EA042669B1 - MODIFIED RNAi AGENTS - Google Patents

Description

Related application

The present application claims priority to US Provisional Application No. 61/561,710, filed Nov. 18, 2011, which is incorporated herein by reference in its entirety.

The field of technology to which the invention belongs

The invention relates to RNAi duplex agents having specific motifs that are effective in inhibiting target gene expression, as well as to RNAi compositions suitable for therapeutic use. In addition, the invention relates to methods for inhibiting target gene expression by administering such RNAi duplex agents, for example, for the treatment of various diseases.

State of the art

RNA interference or RNAi is a term originally coined by Fire and coworkers 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). The short dsRNA directs gene-specific post-transcriptional silencing in many organisms, including vertebrates, and is a novel tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that degrades messenger RNAs homologous to the silencing-triggering sequence. It is known that RISC contains short RNAs (of approximately 22 nucleotides) derived from double-stranded RNA that trigger silencing, but the protein components of this activity remain unknown.

Double-stranded RNA (dsRNA) molecules with effective gene-silencing properties are needed for drug development based on RNA interference (RNAi). The initial stage of RNAi is the activation of the RNA-induced silencing complex (RISC), which requires the destruction of the sense strand of the dsRNA duplex. The sense strand is known to act as the first RISC substrate to be cleaved by argonaut 2 in the middle of the duplex region. Immediately after the cleaved 5' and 3' sense strand fragments are cleared from the Ago2 endonuclease, RISC becomes activated by the antisense strand (Rand et al. (2005), Cell, 123, 621).

It is believed that upon inhibition of sense strand cleavage, endonucleotic cleavage of the target mRNA is attenuated (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 the introduction of 2'-O-Meribose at the Ago2 cleavage site in the sense strand inhibits RNAi in HeLa cells (Leuschner et al. (2006), EMBO Rep., 7, 314). A similar effect is observed with phosphorothioate modifications, indicating that effective RNAi in mammals also requires sense strand cleavage.

Morrissey et al. at the Ago2 cleavage site, among other sites and modifications, an siRNA duplex containing residues modified with 2'-F was also used and consistent silencing comparable to unmodified siRNAs was obtained (Morrissey et al. (2005), Hepatology, 41, 1349). However, the Morrissey modification is not motif-specific, for example, one modification induces 2'-F modifications on all pyrimidines on both sense and antisense strands as long as a pyrimidine residue is present, without any selectivity; and therefore, based on such indications, it is uncertain whether modification of a specific motif at the sense strand cleavage site can actually have an effect on gene silencing activity. Muhonen et al. used an siRNA duplex containing two modified 2'-F residues at the Ago2 cleavage site on the sense or antisense strand and found this to be acceptable (Muhonen et al. (2007), Chemistry & Biodiversity, 4, 858-873). However, the Muhonen modification is also sequence specific, for example, for any given chain, Muhonen modifies only or all pyrimidines or all purines without any selectivity.

Choung et al. used an siRNA duplex containing alternative modifications at the expense of 2'-OMe or various combinations of 2'-F, 2'-OMe and phosphorothioate modifications to stabilize serum siRNA to Surl0058 (Choung et al. (2006), Biochemical and Biophysical Research Communications, 342, 919-927). Choung suggested that residues at the antisense strand cleavage site should not be modified with 2'-ome in order to increase the stability of the siRNA.

Thus, there is a need for mRNA duplex agents to improve the gene silencing efficiency of miRNA gene therapy agents. The present invention addresses such a need.

The essence of the invention

The present invention relates to effective nucleotides or chemical motifs for dsRNA agents, optionally conjugated to at least one ligand, that are effective to inhibit target gene expression, as well as RNAi compositions suitable for therapeutic use.

The inventors unexpectedly found that the introduction of one or more motifs from

- 1 042669 three identical modifications of three consecutive nucleotides at or near the cleavage site of the dsRNA agent, where the dsRNA agent consists 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 tool contains a sense strand and an antisense strand, where each strand has 14 to 30 nucleotides. The dsRNA duplex is represented by formula (III) sense: 5' np-N _a -(XXX) ₁ -N _b -YYY-Nb-(ZZZ) _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' (III)

In formula (III), i, j, k, and l are each independently 0 or 1;

p and q are each independently 0-6;

n is a nucleotide;

each N _a and N _a ' independently represents an oligonucleotide sequence containing 0-25 nucleotides that are either modified or unmodified, or combinations thereof, where each sequence contains at least two differently modified nucleotides;

each N _b and N _b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, which are either modified or unmodified, or combinations thereof;

each np and nq is independently an overhanging nucleotide sequence of 0-6 nucleotides; and each XXX, YYY, ZZZ, X'X'X', ΎΎΎ' and Z'Z'Z' independently represents one motif of three identical modifications on three consecutive nucleotides;

where modifications in N _b differ from modifications in Ύ and modifications in N _b ' differ from modifications in Ύ'.

At least one of the Ύ nucleotides forms a base pair with its complementary Ύ' nucleotides, where the modification at the Ύ nucleotide is different from the modification at the Ύ' nucleotide.

Each n _p and n _q is independently an overhanging nucleotide sequence of 0-6 nucleotides;

each n and n' is an overhanging nucleotide; and each p and q are independently 0-6.

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

In another aspect, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA tool contains a sense strand and an antisense strand, where each strand has 14 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 or near a cleavage site in the strand. 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 another aspect, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA tool contains a sense strand and an antisense strand, with each strand having 14 to 30 nucleotides. 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 another aspect, the invention relates to a method for delivering dsRNA to a specific target in an individual by subcutaneous or intravenous administration.

Detailed description

By introducing one or more motifs of three identical modifications at three consecutive nucleotides into the sense strand and/or antisense strand of the dsRNA agent, in particular at or near the cleavage site, an excellent result can be obtained. The sense strand and the antisense strand of the dsRNA agent may otherwise be completely modified.

The introduction of such motifs interrupts the set of modifications, if any, of the sense strand and/or the antisense strand. The dsRNA agent is optionally conjugated to a GalNAc derivative ligand, for example, on the sense strand. The resulting dsRNA tools exhibit excellent gene silencing activity.

The inventors surprisingly found that the presence of one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNA agent excellently enhances the gene silencing activity of the dsRNA agent.

Accordingly, the invention relates to a double-stranded RNAi agent (dsRNA) capable of inhibiting the expression of a target gene. The dsRNA tool contains a sense strand and an antisense strand. The length of each dsRNA agent chain can vary from 12 to 30 nucleotides. For example, the length of each chain can be 14-30, 17-30, 25-30, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25 or 21-23 nucleotides.

The sense strand and the antisense strand typically form a dsRNA duplex. The length of the duplex region of the dsRNA agent can be 12-30 base pairs. For example, the length of the duplex section can be 14-30, 17-30, 25-30, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25 or 21-23 base pairs. 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 may contain one or more overhangs and/or capping groups of the dsRNA agent at the 3' end or 5' end, or both ends of the strand. The length of the overhang can be 1-6 nucleotides, such as 2-6, 1-5, 2-5, 1-4, 2-4, 1-3, 2-3 or 1-2 nucleotides. The protrusions can be the result of one chain being longer than the other, or the result of two chains of the same length being unevenly spaced. The protrusion may mismatch with the target mRNA, or it may be complementary to the target gene sequences, or may be a different sequence. The first and second strands may also be connected, for example, by additional bases to form a hairpin or other non-base linkers.

In one embodiment, each nucleotide in the overhang of the dsRNA agent of the invention may independently be modified or unmodified nucleotides, including, but not limited to, modified to a 2'-sugar, e.g., 2-F-,2'-O-methylthymidine (T), 2'-O-methoxyethyl-5-methylureide (Theo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, a TT may be an overhang at either end of any strand. The protrusion may mismatch with the target mRNA, or it may be complementary to the target gene sequences, or may be a different sequence.

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

The dsRNA agent of the invention contains only one protrusion, which can enhance the interfering activity of dsRNA without affecting its overall stability. For example, the single-stranded overhang is located at the 3' end of the sense strand, or alternatively at the 3' end of the antisense strand. The dsRNA can also have a blunt end located at the 5' end of the antisense strand (or at the 3' end of the sense strand) or vice versa. Typically, the antisense dsRNA strand has a nucleotide overhang at the 3' end and the 5' end is blunt. Without wishing to be bound by any theory, the asymmetric blunt end at the 5' end of the antisense strand and the 3' overhang of the antisense strand favor loading the guide strand into the 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 19 nc (nt) double-ended bluntmer, where the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, 9 from 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 20 nc double-ended bluntmer, where the sense strand contains at least one of the three 2'-F motifs.

- 3,042,669 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 21 nc double-ended bluntmer, 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 contains a 21 nucleotide (nc) sense strand and a 23 nucleotide (nc) antisense strand, 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, with one end of the dsRNA blunt while the other end includes a protrusion at 2 nc. Preferably, the 2 nc overhang is located at the 3' end of the antisense strand. Optionally, the dsRNA also contains a ligand (preferably GalNAc ₃ ).

In one embodiment, the dsRNA agent of the invention comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues long, starting from the 5'-terminal nucleotide (position 1), positions 1-23 of said first strand contain at least 8 ribonucleotides; the length of the antisense strand is 36-66 nucleotide residues, and, starting from the 3'-terminal nucleotide, contain at least 8 ribonucleotides in positions paired with positions 1-23 of the sense strand, forming a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, whereby a 3'-single-stranded overhang of 1-6 nucleotides is formed; wherein the 5' end of the antisense strand contains 10-30 consecutive nucleotides that are not paired with the sense strand, whereby a single-stranded 5' overhang of 10-30 nucleotides is formed; where at least the 5'-terminal and 3'-terminal nucleotides of the sense strand are base paired with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, whereby a substantially duplex region is formed between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand to reduce expression of the target gene 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 is 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 comprises sense and antisense strands, wherein said dsRNA agent comprises a first strand of at least 25 and a maximum of 29 nucleotides and a second strand of at least 30 nucleotides with at least one motif of three 2'- O-methyl on three consecutive nucleotides at positions 11, 12, 13 from the 5' end; wherein said 3'-end of said first chain and said 5'-end of said second chain form a blunt end and said second chain is 1-4 nucleotides longer at its 3'-end than the first chain, wherein the duplex region, which has at least 25 nucleotides in length and said second strand is sufficiently complementary to the target mRNA along at least 19 nc of the length of said second strand to reduce expression of the target gene when said dsRNA agent is introduced into a mammalian cell; and wherein dicer cleavage of said dsRNA advantageously results in an siRNA containing said 3' end of said second strand, whereby expression of the target gene in the mammal is reduced. Optionally, said dsRNA agent also contains a ligand.

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

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

In the case of a dsRNA agent having a 17-23 nc duplex region, the antisense strand cleavage site is typically located near positions 10, 11 and 12 from the 5' end. Thus, motifs from three identical modifications can be in positions 9, 10, 11; provisions 10, 11, 12; provisions 11, 12, 13; positions 122, 13, 14 or positions 13, 14, 15 of the antisense strand, and the count starts from the 1st nucleotide from the 5' end of the antisense strand or the count starts from the 1st paired nucleotide in the duplex region from the 5' end of the antisense strand . Cleavage site

- 4 042669 in the antisense strand can also vary according to the length of the dsRNA duplex region from

5' end.

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

In one embodiment, the sense strand of the dsRNA agent contains more than one motif of three identical modifications on three consecutive nucleotides. The first motif must be at or near the chain cleavage site, and other motifs may be wing-flank modifications. The term flank modification as used herein refers to a motif located in a different part of the strand that is separated from the motif at or near a cleavage site in the same strand. The flank modification is located either next to the first motif or separated from it by at least one or more nucleotides. If the motifs are located directly next to each other, then the chemical groups of the motifs are different for each, and if the motifs are separated by one or more nucleotides, then the chemical groups of the motifs may be the same or different. Two or more flank modifications may be present. For example, if two flanking modifications are present, then both flanking modifications may occur at one end of the duplex region relative to the first motif that is at or near the cleavage site, or each of the flanking modifications may occur on either side of the first motif.

Similar to the sense strand, the antisense strand of the dsRNA agent contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs is located at or near the strand cleavage site. Such an antisense strand may also contain one or more flank modifications in alignment similar to the flank modifications that are present in the sense strand.

In one embodiment, the flank modification in the sense strand of the antisense strand, or both strands of the dsRNA agent, typically does not contain one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

In another embodiment, the flank modification in the sense strand, the antisense strand, or both, typically does not contain one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

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

If the sense strand and antisense strand of the dsRNA agent each contain at least two flank modifications, the sense strand and antisense strand can be aligned such that two flank modifications, each from the same strand, fall on one end of the duplex region, overlapping with one, two, or three nucleotides; two modifications, each from one strand, fall to the other end of the duplex region, overlapping by one, two, or three nucleotides.

In one embodiment, each nucleotide in the sense strand and antisense strand of the dsRNA agent can be modified, including nucleotides that are part of the motifs. Each nucleotide may be modified with the same or different modifications, which may include one or more changes to one or two non-binding phosphate oxygens and/or one or more binding phosphate oxygens; changing the constituent of the ribose sugar, for example, the 2'-hydroxyl in the ribose sugar; mass replacement of the phosphate part with dephospho-linkers; modification or replacement of the natural base; and replacement or modification of the ribose phosphate backbone.

Since nucleic acids are polymers made up of units, a large number of modifications occur at the position that is repeated in the nucleic acid, such as modification of the base, or the phosphate moiety, or the non-binding O phosphate moiety. In some cases, the modification will occur at all dependent positions in the nucleic acid, but in many cases it will not. As an example, the modification may only take place at the 3' or 5' end position, may only occur at the terminal site, for example, at a position on the terminal nucleotide, or at the last 2, 3, 4, 5, or 10 nucleotides of the chain. The modification may take place in the double strand section, the single strand section, or both sections. The modification may only take place in a double-stranded RNA region, or it may only take place in a single-stranded RNA region. For example, a phosphorothioate modification at the non-binding O position may occur only at one or both ends, may occur only at the terminal site, for example, at a position in the terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the chain , or may occur in the double and single strand sections, in particular at the ends. The 5' end may be phosphorylated.

It is possible, for example, to enhance stability by introducing certain bases into the overhangs or incorporating modified nucleotides or nucleotide substitutes into single-stranded overhangs, for example into the 5' or 3' overhang or both. For example, it may be desirable to introduce purine nucleotides into overhangs. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, by the modification described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar, which are known in the art, such as the use of 2'-deoxy-2'-fluoro(2'-P)- or 2'-O-methyl-modified deoxyribonucleotides instead of the nucleotide ribose sugar bases, and modifications to the phosphate group, such as phosphothioate modifications. The projections may be homologous to 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'-O-allyl, 2'-C-allyl, 2'-deoxy or 2'-fluorine. Chains 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.

Typically, at least two different modifications are present in the sense strand and the antisense strand. These two modifications may be 2'-O-methyl or 2'-fluoro modifications or other modifications.

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

In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2'-O-methylnucleotide, 2'-deoxyfluoronucleotide, 2'-O-N-methylacetamido(2'O-NMA)nucleotide, 2'-O-dimethylaminoethoxyethyl(2' -O-DMAEOE) nucleotide, 2'-O-aminopropyl (2'O-AP) nucleotide or 2'-ara-F-nucleotide.

In one embodiment, Na and/or N _b contains alternating modifications. The terms alternating motif or alternating pattern, as used herein, refers to a motif having one or more modifications, where each modification is in alternating nucleotides of the same strand. An alternating nucleotide can be referred to as every second or one of every three nucleotides, or a similar structure. For example, if A, B, and C each represent one type of modification for a nucleotide, the alternating motif could be ABABAABABA..., AABBAABBAABB..., AABAAABAAABA..., AAAAAAAAAAAA..., AAABBBAAAABB... or ABCABCABCABC. .. and so on.

In one embodiment, Na' and/or N _b ' contains alternating modifications. The terms alternating motif or alternating pattern, as used herein, refers to a motif having one or more modifications, where each modification is in alternating nucleotides of the same strand. An alternating nucleotide can be referred to as every second or one of every three nucleotides, or a similar structure. For example, if A, B, and C each represent one type of modification for a nucleotide, the alternating motif could be ABABAABABA..., AABBAABBAABB..., AABAAABAAABA..., AAAAAAAAAAAA..., AAABBBAAAABB... or ABCABCABCABC. .. and so on.

The type of modifications contained in an alternating motif may be the same or different. For example, if A, B, C, and D are each one type of nucleotide modification, the alternating structure, i.e. the modifications at every other nucleotide may be the same, but each strand of the sense strand or antisense strand may be selected from several possible modifications within the alternating motif, e.g. ABABAB..., ACACAC..., BDBDBD... or CDCDCD... etc.

In one embodiment, the dsRNA agent of the invention comprises a pattern of alternating motif modifications in the sense strand that is offset from the pattern of alternating motif modifications in the antisense strand. The offset may be such that the modified nucleotide group of the sense strand corresponds to the otherwise modified nucleotide group of the antisense strand, and vice versa. For example, if a sense strand is paired with an antisense strand in a dsRNA duplex, an alternating motif in the sense strand may start with ABABAB from the 5'-3' strand and an alternating motif in the antisense strand may start with BABABA from the 3'-5' strand within the duplex. site. As another example, an alternating motif in the sense strand may start with AABBAABB from the 5'-3' strand, and an alternating motif in the antisense strand may begin with BBAABBAA from the 3'-5' strand within the duplex region, so that there is complete or partial displacement of the modification structures between the sense strand and the antisense strand.

- 6 042669

In one embodiment, the dsRNA agent of the invention comprises a 2'-O-methyl modification and 2'III modification alternating motif structure in the sense strand, initially having an offset relative to the 2'-O-methyl modification and 2'III modification alternating motif structure in the antisense strand. chains, i.e. The 2'-O-methyl-modified nucleotide in the sense strand forms a base pair with the 2'-III-modified nucleotide in the antisense strand and vice versa. Position 1 of the sense strand may begin with the 2'-F modification, and position 1 of the antisense strand may begin with the 2'-O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides in the sense strand and/or antisense strand interrupts the initial structure of the modification present in the sense strand and/or antisense strand. Such disruption of the design of the modification of the sense strand and/or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides in the sense strand and/or antisense strand surprisingly enhances gene silencing activity against the target gene.

In one embodiment, if a motif of three identical modifications on three consecutive nucleotides is introduced into any of the strands, the modification of the nucleotide following the motif is a different modification than the motif modification. For example, the part of the sequence containing the motif is ...N _a YYYN _b ..., where Y is the motif modification of three identical modifications on three consecutive nucleotides and N _a and N _b are the modification for the nucleotide following the motif YYY, which is a different modification than Y and where N _a and N _b may be the same or different modifications. On the other hand, N _a and/or N _b may or may not be present when a flank modification is present.

