGfaGfcAfaGfaA
0.09
0.27
0.73

fcAfcUfgUfusUfsUf

D1397
51397
AfaCfaAfuGfuUfcUfuGfcdAdCd
AS1397
uUfadTdAdGdAGfcAfaG
0.20
0.51
0.73

TdAUfasAf

faGfcAfcAfgUfusUfsu

D1398
51398
AfacaGfuguUfcuuGfcucUfauasAf
AS1398
uUfAfUfaGfAfGfcAfAf
0.13
0.34
0.86

GfaAfCfAfcUfGfUfusUfsu

D1399
51399
dAacadGugudTcuudGcucdTauasdA
AS1399
udTdAdTadGdAdGcdAdAdGa
0.24
0.42
0.82

dAdCdAcdTdGdTusdTsu

D1400
51400
AfaCfaAfuGfuUfcUfuGfdCdAdC
AS1400
uUfaUfdAdGdAdGcAfaGf
0.49
0.85
0.78

dTaUfasAf

aGfcAfcAfgUfusUfsu

D1401
51401
AfaCfaAfuGfuUfcUfudGdCdAdCU
AS1401
uUfaUfadGdAdGdCAfaGf
0.67
0.83
0.85

faUfasAf

aGfcAfcAfgUfusUfsu

D1402
51402
aaCfAfguGfUfucUfUfgcUfCfuaUfAfsa
AS1402
uUfaUfAfgaGfCfaaG
0.18
0.47
0.80

fAfacAfCfugUfUfsusu

D1403
51403
AfaCfaAfuGfuUfcUfuGfcdAdCUfa
AS1403
udTdAUfadGdAGfcAfaGf
0.73
0.89
0.77

dTdAsAf

aGfcAfcAfgUfusUfsu

D1404
51404
aacAgugUucuUgcuCuauAsa
AS1404
uUaUAgAGCaAGAaCAC
0.12
0.39
0.79

uGUUsusu

D1405
51405
AacaGuguUcuuGcucUauasA
AS1405
uUAUaGAGcAAGaACAc
0.12
0.37
0.77

UGUusUsu

D1406
51406
AfaCfaAfuGfuUfcUfudGdCAfcUfa
AS1406
udTdAUfaGfadGdCAfaGf
0.59
0.93
0.89

dTdAsAf

aGfcAfcAfgUfusUfsu

D1407
51407
aACagUGuuCUugCUcuAUasa
AS1407
UUauAGagCAagAAcaCU
0.09
0.16
0.55

guUsUsu

D1408
51408
AfaCfaAfuGfuUfcUfuGfcAfcdTd
AS1408
udTdAdTdAGfaGfcAfaGfa
0.22
0.64
0.86

AdTdAsAf

AfcAfcAfgUfusUfsu

D1409
51409
aaCAguGUucUUgcUCuaUAsa
AS1409
uUaUAgaGCaaGAacACu
0.13
0.31
0.76

gUUsusu

D1410
51410
AfaCfaAfuGfuUfcUfuGfcAfdCdTdA
AS1410
udTdAdTdAdGaGfcAfaGfa
0.77
0.94
0.93

dTdAsAf

GfcAfcAfgUfusUfsu

D1411
51411
aacAfgugUfucuUfgcuCfuauAfsa
AS1411
uUfaUfAfgAfGfCfaAfGf
0.23
0.53
1.04

AfaCfAfCfuGfUfUfsusu

D1412
51412
aacdAgugdTucudTgcudCuaudAsa
AS1412
udTadTdAgdAdGdCadAdGd
0.30
0.64
0.90

AadCdAdCudGdTdTsusu

D1413
51413
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasa
AS1413
UfUfaUfaGfaGfcAfaGfaA
0.09
0.19
0.63

fcAfcUfgUfusUfsu

D1414
51414
AfaCfaGfuGfUfUfcUfuGfcUfcUfaUfasa
AS1414
UfUfaUfaGfaGfcAfaGfaa
0.11
0.28
0.66

cAfcUfgUfusUfsu

D1415
51415
AfaCfaGfuGfuUfcUfuGfCfUfcUfaUfasa
AS1415
UfUfaUfaGfagcAfaGfaA
0.06
0.13
0.53

fcAfcUfgUfusUfsu

D1416
51416
aacaguguucuugcucuauasa
AS1416
UfUfAfUfAfGfAfG
0.20
0.53
0.99

fCfAfAfGfAfAfCfAfC

fUfGfUfUfsusu

D1417
51417
AfaCfaGfuGfuUfcUfuGfcUfcUfAfUfasa
AS1417
UfUfauaGfaGfcAfaGfaAf
0.07
0.17
0.53

cAfcUfgUfusUfsu

D1418
51418
aAfCfagUfGfuuCfUfugCfUfcuAfUfasa
AS1418
UfUfauAfGfagCfAfagAf
0.08
0.20
0.70

AfcaCfUfguUfsUfsu

D1419
51419
AfaCfAfGfuGfuUfcUfuGfcUfcU
AS1419
uUfaUfaGfaGfcAfaGfa
0.08
0.20
0.70

faUfasAf

AfcAfcugUfusUfsUf

Example 3. In Vitro Silencing Activity with Various Chemical Modifications on TTR siRNA

The IC₅₀for each modified siRNA is determined in Hep3B cells by standard reverse transfection using Lipofectamine RNAiMAX. In brief, reverse transfection is carried out by adding 5 μl of Opti-MEM to 5 μl of siRNA duplex per well into a 96-well plate along with 10 μl of Opti-MEM plus 0.5 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and incubating at room temperature for 15-20 minutes. Following incubation, 100 μl of complete growth media without antibiotic containing 12,000-15,000 Hep3B cells is then added to each well. Cells are incubated for 24 hours at 37° C. in an atmosphere of 5% CO2 prior to lysis and analysis of ApoB and GAPDH mRNA by bDNA (Quantigene). Seven different siRNA concentrations ranging from 10 nM to 0.6 μM are assessed for IC₅₀determination and ApoB/GAPDH for ApoB transfected cells is normalized to cells transfected with 10 nM Luc siRNA.