The dsRNA agent of the invention may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. The modification of the phosphorothioate or methylphosphonate internucleotide linkage can be at any nucleotide of the sense strand or the antisense strand, or both strands, at any position in the strand. For example, an internucleotide bond modification can be at any nucleotide of the sense strand and/or antisense strand; each internucleotide bond modification may occur in an alternating pattern in the sense strand or antisense strand; or the sense strand or the antisense strand contains both modifications of the internucleotide bond in an alternating pattern. The internucleotide bond modification alternating pattern in the sense strand may be the same as or different from the antisense strand, and the internucleotide bond modification alternating pattern in the sense strand may be offset from the internucleotide bond modification alternating pattern in the antisense strand.

In one embodiment, the dsRNA contains a modification of the phosphorothioate or methylphosphonate internucleotide bond in the overhang. For example, the overhang contains two nucleotides having a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. Internucleotide bond modifications can be made to join overhang nucleotides to terminal paired nucleotides in a duplex region. For example, at least 2, 3, 4, or all of the overhang nucleotides may be linked via a phosphorothioate or methylphosphonate internucleotide bond, and optionally, additional phosphorothioate or methylphosphonate internucleotide bonds may exist connecting the overhang nucleotide to the paired nucleotide that follows the overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, of which two of the three nucleotides are overhang nucleotides and the third is a paired nucleotide following the overhang nucleotide. Preferably, such terminal three nucleotides may be at the 3' end of the antisense strand.

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

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

- 7 042669

In one embodiment, the dsRNA antisense strand contains two blocks of three phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide bonds, where one of the phosphorothioate or methylphosphonate internucleotide bonds is located at any position in the oligonucleotide sequence and the specified antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate and phosphate internucleotide bonds, or an antisense strand containing any phosphorothioate or methylphosphonate or phosphate bond.

In one embodiment, the antisense dsRNA strand contains two blocks of four phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide bonds , where one of the phosphorothioate or methylphosphonate internucleotide bonds is located at any position in the oligonucleotide sequence and the specified antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate and phosphate internucleotide bonds, or an antisense strand containing any phosphorothioate or methylphosphonate or phosphate bond.

In one embodiment, the dsRNA antisense strand contains two blocks of five phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide bonds, with one of phosphorothioate or methylphosphonate internucleotide bonds located at any position in the oligonucleotide sequence and said antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate and phosphate internucleotide bonds, or an antisense strand containing any phosphorothioate or methylphosphonate or phosphate bond.

In one embodiment, the dsRNA antisense strand contains two blocks of six phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide bonds, with one of the phosphorothioate or methylphosphonate internucleotide bonds internucleotide bonds is located at any position in the oligonucleotide sequence and the specified antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate and phosphate internucleotide bonds, or an antisense strand containing any phosphorothioate or methylphosphonate or phosphate bond.

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

In one embodiment, the dsRNA antisense strand contains two blocks of eight phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide bonds, where one of the phosphorothioate or methylphosphonate internucleotide bonds is located at any position in the oligonucleotide sequence and said antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate and phosphate internucleotide bonds, or an antisense strand containing any phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment, the dsRNA antisense strand contains two blocks of nine phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3 or 4 phosphate internucleotide bonds, where one of the phosphorothioate or methylphosphonate internucleotide bonds is located at any position in the oligonucleotide sequence and the specified antisense strand paired with a sense strand containing any combination of phosphorothioate, methylphosphonate and phosphate internucleotide bonds, or an antisense strand containing any phosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment, the dsRNA of the invention also contains one or more modifications of the phosphorothioate or methylphosphonate internucleotide bond within the 1-10 terminal positions of the sense and/or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be connected via a phosphorothioate or methylphosphonate internucleotide bond at one or both ends of the sense and/or antisense strand.

In one embodiment, the dsRNA of the invention also contains one or more phosphorothioate or methylphosphonate internucleotide bond modifications within 1-10 of the internal duplex region of each of the sense and/or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be connected via a phosphorothioate or methylphosphonate internucleotide bond at position 8-16 of the duplex region, measured from the 5' end of the sense strand; The dsRNA may also optionally contain one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1-10 terminal positions.

In one embodiment, the dsRNA of the invention also contains one to five modifications of the phosphorothioate or methylphosphonate internucleotide bond at position 1-5, one to five modifications of the phosphorothioate or methylphosphonate internucleotide bond at position 18-23 of the sense strand (counting from 5' -end) and one to five modifications of the phosphorothioate or methylphosphonate internucleotide bond at positions 1 and 2 and one to five at positions 18-23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains one modification of the phosphorothioate internucleotide bond at position 1-5, one modification of the phosphorothioate or methylphosphonate internucleotide bond at position 18-23 of the sense strand (counting from the 5' end), and one modification of the phosphorothioate internucleotide bond at position 1-5 positions 1 and 2 and two modifications of the phosphorothioate or methylphosphonate internucleotide bond at positions 18-23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains two modifications of the phosphorothioate internucleotide bond at positions 1-5, one modification of the phosphorothioate internucleotide bond at positions 18-23 of the sense strand (counting from the 5' end), one modification of the phosphorothioate internucleotide bond at positions 1 and 2 and 2 modifications of the phosphorothioate internucleotide bond at positions 18-23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains two modifications of the phosphorothioate internucleotide bond at positions 1-5, two modifications of the phosphorothioate internucleotide bond at positions 18-23 of the sense strand (counting from the 5' end), one modification of the phosphorothioate internucleotide bond at positions 1 and 2 and 2 modifications of the phosphorothioate internucleotide bond at positions 18-23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains two modifications of the phosphorothioate internucleotide bond at positions 1-5, two modifications of the phosphorothioate internucleotide bond at positions 18-23 of the sense strand (counting from the 5' end), one modification of the phosphorothioate internucleotide bond at positions 1 and 2 and one modification of the phosphorothioate internucleotide bond at positions 18-23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains one modification of the phosphorothioate internucleotide bond at positions 1-5, one modification of the phosphorothioate internucleotide bond at positions 18-23 of the sense strand (counting from the 5' end), two modifications of the phosphorothioate internucleotide bond at positions 1 and 2 and 2 modifications of the phosphorothioate internucleotide bond at positions 18-23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains one modification of the phosphorothioate internucleotide bond at positions 1-5, one modification at positions 18-23 of the sense strand (counting from the 5' end), two modifications of the phosphorothioate internucleotide bond at positions 1 and 2, and one modification of the phosphorothioate internucleotide bond at positions 18-23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains one modification of the phosphorothioate internucleotide bond at positions 1-5 (counting from the 5' end), two modifications of the phosphorothioate internucleotide bond at positions 1 and 2, and one modification of the phosphorothioate internucleotide bond at positions 18-23 antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains two modifications of the phosphorothioate internucleotide bond at positions 1-5 (counting from the 5' end), one modification of the phosphorothioate internucleotide bond at positions 1 and 2, and two modifications of the phosphorothioate internucleotide bond at positions 18-23 antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains two phosphorothioate internucleotide bond modifications at positions 1-5 and one at positions 18-23 of the sense strand (counting from the 5' end), two phosphorothioate internucleotide bond modifications at positions 1 and 2, and one modification of the phosphorothioate internucleotide bond at positions 18-23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains two modifications of the phosphorothioate internucleotide bond at positions 1-5, one modification of the phosphorothioate internucleotide bond at positions 18-23 of the sense strand (counting from the 5' end), two modifications of the phosphorothioate internucleotide bond at positions 1 and 2 and 2 modifications of the phosphorothioate internucleotide bond at positions 18-23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains two modifications of the phosphorothioate internucleotide bond at positions 1-5, one modification of the phosphorothioate internucleotide bond at positions 18-23 of the sense strand (counting from the 5' end), one modification of the phosphorothioate internucleotide bond at positions 1 and 2 and 2 modifications of the phosphorothioate internucleotide bond at positions 18-23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains two modifications of the phosphorothioate internucleotide bond at positions 1 and 2, two modifications of the phosphorothioate internucleotide bond at positions 20 and 21 of the sense strand (counting from the 5' end), one modification

- 9 042669 phosphorothioate internucleotide bond at position 1 and one at position 21 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains one modification of the phosphorothioate internucleotide bond at position 1, one modification of the phosphorothioate internucleotide bond at position 21 of the sense strand (counting from the 5' end), two modifications of the phosphorothioate internucleotide bond at positions 1 and 2, and two modification of the phosphorothioate internucleotide bond at positions 20 and 21 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains two modifications of the phosphorothioate internucleotide bond at positions 1 and 2, two modifications of the phosphorothioate internucleotide bond at positions 21 and 22 of the sense strand (counting from the 5' end), one modification of the phosphorothioate internucleotide bond at position 1 and one modification of the phosphorothioate internucleotide bond at position 21 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains one modification of the phosphorothioate internucleotide bond at position 1, one modification of the phosphorothioate internucleotide bond at position 21 of the sense strand (counting from the 5' end), two modifications of the phosphorothioate internucleotide bond at positions 1 and 2, and two modification of the phosphorothioate internucleotide bond at positions 21 and 22 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains two modifications of the phosphorothioate internucleotide bond at positions 1 and 2, two modifications of the phosphorothioate internucleotide bond at positions 22 and 23 of the sense strand (counting from the 5' end), one modification of the phosphorothioate internucleotide bond at position 1 and one modification of the phosphorothioate internucleotide bond at position 21 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA of the invention also contains one modification of the phosphorothioate internucleotide bond at position 1, one modification of the phosphorothioate internucleotide bond at position 21 of the sense strand (counting from the 5' end), two modifications of the phosphorothioate internucleotide bond at positions 1 and 2, and two modification of the phosphorothioate internucleotide bond at positions 23 and 23 of the antisense strand (counting from the 5' end).

In one embodiment, the dsRNA agent of the invention comprises mismatch(es) with a target within a duplex, or a combination thereof. Mismatching may take place in the raised portion of the duplex portion. A base pair can be classified based on their propensity to promote dissociation or melting (e.g. due to the free energy of association or dissociation of a particular pairing, the simplest approach is to test pairs based on a single base pair, although nearest neighbor or similar analysis can also be used) . In terms of promoting dissociation, A:U dominates G:C, G:U dominates G:C, and T:C dominates G:C (1=inosine). Mismatches, such as non-canonical or other than canonical pairings, are preferred over canonical (A:T, A:U, G:C) pairings; and pairings that 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 in the duplex regions from the 5' end of the antisense strand, which can be independently selected from the group A:U, G :U, I:C, and mismatches, such as non-canonical or other than canonical, pairings, or pairings that contain a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

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

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

5' np-N _a -(XXX)i-Nb-YYY-Nb-(ZZZ)jN _a -nq 3' (I), where i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N _a is independently an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two differently modified nucleotides;

each Nb is independently an oligonucleotide sequence containing 0-10 modified nucleotides;

each np and nq is independently an overhang nucleotide;

while Nb and 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.

- 10 042669

Preferably YYY is all 2'-F modified nucleotides.

In one embodiment, N _a and/or Nb contain alternating structure modifications.

In one embodiment, the YYY motif is located at or near the sense strand cleavage site. For example, when a dsRNA agent has a duplex region of 17-23 bp, the YYY motif may occur at or near the cleavage site (e.g., may 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, with counting starting from the 1st nucleotide from the 5' end, or optionally counting starting from the 1st paired nucleotide in the duplex region from 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 equal to 1. Therefore, the semantic chain can be represented by the following formulas

5' np-N _a -YYY-Nb-ZZZ-N _a -n _q 3' (Ia),

5' np-Na-XXX-Nb-YYY-Na-nq 3' (Ib), or

5'n _p -N _a -XXX-N _b -YYY-N _b -ZZZ-N _a -n _q 3' (Ic)

When the sense strand is represented by formula (Ia), N _b is an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N _a may be independently an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the sense strand is represented by formula (Ib), N _b is an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na may be independently an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the sense strand is represented by formula (Ic), each Nb is independently an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably N _b is 0, 1, 2, 3, 4, 5 or 6 modified nucleotides. Each N _a may be independently an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

Each of X, Y, and Z can be the same as or different from the other.

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

5' nq'-Na'-(Z'Z'Z')k-Nb'-Y'Y'Y'-Nb'-(X'X'X')l-N'a-np' 3' ( II), wherein k and l are each independently 0 or 1;

p and q are each independently 0-6;

each Na' is independently an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two differently modified nucleotides;

each N _b ' is independently an oligonucleotide sequence containing 0-10 modified nucleotides;

each n _p ' and n _q ' is independently a nucleotide overhang containing 0-6 nucleotides;

where N _b ' and Y' do not have the same modification; and each X'X'X', YYY' and Z'Z'Z' independently represents one motif of their three identical modifications on three consecutive nucleotides.

In one embodiment, N _a ' and/or N _b ' contains alternating structure modifications.

The YYY' motif occurs at or near the antisense strand cleavage site. For example, when the dsRNA agent has a 17-23 nc duplex region, the YYY' motif may occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14 or 13, 14, 15 of the antisense strand, counting starting from the 1st nucleotide from the 5' end; or optionally counting starts from the 1st paired nucleotide in the duplex region from the 5' end. Preferably the Y'Y'Y' motif occurs at positions 11, 12, 13.

In one embodiment, the YYY' motif is all 2'-OM modified nucleotides.

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

Therefore, the antisense strand can be represented by the following formulas

5'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-N'a-np' 3' (IIa),

5' n _q '-N _a '-YYY'-N _b '-X'X'X'-n _p '3' (IIb), or

5' n _q '-N _a '-Z'Z'Z'-N _b '-YYY'-N _b '-X'X'X'-N' _a -n _p '3' (IIc)

When the antisense strand is represented by formula (IIa), Nb' is an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N _a ' is independently an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented by formula (IIb), N _b ' is an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N _a ' is independently an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the antisense strand is represented by formula (IIc), each N _b ' is independently an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N _a ' is independently an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides. Preferably N _b ' represents 0, 1, 2, 3, 4, 5 or 6 modified nucleotides.

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

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

In one embodiment, the sense strand of the dsRNA agent contains the YYY motif occurring at positions 9, 10, and 11 of the sense strand when the duplex region is 21 nc, with counting starting from 1 nucleotide from the 5' end, or optionally counting starting at 1st paired nucleotide in the duplex region from the 5'-end; and Y is the 2'L modification. The sense strand may additionally contain the XXX motif or ZZZ motifs as flank modifications at the opposite end of the duplex region; and each of XXX and ZZZ is independently a 2'-OMe modification or a 2'-F modification.

In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, and 13 of the strand, counting from the 1st nucleotide from the 5' end, or optionally counting from the 1st paired nucleotide in the duplex region from the 5'-end; and Y' is the 2'-O-methyl modification. The antisense strand may further contain the X'X'X' motif or Z'Z'Z' motifs as flanking modifications at the opposite end of the duplex region; and each of X'X'X' and Z'Z'Z' is independently a 2'-OMe modification or a 2'-F modification.

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

Accordingly, the dsRNA agent may comprise a sense strand and an antisense strand, with each strand having 14-30 nucleotides, i.e. be a dsRNA duplex represented by the formula sense: 5' np-N _a -(XXX) _i -N _b -YYY-N _b -(ZZZ) _j -N _a -n _q 3' antisense: 3'np'-N _a '-(X'X'X') _k -N _b '-Y'Y'Y'-N _b '-(Z'Z'Z') _i -N _a '-n _q '5' (III) where i, j, k, and l are each independently 0 or 1;

p and q are each independently 0-6;

each N _a and N _a ' is independently an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each Nb and Nb' is independently an oligonucleotide sequence containing 0-10 modified nucleotides;

where each np', np, nq' and nq is independently an overhanging nucleotide sequence; and each XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' 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, with each strand having 14-30 nucleotides, i. is a dsRNA duplex represented by formula (V) sense: 5' N _a -(XXX) _i -N _b -YYY-N _b -(ZZZ) _j -N _a -n _q 3' antisense: 3'np'- N _a '-(X'X'X') _k -N _b '-Y'Y'Y'-N _b '-(Z'Z'Z') _l -N _a '5' (V), where i , j , k and l are each independently 0 or 1;

p and q are each independently 2;

each N _a and N _a ' is independently an oligonucleotide sequence containing 0-25 modified nucleotides, with each sequence containing at least

- 12 042669 at least two differently modified nucleotides;

each N _b and Nb' independently represents an oligonucleotide sequence containing 0-10 modified nucleotides;

where each np' and n _q independently represent a protruding nucleotide sequence; And

XXX, YYY, ZZZ, X'X'X', YYY' 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 1 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, with each strand having 14-30 nucleotides, i. is a dsRNA duplex represented by the formula (Va) sense: 5' N _a -(XXX) ₁ -N _b -YYY-N _b -(ZZZ) _j -N _a 3' antisense: 3'np'-N _a '-(X'X'X') _k -N _b '-YYY'-N _b '-(Z'Z'Z') _r N _a '5' (Va), where i, j, k and l are each independently is 0 or 1;

p and q are each independently 2;

each Na and Na' is independently an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two differently modified nucleotides;

each Nb and Nb' is independently an oligonucleotide sequence containing 0-10 modified nucleotides;

where np' represents the protruding nucleotide sequence; and each XXX, ΎΎΎ, ZZZ, X'X'X', Ύ'Ύ'Ύ' and Z'Z'Z' independently represent one motif of three identical modifications on three consecutive nucleotides.

Examples of combinations of sense strand and antisense strand that form a dsRNA duplex include the formulas below.

5'np-NaYYY-Nb-ZZZ-Na-nq 3'

3'np'-N _a '-YYY'-N _b '-Z'Z'Z'-N _a '-n _q '5' (IIIa),

5'np-Na-XXX-Nb-YYY-Na-nq 3'

3' n _p '-N _a '-X'X'X'-N _b '-YYY'-N _a '-n _q '5' (IIIb),

5' np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq 3' 3' np'-Na'-X'X'X'-Nb'-YYY'-Nb'-Z'Z'Z '-Na-nq' 5' (IIIc)

When the dsRNA agent is represented by formula (IIIa), each N _b and N _b ' is independently an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. Each Na and Na' is independently an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the dsRNA agent is represented by formula (IIIb), each N _b and Nb' is independently an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na and Na' is independently an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the dsRNA agent is represented by formula (IIIc), each N _b and N _b ' is independently an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na and Na' is independently an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides. Each of Na, Na', N _b and N _b ' independently contains alternating structure modifications.

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

When the dsRNA agent is represented by formula (III), (IIIa), (IIIb) or (IIIc), at least one of the Y nucleotides may base pair with one Y' nucleotide. On the other hand, at least two of the Y nucleotides form base pairs with the corresponding Y' nucleotides, or all three Y nucleotides form base pairs with the corresponding Y' nucleotides.

It is clear that when Na nucleotides base pair with Na', N _b nucleotides base pair with N _b ', X nucleotides base pair with X', Y nucleotides base pair with Y', and Z nucleotides base pair with Z' .

When the dsRNA agent is represented by formula (IIIa) or (IIIc), at least one of the Z nucleotides may base pair with one Z' nucleotide. On the other hand, at least two of the Z nucleotides form base pairs with the corresponding Z' nucleotides, or all three Z nucleotides form base pairs with the corresponding Z' nucleotides.

When the dsRNA agent is represented by formula (IIIb) or (IIIc), at least one of the X nucleotides may base pair with one X' nucleotide. On the other hand, at least two of the X nucleotides form base pairs with the corresponding X' nucleotides; or all three X nucleotides form base pairs with the corresponding X' nucleotides.

In one embodiment, the modification at nucleotide Y is other than the modification at nucleotide Y', the modification at nucleotide Z is other than the modification at nucleotide Z', and/or the modification at nucleotide X is other than the modification at nucleotide X'.

In one embodiment, the dsRNA agent is a polymer containing at least two duplexes represented by formulas (III), (IIIa), (IIIb) or (IIIc), said duplexes being connected by a linker. The linker may or may not be cleavable. Optionally, said polymer also contains a ligand. Each of the dsRNAs may target the same gene or two different genes, or each of the dsRNAs may target the same gene at two different target sites.

In one embodiment, the dsRNA agent is a polymer containing three, four, five, six or more duplexes represented by formulas (III), (IIIa), (IIIb) or (IIIc), said duplexes being connected by a linker. The linker may or may not be cleavable. Optionally, said polymer also contains a ligand. Each of the dsRNAs may target the same gene or two different genes, or each of the dsRNAs may target the same gene at two different target sites.

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

Many publications describe polymeric siRNA, and all can be used in relation to dsRNA according to the invention. Such publications include WO 2007/091269, US Patent No. 7858769, WO 2010/141511, WO 2007/117686, WO 2009/014887 and WO 2011/031520, which are incorporated herein by reference.

A dsRNA agent that contains conjugations of one or more carbohydrate moieties to the dsRNA agent can optimize one or more properties of the dsRNA agent. In many cases, the carbohydrate moiety will attach to the modified unit of the dsRNA agent. For example, the ribose sugar of one or more ribonucleotide units of the dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier, to which a carbohydrate ligand is attached. A ribonucleotide unit in which the ribose sugar of the unit is replaced in this way is referred to herein as a ribose replacement modified unit (RRMS). The cyclic carrier may be a carbocyclic system, i. e. all ring atoms are carbon atoms, or a heterocyclic system, i.e. one or more ring atoms may be heteroatoms, eg nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic system or may contain two or more rings, such as 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 supports include (i) at least one main chain attachment, preferably two main chain attachments, and (ii) at least one intermediate attachment. As used herein, the term main chain attachment point refers to a functional group, such as a hydroxyl group, or generally available for bonding, and which is suitable for introducing a carrier into the main chain, such as a phosphate group or a modified phosphate group, such as a sulfur-containing group of the ribonucleic acid main chain. The term intermediate attachment point (TAP) in some embodiments refers to a ringing cyclic carrier atom, such as a carbon atom or a heteroatom (other than the atom that provides the main chain attachment point) that attaches the selected moiety. The portion may be, for example, a carbohydrate, such as a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide. Optionally, the selected part is connected to the cyclic carrier through an intermediary link. Thus, the cyclic carrier will typically include a functional group, such as an amino group, or, as a rule, provide a link that is suitable for inclusion or for an intermediate link for another chemical species, such as a ligand, with a constituent ring.

In an embodiment, the dsRNA of the invention is conjugated to a ligand via a carrier, wherein the carrier may be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalininl, pyridazinonyl, tetrahydro- 14 042669 and decalin furyl; preferably the acyclic group is selected from the serinol backbone or the diethanolamine backbone.

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

Ligands.

A wide range of particles can be combined with the oligonucleotides of the present invention. Preferred particles are ligands which are attached, preferably covalently, either directly or indirectly via an intermediate linkage.

In preferred embodiments, the implementation of the ligand changes the distribution, targeting or lifetime of the molecule in which it is included. In preferred embodiments, the ligand provides enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, receptor, e.g. . Ligands that provide enhanced affinity for a selected target are also referred to as targeting ligands.

Some ligands may have endosomolytic properties. Endosomolytic ligands promote lysis of endosomes and/or transfer of the composition of the invention or components thereof from the endosome into the cytoplasm of the cell. The endosomolytic ligand can be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand exhibits its active conformation at endosomal pH. The active conformation is one in which the endosomolytic ligand promotes lysis of the endosome and/or transfer of the composition of the invention or its components from the endosome into the cytoplasm of the cell. Examples of 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 derivatives thereof. (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68). In one embodiment, the endosomolytic component may contain a chemical group (eg, an amino acid) that 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 transfer, hybridization, and specificity properties, and can also improve the nuclease resistance of the resulting natural or modified oligonucleotide or polymer molecule containing any combination of the monomers described herein and/or natural or modified ribonucleotides.

Typically, the ligands may contain therapeutic modifiers, for example to enhance absorption; diagnostic compounds or reporter groups, for example, to control distribution; cross-linking agents and portions conferring nuclease resistance. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

Ligands may include a natural substance such as a protein (eg, human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL) or globulin); a carbohydrate (eg dextrin, 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, eg a synthetic polyamino acid, an oligonucleotide (eg an aptamer). Examples of polyamino acids include polylysine (PLL), poly-L-aspartamic acid, poly-E-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide, glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) copolymer )methacrylamide (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendritic polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, polyamine quaternary salt, or alpha-helical peptide.

Ligands may also include targeting groups, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type, such as a kidney cell. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surface active protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate , polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptide mimetic, or aptamer. Tab. 1 shows some examples of targeting ligands and their associated receptors.

Other examples of ligands include dyes, intercalators (eg acridines),

- 15 042669 crosslinkers (eg psoralen, mitomycin C), porphyrins (TPPC4, texafirin, sapphirin), polycyclic aromatic hydrocarbons (eg phenazine, dihydrophenazine), artificial endonucleases or chelators (eg EDTA), lipophilic molecules, eg cholesterol , cholic acid, adamantanacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bisO(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. antennapediapeptide, Tat-peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g. PEG-40K), MPEG, [MPEG]2, polyamines, alkyls, substituted alkyls, isotopically labeled markers, enzymes, haptens (eg biotin), transport/absorption agents (eg aspirin, vitamin E, folic acid), synthetic ribonucleases (eg imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HPR or AP.

Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with a specific affinity for a coligand, or antibodies, such as an antibody that binds to a specific cell type, such as a cancer cell, endothelial cell, or bone cell. Ligands can also include hormones and hormone receptors. They may also include non-peptidic substances such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, or aptamers. The ligand may be, for example, a lipopolysaccharide, a p38 MAP kinase activator, or an NF-kB activator.

The ligand may be a substance, eg a drug, which can enhance the uptake of the mRNA agent into a cell, eg by disrupting the cell's cytoskeleton, eg by disrupting the cell's microtubules, microfilaments and/or intermediate strands. The drug may be, for example, a taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, larunculin A, phallodin, swincholide A, indanocin or myoservin.

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

In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, such as human serum albumin (HSA). The HSA binding ligand allows the conjugate to be delivered to a target tissue, such as a non-renal target tissue of the body. For example, the target tissue may be liver tissue, including parenchymal liver cells. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase resistance to degradation of conjugates;

(b) increase targeting or transport to a target cell or cell membrane; and/or (c) be used to regulate serum protein binding, eg HSA.

A lipid-based ligand can be used to modulate, for example, regulate, the binding of a conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidneys and therefore less likely to be excreted from the body. A lipid or lipid-based ligand that binds to HSA with less force can be used to target the conjugate to the kidneys.

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

In another preferred embodiment, the lipid-based ligand binds little or no HSA, such that the conjugate will preferably be delivered to the kidneys. Other substances that target kidney cells can also be used instead of or in addition to the lipid based ligand.

In another aspect, the ligand is a particle, such as a vitamin, that is taken up by a target cell, such as a proliferating cell. This is particularly applicable to the treatment of disorders characterized by undesired cell proliferation, eg of a malignant or non-cancerous type, such as cancer cells. Examples of vitamins include vitamin A, E, and K. Other examples of vitamins include B vitamins such as 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-penetrating agent, preferably a helical cell-penetrating agent. Preferably the agent is amphipathic. An example of an agent is the tat peptide or antennopedia. If the agent is a peptide, it may be modified, including a peptidomimetic, invertomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. The helical agent is preferably an alpha helical agent which preferably has a lipophilic and lipophobic phase.

The ligand may be a peptide or a peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a specific three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic portion may be about 5-50 amino acids long, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long. The peptide or peptidomimetic may be, for example, a cell-penetrating peptide, a cationic peptide, an amphiphatic peptide, or a hydrophobic peptide (eg, consisting mainly of Tyr, Trp or Phe). The peptide portion may be a dendritic peptide, a folded peptide, or a cross-linked peptide. Alternatively, the peptide portion may include a hydrophobic membrane translocation sequence (MTS). An example of a hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP. An RFGF analog (eg, the amino acid sequence AALLPVLLAAP) containing a hydrophobic MTS may also be a targeting moiety. The peptide may be a delivery peptide that can carry large polar molecules, including peptides, olinonucleotides, and protein, across cell membranes. For example, sequences from the HIV-Tat protein (GRKKRRQRRRPPQ) and Drosophila antennapedia protein (RQIKIWFQNRRMKWKK) were found to be able to function as delivery peptides. The peptide or peptidomimetic can be encoded by a random DNA sequence such that the peptide is identified from a phage display library, or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably, the peptide or peptidomimetic linked to the mRNA agent via an inserted monomeric unit is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD) peptide or an RGD mimetic. The length of the peptide portion can vary from about 5 amino acids to about 40 amino acids. The peptide portion may have a structural modification in order to increase stability or enhance the immediate conformational properties. You can use any structural modifications described below.

The RGD peptide moiety can be used to target a tumor cell such as an endothelial tumor cell or a breast cancer cell (Zizmann et al., Cancer Res., 62:513943, 2002). The RGD peptide can facilitate targeting of mRNA agents to tumors of various other tissues, including lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy, 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of mRNA agents to the kidneys. The RGD peptide may be linear or cyclic and may be modified, such as glycosylated or methylated, to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an mRNA agent to a tumor cell expressing α _¥ β ₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). You can use peptides that target markers enriched in proliferating cells. For example, RGD-containing peptides and peptidomimetics can target cancer cells, in particular cells that expose integrin. Thus, RGD peptides, RGD-containing cyclic peptides, RGD peptides that include D-amino acids, as well as synthetic RGD mimetics can be used. In addition to RGD, other moieties that target the integrin ligand can be used. Typically, such ligands can be used to regulate cell proliferation and angiogenesis. Ligand conjugates of the type that target PECAM-1, VEGF, or another cancer gene, such as the cancer gene described herein, are preferred.

The cell penetrating peptide is capable of penetrating a cell, for example a microbial cell such as a bacterial cell or a fungal cell, or a mammalian cell such as a human cell. The microbial cell penetrating peptide can be, for example, an α-helical linear peptide (eg LL-37 or ceropin P1), a peptide containing a disulfide bond (eg α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominant amino acids (for example, PR-39 or indolicidin).

A cell-penetrating peptide may also include a nucleolar localization signal (NLS). For example, the cell-penetrating peptide can be a two-part amphipathic peptide, such as MPG, which is formed from the HIV-1 gp41 fusion peptide domain and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

In one embodiment, the targeting peptide may be an amphipathic α-helical peptide. Examples of amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, myxin intestinal antimicrobial peptides (HFIAP), magainins, brevinins- 2, dermaseptins, melittins, pleurocidin, H ₂ A peptides, Xenopus peptides, esculentin-1 and caerins. Preferably, a number of factors will be considered to maintain the integrity and stability of the helix. For example, the maximum number of helix-stabilizing residues (eg, leu, ala, or lys) and the minimum number of helix-destabilizing residues (eg, proline or cyclic monomer units) will be used. A capping residue will be considered (eg, Gly is an example of an N-capping residue), and/or C-terminal amidation may be used to provide an additional H-bond to stabilize the helix. The 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, homoarginine, ornithine, or histidine can form salt bridges with anionic glutamate or aspartate residues.

Peptide ligands and peptidomimetics include naturally occurring or modified peptides such as D- or L-peptides; α, β or γ-peptides; N-methylpeptides; azapeptides; peptides with one or more amide, i.e. peptide bonds replaced by one or more urea, thiourea, carbamate or sulfonylurea bonds; or cyclic peptides.

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

Endosome release agents include imidazoles, poly- or oligoimidazoles, PEI, peptides, fusogenic peptides, polycarboxylates, polyacations, masked oligo- or polycations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, polymers with masked or unmasked cationic or anionic charges , dendrites with masked or unmasked cationic or anionic charges.

PK modulator refers to a pharmacokinetic modulator. The PK modulator includes lipophilic compounds, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, vitamins, and the like. Examples of PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. It is also known that oligonucleotides that include multiple phosphorothioate linkages bind to whey protein, thus short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, containing multiple phosphorothioate linkages in the backbone are also in accordance with the present invention in as ligands (eg, as PK modulating ligands).

In addition, aptamers that bind serum components (eg, serum proteins) are also suitable as PK modulating ligands of the present invention.

Other ligand conjugates corresponding to the invention are described in US patent applications USSN: 10/916185, registered August 10, 2004; USSN: 10/946873, registered September 21, 2004; USSN: 10/833934, registered August 3, 2007; USSN: 11/115989, registered April 27, 2005; and USSN: 11/944227, registered Nov. 21, 2007, which are incorporated herein by reference.

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

Ligands can be linked to oligonucleotides at various points, for example at the 3' end, 5' end and/or intermediate position. In preferred embodiments, the ligand is attached to the oligonucleotides via an intermediate linkage, such as the carrier described herein. The ligand or tethered ligand may be present in the monomer when said monomer is included in the growing chain. In some embodiments, a ligand may be incorporated via coupling with a precursor monomer after said precursor monomer has been incorporated into the growing chain. For example, a monomer having, for example, an amino terminal group (ie, no associated ligand), such as TAP-(CH2) _n NH2, can be included in the growing oligonucleotide chain. Then, during the subsequent operation, i.e. after incorporating the precursor monomer into the chain, a ligand having an electrophilic group such as a pentafluorophenyl ether or an aldehyde group can be attached to the precursor monomer by coupling the electrophilic group of the ligand to the terminal nucleophilic group of the linkage of the precursor monomer.

In another example, a monomer having a suitable group to participate in a click chemistry reaction,

- 18 042669 can be included, for example, azide - or alkyne-terminal ligament/linker. During the subsequent operation, i.e. after incorporating the precursor monomer into the chain, a ligand having a complementary chemical group, such as alkyne or azide, can be attached to the precursor monomer by a combination of an alkyne and an azide.

In the case of double stranded oligonucleotides, the ligands may be attached to one or both strands. In some embodiments, the double-stranded mRNA agent contains a ligand conjugated to the sense strand. In other embodiments, the double-stranded mRNA agent comprises a ligand conjugated to an antisense strand.

In some embodiments, the ligand may be conjugated to nucleotide bases, sugar moieties, or internucleoside bonds of nucleic acid molecules. Conjugation to purine nucleotide bases or derivatives thereof can take place at any position, including endocyclic and exocyclic atoms. In some embodiments, positions 2, 6, 7, or 8 of the purine nucleotide base are attached to the conjugate portion. Conjugation to pyrimidine nucleotide bases or derivatives thereof can also occur at any position. In some embodiments, positions 2, 5, and 6 of the pyrimidine nucleotide base may be replaced by a portion of the conjugate. Conjugation to the sugar moieties of nucleosides can occur at any carbon atom. Examples of the carbon atoms of the sugar portion that may be attached to the conjugate portion include 2', 3' and 5' carbon atoms. The 1' position can also be linked to a portion of the conjugate, such as a minor residue. Internucleoside bonds can also carry parts of the conjugate. In the case of phosphorus-containing bonds (eg, phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), part of the conjugate may be attached directly to the phosphorus atom or to the O, N or S atom associated with the phosphorus atom. In the case of amine- or amide-containing internucleoside bonds (eg, PNA), part of the conjugate may 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 can be used, although the ligand is usually a carbohydrate, eg a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, a polysaccharide.

Linkers that conjugate a ligand to a nucleic acid include the linkers discussed above. For example, the ligand may be one or more GalNAc (N-acetylglucosamine) derivatives attached via a divalent or trivalent branched linker.

In one embodiment, the dsRNA of the invention is conjugated to a divalent and trivalent linker comprising the structures shown in any of formulas (IV)-(VII)

while q ^2A , q ^2B , q ^3A , q ^3B , q ^4A , q ^4B , q ^5A , q ^5B and q ^5C are independently equal in the case of each occurrence of 0-20, while the repeating link may be the same or different ;

each P ^2A , R ^2B , R ^3A , P ^3B , R ^4A , R ^4B , R ^5A , R ^{5B , R 5C ,} T ^{2A , T 2B ,} T ^3A , T ^3B , T ^4A , T ^4B , T ^5A , T ^5B , T ^5C independently for each occurrence is absent or is CO, NH, O, S, OC(O), NHC(O), CH ₂ , CH2NH or CH ₂ O;

Q ^2A , Q ^2b , Q ^3A , Q ^3b , Q ^4A , Q ^4b , Q ^5A , Q ^5B , Q ^5C independently in each occurrence are absent or represent alkylene, substituted alkylene, while one or more methylenes may be interrupted or terminated one or more O, S, S(O), SO ₂ , N(R ⁿ ), C(R')=C(R), C=C or C(O);

Each R ^2A , R ^2b , R ^3A , R 3B , R ^4A , ^{R 4b ,} R ^5A , R ^5B , R ^5C independently in each occurrence is absent or represents NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R ^a )C(O), -C(O)-CH(R ^a )-NH-, CO, o ^H0 A >S-S _ /S-S o O ΟΌOK. Of CH=NO, ' ⁿ '' ¹ ' or heterocyclyl;

L ^2A , L ^2b , L ^3A , L ^3b , L ^4A , L ^4b , L ^5A , L ^5B and L ^5C are a ligand, i.e. each independently at each occurrence is a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide,

- 19 042669 tetrasaccharide, oligosaccharide or polysaccharide; And

R ^a is H or an amino acid side chain.

In particular, trivalent GalNAc conjugating derivatives such as those of formula (VII) are useful for use with RNAi agents for inhibiting target gene expression.

wherein L ^5A , L ^5B and L ^5C are a monosaccharide such as a GalNAc derivative.

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

NO / ^0N

NO ¹ >

VV-o f

Η O - - ° - N N O

AcHN ₀ Η H

n

NHAc, NHAc

- 20 042669

Definitions.