Abbreviation
Nucleotide(s)

Af
2′-F-adenosine

Cf
2′-F-cytidine

Gf
2′-F-guanosine

Uf
2′-F-uridine

A
adenosine

C
cytidine

G
guanosine

U
uridine

a
2′-O-methyladenosine

c
2′-O-methylcytidine

g
2′-O-methylguanosine

u
2′-O-methyluridine

dT
2′-deoxythymidine

s
phosphorothioate linkage

TABLE 3

ANGPTL3 modified duplex

SS seq (SEQ ID NOS 845-

RNAimax, Hep3b

Duplex
Sense
1025, respectively, in order

AS seq (SEQ ID NOS 1026-1206,
10
0.1
0.025

ID
ID
of appearance)
AS ID
respectively, in order of appearance)
nM
nM
nM

D2000
S2000
UfcAfcAfaUfuAfAfGfcUfcCfuUfcUfuUf
A2000
aAfaGfaAfgGfaGfcuuAfaUfuGfuGfasAfsc
0.036
0.274
0.233

D2001
S2001
UfuAfuUfgUfuCfCfUfcUfaGfuUfaUfuUf
A2001
aAfaUfaAfcUfaGfaggAfaCfaAfuAfasAfsa
0.044
0.278
0.247

D2002
S2002
GfcUfaUfgUfuAfGfAfcGfaUfgUfaAfaAf
A2002
uUfuUfaCfaUfcGfucuAfaCfaUfaGfcsAfsa
0.062
0.474
0.449

D2003
S2003
GfgAfcAfuGfgUfCfUfuAfaAfgAfcUfuUf
A2003
aAfaGfuCfuUfuAfagaCfcAfuGfuCfcsCfsa
0.303
1.042
0.912

D2004
S2004
CfaAfaAfaCfuCfAfAfcAfuAfuUfuGfaUf
A2004
aUfcAfaAfuAfuGfuugAfgUfuUfuUfgsAfsa
0.102
0.623
0.499

D2005
S2005
AfcCfaGfuGfaAfAfUfcAfaAfgAfaGfaAf
A2005
uUfcUfuCfuUfuGfauuUfcAfcUfgGfusUfsu
0.124
0.901
0.756

D2006
S2006
CfaCfaAfuUfaAfGfCfuCfcUfuCfuUfuUf
A2006
aAfaAfgAfaGfgAfgcuUfaAfuUfgUfgsAfsa
0.069
0.269
0.244

D2007
S2007
CfuAfuGfuUfaGfAfCfgAfuGfuAfaAfaAf
A2007
uUfuUfuAfcAfuCfgucUfaAfcAfuAfgsCfsa
0.052
0.622
0.589

D2008
S2008
UfcAfaCfaUfaUfUfUfgAfuCfaGfuCfuUf
A2008
aAfgAfcUfgAfuCfaaaUfaUfgUfuGfasGfsu
0.133
0.798
0.785

D2009
S2009
AfaCfuGfaGfaAfGfAfaCfuAfcAfuAfuAf
A2009
uAfuAfuGfuAfgUfucuUfcUfcAfgUfusCfsc
0.097
0.671
0.528

D2010
S2010
AfcAfaUfuAfaGfCfUfcCfuUfcUfuUfuUf
A2010
aAfaAfaGfaAfgGfagcUfuAfaUfuGfusGfsa
0.145
0.308
0.293

D2011
S2011
CfuCfcAfgAfgCfCfAfaAfaUfcAfaGfaUf
A2011
aUfcUfuGfaUfuUfuggCfuCfuGfgAfgsAfsu
0.122
0.882
0.938

D2012
S2012
CfgAfuGfuAfaAfAfAfuUfuUfaGfcCfaAf
A2012
uUfgGfcUfaAfaAfuuuUfuAfcAfuCfgsUfsc
0.102
0.843
0.733

D2013
S2013
GfuCfuUfaAfaGfAfCfuUfuGfuCfcAfuAf
A2013
uAfuGfgAfcAfaAfgucUfuUfaAfgAfcsCfsa
1.133
1.105
1.022

D2014
S2014
CfaAfcAfuAfuUfUfGfaUfcAfgUfcUfuUf
A2014
aAfaGfaCfuGfaUfcaaAfuAfuGfuUfgsAfsg
0.077
0.413
0.450

D2015
S2015
AfcUfgAfgAfaGfAfAfcUfaCfaUfaUfaAf
A2015
uUfaUfaUfgUfaGfuucUfuCfuCfaGfusUfsc
0.055
0.293
0.364

D2016
S2016
CfcAfgAfgCfcAfAfAfaUfcAfaGfaUfuUf
A2016
aAfaUfcUfuGfaUfuuuGfgCfuCfuGfgsAfsg
0.080
0.650
0.499

D2017
S2017
GfaUfgUfaAfaAfAfUfuUfuAfgCfcAfaUf
A2017
aUfuGfgCfuAfaAfauuUfuUfaCfaUfcsGfsu
0.076
0.605
0.579

D2018
S2018
UfcUfuAfaAfgAfCfUfuUfgUfcCfaUfaAf
A2018
uUfaUfgGfaCfaAfaguCfuUfuAfaGfasCfsc
1.326
1.098
0.927

D2019
S2019
AfaCfaUfaUfuUfGfAfuCfaGfuCfuUfuUf
A2019
aAfaAfgAfcUfgAfucaAfaUfaUfgUfusGfsa
0.047
0.560
0.477

D2020
S2020
CfuGfaGfaAfgAfAfCfuAfcAfuAfuAfaAf
A2020
uUfuAfuAfuGfuAfguuCfuUfcUfcAfgsUfsu
0.066
0.690
0.681

D2021
S2021
AfaUfuAfaGfcUfCfCfuUfcUfuUfuUfaUf
A2021
aUfaAfaAfaGfaAfggaGfcUfuAfaUfusGfsu
0.041
0.611
0.251

D2022
S2022
AfaAfuCfaAfgAfUfUfuGfcUfaUfgUfuAf
A2022
uAfaCfaUfaGfcAfaauCfuUfgAfuUfusUfsg
0.053
0.555
0.516

D2023
S2023
UfuCfaGfuUfgGfGfAfcAfuGfgUfcUfuAf
A2023
uAfaGfaCfcAfuGfuccCfaAfcUfgAfasGfsg
0.779
1.045
0.963

D2024
S2024
GfgGfcCfaAfaUfUfAfaUfgAfcAfuAfuUf
A2024
aAfuAfuGfuCfaUfuaaUfuUfgGfcCfcsUfsu
1.487
0.949
0.883

D2025
S2025
AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf
A2025
aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg
0.043
0.432
0.477

D2026
S2026
AfgAfaCfuAfcAfUfAfuAfaAfcUfaCfaAf
A2026
uUfgUfaGfuUfuAfuauGfuAfgUfuCfusUfsc
0.324
1.042
0.905