As used herein, the terms dsRNA, siRNA, and mRNA agent are used interchangeably for agents that can mediate the silencing of a target RNA, such as an mRNA, such as a gene transcript that encodes a protein. For convenience, such mRNA is also referred to herein as silencing mRNA. Such a gene is also referred to herein as a genotarget. Typically, the RNA that is silenced is an endogenous gene or a pathogenic gene. In addition, RNAs other than mRNAs, such as tRNAs, and viral RNAs can also be targets.

As used herein, the expression mediates RNAi refers to the ability to silence a target RNA sequence-specifically. Without wishing to be bound by any theory, it is believed that silencing uses an RNAi mechanism or process and a guide RNA, such as a 21-23 nt siRNA means.

As used herein, specifically hybridizable and complementary are terms that are used to indicate a sufficient degree of complementarity such that there is a stable and specific binding 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 where specific binding is desired, ie. under physiological conditions in the case of assays or therapeutic treatment, or in the case of in vitro assays, under the conditions under which the assays are performed. Non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, the dsRNA agent of the invention is sufficiently complementary to the target RNA, eg the target mRNA, such that the dsRNA agent mutes the production of the protein encoded by the target mRNA. In another embodiment, the dsRNA agent of the invention

- 21 042669 is exactly complementary to the target RNA, eg the target RNA and the dsRNA duplex agent are annealed, eg to form an all Watson-Crick base-pair hybrid in the exact complementarity region. Sufficiently complementary target RNA may include an intermediate region (eg, at least 10 nucleotides) that is exactly complementary to the target RNA. Moreover, in some embodiments, the dsRNA tool of the invention specifically discriminates a single nucleotide difference. In such a case, the dsRNA agent mediates RNAi only if exact complementarity is found in a region (eg, within 7 nucleotides) with a difference of one nucleotide.

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

The term halo refers to any fluorine, chlorine, bromine or iodine radical. The term alkyl refers to saturated and unsaturated non-aromatic hydrocarbon chains, which may be straight chains or branched chains containing the specified number of carbon atoms (which include, but is not limited to, propyl, allyl, or propargyl), into which N, O, or S may optionally be inserted. For example , Q-Cu indicates that the group can have from 1 to 10 (inclusive) carbon atoms. The term alkoxy refers to the -O-alkyl radical. The term alkylene refers to divalent alkyl (ie -R-). The term alkylenedioxo refers to divalent groups of structure -O-R-O-, in which R is alkylene. The term aminoalkyl refers to alkyl substituted with amino. The term mercapto refers to the -SH radical. The term thioalkoxy refers to the -S-alkyl radical.

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

The term cycloalkyl as used herein includes saturated and partially unsaturated cyclic hydrocarbon groups with 3-12 carbon atoms, such as 3-8 carbon atoms, and, for example, 3-6 carbon atoms, while the cycloalkyl group may optionally be additionally replaced. 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 system with 1-3 heteroatoms if the system is monocyclic, 1-6 heteroatoms if the system is bicyclic, or 1-9 heteroatoms , if the system is tricyclic, these heteroatoms are selected from O, N or S (for example, carbon atoms and 1-3, 1-6 or 1-9 heteroatoms O, N or S, if the system is monocyclic, bicyclic or tricyclic, respectively), while 0, 1, 2, 3 or 4 atoms of each ring may be substituted with 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 alkyl substituted with heteroaryl. The term heteroarylalkoxy refers to alkoxy substituted with heteroaryl.

The term heterocyclyl refers to a non-aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic system with 1-3 heteroatoms if the system is monocyclic, 1-6 heteroatoms if the system is bicyclic, or 1-9 heteroatoms , if the system is tricyclic, these heteroatoms are selected from O, N or S (for example, carbon atoms and 1-3, 1-6 or 1-9 heteroatoms O, N or S, if the system is monocyclic, bicyclic or tricyclic, respectively), while 0, 1, 2 or 3 atoms of each ring may be substituted with a substituent. Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term oxo refers to the oxygen atom that 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 with a substituent.

The term substituted refers to the replacement of one or more hydrogen radicals in a given structure by a radical of a specific substituent, including, but not limited to, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, bonhydroxy, alkoxycarboxyalkyl, carboxylamino, alkyl, alkoxy an aralkoxycarbonyl, a carboxyl group, a sulfonic acid residue, a sulfonyl, a phosphonic acid residue, an aryl, a heteroaryl, a heterocyclyl, or an aliphatic group. It should be borne in mind that replace

- 22 042669 body can also be substituted.

Split linking groups.

A cleavable linking group is a group that is sufficiently stable outside the cell, but which, upon introduction into the target cell, is cleaved to release two moieties held together by the linker. 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 in the first standard condition (which can be chosen to mimic or represent intracellular conditions, for example) than in the individual's blood, or in a second standard condition (which may be selected, for example, to mimic or represent conditions in blood or serum).

Cleavable linking groups are sensitive to cleavage agents, such as pH, redox potential, or the presence of destructive molecules. As a rule, cleavage agents within cells predominate or are found at higher levels or activities than in serum or blood. Examples of such disruptive agents include redox agents that are selected for particular substrates or that are not substrate specific, including, for example, oxidizing or reducing enzymes or reducing agents, such as mercaptans, present in cells that can degrade the redox-cleavable linking group. by recovery; esterases; endosomes or agents that can create an acidic environment, such as agents that give a pH of five or less; enzymes that can hydrolyze or degrade an acid-cleavable linking group by acting like normal acid, peptidases (which may be substrate-specific) and phosphatases.

A cleavable linking group, such as a disulfide bond, may be pH sensitive. The pH value of human serum is 7.4, while the average intracellular pH is slightly lower, fluctuating in the range of 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 of about 5.0. Some linkers will have a cleavable linking group that cleaves at a preferred pH, thereby releasing the cationic lipid from the ligand into the interior of the cell or into the desired compartment of the cell.

The linker may include a cleavable linking group that is cleavable by a specific enzyme. The type of cleavable linking group included in the linker may depend on the target cell. For example, liver-targeting ligands can be linked to cationic lipids via 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 rich in esterases. Other cell types rich in esterases include lung cells, renal cortex, and testes.

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

In general, the suitability of a cleavable linking group candidate can be assessed by testing the ability of a disruptive agent (or condition) to cleave the linking group candidate. It would also be desirable to test the cleavable linking group candidate also for its ability to resist cleavage in the blood or upon contact with other non-target tissue. In this way, the relative sensitivity to cleavage under the first and second condition can be determined, where the first is chosen to indicate cleavage in the target cell and the second is chosen to indicate cleavage in other tissues or body fluids, eg blood or serum. Assessments can be made in cell-free systems, in cells, in cell culture, in organ or tissue culture, or in intact animals. It may be useful to conduct an initial assessment in cell-free or culture conditions and confirm with additional assessments in intact animals. In preferred embodiments, candidate candidate compounds are cleaved at least 2, 4, 10, or 100 times faster in a cell (or under in vitro conditions selected to mimic cellular conditions) compared to blood or serum (or under in vitro conditions). , chosen to simulate conditions outside the cell).

Linking groups cleavable under redox conditions.

One class of cleavable linking groups are linking groups that are cleavable under redox conditions. An example of a linking group cleavable upon reduction is a disulfide linking group (-S-S-). In order to determine whether a candidate cleavable linking group is suitable for a linking group that is cleavable upon reduction, or, for example, suitable for use with a certain part of the mRNA and a certain targeting agent, one can consider the methods described in the present description. For example, a candidate can be assessed by incubation with dithiothreitol (DTT) or other reducing agent using reagents known in the art that mimic the rate of degradation that would be observed in a cell, eg a target cell. Candidates can also be evaluated under conditions that are chosen to mimic conditions in blood or serum. In preferred

- 23 042669 In a potent embodiment, the candidate compounds are cleaved in the blood by at most 10%. In preferred embodiments, candidate candidate compounds are degraded at least 2, 4, 10, or 100 times faster in the cell (or under in vitro conditions selected to mimic cellular conditions) compared to blood (or under in vitro conditions selected to mimic conditions outside the cell). The rate of degradation of candidate compounds can be determined using standard enzymatic kinetic assays under conditions chosen to mimic the intracellular environment and compared to conditions chosen to mimic the extracellular environment.

Cleavable linking groups based on phosphates.

Cleavable phosphate-based linking groups are cleaved by agents that destroy or hydrolyze the phosphate group. Examples of agents that cleave the phosphate group in cells are enzymes in cells such as phosphatases. 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 are -OP(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-. Preferred is -O-P(O)(OH)-O-. Such candidates may be evaluated using methods similar to those described above.

Linking groups cleavable by acid.

Acid-cleavable linking groups are groups that are cleavable in an acidic environment. In preferred embodiments, acid-cleavable linking groups are cleaved in an acidic environment at a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by means such as enzymes that can act like a normal acid. In the cell, specific low pH organelles such as endosomes and lysosomes can provide cleavage conditions for acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. Acid cleavable groups may have the general formula -C=NN-, C(O)O or -OC(O). It is preferred that the carbon attached to the oxygen of the ester (or alkoxy) group is an aryl, substituted alkyl or tertiary alkyl group such as dimethylpentyl or t-butyl. Such candidates may be evaluated using methods similar to those described above.

Linking groups based on esters.

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

Cleavable groups based on peptides

Cleavable peptide-based linking groups are cleaved in cells by enzymes such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to form oligopeptides (eg, dipeptides, tripeptides, etc.) and polypeptides.

Cleavable groups based on peptides do not include an amino group (-C(O)NH-). The amide group may be formed between any alkylene, alkenylene or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to form peptides and proteins. A peptide-based cleavable group is generally limited to the peptide bond (ie, amide bond) formed between amino acids to form peptides and proteins, and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula -NHCHR ^a C(O)NHCR ^b C(O)-, where R ^A and R ^B are the R groups of two adjacent amino acids. Such candidates can be evaluated using methods similar to those described above. As used herein, the term carbohydrate refers to a compound which, either by itself, is a carbohydrate consisting 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 associated with each carbon atom; or a compound having as its part a carbohydrate moiety consisting of one or more monosaccharide units, each having at least 6 carbon atoms (which may be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom associated with each a carbon atom. Representative carbohydrates include sugars (mono-, di-, tri-, and oligosaccharides containing about 4-9 monosaccharide units) and polysaccharides such as starches, glycogen, cellulose, and polysaccharide gums. Specific saccharides include C ₅ and higher (preferably C5-C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (preferably C ₅ -C ₈ ).

- 24 042669

Alternative embodiments.

In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA tool contains a sense strand and an antisense strand, with each strand having 14-30 nucleotides. The sense strand contains at least one motif of three identical modifications of three consecutive nucleotides, where at least one of the motifs occurs at or near a cleavage site in the antisense strand. Each nucleotide in the sense strand and antisense strand is modified. The modifications in the sense strand and the antisense strand, each independently, include at least two different modifications.

In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA tool contains a sense strand and an antisense strand, with each strand having 14-30 nucleotides. The sense 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 a cleavage site in the antisense strand. The antisense strand contains at least one motif of three identical modifications on three consecutive nucleotides. The structure of the modification of the antisense strand is shifted by one or more nucleotides relative to the structure of the modification of the sense strand.

In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA tool contains a sense strand and an antisense strand, with each strand having 14-30 nucleotides. A sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides when at least one of the motifs occurs at or near a cleavage site in the strand and at least one of the motifs occurs in another part 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 is located at or near a cleavage site in the strand and at least one of the motifs is located on another portion of the strand that is separated from the motif at or near the cleavage site by at least one nucleotide.

In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA tool contains a sense strand and an antisense strand, with each strand having 14-30 nucleotides. The sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs is located at or near a cleavage site in the strand and at least one of the motifs is located on another part 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 is located at or near a cleavage site in the strand and at least one of the motifs is located on another portion of the strand that is separated from the motif at or near the cleavage site by at least one nucleotide. A modification in a motif located at a cleavage site in the sense strand is different from a modification in a motif located 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 tool contains a sense strand and an antisense strand, with each strand having 12-30 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 is located at a 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 also contain one or more motifs of three identical modifications on three consecutive nucleotides, where one or more additional motifs occur on 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 also contain one or more motifs of three identical modifications on three consecutive nucleotides, where one or more additional motifs are located on another portion of the strand that is separated from the three 2'-O-methyl modifications at the cleavage site by at least one nucleotide. At least one of the nucleotides having the 2'-F modification can form a base pair with one nucleotide having the 2'-O-methyl modification.

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

In one embodiment, the siRNA compounds described herein may be formulated for administration to an individual. The siRNA composition can be in different states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (eg, less than 80%, 50%, 30%, 20%, or 10% water). In another example, the siRNA is in an aqueous phase, such as in a solution that includes water.

Aqueous or crystalline compositions can, for example, be incorporated into a delivery medium, such as a liposome (especially in the case of an aqueous phase) or a particle (e.g., a microparticle, which can

- 25 042669 be suitable for the crystalline composition). Typically, an siRNA composition is prepared in a manner that is compatible with the intended route of administration described herein. For example, in certain embodiments, the implementation of the composition receive at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, drying in a fluidized bed, or a combination of such methods; or sonication with lipid, freeze-drying, condensation, and other self-assembly.

An siRNA preparation can be made in combination with another agent, such as another therapeutic agent, or an agent that stabilizes the siRNA, such as a protein that complexes with the siRNA to form an mRNA. Other agents also include chelators such as EDTA (eg to remove divalent cations such as Mg ²⁺ ), salts, RNase inhibitors (eg a broad specificity RNase inhibitor such as RNAsin), and the like.

In one embodiment, the siRNA preparation contains another siRNA compound, such as a second siRNA, that mediates RNAi for a second gene and for the same gene. Another preparation may contain at least three, five, ten, twenty, fifty, one hundred or more different types of siRNA. Such miRNAs can mediate RNAi for a similar number of different genes.

In one embodiment, the siRNA preparation contains at least a second therapeutic agent (eg, an agent other than RNA or DNA). For example, an siRNA composition for treating a viral disease, such as HIV, may contain a known antiviral agent (eg, a protease inhibitor or a reverse transcriptase inhibitor). In another example, an siRNA composition for cancer treatment may further comprise a chemotherapeutic agent.

Examples of formulations are discussed below.

Liposomes.

For ease of description, the preparations, compositions, and methods in this section are mostly discussed in relation to unmodified siRNA compounds. However, it should be appreciated that such formulations, compositions, and methods may be carried out with other siRNA compounds, such as modified siRNAs, and such practices are within the scope of the invention. An siRNA compound, such as a double-stranded siRNA compound, or a csiRNA compound (ssiRNA) (e.g., a precursor, such as a larger siRNA compound that can be processed to a csiRNA compound, or a DNA that encodes an siRNA compound, such as a double-stranded siRNA or csiRNA compound, or its precursor) can be formulated to be delivered in an aggregate of membrane molecules, such as a liposome or micelle. As used herein, the term liposome refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as one or more bilayers. Liposomes include unilamellar and polylamellar vesicles, which have a shell formed from a lipophilic material and an aqueous interior. The aqueous portion contains the siRNA composition. The lipophilic material isolates the aqueous interior from the aqueous exterior, which generally does not include the composition of the siRNA, although this may be the case in some instances. Liposomes are useful for carrying and delivering active ingredients to the site of action. Because the liposomal envelope is structurally similar to biological membranes, when liposomes are applied to tissue, the liposome bilayer fuses with cell membranes. As the liposome-cell fusion continues, the internal water content, which includes the siRNA, is delivered to the cell, where the siRNA can specifically bind to the target RNA and can mediate RNAi. In some cases, liposomes are also specifically targeted, for example to target siRNAs to certain cell types.

The siRNA-containing liposome can be obtained in various ways. In one example, the lipid component of the liposome is dissolved in a detergent such that micelles are formed with the lipid component. For example, the lipid component may be an amphipathic cationic lipid or a lipid conjugate. The detergent may have a high critical micelle concentration and may be non-ionic. Examples of detergents include cholates, CHAPS, octylglucoside, deoxycholate, and lauroylsarcosine. The siRNA preparation is then added to micelles that include a lipid component. The cationic groups of the lipid interact with the siRNA and condense around the siRNA to form a liposome. After condensation, the detergent is removed, for example by dialysis, and a siRNA liposomal preparation is obtained.

If necessary, a carrier compound can be added during the condensation reaction which promotes condensation, for example by controlled addition. For example, the carrier may be a polymer other than a nucleic acid (eg, spermine or spermidine). You can also adjust the pH for favorable condensation.

Further description of methods for producing stable polynucleotide delivery vesicles that contain a polynucleotide/cationic lipid complex as a structural component of the delivery vesicle is given, for example, in WO 96/37194. The formation of liposomes may also include one or more aspects of the described exemplary methods from Feigner P.L. et al., Proc. Natl. Acad. Sci. USA 8:74137417, 1987; US patent No. 4897355; US patent No. 5171678; Bangham et al., M. Mol. Biol., 23:238, 1965;

- 26 042669

Olson et al., Biochem. Biophys. Acta, 557:9, 1979; Szoka et al., Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew et al., Biochem. Biophys. Acta, 775:169, 1984; Kim et al., Biochem. Biophys. Acta, 728:339, 1983; and Fukunaga et al., Endocrinol., 115:757, 1984. Commonly used methods to obtain appropriately sized lipid aggregates for use in delivery vesicles include sonication and freeze-thaw plus extrusion (see, for example, Mayer et al., Biochem. Biophys Acta 858:161 (1986). When suitably small (50-200 nm) and relatively uniform aggregates are required, microfluidization can be used (Mayhew et al., Biochem. Biophys. Acta, 775:169, 1984). Such methods are readily adaptable for packaging siRNA preparations into liposomes.

Liposomes that are pH sensitive or negatively charged trap nucleic acid molecules rather than complex with them. Since the nucleic acid molecules and the lipid are similarly charged, repulsion occurs rather than complex formation. However, some nucleic acid molecules are captured by the aqueous interior of such liposomes. Liposomes that are pH sensitive are used to deliver the DNA encoding the thymidine kinase gene to cell layers in monoculture. Exogenous gene expression was found in target cells (Zhou et al., Journal of Controlled Realease, 19 (1992), 269-274).

One main type of liposome composition includes phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome formulations can be prepared, for example, from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposomal formulations are typically prepared from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are predominantly prepared from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is derived from phosphatidylcholine (PC), such as, for example, soy PC and egg PC. Another type is obtained from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods for introducing liposomes into cells in vitro are given in US patent No. 5283185, US patent No. 5171678, WO 94/00569, WO 93/24640, WO 91/16024, Feigner, J. Biol. Chem. 169:2550, 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 have the advantage of being able to fuse with 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 siRNA to macrophages.

Other advantages of liposomes include the following: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can include a wide variety of water- and fat-soluble drugs; liposomes can protect siRNAs encapsulated in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, vol. 1, p. 245). Important considerations in the preparation of liposome compositions are the surface charge of the lipid, the size of the vesicles, and the water volume of the liposomes.

The positively charged synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium chloride (DOTMA) can be used to prepare small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes that are able to fuse with negatively charged cell membrane lipids of tissue culture cells resulting in siRNA delivery (see, e.g., Feigner P. L. et al., Proc. Natl. Acad. Sci., USA, 8:7413-7417, 1987; and US Pat. No. 4,897,355 describing DOTMA and its use with DNA).

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

Other cationic lipids described include compounds that are conjugated to various moieties, including, for example, carboxyspermine, which is conjugated to one of two types of lipids, and includes compounds such as 5-carboxyspermylglycine dioctoleoilamide (DOGS) (transfectam™, Promega, Madison, Wisconsin ) and 5-carboxyspermylamide with dipalmitoylphosphatidylethanols

- 27 042669 to (DPPES) (see, for example, US patent No. 5171678).

Another cationic lipid conjugate includes a cholesterol lipid derivatization product (DCChoi) which is formulated into liposomes in combination with DOPE (see Gao X. and Huang L., Biochem. Biophys. Res. Commun., 179:280, 1991). Lipopolylysin obtained by conjugation of polylysine with DOPE is reported to be effective for transfection in the presence of serum (Zhou X. et al., Biochem. Biophys. Acta, 1065:8, 1991). In other words, for some cell lines, such liposomes containing conjugated cationic lipids show less toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98/39359 and wO 96/37194.

Liposomal preparations are particularly suitable for topical administration, and liposomes have some advantages over other preparations. Such advantages include the accumulation of the injected drug in the desired target and the ability to introduce siRNA into the skin. In some embodiments, liposomes are used to deliver siRNA to epidermal cells and also to enhance siRNA penetration into skin tissues, such as skin. For example, liposomes can be applied topically. Topical delivery to the skin of drugs prepared as liposomes has been documented (see, for example, 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, 84:78517855, 1987).

Non-ionic liposome systems have also been tested to determine their usefulness for drug delivery to the skin, in particular systems comprising a non-ionic surfactant and cholesterol. Non-ionic liposome preparations containing novas I (glyceryl dilaurate/cholesterol/polyoxyethylene 10-stearyl ether) and novas II (glyceryl distearate/cholesterol/polyoxyethylene 10-stearyl ether) were used to deliver the drug to the mouse skin dermis. Such siRNA preparations are useful in the treatment of a dermatological disorder.

Liposomes that include siRNA can be obtained as highly deformable. Such deformability may allow liposomes to penetrate through a pore that is smaller than the average radius of the liposome. For example, transfersomes are a type of liposomes that can be deformed. Transfersomes can be prepared by adding surface field activators, typically surfactants, to a standard liposomal formulation. Transfersomes that include siRNAs can be delivered, for example, subcutaneously, by injection, in order to deliver siRNAs to keratinocytes in the skin. In order to pass through intact mammalian skin, lipid vesicles must pass through a series of fine pores, each less than 50 nm in diameter, under the influence of a suitable transdermal gradient. In addition, due to the properties of lipids, such transfersomes can be self-optimizing (adaptive to the shape of the pores, for example, in the skin), self-healing and can usually reach their targets without fragmentation, and often self-charging.

Other formulations suitable for the present invention are described in U.S. Provisional Applications Ser. , 61/047087, filed April 22, 2008, and 61/051528, filed May 8, 2008. PCT Application No. PCT/US2007/080331, filed October 3, 2007, also describes formulations suitable for the present invention.

Surfactants.

For ease of description, the preparations, compositions, and methods in this section are mostly discussed in relation to unmodified siRNA compounds. However, it should be appreciated that such formulations, compositions, and methods may be carried out with other siRNA compounds, such as modified siRNAs, and such practices are within the scope of the invention. Surfactants find wide use in formulations such as emulsions (including microemulsions) and liposomes (see above). The siRNA (or precursor, eg, larger dsRNA that can be processed into an siRNA, or DNA that encodes the siRNA or precursor) compositions may include a surfactant. In one embodiment, the siRNA is in an emulsion formulation that includes a surfactant. The most common way to classify and evaluate the properties of many different types of surfactants, both natural and synthetic, is to use the hydrophilic-lipophilic balance (HLB). The nature of the hydrophilic group provides the most useful means of categorizing the various surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

If the surfactant molecule is not ionized, it is classified

- 28 042669 is available as a non-ionic surfactant. Nonionic surfactants find wide use in pharmaceutical products and are applicable over a wide pH range. Typically 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 copolymers are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

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

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

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

The use of surfactants in drug products, preparations and emulsions is reviewed by Rieger in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285.

Micelles and other membrane preparations.

For ease of description, micellar and other preparations, compositions and methods in this section are mostly discussed in relation to unmodified siRNA compounds. However, it is understood that such micellar and other preparations, compositions and methods can be carried out with other siRNA compounds, such as modified siRNAs, and such practice is within the scope of the invention. Composition of an siRNA compound, e.g., a double-stranded siRNA compound, or a csiRNA compound (ssiRNA) (e.g., a precursor, e.g., a larger siRNA compound that can be processed to a csiRNA compound, or a DNA that encodes an siRNA compound, such as a double-stranded siRNA, or csiRNA compound , or its predecessor) can be provided in a micellar preparation. As used herein, micelles are defined as a particular type of aggregation of molecules in which amphipathic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecules are directed inwards, leaving the hydrophilic portions in contact with the surrounding aqueous phase. There is a reverse arrangement if the environment is hydrophobic.

A mixed micelle preparation suitable for delivery across skin membranes can be prepared by mixing an aqueous solution of an siRNA composition, an alkali metal (C ₈ -C ₁ o)alkyl sulfate, and a micelle-forming compound. Examples of 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, monolaurates, borage oil, evening primrose oil, menthol, trihydroxyoxocholonylglycine and its pharmaceutically acceptable salts, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ethers and analogs thereof, polydocanol alkyl ethers and analogs thereof, chenodeoxycholate, deoxycholate and mixtures thereof. The micelle-forming compounds may be added simultaneously or after the addition of the alkali metal alkyl sulfate. Mixed micelles will form with essentially any kind of mixing of the ingredients, but with vigorous agitation, smaller micelles are provided.

In one method, a first micellar composition is prepared that contains an siRNA composition and at least an alkali metal alkyl sulfate. The first micellar composition is then mixed with at least three micelle-forming compounds to form a mixed micellar composition. In another method, a micellar composition is prepared by mixing the siRNA composition, alkali metal alkyl sulfate and at least one of the micelle forming compounds with vigorous stirring and then adding the remaining micelle forming compounds.

Phenol and/or m-cresol can be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. On the other hand, phenol and/or m-cresol can be added with micellar ingredients. An isotonic agent such as glycerin may be added at

- 29 042669 after the formation of a mixed micellar composition.

To deliver the micellar composition as a spray, the formulation may be placed in an aerosol dispenser and the propellant loaded into the dispenser. The pressurized propellant in the dispenser is in liquid form. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one phase, i.e. to have one phase. If there are two phases, it is necessary to shake the dispenser before spraying a portion of the contents, for example, through a metering valve. The nebulized dose of the pharmaceutical agent is pushed out of the metering valve in fine sprays.

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

Specific concentrations of the required ingredients can be determined by relatively simple experience. For oral absorption, it is generally desirable to increase the dosage, for example, at least two or three times the dosage for injection or administration through the gastrointestinal tract.

Particles.

For ease of description, the particles, preparations, compositions, and methods in this section are mostly discussed in relation to unmodified siRNA compounds. However, it can be understood that such particles, preparations, compositions and methods can be carried out with other siRNA compounds, such as modified siRNAs, and such practice is within the scope of the invention. In another embodiment, preparations of an siRNA compound, such as a double-stranded siRNA compound or a csiRNA compound (e.g., a precursor, such as a larger siRNA compound that can be processed to a csiRNA compound, or a DNA that encodes an siRNA compound, such as a double-stranded siRNA compound, or a csiRNA compound, or its predecessor) may be included in a particle, such as a microparticle. Microparticles can be obtained by spray drying, but can also be obtained by other methods, including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of such methods.

pharmaceutical compositions.

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

Pharmaceutical compositions may be formulated specifically for administration in solid or liquid form, including forms adapted for the following:

(1) oral administration, eg infusion forms (aqueous or non-aqueous solutions or suspensions), tablets, eg intended 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 epidermal injection, for example, in the form of a sterile solution or suspension or delayed release formulation;

(3) topical administration, for example, in the form of a cream, ointment, or controlled release patch or spray applied to the skin;

(4) intravaginal or intrarectal administration, for example, in the form of a pessary, cream or foam;

(5) sublingual administration;

(6) intraocular administration;

(7) transdermal administration; or (8) nasal administration.

Delivery using the subcutaneous or intravenous route may be particularly preferred.

The term "therapeutically effective amount" as used herein means the amount of a compound, material or composition comprising a compound of the invention that is effective to produce some desired therapeutic effect in at least a subset of cells in an animal at a reasonable benefit/benefit ratio. hazard acceptable for any drug treatment.

The expression pharmaceutically acceptable is used herein to refer to those compounds, materials, compositions and/or dosage forms which, within reasonable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problem, or complications, with a reasonable benefit / danger ratio.

The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid excipient, diluent, excipient, preparation aid (e.g., lubricating

- 30 042669 general substance, talc, magnesium, calcium or zinc stearates or stearic acid) or solvent encapsulating material involved in the movement or transfer of the compound in the body of an individual from one organ or part of the body to another organ or part of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that 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) crushed tragacanth;

(5) malt;

(6) gelatin;

(7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc;

(8) excipients such as cocoa butter and suppository waxes;

(9) oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil;

(10) glycols such as propylene glycol;

(11) polyols such as glycerol, 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 solution;

(18) Ringer's solution;

(19) ethyl alcohol;

(20) pH buffer solutions;

(21) polyesters, polycarbonate and/or polyanhydrides;

(22) excipients such as polypeptides and amino acids;

(23) a serum component such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances used in pharmaceutical preparations.

The formulations may, for convenience, be presented in unit dosage form and may be prepared by any means known in the art of pharmacy. The amount of active ingredient that can be combined with the carrier material to form a single dosage form will vary depending on the recipient being treated and the particular route of administration. The amount of active ingredient that can be combined with the carrier material to form a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Typically, in one hundred percent, such an amount will vary from about 0.1% to about 99% active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.

In some embodiments, the formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, such as bile acids, and polymeric carriers, such as polyesters and polyanhydrides, and a compound of the present invention. In some embodiments, the above formulation imparts oral bioavailability to a compound of the present invention.

An siRNA agent preparation may comprise a combination with another agent, such as another therapeutic agent, or a substance that stabilizes the mRNA, such as a protein that forms a complex with the mRNA to form an iRNP. In addition, other agents include chelators such as EDTA (eg, to remove divalent cations such as Mg ²⁺ ), salts, RNase inhibitors (eg, a broad specificity RNase inhibitor such as RNAsin), and the like.

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

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 can be done by using a liquid suspension of crystalline or amorphous material having poor water solubility. Then the rate of absorption of the drug depends on the rate of dissolution in water, which, in turn, may depend on the size of the crystals and the crystalline form. On the other hand, delayed absorption of a parenterally administered drug

- 31 042669 funds can be obtained by dissolving or suspending the drug in an oil medium.

The compounds of the invention may be formulated for administration by any convenient route for human or veterinary use in analogy to other pharmaceutical preparations.

The term treatment is intended to mean also prevention, treatment and healing. The patient receiving such treatment is any animal in need thereof, including primates, in particular humans, and other mammals such as horses, cattle, pigs and sheep, and poultry and all domestic animals.

Double-stranded RNAi agents are produced in the cell in vivo, for example, from exogenous DNA templates that are delivered to the cell. For example, DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to the individual, for example, by intravenous injection, topical administration (US Pat. No. 5,328,470), or stereotaxic injection (see, for example, Chen et al. (1994), Proc. Natl. Acad. Sci., USA, 91 : 3054-3057). A pharmaceutical formulation of a gene therapy vector may include the gene therapy vector in a suitable diluent, or may include a slow release matrix into which a gene delivery medium is inserted. DNA templates, for example, may include two transcription units, of which one produces a transcript that includes the upper strand of the dsRNA agent and the other produces a transcript that includes the lower strand of the dsRNA agent. When the templates are transcribed, the dsRNA agent is produced and processed into fragments of the siRNA agent that mediate gene silencing.

Shipping ways.

A composition that includes an mRNA can be delivered to an individual in a variety of ways. Examples of routes include intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

The mRNA molecules of the invention may be included in pharmaceutical compositions suitable for administration. Such compositions typically contain one or more types of mRNA and a pharmaceutically acceptable carrier. As used herein, the formulation of a pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents with pharmaceutically active substances is well known in the art. Their use in the composition is contemplated unless any conventional medium or agent is incompatible with the active compound. Additional active compounds may also be included in the compositions.

The compositions of the present invention can be administered in a number of ways depending on whether local or systemic treatment is desired and the site being 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 can be chosen to enhance targeting. For example, in order to reach muscle cells, an intramuscular injection into the muscles of interest may be a logical choice. Lung cells can be accessed by injecting mRNA in aerosol form. Vascular endothelial cells can be accessed by coating with mRNA on the catheter and mechanically introducing DNA.

Dosage.

In one aspect, a feature of the invention is a method of administering to an individual (eg, a human) a dsRNA agent, such as an siRNA agent. The method includes administering a standard dose of a dsRNA agent, e.g. an siRNA agent, e.g. a double-stranded siRNA agent, which (a) has a double-stranded portion of 14-30 nucleotides (nc), e.g. 21-23 nc, (b) is complementary to the target RNA (e.g. pathogenic target RNA) and optionally (c) includes at least one 3' overhang of 1-5 nucleotides in length. In one embodiment, the unit dose is less than 10 mg/kg body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, or 0 .00001 mg/kg body weight and less than 200 nmol RNA agent (e.g., about 4.4 x 10 ¹⁶ copies) per kg body weight 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, or 0.00015 nmol of RNA agent per kg of body weight.

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

In some embodiments, the unit dose is administered less frequently than once a day, such as less than every 2, 4, 8, or 30 days. In another embodiment, the unit dose is administered

- 32 042669 irregularly. For example, a unit dose may be administered once.

In one embodiment, the effective dose is administered with other conventional therapeutic methods. In one embodiment, the individual has a viral infection and the agent is an antiviral agent other than a dsRNA agent, such as other than an siRNA agent. In another embodiment, the subject has atherosclerosis and an effective dose of a dsRNA agent, eg an siRNA agent, is administered in combination, eg, following a surgical procedure, eg, angioplasty.

In one embodiment, an individual is administered an initial dose and one or more maintenance doses of a dsRNA agent, e.g. an siRNA agent (e.g. miRNA, or its precursor). The maintenance dose or doses may be the same or less than the initial dose, eg half the initial dose. The maintenance regimen may include treating the individual with a dose or doses ranging from 0.01 to 15 mg/kg of body weight per day, for example 10, 1, 0.1, 0.01, 0.001 or 0.00001 mg per kg of body weight in day. Maintenance doses are administered, for example, not more frequently than once every 2, 5, 10 or 30 days. In addition, the treatment regimen may last for a period of time, which will vary depending on the nature of the particular disease, its severity and the general condition of the patient. In some embodiments, the dosage may be delivered no more than once per day, such as no more than once every 24, 36, or 48 or more hours, such as no more than once every 5 or 8 days. After treatment of the patient, changes in his condition and relief of the symptoms of the disease state can be monitored. The dosage of the compound may either be increased if the patient does not respond sufficiently to current dosage levels, or the dose may be reduced if there is improvement in the symptoms of the disease state, if the disease state persists, or if a side effect is observed.

An effective dose may be administered in a single dose or in two or more doses, as desired or considered appropriate in the particular circumstances. If it is desired to facilitate repeated or frequent infusions, implantation of a delivery device such as a pump, a temporary stent (eg, intravenous, intraperitoneal, intracisternal, or intracapsular) or reservoir may be beneficial.

In one embodiment, the composition includes several types of dsRNA agent. In another embodiment, a species of dsRNA agent has sequences that do not overlap or coexist with other species with respect to a naturally occurring target sequence. In another embodiment, several types of dsRNA agent are specific for different natural target genes. In another embodiment, the dsRNA agent is allele specific.

The dsRNA agents of the invention described herein can be administered to mammals, in particular large mammals such as non-human or human primates, in a variety of ways.

In one embodiment, administration of a composition of a dsRNA agent, such as an siRNA agent, is parenteral, such as intravenous (eg, bolus or diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal , sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, or ophthalmic. The introduction can be carried out by an individual or another person, such as a medical professional. The drug may be provided in metered doses or in a device that delivers a metered dose. Selected delivery types are discussed in more detail below.

The invention relates to methods, compositions and kits for rectal administration or delivery of dsRNA agents described in the present description.

Methods for inhibiting target gene expression.

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

Another aspect of the invention relates to a method for modulating the expression of a target gene in a cell, comprising providing said cell with a dsRNA agent of the present 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, CRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erc1/2 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 gene alpha, mutations in the p73 gene, mutations in the p21 gene (WAFI/CIPI), mutations in the p27 gene (KIP1), mutations in the PPMID gene, mutations in the RAS gene, mutations in the gene

- 33 042669 caveolin I, 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 limiting. All references, pending patent applications and published patents cited in the present description, are incorporated into the present description by reference.

Examples

Example 1 Screening for miRNA duplexes in vitro.

Cell culture and transfections.

Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, VA) are grown to near confluence at 37°C in 5% CO2 in RPMI (ATCC) supplemented with 10% FBS, streptomycin and glutamine (ATCC ) and then removed from the plate by trypsinization. Transfection is performed by adding 14.8 µl of Opti-MEM plus 0.2 µl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad, CA, cat. no. 13788-150) to 5 µl of siRNA duplexes per well in a 96-well plate, and incubated at room temperature for 15 min. Then, 80 µl of complete growth medium without antibiotics containing ~2x10 ⁴ Hep3B cells is added to the siRNA mixture. Cells are incubated for either 24 or 120 hours and then the RNA is purified. Single dose experiments are performed at a final concentration of duplexes of 10 and 0.1 nM, and dose-response experiments are performed using 8.4-fold serial dilutions with a maximum dose of duplexes at a final concentration of 10 nM.

Total RNA isolation using the DYNABEADS mRNA kit (Invitrogen, part #: 610-12).