D2027
S2027
AfuUfaAfgCfuCfCfUfuCfuUfuUfuAfuUf
A2027
aAfuAfaAfaAfgAfaggAfgCfuUfaAfusUfsg
0.042
0.283
0.224

D2028
S2028
AfgAfuUfuGfcUfAfUfgUfuAfgAfcGfaUf
A2028
aUfcGfuCfuAfaCfauaGfcAfaAfuCfusUfsg
0.349
0.936
0.896

D2029
S2029
UfcAfgUfuGfgGfAfCfaUfgGfuCfuUfaAf
A2029
uUfaAfgAfcCfaUfgucCfcAfaCfuGfasAfsg
0.914
0.907
0.944

D2030
S2030
GfgCfcAfaAfuUfAfAfuGfaCfaUfaUfuUf
A2030
aAfaUfaUfgUfcAfuuaAfuUfuGfgCfcsCfsu
0.047
0.353
0.326

D2031
S2031
CfaUfaUfuUfgAfUfCfaGfuCfuUfuUfuAf
A2031
uAfaAfaAfgAfcUfgauCfaAfaUfaUfgsUfsu
0.110
0.867
0.842

D2032
S2032
UfaCfaUfaUfaAfAfCfuAfcAfaGfuCfaAf
A2032
uUfgAfcUfuGfuAfguuUfaUfaUfgUfasGfsu
0.200
0.699
0.656

D2033
S2033
UfuUfuAfuUfgUfUfCfcUfcUfaGfuUfaUf
A2033
aUfaAfcUfaGfaGfgaaCfaAfuAfaAfasAfsg
0.050
0.218
0.192

D2034
S2034
UfuGfcUfaUfgUfUfAfgAfcGfaUfgUfaAf
A2034
uUfaCfaUfcGfuCfuaaCfaUfaGfcAfasAfsu
0.096
0.792
0.640

D2035
S2035
CfaGfuUfgGfgAfCfAfuGfgUfcUfuAfaAf
A2035
uUfuAfaGfaCfcAfuguCfcCfaAfcUfgsAfsa
0.127
0.936
0.890

D2036
S2036
AfaAfuUfaAfuGfAfCfaUfaUfuUfcAfaAf
A2036
uUfuGfaAfaUfaUfgucAfuUfaAfuUfusGfsg
0.061
0.683
0.668

D2037
S2037
GfaUfcAfgUfcUfUfUfuUfaUfgAfuCfuAf
A2037
uAfgAfuCfaUfaAfaaaGfaCfuGfaUfcsAfsa
0.157
1.010
0.723

D2038
S2038
AfcAfuAfuAfaAfCfUfaCfaAfgUfcAfaAf
A2038
uUfuGfaCfuUfgUfaguUfuAfuAfuGfusAfsg
0.047
0.532
0.525

D2039
S2039
UfuUfaUfuGfuUfCfCfuCfuAfgUfuAfuUf
A2039
aAfuAfaCfuAfgAfggaAfcAfaUfaAfasAfsa
0.031
0.505
0.238

D2040
S2040
UfgCfuAfuGfuUfAfGfaCfgAfuGfuAfaAf
A2040
uUfuAfcAfuCfgUfcuaAfcAfuAfgCfasAfsa
0.056
0.484
0.408

D2041
S2041
GfgGfaCfaUfgGfUfCfuUfaAfaGfaCfuUf
A2041
aAfgUfcUfuUfaAfgacCfaUfgUfcCfcsAfsa
0.570
0.999
0.994

D2042
S2042
UfgAfcAfuAfuUfUfCfaAfaAfaCfuCfaAf
A2042
uUfgAfgUfuUfuUfgaaAfuAfuGfuCfasUfsu
0.065
0.870
0.728

D2043
S2043
AfuCfaGfuCfuUfUfUfuAfuGfaUfcUfaUf
A2043
aUfaGfaUfcAfuAfaaaAfgAfcUfgAfusCfsa
0.048
0.362
0.282

D2044
S2044
CfaUfaUfaAfaCfUfAfcAfaGfuCfaAfaAf
A2044
uUfuUfgAfcUfuGfuagUfuUfaUfaUfgsUfsa
0.314
0.904
0.937

D2045
S2045
CfuUfgAfaCfuCfAfAfcUfcAfaAfaCfuUf
A2045
aAfgUfuUfuGfaGfuugAfgUfuCfaAfgsUfsg
0.060
0.295
0.251

D2046
S2046
CfuAfcUfuCfaAfCfAfaAfaAfgUfgAfaAf
A2046
uUfuCfaCfuUfuUfuguUfgAfaGfuAfgsAfsa
0.052
0.570
0.599

D2047
S2047
AfaGfaGfcAfaCfUfAfaCfuAfaCfuUfaAf
A2047
uUfaAfgUfuAfgUfuagUfuGfcUfcUfusCfsu
0.028
0.369
0.381

D2048
S2048
AfaAfcAfaGfaUfAfAfuAfgCfaUfcAfaAf
A2048
uUfuGfaUfgCfuAfuuaUfcUfuGfuUfusUfsu
0.039
0.227
0.204

D2049
S2049
GfcAfuAfgUfcAfAfAfuAfaAfaGfaAfaUf
A2049
aUfuUfcUfuUfuAfuuuGfaCfuAfuGfcsUfsg
0.032
0.437
0.422

D2050
S2050
AfuAfuAfaAfcUfAfCfaAfgUfcAfaAfaAf
A2050
uUfuUfuGfaCfuUfguaGfuUfuAfuAfusGfsu
0.297
0.946
0.850

D2051
S2051
GfaAfcUfcAfaCfUfCfaAfaAfcUfuGfaAf
A2051
uUfcAfaGfuUfuUfgagUfuGfaGfuUfcsAfsa
0.179
0.929
0.884

D2052
S2052
UfaCfuUfcAfaCfAfAfaAfaGfuGfaAfaUf
A2052
aUfuUfcAfcUfuUfuugUfuGfaAfgUfasGfsa
0.091
0.536
0.524

D2053
S2053
AfgAfgCfaAfcUfAfAfcUfaAfcUfuAfaUf
A2053
aUfuAfaGfuUfaGfuuaGfuUfgCfuCfusUfsc
0.086
0.611
0.621

D2054
S2054
GfaUfaAfuAfgCfAfUfcAfaAfgAfcCfuUf
A2054
aAfgGfuCfuUfuGfaugCfuAfuUfaUfcsUfsu
0.058
0.676
0.591