Cells are harvested and lysed in 150 μl of lysis/binding buffer, then stirred for 5 min at 850 rpm using an Eppendorf thermomixer (mixing speed is the same throughout the process). A mixture of 10 µl of magnetic beads and 80 µl of lysis/binding buffer is added to the wells of a round bottom well plate and mixed for 1 minute. The magnetic beads are fixed using a magnetic stand and the supernatant is removed without affecting the beads. After removal of the supernatant, lysed cells are added to the remaining beads and mixed for 5 minutes. After removal of the supernatant, the magnetic beads are washed 2 times with 150 µl of wash buffer A and mixed for 1 min. The beads are fixed again and the supernatant removed. The beads are then washed with 150 µl of wash buffer B, fixed and the supernatant discarded. The beads are then washed with 150 µl of elution buffer, fixed and the supernatant discarded. The granules are left to dry for 2 minutes. After drying, 50 µl of elution buffer is added and stirred for 5 min at 70°C. The granules are fixed with a magnet for 5 minutes. Remove 40 µl of the supernatant and add to another 96-well plate.

Synthesis of cDNA using ABI High Throughput cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat # 4368813).

To 5 µl total RNA is added a master mix of 1 µl 10X buffer, 0.4 µl 25X dNTP, 1 µl random primers, 0.5 µl reverse transcriptase, 0.5 µl RNase inhibitor and 1.6 µl H ₂ O per reaction. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cell (Hercules, CA) through the following steps: 25°C 10 min, 37°C 120 min, 85°C 5 s, 4°C hold.

PCR in real time.

To a master mix containing 0.5 µl GAPDH TaqMan probe (Applied Biosystems, cat. no. 4236317E (human), cat. no. 4308313 (rodent)), 0.5 µl TTR TaqMan probe (Applied Biosystems, cat. no. HS00174914_m1 (human ), cat.# Rn00562124_m1 (rats)) and 5 µl of Lightcycler 480 Probe Master Mix (Roche, cat. no. 04887301001), 2 µl of cDNA are added per well in a 384-well plate (Roche, cat. no. 04887301001). Real-time PCR was performed on a Roche LC 480 real-time PCR machine (Roche). Each duplex was tested in at least two independent transfections and each transfection was analyzed in duplicate unless otherwise noted.

In order to calculate the relative fold change, real-time data are analyzed using the AACt method and normalized to analyzes performed with cells transfected with 10 nM AD-1955 or sham-transfected cells. IC ₅₀ is calculated using a 4-parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naive cells at the same dose range or their own lowest dose. IC ₅₀ is calculated for each individual transfection, as well as in combination, where one IC ₅₀ corresponds to data from both transfections.

The results of gene silencing of an example miRNA duplex with various motif modifications according to the invention are shown in the table below.

Example 2. Synthesis of RNA and annealing of duplexes.

1. Synthesis of oligonucleotides.

All oligonucleotides are synthesized on an AKTAoligopilot synthesizer or an ABI 394 synthesizer. Oligonucleotides are synthesized using, unless otherwise indicated, a commercially available porous glass solid support with defined pore size (dT-CPG, 500A, Prime Synthesis) and phosphoramidites

- 34 042669

RNA with standard protective groups -3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-Odimethoxytrityl-N2-isobutyryl-2'-tert-butyldimethylsilylguanosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite and 5'Ό-dimethoxytrityl-2 '-tert-butyldimethylsilyluridin-3'-O-N,N'diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies). 2'-P-Phosphoramidites 5'-Odimethoxytrityl-N4-acetyl-2'-fluorocytidine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite and 5'-Odimethoxytrityl-2'-fluorouridine-3'-O-N,N '-diisopropyl-2-cyanoethylphosphoramidite is purchased from Promega. All phosphoramidites are used at 0.2M in acetonitrile (CH3CN) except for guanosine, which is used at 0.2M in 10% THF/ANC (v/v). An addition/recycling time of 16 minutes was used. The activator is 5-ethylthiotetrazole (0.75 M, American International Chemicals) for PO oxidation using iodine/water/pyridine and for PS oxidation using PADS (2%) in 2,6-lutidine/ACN (1: 1, v/v).

Ligand-conjugated chains are synthesized using a solid support containing the appropriate ligand. For example, insertion of a carbohydrate moiety/ligand (in the case of, for example, GalNAc) at the 3' end of the sequence is achieved by starting the synthesis with an appropriate carbohydrate solid support. Similarly, the cholesterol moiety is introduced at the 3' end, starting the synthesis on the cholesterol carrier. Typically, the ligand moiety is coupled to trans-4-hydroxyprolinol via a linkage chosen as described in the previous examples to give the hydroxyprolinol ligand moiety. The hydroxyprolinol ligand portion is then coupled to a solid support via a succinate linker or converted to phosphoramidite under standard phosphorylation conditions to provide the desired carbohydrate conjugate building blocks. Fluorophore labeled siRNAs are synthesized from the appropriate phosphoramidite or solid support purchased from Biosearch Technologies. The oleyllithocholic (GalNAc) ₃ polymer carrier was prepared in-house at a loading of 38.6 µmol/g. A mannose (Man) ₃ polymer carrier is also prepared in-house at a loading of 42.0 µmol/g.

Conjugation of the selected ligand to the desired position, eg the 5' end of the sequence, is achieved by attaching the appropriate phosphoramidite to the growing strand under standard phosphoramidite coupling conditions, unless otherwise indicated. Over 15 min, the combination of a 0.1 M solution of phosphoramidite in anhydrous CH ₃ CN in the presence of the activator 5-(ethylthio)-1H-tetrazole binds it to the oligonucleotide. Oxidation of the internucleoside phosphite to phosphate is carried out using standard iodine water as reported in (1) or by treatment with tert-butylhydroxyperoxide/acetonitrile/water (10:87:3) with a 10 minute holding time to oxidize the conjugated oligonucleotide. Phosphorothioate is introduced by oxidizing phosphite to Phosphorothioate using a sulfur transfer agent such as DDTT (purchased from AM Chemicals), PADS and/or Bocage's reagent. Cholesterolphosphoramidite is synthesized by the company and used at a concentration of 0.1 M in dichloromethane. The coupling time for cholesterolphosphoramidite is 16 minutes.

2. Removal of the protective group-I (deprotection of the nucleotide base).

Upon completion of the synthesis, the carrier is transferred to a 100 ml glass vial (VWR). The oligonucleotide is cleaved from the carrier while removing the base protecting group and phosphate groups with 80 ml of a mixture of ethanol and ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55°C. The vial is cooled briefly on ice and then the ethanol-ammonia mixture is filtered into another 250 ml vial. The CPG is washed with ethanol/water (1:1, v/v) 2x40 ml. The volume of the mixture is then reduced to ~30 ml by rotary evaporation. The mixture is then frozen on dry ice and dried under vacuum at speed vac.

3. Removal of the protective group-II (deletion of the 2'-TBDMS group).

The dried residue is taken up in 26 ml of a mixture of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60°C for 90 min to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' position . The reaction is then quenched with 50 ml of 20 mM sodium acetate solution, adjusted to pH 6.5 and stored frozen until purified.

4. Analysis.

Oligonucleotides are analyzed by high performance liquid chromatography (HPLC) before purification, and the choice of buffer and column depends on the nature of the sequence and or conjugated ligand.

5. Purification by HPLC.

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

6. Obtaining siRNA.

- 35 042669

To obtain siRNA, equimolar amounts of sense and antisense strand are heated in IxPBS at 95°C for 5 min and slowly cooled to room temperature. The integrity of the duplex is confirmed by HPLC.

Table 1

Modified duplex ANGPTL3

Duplex ID SID Sense strand (S) AS ID Antisense strand (AS) % mRNA remaining conc. miRNA IC50 (NM) 1 nM 0.1 nM 0.01 nM D1000 S1000 Af and Gf and Af a Cf cAf Af Gf aGf and Af and Uf cCf a su AS 1000 AfUfgGfaAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.1 0.47 0.006 D1001 S1001 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS 1001 aUfsgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.10 0.49 0.0065 D1002 S1002 AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1002 aUfgGfAfAfuAfcUfcuuGfgsUfuAfcAfusGfsa 0.04 0.10 0.46 0.0068 D1003 S1003 AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1003 aUfgGfAfAfuAfcUfcuuGfgUfsuAfcAfusGfsa 0.05 0.12 0.56 0.0073 D1004 S1004 aUGuaACccAGagUAuuCCasu AS 1004 AUggAAuaCUcuUGguUAcaUsGsa 0.07 0.13 0.44 0.008 D1005 S1005 AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1005 aUfgGfAfAfuAfcUfcuuGfgsUfsuAfcAfusGfsa 0.06 0.11 0.53 0.0093 D1006 S1006 AfuGfuAfAfccAfAfGfaGfuAfuUfcCfasUf AS1006 aUfgGfaAfuAfcUfcuuGfGfuuAfcAfusGfsa 0.05 0.16 0.55 0.0095 D1007 S1007 AfuGfuAfAfCfcAfAfGfaGfuAfuUfcCfasUf AS1007 aUfgGfaAfuAfcUfcuuGfguuAfcAfusGfsa 0.05 0.14 0.48 0.0098 D1008 S1008 auguaaccaadGadGudAudAcdGasu AS 1008 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.07 0.11 0.33 0.010 D1009 S1009 UfgGfGfAfuUfuCfAfUfgUfaAfcCfAfAfgsAf AS 1009 uCfuugGfuUfaCfaugAfaAfuccCfasUfsc 0.03 0.14 0.56 0.0101 D1010 S1010 UfgGfgauUfuCfAfUfgUfaAfcCfaAfgsAf AS1010 uCfuUfgGfuUfaCfaugAfaAfUfCfcCfasUfsc 0.03 0.14 0.65 0.0101 D1O11 S1O11 aUfGfuAfAfccAfAfGfaGfuAfuUfcCfasUf AS1011 aUfgGfaAfuAfcUfcuuGfGfuuAfcaUfsgsa 0.06 0.10 0.55 0.011 D1012 S1012 UfgGfgAfuUfuCfAfUfgUfaacCfaAfgsAf AS1012 uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc 0.04 0.13 0.54 0.0114 D1013 S1013 auguaaccaadGadGudAudAcdGasu AS1013 aUfgGfaAfuAfcUfcUfugdGudTadCadTsgsa 0.11 0.19 0.49 0.011 D1014 S1014 AfuGfuaaCfcAfAfGfaGfuAfuUfcCfasUf AS1014 aUfgGfaAfuAfcUfcuuGfgUfUfAfcAfusGfsa 0.04 0.16 0.59 0.013 D1015 S1015 AfuguAfaccAfaGfdAGfdTAdTudCcdAsu AS1015 dAUdGgdAadTAfdCUfcUfuGfgUfuAfcAfusGfsa 0.07 0.15 0.51 0.013 D1016 S1016 auGfuAfaCfcAfAfGfaGfuAfuUfcCfasUf AS1016 aUfgGfaAfuAfcUfcuuGfgUfuAfcAfUfsGfsa 0.05 0.14 0.64 0.013 D1017 S1017 UfGfggAfuUfuCfAfUfgUfAfAfcCfaAfgsAf AS1017 uCfuUfgGfuuaCfaugAfaAfuCfCfcasUfsc 0.09 0.41 0.74 0.0133 D1018 S1018 AfuguAfaCfcAfAfGfaGfuAfuUfcCfasUf AS1018 aUfgGfaAfuAfcUfcuuGfgUfuAfCfAfusGfsa 0.03 0.14 0.61 0.014 D1019 S1019 AfuGfuAfaccAfAfGfaGfuAfuUfcCfasUf AS1019 aUfgGfaAfuAfcUfcuuGfGfUfuAfcAfusGfsa 0.02 0.2 0.7 0.014 D1020 S1020 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1020 asUfsgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.04 0.16 0.67 0.0156 D1021 S1021 aUfguAfAfccAfAfgagUfaUfuCfcasUf AS 1021 aUfGfgAfaUfaCfUfCfuuGfGfuuAfCfaUfsgsa 0.11 0.24 0.64 0.016 D1022 S1022 dTdGggdAdTuudCdAugdTdAacdCdAagsdA AS1022 udCdTugdGdTuadCdAugdAdAaudCdCcasdTsc 0.08 0.27 0.64 0.0161 D1023 S1023 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1023 aUfgsGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.19 0.63 0.0163 D1024 S1024 UfgGfgAfuUfuCfAfUfguaAfcCfaAfgsAf AS1024 uCfuUfgGfuUfAfCfaugAfaAfuCfcCfasUfsc 0.05 0.25 0.69 0.0164 D1025 S1025 UfgGfgAfuUfuCfAfUfgUfAfAfcCfaAfgsAf AS1025 uCfuUfgGfuuaCfaugAfaAfuCfcCfasUfsc 0.04 0.18 0.75 0.0166 D1026 S1026 UfgGfgAfuUfuCfAfUfgUfaAfcCfaAfgsAf AS1026 uCf and UfgGf and UfaCfa ugAfa Af uCfcCfasUfsc 0.04 0.19 0.66 0.0178 D1027 S1027 UfgGfgAfuUfuCfAfUfgUfaAfccaAfgsAf AS1027 uCfuUfGfGfuUfaCfaugAfaAfuCfcCfasUfsc 0.04 0.19 0.69 0.018 D1028 S1028 dAdTgudAdAccdAdAgadGdTaudTdCcasdT AS1028 adTdGgadAdTacdTdCuudGdGuudAdCausdGsa 0.15 0.29 0.72 0.018 D1029 S1029 AdTGdTAdACdCAdAGdAGdTAdTUdCCdAsU AS 1029 dAUdGGdAAdTAdCUdCUdTGdGUdTAdCAdTsGsdA 0.1 0.27 0.61 0.018 D1030 S1030 UfgGfGfAfuuuCfAfUfgUfaAfcCfaAfgsAf AS1030 uCf and UfgGf and UfaCfa ugAfAfAf uccCfasUfsc 0.04 0.21 0.64 0.0187 D1031 S1031 AfuGfuAfAfccAfAfGfAfGfuAfuuccAfsu AS1031 AfUfGfGfAfAfuAfCfUfCfUfuGfGfuuAfcAfusGfsa 0.06 0.15 0.62 0.019 D1032 S1032 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1032 asUfgGfAfAfuAfcUfcuuGfgUfsuAfcAfusGfsa 0.09 0.34 0.78 0.021 D1O33 S1O33 UfgGfgAfuUfuCfaUfGfUfaacCfaAfgsAf AS1033 uCfuUfgGfUfUfacaUfgAfaAfuCfcCfasUfsc 0.06 0.26 0.57 0.0212 D1034 S1034 AfuGfuAfAfccAfaGfaGfuAfuUfcCfasUf AS1034 aUfgGfaAfuAfcUfcUfuGfGfuuAfcAfusGfsa 0.11 0.39 0.82 0.0216 D1035 S1035 UfgGfgAfuuuCfAfUfgUfaAfcCfaAfgsAf AS1035 uCfuUfgGfuUfaCfaugAfAfAfuCfcCfasUfsc 0.04 0.16 0.56 0.0222 D1036 S1036 UfgGfGfAfuUfuCfaUfgUfaAfcCfAfAfgsAf AS1036 uCfuugGfuUfaCfaUfgAfaAfuccCfasUfsc 0.06 0.31 0.78 0.0234 D1037 S1037 UfgGfGfAfuUfuCfAfUfgUfaAfcCfaAfgsAf AS1037 uCf and UfgGf and UfaCfa ugAfa Af uccCfasUfsc 0.03 0.14 0.62 0.0235 D1038 S1038 UfGfggAfUfuuCfAfugUfAfacCfAfagsAf AS1038 uCfUfugGfUfuaCfAfugAfAfauCfCfcasUfsc 0.09 0.39 0.78 0.0239 D1039 S1039 AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS 1039 aUfgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.14 0.59 0.025 D1040 S1040 AfuGfuAfaCfcAfAfGfaGfuAfuUfccasUf AS1040 aUfGfGfaAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.13 0.56 0.025 D1041 S1041 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1041 asUfgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.06 0.27 0.79 0.0252 D1042 S1042 UfgGfgAfuuuCfAfUfgUfAfAfcCfaAfgsAf AS1042 uCf and UfgGf and uaCfa UgAfAfAf uCfcCfasUfsc 0.05 0.27 0.67 0.0259 D1043 S1043 AfuGfuAfaCfcAfAfGfaGfuauUfcCfasUf AS1043 aUfgGfaAfUfAfcUfcuuGfgUfuAfcAfusGfsa 0.02 0.16 0.63 0.027 D1044 S1044 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1044 asUfgGfAfAfuAfcUfcuuGfgsUfsuAfcAfusGfsa 0.06 0.30 0.81 0.0271 D1045 S1045 aUfguAfAfccAfAfgaGfGfauUfCfcasUf AS1045 aUfGfgaAfUfacUfCfuuGfGfuuAfCfaUfsgsa 0.12 0.29 0.8 0.028 D1046 S1046 AfuGfuAfaCfcAfAfGfaguAfuUfcCfasUf AS1046 aUfgGfaAfuAfCfUfcuuGfgUfuAfcAfusGfsa 0.03 0.15 0.59 0.030 D1047 S1047 UfgGfGfAfuUfuCfaUfgUfAfAfcCfaAfgsAf AS 1047 uCf and UfgGf and uaCfa UfgAfa Af uccCfasUfsc 0.08 0.44 0.83 0.0324 D1048 S1048 AfuGfuAfaCfcAfAfGfaGfuAfuUfcCfasUf AS1048 aUfgGfaAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.07 0.23 0.67 0.036 D1049 S1049 AfuGfuAfAfccAfAfGfAfGfuAfuuccAfsu AS1049 AfUfGfGfAfAfuAfCfUfCfUfUfGfGfUfuAfCfAfusGfsa 0.08 0.23 0.73 0.037

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D1O5O S1050 UfgGfgAf u uuCfa UfgUfaAfcCfAf AfgsAf AS 1050 uCfuugGfuUfaCfaUfgAfAfAfuCfcCfasUfsc 0.06 0.29 0.78 0.0372 D1O51 S1051 AfuGfuAfaccaagaguAfuUfcCfasUf AS 1051 aUfgGfaAfudAcdTcdTudGgdTuAfcAfusgsa 0.12 0.41 0.86 0.040 D1O52 S1052 AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu AS1052 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.1 0.22 0.72 0.042 D1O53 S1053 AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu AS1053 dAUdGGdAAfuAfcUfcUfuGfGfUfuAfCfAfusGfsa 0.09 0.31 0.69 0.044 D1054 S1054 AfuGfuAfaCfcAfaGfadGdTAfuUfcdCdAsUf AS 1054 adTdGGfaAfudAdCUfcUfuGfgUfuAfcAfusGfsa 0.1 0.45 0.75 0.047 D1O55 S1055 AfuguAfaccAfaGfaGfdTAdTUdCcdAsu AS1055 dAUdGGdAadTAfcUfcUfuGfgUfuAfcAfusGfsa 0.12 0.26 0.7 0.049 D1056 S1056 AuGuAaCcAaGaGuAuUcCasU AS1056 □UgGaAuAcUcUuGgUuAcAusGsa 0.08 0.24 0.65 0.050 D1O57 S1057 AfuguAfaccAfagaGfuauUfccasUf AS1057 aUfGfGfaAfUfAfcUfCfUfuGfGfUfuAfCfAfusGfsa 0.14 0.42 0.62 0.051 D1058 S1058 AfuGfuAfaccaagaguAfuUfcCfasUf AS 1058 aUfgGfaAfudAcdTcdTudGgdTuAfcAfusGfsa 0.12 0.36 0.86 0.053 D1059 S1059 AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu AS1059 dAUdGGdAadTAfdCUfcUfuGfgUfuAfcAfusGfsa 0.09 0.27 0.7 0.054 D1060 S1060 adTgudAdAccdAdAgagdTadTudCcasdT AS1060 adTdGgdAadTadCdTdCuudGdGuudAdCadTsgsa 0.11 0.37 0.66 0.056 D1061 S1061 AfuGfuAfaCfcAfaGfdAdGuAfuUfcdCdAsUf AS1061 adTdGGfaAfuAfdCdTcUfuGfgUfuAfcAfusGfsa 0.1 0.31 0.77 0.059 D1062 S1062 AfuguAfaccAfaGfdAGfdTAdTudCcdAsu AS1062 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.1 0.27 0.65 0.059 D1063 S1063 adTdGuadAdCccdAdGagdTdAuudCdCasu AS1063 dAdTggdAdAuadCdTcudTdGgudTdAcadTsdGsa 0.12 0.44 0.82 0.064 D1064 S1064 AfuGfuAfaCfcAfaGfaGfdTdAuUfcdCdAsUf AS1064 adTdGGfaAfdTdAcUfcUfuGfgUfuAfcAfusGfsa 0.12 0.32 0.83 0.064 D1065 S1065 AfuguAfaccAfaGfaGfdTAdTudCcdAsu AS1065 dAUdGgdAadTAfcUfcUfuGfgUfuAfcAfusGfsa 0.13 0.34 0.72 0.066 D1066 S1066 AfuGfuAfaCfcAfaGfaGfudAdTUfcdCdAsUf AS1066 adTdGGfadAdTAfcUfcUfuGfgUfuAfcAfusGfsa 0.11 0.33 0.72 0.067 D1067 S1067 AfuguAfaccAfaGfaGfdTAdTUdCcdAsu AS1067 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.11 0.37 0.62 0.070 D1068 S1068 AfuguAfaccAfaGfaGfdTAdTUdCcdAsu AS1068 dAUdGGdAAuAfcUfcUfuGfGfUfuAfCfAfusGfsa 0.16 0.33 0.64 0.072 D1069 S1069 aUfGfuaAfCfccAfGfagUfAfuuCfCfasu AS1069 AfUfggAfAfuaCfUfcuUfGfguUfAfcaUfsGfsa 0.14 0.43 0.73 0.074 D1O7O S1070 AfuGfuAfaCfCfAfaGfaguAfuUfcCfasUf AS 1070 aUfgGfaAfuAfCfUfcUfuggUfuAfcAfusGfsa 0.08 0.42 0.94 0.075 D1O71 S1071 UfgGfgAfuuuCfaUfgUfaAfcCfaAfgsAf AS 1071 u Cf u UfgGf u UfaCfa UfgAf Af Af u Cf cCf a sUf sc 0.14 0.28 0.83 0.0797 D1O72 S1072 AfuGfuAfaCfcAfaGfAfGfuauUfcCfasUf AS1072 aUfgGfaAfUfAfcucUfuGfgUfuAfcAfusGfsa 0.05 0.26 0.8 0.082 D1O73 S1073 AfuGfuAfaCfcAfaGfadGdTdAdTUfcCfasUf AS1073 aUfgGfadAdTdAdCUfcUfuGfgUfuAfcAfusGfsa 0.12 0.41 0.73 0.083 D1074 S1074 AfUfguAfAfccAfAfgaGfUfauUfCfcasUf AS1074 aUfGfgaAfUfacUfCfuuGfGfuuAfCfausGfsa 0.14 0.44 0.75 0.086 D1O75 S1075 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1075 aUfgGfdAdAdTdAcUfcUfuGfgUfuAfcAfusGfsa 0.1 0.41 0.72 0.088 D1076 S1076 AfuGfuAfaCfcAfaGfaGfudAdTdTdCCfasUf AS1076 aUfgdGdAdAdTAfcUfcUfuGfgUfuAfcAfusGfsa 0.15 0.45 0.86 0.088 D1O77 S1077 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasu AS1077 AfUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.08 0.46 0.95 0.092 D1078 S1078 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1078 dAUdGGdAadTAfcUfcUfuGfgUfuAfcAfusGfsa 0.09 0.32 0.76 0.093 D1079 S1079 AfuguAfaccAfaGfaGfdTadTudCcdAsu AS 1079 dAudGgdAadTAfcUfcUfuGfgUfuAfcAfusGfsa 0.14 0.38 0.76 0.095 D1080 S1080 Af u Gf u Af a Cfc Af a Gf Af Gf u Af u ucCf a sUf AS 1080 aUfgGfAfAfuAfcucUfuGfgUfuAfcAfusGfsa 0.05 0.42 0.86 0.099 D1081 S1081 Af u Gf u Af a Cf cAf a Gf a Gf u Af u Ufd CdCd AsdT AS 1081 dAdTdGdGaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.17 0.47 0.9 0.105 D1082 S1082 AfuGfuAfaccaagaguAfuUfcCfasUf AS 1082 aUfgGfaAfudACfudCUfudGGfudTAfcAfusgsa 0.12 0.44 0.83 0.106 D1083 S1083 Af u Gf u Af a Cf cAf a Gf a Gf u Af u UfcCf a sUf AS1083 adTdGGfaAfdTdAcUfcUfuGfgUfuAfcAfusGfsa 0.11 0.34 0.74 0.109 D1084 S1084 AfuGfuAfAfCfcAfaGfaGfuauUfcCfasUf AS 1084 aUfgGfaAfUfAfcUfcUfuGfguuAfcAfusGfsa 0.1 0.45 0.93 0.117 D1085 S1085 AfuGfUfAfaCfcAfaGfaGfuauUfcCfasUf AS1085 aUfgGfaAfUfAfcUfcUfuGfgUfuacAfusGfsa 0.07 0.42 0.78 0.120 D1086 S1086 aUfguAfAfccAfAfgaGfuAfuUfcCfasUf AS 1086 aUfgGfaAfuAfcUfCfuuGfGfuuAfCfaUfsgsa 0.17 0.45 0.83 0.1197 D1087 S1087 AfuGfuAfaCfcAfaGfaGfUfAfuUfcCfasu AS 1087 AfUfgGfaAfuacUfcUfuGfgUfuAfcAfusGfsa 0.05 0.3 0.7 0.120 D1088 S1088 Af u Gf u Af a Cf cAf a Gf a Gf u Af u UfcCf a sUf AS 1088 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusgsa 0.11 0.46 0.8 0.120 D1089 S1089 AfuGfuAfaCfcAfaGfaGfUfAfuUfcCfasUf AS 1089 aUfgGfaAfuacUfcUfuGfgUfuAfcAfusGfsa 0.14 0.49 0.85 0.122 D1090 S1090 AfuGfuAfaCfcAfaGfaGfuauUfcCfasUf AS 1090 aUfgGfaAfUfAfcUfcUfuGfgUfuAfcAfusGfsa 0.1 0.41 0.85 0.125 D1091 S1091 AfuguAfaccAfaGfaGfdTAdTudCcdAsu AS 1091 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.16 0.38 0.77 0.125 D1092 S1092 Af u Gf u Af a Cf cAf a Gf Af Gf u Af u UfcCf a su AS1092 AfUfgGfaAfuAfcucUfuGfgUfuAfcAfusGfsa 0.05 0.31 0.93 0.126 D1093 S1093 a u Gf u Af a Cf cAf a Gf Af Gf u Af u UfcCf a sUf AS1093 aUfgGfaAfuAfcucUfuGfgUfuAfcAfUfsGfsa 0.06 0.33 0.9 0.135 D1094 S1094 AfuGfuAfaCfcAfaGfaGfUfAfuUfccasUf AS 1094 aUfGfGfaAfuacUfcUfuGfgUfuAfcAfusGfsa 0.07 0.39 0.85 0.142 D1095 S1095 AfuGfuAfaCfcAfaGfAfGfuAfuUfcCfasUf AS1095 a UfgGfa Af u Afc u c Uf u GfgUf u Afc Af u sGf sa 0.09 0.39 0.76 0.146 D1096 S1096 AfuGfuAfaCfcAfaGfaGfUfAfuucCfasUf AS1096 aUfgGfAfAfuacUfcUfuGfgUfuAfcAfusGfsa 0.06 0.38 0.85 0.147 D1097 S1097 AfuGfUfAfaCfcAfaGfaGfuAfuucCfasUf AS1097 aUfgGfAfAfuAfcUfcUfuGfgUfuacAfusGfsa 0.12 0.47 0.87 0.147 D1098 S1098 AfuGfuAfaCfcAfaGfaGfuAfUfUfccasUf AS1098 aUfGfGfaauAfcUfcUfuGfgUfuAfcAfusGfsa 0.06 0.42 0.85 0.151 D1099 S1099 Af u Gf u Af a Cf cAf a Gf a Gf u Af u UfcCf a sUf AS 1099 dAUdGGdAadTAfdCUfcUfuGfgUfuAfcAfusGfsa 0.16 0.41 0.85 0.152 DI 100 S1100 AfuguAfaccAfaGfaGfuAfuUfcCfasUf AS 1100 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.15 0.48 0.72 0.152 D1101 S1101 AfuGfuAfaCfcAfaGfAfGfuAfuUfccasUf AS1101 aUfGfGfaAfuAfcucUfuGfgUfuAfcAfusGfsa 0.06 0.38 0.94 0.158 D1102 S1102 AfuGfuAfaccaagaguAfuUfcCfasUf AS 1102 aUfgGfaAfuAfdCuCfdTuGfdGuUfacAfusGfsa 0.21 0.45 0.89 0.162 D1103 S1103 AfuGfuaaCfCfAfaGfaGfuAfuUfcCfasUf AS1103 aUfgGfaAfuAfcUfcUfuggUfUfAfcAfusGfsa 0.14 0.49 0.95 0.163

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D1104 S1104 AfuGfuAfaccAfaGfaGfUfAfuUfcCfasUf AS1104 aUfgGfaAfuacUfcUfuGfGfUfuAfcAfusGfsa 0.06 0.36 0.92 0.163 D11O5 S1105 AfuGfuAfaCfcAfaGfaGfuAfuucCfasUf AS 1105 aUfgGfAfAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.1 0.45 0.84 0.167 D1106 S1106 AfuGfuaaCfcAfaGfAfGfuAfuUfcCfasUf AS 1106 aUfgGfaAfuAfcucUfuGfgUfUfAfcAfusGfsa 0.09 0.43 0.91 0.170 D11O7 S1107 AfuGfuAfaccAfaGfAfGfuAfuUfcCfasUf AS1107 aUfgGfaAfuAfcucUfuGfGfUfuAfcAfusGfsa 0.09 0.46 1 0.171 DI 108 S1108 AfuguAfaccAfaGfaGfdTadTudCcdAsu AS 1108 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.11 0.39 0.71 0.176 DI 109 S1109 AfuGfUfAfaCfcAfaGfaGfuAfuUfccasUf AS1109 aUfGfGfaAfuAfcUfcUfuGfgUfuacAfusGfsa 0.1 0.43 0.9 0.180 D1110 S1110 AfuGfuAfaCfcAfaGfaguAfUfUfcCfasUf AS1110 aUfgGfaauAfCfUfcUfuGfgUfuAfcAfusGfsa 0.06 0.42 0.88 0.182 Dllll Sllll AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1111 dAUdGGdAAuAfcUfcUfuGfGfUfuAfCfAfusGfsa 0.18 0.49 0.79 0.183 D1112 S1112 AfuGfUfAfaccAfaGfaGfuAfuUfcCfasUf AS1112 aUfgGfaAfuAfcUfcUfuGfGfUfuacAfusGfsa 0.14 0.48 0.85 0.195 D1113 S1113 AfuGfuAfaCfcAfaGfaguAfuUfcCfasUf AS1113 aUfgGfaAfuAfCfUfcUfuGfgUfuAfcAfusGfsa 0.09 0.41 0.85 0.201 D1114 S1114 auGfuAfaCfcAfaGfaGfUfAfuUfcCfasUf AS1114 aUfgGfaAfuacUfcUfuGfgUfuAfcAfUfsGfsa 0.05 0.44 0.94 0.201 D1115 S1115 AfuguAfaCfcAfaGfaGfUfAfuUfcCfasUf AS1115 aUfgGfaAfuacUfcUfuGfgUfuAfCfAfusGfsa 0.08 0.41 0.96 0.204 D1116 S1116 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1116 adTdGGfadAdTAfcUfcUfuGfgUfuAfcAfusGfsa 0.15 0.47 0.79 0.208 D1117 S1117 AfuGfuaaCfcAfaGfaGfUfAfuUfcCfasUf AS1117 aUfgGfaAfuacUfcUfuGfgUfUfAfcAfusGfsa 0.08 0.42 0.92 0.224 D1118 S1118 auguaaccaagaguauuccasu AS1118 AfUfGfGfAfAfUfAfCfUfCfUfUfGfGfUfUfAfCfAfUfsgsa 0.19 0.5 0.87 0.303 D1119 S1119 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1119 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.14 0.55 0.89 D1120 S1120 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1120 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.19 0.63 0.72 D1121 S1121 AfuGfuAfaccAfaGfaGfuAfuUfcCfasUf AS1121 aUfgGfaAfuAfcUfcUfuGfGfUfuAfcAfusGfsa 0.14 0.61 0.91 D1122 S1122 AfUfGfuAfaCfcAfaGfaGfuAfuUfccasUf AS1122 aUfGfGfaAfuAfcUfcUfuGfgUfuAfcausGfsa 0.14 0.54 0.95 D1123 S1123 auGfuAfAfCfcAfaGfaGfuAfuUfcCfasUf AS1123 aUfgGfaAfuAfcUfcUfuGfguuAfcAfUfsGfsa 0.13 0.61 0.97 D1124 S1124 AfuGfuAfaCfcAfaGfaGfuAfUfUfcCfasUf AS1124 aUfgGfaauAfcUfcUfuGfgUfuAfcAfusGfsa 0.14 0.56 0.94 D1125 S1125 AfuGfuAfaCfcaaGfaGfuAfuUfcCfasUf AS1125 aUfgGfaAfuAfcUfcUfUfGfgUfuAfcAfusGfsa 0.21 0.74 0.95 D1126 S1126 AfUfGfuAfaCfcAfaGfaGfuAfuucCfasUf AS1126 aUfgGfAfAfuAfcUfcUfuGfgUfuAfcausGfsa 0.2 0.69 0.91 D1127 S1127 AfuguAfAfCfcAfaGfaGfuAfuUfcCfasUf AS1127 aUfgGfaAfuAfcUfcUfuGfguuAfCfAfusGfsa 0.17 0.7 0.96 D1128 S1128 AfUfGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1128 aUfgGfaAfuAfcUfcUfuGfgUfuAfcausGfsa 0.19 0.62 0.85 D1129 S1129 AfuGfuAfaCfcAfaGfaGfuAfuUfCfCfasUf AS1129 aUfggaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.23 0.76 0.98 D1130 S1130 AfuGfuAfaCfcAfagaGfuAfuUfcCfasUf AS1130 aUfgGfaAfuAfcUfCfUfuGfgUfuAfcAfusGfsa 0.21 0.64 0.9 D1131 S1131 AfuGfuAfAfCfcaaGfaGfuAfuUfcCfasUf AS1131 aUfgGfaAfuAfcUfcUfUfGfguuAfcAfusGfsa 0.17 0.7 1.01 D1132 S1132 AfuGfUfAfaCfcAfaGfaGfuAfuUfcCfasUf ASI132 aUfgGfaAfuAfcUfcUfuGfgUfuacAfusGfsa 0.17 0.58 0.87 D1133 S1133 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfAfsUf ASI133 augGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.33 0.89 1.05 D1134 S1134 AfUfGfuAfaCfcAfaGfaguAfuUfcCfasUf ASI134 aUfgGfaAfuAfCfUfcUfuGfgUfuAfcausGfsa 0.16 0.64 0.96 D1135 S1135 AfuGfUfAfaCfcAfaGfaguAfuUfcCfasUf AS1135 aUfgGfaAfuAfCfUfcUfuGfgUfuacAfusGfsa 0.12 0.53 0.96 D1136 S1136 AfuGfuAfAfCfcAfagaGfuAfuUfcCfasUf AS1136 aUfgGfaAfuAfcUfCfUfuGfguuAfcAfusGfsa 0.16 0.58 0.98 D1137 S1137 AfuGfuAfAfCfcAfaGfaGfuAfuUfcCfasUf ASI137 a UfgGf a Af u Af c Uf cU f u Gfgu u Afc Af u sGf sa 0.16 0.6 0.91 D1138 S1138 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf ASI138 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsAf 0.1 0.54 0.91 D1139 S1139 AfUfGfuAfaCfcAfagaGfuAfuUfcCfasUf ASI139 aUfgGfaAfuAfcUfCfUfuGfgUfuAfcausGfsa 0.24 0.68 0.98 D1140 S1140 AfuGfUfAfaCfcAfagaGfuAfuUfcCfasUf ASI140 aUfgGfaAfuAfcUfCfUfuGfgUfuacAfusGfsa 0.13 0.75 0.9 D1141 S1141 AfuGfuAfAfCfcAfaGfaguAfuUfcCfasUf ASI141 aUfgGfaAfuAfCfUfcUfuGfguuAfcAfusGfsa 0.15 0.52 1.05 D1142 S1142 AfuGfuAfaCfCfAfaGfaGfuAfuUfcCfasUf ASI142 aUfgGfaAfuAfcUfcUfuggUfuAfcAfusGfsa 0.16 0.66 0.89 D1143 S1143 auGfuAfaCfcAfaGfaGfuAfuUfcCfasUf ASI143 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfUfsGfsa 0.12 0.51 0.89 D1144 S1144 AfUfGfuAfaCfcaaGfaGfuAfuUfcCfasUf ASI144 aUfgGfaAfuAfcUfcUfUfGfgUfuAfcausGfsa 0.25 0.71 0.95 D1145 S1145 AfuGfUfAfaCfcaaGfaGfuAfuUfcCfasUf ASI145 aUfgGfaAfuAfcUfcUfUfGfgUfuacAfusGfsa 0.17 0.74 0.98 D1146 S1146 AfuguAfaCfcAfaGfaGfuAfuUfcCfasUf ASI146 aUfgGfaAfuAfcUfcUfuGfgUfuAfCfAfusGfsa 0.11 0.51 0.86 D1147 S1147 AfuGfuAfaCfcAfaGfaGfuAfuUfccasUf ASI147 aUfGfGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.1 0.52 0.83 DI 148 SI 148 AfUfGfuAfaccAfaGfaGfuAfuUfcCfasUf ASI148 a UfgGfaAf u AfcUfcUf uGfGfUfu Afca usGfsa 0.14 0.63 0.98 DI 149 SI 149 AfuGfuAfAfCfcAfaGfaGfuAfuucCfasUf ASI149 aUfgGfAfAfuAfcUfcUfuGfguuAfcAfusGfsa 0.13 0.58 0.88 D1150 S1150 AfuGfuaaCfcAfaGfaGfuAfuUfcCfasUf ASI150 aUfgGfaAfuAfcUfcUfuGfgUfUfAfcAfusGfsa 0.15 0.62 0.94 D1151 S1151 AfUfGfuaaCfcAfaGfaGfuAfuUfcCfasUf AS1151 aUfgGfaAfuAfcUfcUfuGfgUfUfAfcausGfsa 0.18 0.73 0.94 D1152 S1152 auGfUfAfaCfcAfaGfaGfuAfuUfcCfasUf ASI152 aUfgGfaAfuAfcUfcUfuGfgUfuacAfUfsGfsa 0.13 0.53 0.97 D1153 S1153 AfuGfuAfAfCfcAfaGfaGfuAfuUfccasUf ASI153 aUfGfGfaAfuAfcUfcUfuGfguuAfcAfusGfsa 0.13 0.53 0.98 D1154 S1154 UfgGfgAfuUfuCfaUfgUfaAfcCfaAfgsAf ASI154 uCfuUfgGfuUfaCfaUfgAfaAfuCfcCfasUfsc 0.09 0.5 0.78 D1155 S1155 UfgGfGf Afu u uCfa UfgUf Af AfcCfa AfgsAf ASI155 uCfuUfgGfuuaCfaUfgAfAfAfuccCfasUfsc 0.13 0.62 0.89 D1156 S1156 UfgGfgAfuuuCfaUfGfUfaAfcCfaAfgsAf AS1156 uCfuUfgGfuUfacaUfgAfAfAfuCfcCfasUfsc 0.12 0.65 0.85 D1157 S1157 UfgGfgAfuUfuCfaUfgUfAfAfcCfaAfgsAf ASI157 uCfuUfgGfuuaCfaUfgAfaAfuCfcCfasUfsc 0.11 0.54 0.85