D2055
S2055
CfaUfaGfuCfaAfAfUfaAfaAfgAfaAfuAf
A2055
uAfuUfuCfuUfuUfauuUfgAfcUfaUfgsCfsu
0.048
0.630
0.674

D2056
S2056
UfaUfaAfaCfuAfCfAfaGfuCfaAfaAfaUf
A2056
aUfuUfuUfgAfcUfuguAfgUfuUfaUfasUfsg
0.072
0.534
0.459

D2057
S2057
AfaCfuCfaAfcUfCfAfaAfaCfuUfgAfaAf
A2057
uUfuCfaAfgUfuUfugaGfuUfgAfgUfusCfsa
0.161
0.864
0.775

D2058
S2058
AfcUfuCfaAfcAfAfAfaAfgUfgAfaAfuAf
A2058
uAfuUfuCfaCfuUfuuuGfuUfgAfaGfusAfsg
0.198
0.969
0.865

D2059
S2059
GfaGfcAfaCfuAfAfCfuAfaCfuUfaAfuUf
A2059
aAfuUfaAfgUfuAfguuAfgUfuGfcUfcsUfsu
0.031
0.253
0.210

D2060
S2060
AfaCfcAfaCfaGfCfAfuAfgUfcAfaAfuAf
A2060
uAfuUfuGfaCfuAfugcUfgUfuGfgUfusUfsa
0.035
0.561
0.569

D2061
S2061
AfgUfcAfaAfuAfAfAfaGfaAfaUfaGfaAf
A2061
uUfcUfaUfuUfcUfuuuAfuUfuGfaCfusAfsu
0.057
0.668
0.386

D2062
S2062
AfgUfcAfaAfaAfUfGfaAfgAfgGfuAfaAf
A2062
uUfuAfcCfuCfuUfcauUfuUfuGfaCfusUfsg
0.720
1.017
0.924

D2063
S2063
CfuUfgAfaAfgCfCfUfcCfuAfgAfaGfaAf
A2063
uUfcUfuCfuAfgGfaggCfuUfuCfaAfgsUfsu
0.324
1.020
0.963

D2064
S2064
CfuUfcAfaCfaAfAfAfaGfuGfaAfaUfaUf
A2064
aUfaUfuUfcAfcUfuuuUfgUfuGfaAfgsUfsa
0.048
0.549
0.531

D2065
S2065
CfaAfcUfaAfcUfAfAfcUfuAfaUfuCfaAf
A2065
uUfgAfaUfuAfaGfuuaGfuUfaGfuUfgsCfsu
0.046
0.739
0.649

D2066
S2066
AfcCfaAfcAfgCfAfUfaGfuCfaAfaUfaAf
A2066
uUfaUfuUfgAfcUfaugCfuGfuUfgGfusUfsu
0.076
0.840
0.777

D2067
S2067
GfaAfcCfcAfcAfGfAfaAfuUfuCfuCfuAf
A2067
uAfgAfgAfaAfuUfucuGfuGfgGfuUfcsUfsu
0.103
0.916
0.808

D2068
S2068
GfaAfuAfuGfuCfAfCfuUfgAfaCfuCfaAf
A2068
uUfgAfgUfuCfaAfgugAfcAfuAfuUfcsUfsu
0.046
0.532
0.S20

D2069
S2069
UfgAfaAfgCfcUfCfCfuAfgAfaGfaAfaAf
A2069
uUfuUfcUfuCfuAfggaGfgCfuUfuCfasAfsg
0.067
0.894
0.822

D2070
S2070
UfuCfaAfcAfaAfAfAfgUfgAfaAfuAfuUf
A2070
aAfuAfuUfuCfaCfuuuUfuGfuUfgAfasGfsu
0.052
0.557
0.395

D2071
S2071
AfaCfuAfaCfuAfAfCfuUfaAfuUfcAfaAf
A2071
uUfuGfaAfuUfaAfguuAfgUfuAfgUfusGfsc
0.025
0.220
0.232

D2072
S2072
CfcAfaCfaGfcAfUfAfgUfcAfaAfuAfaAf
A2072
uUfuAfuUfuGfaCfuauGfcUfgUfuGfgsUfsu
0.293
0.923
0.899

D2073
S2073
AfaCfcCfaCfaGfAfAfaUfuUfcUfcUfaUf
A2073
aUfaGfaGfaAfaUfuucUfgUfgGfgUfusCfsu
0.021
0.375
0.356

D2074
S2074
UfgUfcAfcUfuGfAfAfcUfcAfaCfuCfaAf
A2074
uUfgAfgUfuGfaGfuucAfaGfuGfaCfasUfsa
0.052
0.402
0.513

D2075
S2075
GfaAfaGfcCfuCfCfUfaGfaAfgAfaAfaAf
A2075
uUfuUfuCfuUfcUfaggAfgGfcUfuUfcsAfsa
0.171
0.904
0.893

D2076
S2076
AfaUfaUfuUfaGfAfAfgAfgCfaAfcUfaAf
A2076
uUfaGfuUfgCfuCfuucUfaAfaUfaUfusUfsc
0.142
0.614
0.688

D2077
S2077
AfcUfaAfcUfaAfCfUfuAfaUfuCfaAfaAf
A2077
uUfuUfgAfaUfuAfaguUfaGfuUfaGfusUfsg
0.020
0.312
0.316

D2078
S2078
CfaAfcAfgCfaUfAfGfuCfaAfaUfaAfaAf
A2078
uUfuUfaUfuUfgAfcuaUfgCfuGfuUfgsGfsu
0.026
0.313
0.393

D2079
S2079
CfcAfcAfgAfaAfUfUfuCfuCfuAfuCfuUf
A2079
aAfgAfuAfgAfgAfaauUfuCfuGfuGfgsGfsu
0.012
0.596
0.345

D2080
S2080
GfuCfaCfuUfgAfAfCfuCfaAfcUfcAfaAf
A2080
uUfuGfaGfuUfgAfguuCfaAfgUfgAfcsAfsu
0.054
0.503
0.456

D2081
S2081
CfuCfcUfaGfaAfGfAfaAfaAfaUfuCfuAf
A2081
uAfgAfaUfuUfuUfucuUfcUfaGfgAfgsGfsc
0.050
0.596
0.531

D2082
S2082
AfuUfuAfgAfaGfAfGfcAfaCfuAfaCfuAf
A2082
uAfgUfuAfgUfuGfcucUfuCfuAfaAfusAfsu
0.064
0.806
0.928

D2083
S2083
CfuAfaCfuAfaCfUfUfaAfuUfcAfaAfaUf
A2083
aUfuUfuGfaAfuUfaagUfuAfgUfuAfgsUfsu
0.056
0.844
0.761

D2084
S2084
CfaGfcAfuAfgUfCfAfaAfuAfaAfaGfaAf
A2084
uUfcUfuUfuAfuUfugaCfuAfuGfcUfgsUfsu
0.046
0.859
0.756

D2085
S2085
GfaAfaUfaAfgAfAfAfuGfuAfaAfaCfaUf
A2085
aUfgUfuUfuAfcAfuuuCfuUfaUfuUfcsAfsu
0.039
0.615
0.612

D2086
S2086
UfcAfcUfuGfaAfCfUfcAfaCfuCfaAfaAf
A2086
uUfuUfgAfgUfuGfaguUfcAfaGfuGfasCfsa
0.057
0.724
0.663

D2087
S2087
UfcUfaCfuUfcAfAfCfaAfaAfaGfuGfaAf
A2087
uUfcAfcUfuUfuUfguuGfaAfgUfaGfasAfsu
0.732
1.028
0.915

D2088
S2088
UfuUfaGfaAfgAfGfCfaAfcUfaAfcUfaAf
A2088
uUfaGfuUfaGfuUfgcuCfuUfcUfaAfasUfsa
0.061
0.795
0.785

D2089
S2089
AfaAfaCfaAfgAfUfAfaUfaGfcAfuCfaAf
A2089
uUfgAfuGfcUfaUfuauCfuUfgUfuUfusUfsc
0.330
1.017
0.865

D2090
S2090
AfgCfaUfaGfuCfAfAfaUfaAfaAfgAfaAf
A2090
uUfuCfuUfuUfaUfuugAfcUfaUfgCfusGfsu
0.038
0.606
0.589

D2091
S2091
AfgAfcCfcAfgCfAfAfcUfcUfcAfaGfuUf
A2091
aAfcUfuGfaGfaGfuugCfuGfgGfuCfusGfsa
0.301
0.850
0.753

D2092
S2092
AfgUfcCfaUfgGfAfCfaUfuAfaUfuCfaAf
A2092
uUfgAfaUfuAfaUfgucCfaUfgGfaCfusAfsc
0.407
0.791
0.726

D2093
S2093
GfaUfgGfaUfcAfCfAfaAfaCfuUfcAfaUf
A2093
aUfuGfaAfgUfuUfuguGfaUfcCfaUfcsUfsa
0.120
0.658
0.654

D2094
S2094
CfuAfgAfgAfaGfAfUfaUfaCfuCfcAfuAf
A2094
uAfuGfgAfgUfaUfaucUfuCfuCfuAfgsGfsc
0.071
0.610
0.645

D2095
S2095
AfaAfgAfcAfaCfAfAfaCfaUfuAfuAfuUf
A2095
aAfuAfuAfaUfgUfuugUfuGfuCfuUfusCfsc
0.029
0.306
0.461

D2096
S2096
CfaUfuAfuAfuUfGfAfaUfaUfuCfuUfuUf
A2096
aAfaAfgAfaUfaUfucaAfuAfuAfaUfgsUfsu
0.031
0.510
0.595

D2097
S2097
GfaCfcCfaGfcAfAfCfuCfuCfaAfgUfuUf
A2097
aAfaCfuUfgAfgAfguuGfcUfgGfgUfcsUfsg
0.075
0.697
0.845

D2098
S2098
GfgAfuCfaCfaAfAfAfcUfuCfaAfuGfaAf
A2098
uUfcAfuUfgAfaGfuuuUfgUfgAfuCfcsAfsu
0.130
0.831
0.951

D2099
S2099
GfaAfgAfuAfuAfCfUfcCfaUfaGfuGfaAf
A2099
uUfcAfcUfaUfgGfaguAfuAfuCfuUfcsUfsc
0.058
0.828
0.938

D2100
S2100
GfaCfaAfcAfaAfCfAfuUfaUfaUfuGfaAf
A2100
uUfcAfaUfaUfaAfuguUfuGfuUfgUfcsUfsu
0.026
0.564
0.856

D2101
S2101
GfgGfaAfaUfcAfCfGfaAfaCfcAfaCfuAf
A2101
uAfgUfuGfgUfuUfcguGfaUfuUfcCfcsAfsa
0.314
0.948
1.033

D2102
S2102
AfcCfcAfgCfaAfCfUfcUfcAfaGfuUfuUf
A2102
aAfaAfcUfuGfaGfaguUfgCfuGfgGfusCfsu
0.033
0.448
0.675

D2103
S2103
GfgAfcAfuUfaAfUfUfcAfaCfaUfcGfaAf
A2103
uUfcGfaUfgUfuGfaauUfaAfuGfuCfcsAfsu
0.156
0.897
0.912

D2104
S2104
GfaUfcAfcAfaAfAfCfuUfcAfaUfgAfaAf
A2104
uUfuCfaUfuGfaAfguuUfuGfuGfaUfcsCfsa
0.056
0.619
0.769

D2105
S2105
AfcUfcCfaUfaGfUfGfaAfgCfaAfuCfuAf
A2105
uAfgAfuUfgCfuUfcacUfaUfgGfaGfusAfsu
0.100
0.823
0.925

D2106
S2106
AfcAfaCfaAfaCfAfUfuAfuAfuUfgAfaUf
A2106
aUfuCfaAfuAfuAfaugUfuUfgUfuGfusCfsu
0.035
0.565
0.843

D2107
S2107
GfgAfaAfuCfaCfGfAfaAfcCfaAfcUfaUf
A2107
aUfaGfuUfgGfuUfucgUfgAfuUfuCfcsCfsa
0.076
0.701
0.890

D2108
S2108
CfcCfaGfcAfaCfUfCfuCfaAfgUfuUfuUf
A2108
aAfaAfaCfuUfgAfgagUfuGfcUfgGfgsUfsc
0.057
0.626
0.884

D2109
S2109
GfaCfaUfuAfaUfUfCfaAfcAfuCfgAfaUf
A2109
aUfuCfgAfuGfuUfgaaUfuAfaUfgUfcsCfsa
0.160
0.873
1.012

D2110
S2110
AfaCfgUfgGfgAfGfAfaCfuAfcAfaAfuAf
A2110
uAfuUfuGfuAfgUfucuCfcCfaCfgUfusUfsc
0.101
0.881
0.981

D2111
S2111
CfuCfcAfuAfgUfGfAfaGfcAfaUfcUfaAf
A2111
uUfaGfaUfuGfcUfucaCfuAfuGfgAfgsUfsa
0.026
0.435
0.691

D2112
S2112
CfaAfcAfaAfcAfUfUfaUfaUfuGfaAfuAf
A2112
uAfuUfcAfaUfaUfaauGfuUfuGfuUfgsUfsc
0.154
0.882
1.091

D2113
S2113
GfaAfaUfcAfcGfAfAfaCfcAfaCfuAfuAf
A2113
uAfuAfgUfuGfgUfuucGfuGfaUfuUfcsCfsc
0.045
0.764
1.004

D2114
S2114
CfuCfuCfaAfgUfUfUfuUfcAfuGfuCfuAf
A2114
uAfgAfcAfuGfaAfaaaCfuUfgAfgAfgsUfsu
0.105
0.925
0.988

D2115
S2115
AfcAfuUfaAfuUfCfAfaCfaUfcGfaAfuAf
A2115
uAfuUfcGfaUfgUfugaAfuUfaAfuGfusCfsc
0.114
0.919
0.905

D2116
S2116
GfgGfaGfaAfcUfAfCfaAfaUfaUfgGfuUf
A2116
aAfcCfaUfaUfuUfguaGfuUfcUfcCfcsAfsc
0.234
1.023
0.951

D2117
S2117
UfcCfaUfaGfuGfAfAfgCfaAfuCfuAfaUf
A2117
aUfuAfgAfuUfgCfuucAfcUfaUfgGfasGfsu
0.033
0.566
0.778

D2118
S2118
AfaCfaAfaCfaUfUfAfuAfuUfgAfaUfaUf
A2118
aUfaUfuCfaAfuAfuaaUfgUfuUfgUfusGfsu
0.031
0.535
0.785

D2119
S2119
UfgGfcAfaUfgUfCfCfcCfaAfuGfcAfaUf
A2119
aUfuGfcAfuUfgGfggaCfaUfuGfcCfasGfsu
0.065
0.815
0.967

D2120
S2120
UfcAfgGfuAfgUfCfCfaUfgGfaCfaUfuAf
A2120
uAfaUfgUfcCfaUfggaCfuAfcCfuGfasUfsa
0.223
0.825
0.924

D2121
S2121
UfuAfaUfuCfaAfCfAfuCfgAfaUfaGfaUf
A2121
aUfcUfaUfuCfgAfuguUfgAfaUfuAfasUfsg
0.083
0.781
0.915

D2122
S2122
GfgAfgAfaCfuAfCfAfaAfuAfuGfgUfuUf
A2122
aAfaCfcAfuAfuUfuguAfgUfuCfuCfcsCfsa
0.079
0.680
0.767

D2123
S2123
CfcAfuAfgUfgAfAfGfcAfaUfcUfaAfuUf
A2123
aAfuUfaGfaUfuGfcuuCfaCfuAfuGfgsAfsg
0.026
0.537
0.793

D2124
S2124
AfcAfaAfcAfuUfAfUfaUfuGfaAfuAfuUf
A2124
aAfuAfuUfcAfaUfauaAfuGfuUfuGfusUfsg
0.044
0.680
0.828

D2125
S2125
AfaUfgCfaAfuCfCfCfgGfaAfaAfcAfaAf
A2125
uUfuGfuUfuUfcCfgggAfuUfgCfaUfusGfsg
0.349
0.971
1.005

D2126
S2126
CfaGfgUfaGfuCfCfAfuGfgAfcAfuUfaAf
A2126
uUfaAfuGfuCfcAfuggAfcUfaCfcUfgsAfsu
0.070
0.548
0.546

D2127
S2127
UfuCfaAfcAfuCfGfAfaUfaGfaUfgGfaUf
A2127
aUfcCfaUfcUfaUfucgAfuGfuUfgAfasUfsu
0.225
0.958
0.967

D2128
S2128
GfuUfgGfgCfcUfAfGfaGfaAfgAfuAfuAf
A2128
uAfuAfuCfuUfcUfcuaGfgCfcCfaAfcsCfsa
0.765
0.969
0.922

D2129
S2129
CfaUfaGfuGfaAfGfCfaAfuCfuAfaUfuAf
A2129
uAfaUfuAfgAfuUfgcuUfcAfcUfaUfgsGfsa
0.028
0.583
0.777

D2130
S2130
AfaCfaUfuAfuAfUfUfgAfaUfaUfuCfuUf
A2130
aAfgAfaUfaUfuCfaauAfuAfaUfgUfusUfsg
0.249
0.916
0.981

D2131
S2131
GfcAfaUfcCfcGfGfAfaAfaCfaAfaGfaUf
A2131
aUfcUfuUfgUfuUfuccGfgGfaUfuGfcsAfsu
0.435
1.002
1.019

D2132
S2132
GfgUfaGfuCfcAfUfGfgAfcAfuUfaAfuUf
A2132
aAfuUfaAfuGfuCfcauGfgAfcUfaCfcsUfsg
0.427
0.988
0.918

D2133
S2133
AfuCfgAfaUfaGfAfUfgGfaUfcAfcAfaAf
A2133
uUfuGfuGfaUfcCfaucUfaUfuCfgAfusGfsu
0.170
0.706
0.890

D2134
S2134
CfcUfaGfaGfaAfGfAfuAfuAfcUfcCfaUf
A2134
aUfgGfaGfuAfuAfucuUfcUfcUfaGfgsCfsc
0.033
0.543
0.733

D2135
S2135
GfuUfgGfaAfgAfCfUfgGfaAfaGfaCfaAf
A2135
uUfgUfcUfuUfcCfaguCfuUfcCfaAfcsUfsc
0.137
0.975
0.944

D2136
S2136
AfcAfuUfaUfaUfUfGfaAfuAfuUfcUfuUf
A2136
aAfaGfaAfuAfuUfcaaUfaUfaAfuGfusUfsu
0.114
0.882
0.940

D2137
S2137
CfaAfuCfcCfgGfAfAfaAfcAfaAfgAfuUf
A2137
aAfuCfuUfuGfuUfuucCfgGfgAfuUfgsCfsa
0.155
0.755
0.686

D2138
S2138
CfuAfcUfuGfgGfAfUfcAfcAfaAfgCfaAf
A2138
uUfgCfuUfuGfuGfaucCfcAfaGfuAfgsAfsa
0.196
0.825
0.658

D2139
S2139
AfcAfaCfcUfaAfAfUfgGfuAfaAfuAfuAf
A2139
uAfuAfuUfuAfcCfauuUfaGfgUfuGfusUfsu
0.133
0.704
0.671

D2140
S2140
AfuCfcAfuCfcAfAfCfaGfaUfuCfaGfaAf
A2140
uUfcUfgAfaUfcUfguuGfgAfuGfgAfusCfsa
0.184
0.775
0.658

D2141
S2141
AfaCfuGfaGfgCfAfAfaUfuUfaAfaAfgAf
A2141
uCfuUfuUfaAfaUfuugCfcUfcAfgUfusCfsa
0.076
0.682
0.777

D2142
S2142
AfgAfgUfaUfgUfGfUfaAfaAfaUfcUfgUf
A2142
aCfaGfaUfuUfuUfacaCfaUfaCfuCfusGfsu
0.448
0.659
0.761

D2143
S2143
AfaUfcCfcGfgAfAfAfaCfaAfaGfaUfuUf
A2143
aAfaUfcUfuUfgUfuuuCfcGfgGfaUfusGfsc
0.097
0.844
0.924

D2144
S2144
UfaCfuUfgGfgAfUfCfaCfaAfaGfcAfaAf
A2144
uUfuGfcUfuUfgUfgauCfcCfaAfgUfasGfsa
0.084
0.875
0.947

D2145
S2145
CfaAfcCfuAfaAfUfGfgUfaAfaUfaUfaAf
A2145
uUfaUfaUfuUfaCfcauUfuAfgGfuUfgsUfsu
0.104
0.811
0.814

D2146
S2146
UfuGfaAfuGfaAfCfUfgAfgGfcAfaAfuUf
A2146
aAfuUfuGfcCfuCfaguUfcAfuUfcAfasAfsg
0.046
0.549
0.680

D2147
S2147
AfcUfgAfgGfcAfAfAfuUfuAfaAfaGfgAf
A2147
uCfcUfuUfuAfaAfuuuGfcCfuCfaGfusUfsc
0.079
0.890
1.005

D2148
S2148
GfaGfuAfuGfuGfUfAfaAfaAfuCfuGfuAf
A2148
uAfcAfgAfuUfuUfuacAfcAfuAfcUfcsUfsg
0.497
0.676
0.783

D2149
S2149
AfcUfuGfgGfaUfCfAfcAfaAfgCfaAfaAf
A2149
uUfuUfgCfuUfuGfugaUfcCfcAfaGfusAfsg
0.049
0.699
0.907

D2150
S2150
AfuGfgUfaAfaUfAfUfaAfcAfaAfcCfaAf
A2150
uUfgGfuUfuGfuUfauaUfuUfaCfcAfusUfsu
0.093
0.928
0.941

D2151
S2151
UfgAfaUfgAfaCfUfGfaGfgCfaAfaUfuUf
A2151
aAfaUfuUfgCfcUfcagUfuCfaUfuCfasAfsa
0.201
0.736
0.885

D2152
S2152
CfuGfaGfgCfaAfAfUfuUfaAfaAfgGfcAf
A2152
uGfcCfuUfuUfaAfauuUfgCfcUfcAfgsUfsu
0.071
0.938
0.872

D2153
S2153
AfgUfaUfgUfgUfAfAfaAfaUfcUfgUfaAf
A2153
uUfaCfaGfaUfuUfuuaCfaCfaUfaCfusCfsu
0.504
0.816
0.689

D2154
S2154
GfaAfaAfcAfaAfGfAfuUfuGfgUfgUfuUf
A2154
aAfaCfaCfcAfaAfucuUfuGfuUfuUfcsCfsg
0.061
0.723
0.922

D2155
S2155
AfgUfgUfgGfaGfAfAfaAfcAfaCfcUfaAf
A2155
uUfaGfgUfuGfuUfuucUfcCfaCfaCfusCfsa
0.071
0.689
0.869

D2156
S2156
GfuCfuCfaAfaAfUfGfgAfaGfgUfuAfuAf
A2156
uAfuAfaCfcUfuCfcauUfuUfgAfgAfcsUfsu
0.133
0.643
0.974

D2157
S2157
GfaAfuGfaAfcUfGfAfgGfcAfaAfuUfuAf
A2157
uAfaAfuUfuGfcCfucaGfuUfcAfuUfcsAfsa
0.204
0.751
1.008

D2158
S2158
UfgAfgGfcAfaAfUfUfuAfaAfaGfgCfaAf
A2158
uUfgCfcUfuUfuAfaauUfuGfcCfuCfasGfsu
0.089
0.820
0.937

D2159
S2159
GfuAfuGfuGfuAfAfAfaAfuCfuGfuAfaUf
A2159
aUfuAfcAfgAfuUfuuuAfcAfcAfuAfcsUfsc
0.535
0.697
0.788

D2160
S2160
AfaAfaCfaAfaGfAfUfuUfgGfuGfuUfuUf
A2160
aAfaAfcAfcCfaAfaucUfuUfgUfuUfusCfsc
0.297
0.954
1.004

D2161
S2161
GfuGfuGfgAfgAfAfAfaCfaAfcCfuAfaAf
A2161
uUfuAfgGfuUfgUfuuuCfuCfcAfcAfcsUfsc
0.178
0.872
0.918

D2162
S2162
AfuGfgAfaGfgUfUfAfuAfcUfcUfaUfaAf
A2162
uUfaUfaGfaGfuAfuaaCfcUfuCfcAfusUfsu
0.026
0.489
0.890

D2163
S2163
AfaUfgAfaCfuGfAfGfgCfaAfaUfuUfaAf
A2163
uUfaAfaUfuUfgCfcucAfgUfuCfaUfusCfsa
0.111
0.789
0.859

D2164
S2164
GfaGfgCfaAfaUfUfUfaAfaAfgGfcAfaUf
A2164
aUfuGfcCfuUfuUfaaaUfuUfgCfcUfcsAfsg
0.241
0.956
0.869

D2165
S2165
UfaUfgUfgUfaAfAfAfaUfcUfgUfaAfuAf
A2165
uAfuUfaCfaGfaUfuuuUfaCfaCfaUfasCfsu
0.571
0.762
0.931

D2166
S2166
AfcAfaAfgAfuUfUfGfgUfgUfuUfuCfuAf
A2166
uAfgAfaAfaCfaCfcaaAfuCfuUfuGfusUfsu
0.106
0.981
0.924

D2167
S2167
UfgUfgGfaGfaAfAfAfcAfaCfcUfaAfaUf
A2167
aUfuUfaGfgUfuGfuuuUfcUfcCfaCfasCfsu
0.064
0.765
0.902

D2168
S2168
UfgGfaAfgGfuUfAfUfaCfuCfuAfuAfaAf
A2168
uUfuAfuAfgAfgUfauaAfcCfuUfcCfasUfsu
0.029
0.675
0.859

D2169
S2169
AfuGfaAfcUfgAfGfGfcAfaAfuUfuAfaAf
A2169
uUfuAfaAfuUfuGfccuCfaGfuUfcAfusUfsc
0.054
0.733
0.843

D2170
S2170
AfgGfcAfaAfuUfUfAfaAfaGfgCfaAfuAf
A2170
uAfuUfgCfcUfuUfuaaAfuUfuGfcCfusCfsa
0.075
0.754
0.881

D2171
S2171
AfaGfaUfuUfgGfUfGfuUfuUfcUfaCfuUf
A2171
aAfgUfaGfaAfaAfcacCfaAfaUfcUfusUfsg
0.303
1.065
0.977

D2172
S2172
AfaAfcAfaCfcUfAfAfaUfgGfuAfaAfuAf
A2172
uAfuUfuAfcCfaUfuuaGfgUfuGfuUfusUfsc
0.101
0.855
0.880

D2173
S2173
AfuAfcUfcUfaUfAfAfaAfuCfaAfcCfaAf
A2173
uUfgGfuUfgAfuUfuuaUfaGfaGfuAfusAfsa
0.107
0.961
0.960

D2174
S2174
UfgAfaCfuGfaGfGfCfaAfaUfuUfaAfaAf
A2174
uUfuUfaAfaUfuUfgccUfcAfgUfuCfasUfsu
0.078
0.714
0.878

D2175
S2175
GfgCfaAfaUfuUfAfAfaAfgGfcAfaUfaAf
A2175
uUfaUfuGfcCfuUfuuaAfaUfuUfgCfcsUfsc
0.054
0.767
0.918

D2176
S2176
UfuUfuCfuAfcUfUfGfgGfaUfcAfcAfaAf
A2176
uUfuGfuGfaUfcCfcaaGfuAfgAfaAfasCfsa
0.915
1.030
0.916

D2177
S2177
AfaCfaAfcCfuAfAfAfuGfgUfaAfaUfaUf
A2177
aUfaUfuUfaCfcAfuuuAfgGfuUfgUfusUfsu
0.042
0.260
0.448

D2178
S2178
UfaCfuCfuAfuAfAfAfaUfcAfaCfcAfaAf
A2178
uUfuGfgUfuGfaUfuuuAfuAfgAfgUfasUfsa
0.063
0.897
0.869

D2179
S2179
GfaAfcUfgAfgGfCfAfaAfuUfuAfaAfaAf
A2179
uUfuUfuAfaAfuUfugcCfuCfaGfuUfcsAfsu
0.178
0.858
0.869

D2180
S2180
CfaGfaGfuAfuGfUfGfuAfaAfaAfuCfuUf
A2180
aAfgAfuUfuUfuAfcacAfuAfcUfcUfgsUfsg
0.436
0.677
0.813

Example 4. In Vitro Silencing Activity with Various Chemical Modifications on ANGPTL3 siRNA

Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×10⁴Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nM final duplex concentration unless otherwise stated.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR

2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl ANGPTL TaqMan probe (Applied Biosystems cat #Hs00205581_m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections, and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data was analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose. AD-1955 sequence, used as a negative control, targets luciferase and has the following sequence:

(SEQ ID NO: 1207)

sense: cuuAcGcuGAGuAcuucGAdTsdT;

(SEQ ID NO: 1208)

antisense: UCGAAGuACUcAGCGuAAGdTsdT.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Number	Name	Date	Kind
8586727	Kelnar et al.	Nov 2013	B2
9012623	Rana	Apr 2015	B2
9181551	McSwiggen	Nov 2015	B2
9399775	Rajeev	Jul 2016	B2
20050026160	Allerson	Feb 2005	A1
20050148530	McSwiggen	Jul 2005	A1
20070135372	MacLachlan et al.	Jun 2007	A1
20070167392	Bhat	Jul 2007	A1

Number	Date	Country
2 305 805	Apr 2011	EP
WO03070918	Aug 2003	WO
2004015107	Feb 2004	WO
2005121370	Dec 2005	WO
2009002944	Dec 2008	WO
2009073809	Jun 2009	WO
2009134487	Nov 2009	WO
2010148013	Dec 2010	WO
2012037254	Mar 2012	WO

Modified RNAi agents

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

Field of Search

CPC

International Classifications

Disclaimer

Abstract

Description

Claims

RELATED APPLICATION

PCT Information

US Referenced Citations (8)

Foreign Referenced Citations (9)

Non-Patent Literature Citations (10)

Related Publications (1)

Provisional Applications (1)

Entry
Written Opinion of the International Search Authority PCT/US2012/065601, dated Mar. 2013, pp. 1-13.
Watts et al. Drug Discovery Today 13, 2008, 842-855.
Watt et al. (Drug Discovery Today 13, 2008).
Eberle et al. (The Journal of Immunology, 2008, 180: 3229-3237).
Morrissey et al. Nature Biotechnology 23, 2005, 1002-1007.
Muhonen et al. Chemsitry & Biodiversity 4, 2007, pp. 858-873.
Prakash et al., “Positional Effect of Chemical Modifications on Short Interference RNA Activity in Mammalian Cells,” J. Med. Chem. 48:4247-4253 (2005).
Collingwood et al., “Chemical Modification Patterns Compatible with High Potency Dicer-Substrate Small Interfering RNAs,” Oligonucleotides 18:187-200 (2008).
Bramsen et al., “A Large-Scale Chemical Modification Screen Identifies Design Rules to Generate siRNAs with High Activity, High Stability and Low Toxicity,” Nucleic Acids Res. 37(9):2867-2881 (2009).
Deleavey et al., “Synergistic Effects Between Analogs of DNA and RNA Improve the Potency of siRNA-Mediated Gene Silencing,” Nucleic Acids Res. 38(13):4547-4557 (2010).