- 38 042669

D1158 S1158 UfgGfgAfuuuCfaUfgUfAfAfcCfaAfgsAf AS1158 uCfuUfgGfuuaCfaUfgAfAfAfuCfcCfasUfsc 0.13 0.53 0.8 D1159 S1159 UfGfggAfUfuUfcAfuGfuAfAfccAfAfgsAf AS1159 uCfuuGfGfuuAfcAfuGaAfauCfCfcasUfsc 0.59 0.89 0.81 DI 160 S1160 UfGfggAfUfuuCfaUfgUfAfAfcCfaAfgsAf AS1160 u Cf u UfgGf u u a Cf a UfgAf Af a uCfCfca sUf sc 0.16 0.72 0.9 D1161 S1161 UfgGfgAfuUfucaUfGfUfaAfcCfaAfgsAf AS1161 uCfuUfgGfuUfacaUfGfAfaAfuCfcCfasUfsc 0.27 0.69 0.86 D1162 S1162 AfuGfuAfaCfcaaGfaGfUfAfuUfcCfasUf AS1162 aUfgGfaAfuacUfcUfUfGfgUfuAfcAfusGfsa 0.12 0.6 0.95 D1163 S1163 AfuGfuAfaccAfaGfaGfuAfUfUfcCfasUf AS1163 aUfgGfaauAfcUfcUfuGfGfUfuAfcAfusGfsa 0.05 0.56 1.02 DI 164 S1164 AfuGfuAfaCfcAfagaGfUfAfuUfcCfasUf AS1164 aUfgGfaAfuacUfCfUfuGfgUfuAfcAfusGfsa 0.13 0.55 1 D1165 S1165 AfuGfuAfaCfcaaGfaGfuAfUfUfcCfasUf AS1165 aUfgGfaauAfcUfcUfUfGfgUfuAfcAfusGfsa 0.09 0.6 0.97 D1166 S1166 AfuguAfaCfCfAfaGfaGfuAfuUfcCfasUf AS1166 aUfgGfaAfuAfcUfcUfuggUfuAfCfAfusGfsa 0.15 0.59 0.91 D1167 S1167 AfuGfuAfaCfcAfagaGfuAfUfUfcCfasUf AS1167 aUfgGfaauAfcUfCfUfuGfgUfuAfcAfusGfsa 0.11 0.59 1 DI 168 S1168 AfuGfuAfaCfCfAfagaGfuAfuUfcCfasUf AS1168 aUfgGfaAfuAfcUfCfUfuggUfuAfcAfusGfsa 0.13 0.57 0.94 DI 169 S1169 auGfuAfaCfcAfaGfaGfuAfUfUfcCfasUf AS1169 aUfgGfaauAfcUfcUfuGfgUfuAfcAfUfsGfsa 0.08 0.5 0.9 D1170 S1170 AfuguAfaCfcAfaGfaGfuAfUfUfcCfasUf AS1170 aUfgGfaauAfcUfcUfuGfgUfuAfCfAfusGfsa 0.06 0.53 0.91 D1171 S1171 auGfuAfaCfcAfaGfaGfuAfuUfCfCfasUf AS1171 aUfggaAfuAfcUfcUfuGfgUfuAfcAfUfsGfsa 0.07 0.56 0.89 D1172 S1172 AfuGfuAfaCfCfAfaGfaGfuAfuucCfasUf AS1172 aUfgGfAfAfuAfcUfcUfuggUfuAfcAfusGfsa 0.13 0.59 0.98 D1173 S1173 AfuGfuAfaCfcaaGfAfGfuAfuUfcCfasUf AS1173 aUfgGfaAfuAfcucUfUfGfgUfuAfcAfusGfsa 0.2 0.65 1.03 D1174 S1174 AfuGfuaaCfcAfaGfaGfuAfUfUfcCfasUf AS1174 aUfgGfaauAfcUfcUfuGfgUfUfAfcAfusGfsa 0.07 0.51 0.95 D1175 S1175 AfuguAfaCfcAfaGfaGfuAfuUfCfCfasUf AS1175 aUfggaAfuAfcUfcUfuGfgUfuAfCfAfusGfsa 0.2 0.53 0.76 D1176 S1176 auGfuAfaCfcAfaGfaGfuAfuUfcCfAfsUf AS1176 augGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.74 0.98 0.81 D1177 S1177 AfuGfuAfaCfcAfaGfaGfuAfuucCfAfsUf AS1177 augGfAfAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.43 0.64 0.88 D1178 S1178 auguaaccAfaGfaGfuAfuUfcCfasUf AS1178 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.17 0.49 0.81 D1179 S1179 AfuGfuaaCfcAfaGfaGfuAfuUfCfCfasUf AS1179 aUfggaAfuAfcUfcUfuGfgUfUfAfcAfusGfsa 0.22 0.65 0.73 DI 180 SI 180 AfuguAfaCfcAfaGfaGfuAfuUfcCfAfsUf AS1180 augGfaAfuAfcUfcUfuGfgUfuAfcAfUfsGfsa 0.6 1.09 0.8 D1181 S1181 auGfuAfaCfcAfaGfaGfuAfuUfccasu AS1181 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.3 0.78 0.78 DI 182 S1182 auguaaccaaGfaGfuAfuUfcCfasUf AS1182 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.35 0.73 0.84 DI 183 S1183 AfuGfuAfaccAfaGfaGfuAfuUfCfCfasUf AS1183 aUfggaAfuAfcUfcUfuGfGfUfuAfcAfusGfsa 0.19 0.6 0.94 DI 184 SI 184 AfuGfuaaCfcAfaGfaGfuAfuUfcCfAfsUf AS1184 augGfaAfuAfcUfcUfuGfgUfuAfCfAfusGfsa 0.61 1.08 0.8 DI 185 S1185 auGfuAfaCfcAfaGfaGfuAfuuccasu ASI185 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.16 0.52 0.72 DI 186 S1186 auguaaccaagaGfuAfuUfcCfasUf ASI186 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.2 0.53 0.74 DI 187 S1187 AfuGfuAfaCfcaaGfaGfuAfuUfCfCfasUf ASI187 aUfggaAfuAfcUfcUfUfGfgUfuAfcAfusGfsa 0.34 0.66 0.85 DI 188 SI 188 AfuGfuAfaccAfaGfaGfuAfuUfcCfAfsUf ASI188 augGfaAfuAfcUfcUfuGfgUfUfAfcAfusGfsa 0.61 0.98 1.02 DI 189 SI 189 AfuGfuAfaCfcAfaGfaGfuAfuuccasu ASI189 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.3 0.73 0.85 DI 190 S1190 auguaaccaagaguauuccasu ASI190 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.28 0.69 0.78 D1191 S1191 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1191 aUfgGfaAfuAfcUfcUfugdGudTadCadTsgsa 0.33 0.88 0.64 D1192 S1192 AfuGfuAfaCfcAfagaGfuAfuUfCfCfasUf ASI192 aUfggaAfuAfcUfCfUfuGfgUfuAfcAfusGfsa 0.31 0.64 0.83 D1193 S1193 AfuGfuAfaCfcaaGfaGfuAfuUfcCfAfsUf ASI193 augGfaAfuAfcUfcUfuGfGfUfuAfcAfusGfsa 0.64 0.82 0.92 DI 194 S1194 AfuGfuAfaCfcAfaGfaGfuauuccasu ASI194 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.21 0.62 0.77 D1195 S1195 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf ASI195 aUfgGfaAfuAfcUfcUfuGfGfUfuAfCfAfusGfsa 0.17 0.7 0.95 D1196 S1196 AfuGfuAfaCfcAfaGfaguAfuUfCfCfasUf ASI196 aUfggaAfuAfCfUfcUfuGfgUfuAfcAfusGfsa 0.19 0.71 0.65 D1197 S1197 AfuGfuAfaCfcAfagaGfuAfuUfcCfAfsUf ASI197 augGfaAfuAfcUfcUfUfGfgUfuAfcAfusGfsa 0.64 0.82 0.93 DI 198 S1198 auguAfaCfcAfaGfaGfuAfuUfccasu ASI198 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.19 0.65 0.72 DI 199 S1199 AfuGfuAfaCfcAfaGfaGfuauUfCfCfasUf ASI199 aUfggaAfUfAfcUfcUfuGfgUfuAfcAfusGfsa 0.15 0.52 0.64 D1200 S1200 AfuGfuAfaCfcAfaGfaguAfuUfcCfAfsUf AS1200 augGfaAfuAfcUfCfUfuGfgUfuAfcAfusGfsa 0.48 0.74 0.92 D1201 S1201 auguAfaCfcAfaGfaGfuAfuUfcCfasu AS1201 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.17 0.71 0.77 D1202 S1202 AfuGfuAfaCfcAfaGfaGfuauUfcCfAfsUf AS1202 augGfaAfuAfCfUfcUfuGfgUfuAfcAfusGfsa 0.43 0.69 0.85 D1203 S1203 auguaaCfcAfaGfaGfuAfuUfcCfasUf AS1203 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.14 0.61 0.76 D1204 S1204 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1204 adTdGGfaAfudAdCUfcUfuGfgUfuAfcAfusGfsa 0.16 0.56 0.89 D1205 S1205 AfuGfuAfaCfcAfaGfaGfdTdAdTdTcCfasUf AS1205 aUfgGfdAdAdTdAcUfcUfuGfgUfuAfcAfusGfsa 0.13 0.57 0.9 D1206 S1206 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1206 adTdGdGdAAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.29 0.73 0.89 D1207 S1207 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1207 adTdGGfaAfuAfdCdTcUfuGfgUfuAfcAfusGfsa 0.16 0.56 0.78 D1208 S1208 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1208 aUfdGdGdAdAuAfcUfcUfuGfgUfuAfcAfusGfsa 0.22 0.67 0.89 D1209 S1209 AfuguAfaccAfaGfaGfuAfuUfcCfasUf AS1209 aUfgGfaAfuAfcUfcUfuGfGfUfuAfCfAfusGfsa 0.14 0.55 0.78 D1210 S1210 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1210 aUfgdGdAdAdTAfcUfcUfuGfgUfuAfcAfusGfsa 0.14 0.5 0.84 D1211 S1211 AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf AS1211 aUfgGfadAdTdAdCUfcUfuGfgUfuAfcAfusGfsa 0.14 0.59 0.72

- 39 042669

D1212 51212 auguaaccaaGfaGfuAfuUfcCfasUf AS1212 aUfgGfaAfuAfcUfcUfugdGudTadCadTsgsa 0.21 0.74 0.77 D1213 51213 AfuGfuAfaCfcAfaGfaGfuAfudTdCdCdAsUf AS1213 adTdGdGdAAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.15 0.53 0.91 D1214 51214 aUfgUfaAfcCfaAfgAfgUfaUfuCfcAfsu AS1214 aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.12 0.71 0.87 D1215 51215 AfuGfuAfaCfcAfaGfaGfuAfdTdTdCdCasUf AS1215 aUfdGdGdAdAuAfcUfcUfuGfgUfuAfcAfusGfsa 0.18 0.67 0.97 D1216 51216 AfuGfuAfaccaagaguAfuUfcCfasUf AS1216 aUfgGfaAfuacucuuggUfuAfcAfusgsa 0.36 0.87 1.07 D1217 51217 AfuGfuAfaccaagaguAfuUfcCfasUf AS1217 a UfgGfa Af u AfCfUfCf UfuGfGf u uAfcAf usgsa 0.37 0.73 1.03 D1218 51218 AfUfguAfAfccAfAfgaGfUfauUfCfcasUf AS1218 aUfGfgaAfUfacUfCfuuGfGfuuAfCfausGfsa 0.23 0.42 0.84 D1219 51219 AfuGfuAfaccaagaguAfuUfcCfasUf AS1219 aUfgGfaAfuaCfUfcUfUfgGfuuAfcAfusgsa 0.43 0.71 1.03 D122O S122O AfuGfuAfaccaagaguAfuUfcCfasUf AS122O aUfgGfaAfuAfcUfCfUfuGfGfuuAfcAfusgsa 0.37 0.63 0.99 D1221 S1221 AfuGfuAfaccaagaguAfuUfcCfasUf AS1221 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- 40 042669

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- 41 042669

D132O S132O Af a CfaGf u Gf u Uf cUf u Gf cUf c Ufa Ufa sAf AS132O uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfususu 0.08 0.16 0.61 D1321 S1321 AfaCfaGfuGfuUfcUfuGfcUfCfUfaUfasAf AS1321 uUfaUfagaGfcAfaGfaAfcAfcUfgUfusUfsu 0.06 0.14 0.58 D1322 S1322 Afa CfaGf u Gfu Ufcu u GfcUfc Ufa Ufa sAf AS1322 uUfaUfaGfaGfcAfAfGfaAfcAfcUfgUfusUfsu 0.15 0.49 0.84 D1323 S1323 AfaCfaGfuGfuUfcUfuGfcUfcuaUfasAf AS1323 uUfaUfAfGfaGfcAfaGfaAfcAfcUfgUfususu 0.07 0.20 0.62 D1324 S1324 Af Af CfaGf u Gf u Uf cUf u GfcUfc Ufa Ufa sAf AS1324 uUfaUfaGfaGfcAfaGfaAfcAfcUfguusUfsu 0.08 0.25 0.78 D1325 S1325 AfAfCfaGfuGfuUfcUfuGfcUfcUfaUfasAf AS1325 uUfaUfaGfaGfcAfaGfaAfcAfcUfguusUfsu 0.08 0.18 0.80 D1326 S1326 AfaCfaGfuGfuUfcUfuGfcUfcUfAfUfasAf AS1326 uUfauaGfaGfcAfaGfaAfcAfcUfgUfusUfsu 0.07 0.21 0.66 D1327 S1327 AfaCfaGfuGfuucUfuGfcUfcUfaUfasAf AS1327 uUfaUfaGfaGfcAfaGfAfAfcAfcUfgUfusUfsu 0.10 0.31 0.70 D1328 S1328 AfAfCfaGfuGfuUfcUfuGfcUfcUfauasAf AS1328 uUfAfUfaGfaGfcAfaGfaAfcAfcUfguusUfsu 0.07 0.15 0.55 D1329 S1329 Afa CfAf Gf u GfuUfcUfu GfcUfc Ufa Ufa sAf AS1329 uUfaUfaGfaGfcAfaGfaAfcAfcugUfusUfsu 0.08 0.19 0.71 D133O S133O AfaCfaGfuGfuUfcUfuGfcUfcUfaUfAfsAf AS133O uuaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu 0.09 0.27 0.76 D1331 S1331 AfaCfaGfuguUfcUfuGfcUfcUfaUfasAf AS1331 uUfaUfaGfaGfcAfaGfaAfCfAfcUfgUfusUfsu 0.07 0.21 0.65 D1332 S1332 AfAfCfaGfuGfuUfcUfuGfcUfcuaUfasAf AS1332 uUfaUfAfGfaGfcAfaGfaAfcAfcUfguusUfsu 0.07 0.17 0.53 D1333 S1333 AfaCfaGfUfGfuUfcUfuGfcUfcUfaUfasAf AS1333 uUfaUfaGfaGfcAfaGfaAfcacUfgUfusUfsu 0.08 0.25 0.73 D1334 S1334 AfaCfaguGfuUfcUfuGfcUfcUfaUfasAf AS1334 uUfaUfaGfaGfcAfaGfaAfcAfCfUfgUfusUfsu 0.07 0.18 0.54 D1335 S1335 AfaCfaGfuGfuUfcuuGfcUfcUfaUfasAf AS1335 uUfaUfaGfaGfcAfAfGfaAfcAfcUfgUfususu 0.14 0.38 0.57 D1336 S1336 AfaCfaGfuGfUfUfcUfuGfcUfcUfaUfasAf AS1336 uUfaUfaGfaGfcAfaGfaacAfcUfgUfusUfsu 0.16 0.50 0.96 D1337 S1337 AfaCfaGfuGfuUfcUfuGfcUfcUfauasAf AS1337 uUfAfUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu 0.08 0.19 0.54 D1338 S1338 AfAfCfaGfuGfuUfcUfugcUfcUfaUfasAf AS1338 uUfaUfaGfaGfCfAfaGfaAfcAfcUfguusUfsu 0.08 0.20 0.69 D1339 S1339 Afa CfaGf u Gfu UfCfUfu GfcUfc Ufa Ufa sAf AS1339 uUfaUfaGfaGfcAfagaAfcAfcUfgUfusUfsu 0.07 0.16 0.55 D1340 S1340 AfaCfaGfuGfuUfcUfuGfcUfcuaUfasAf AS1340 uUfaUfAfGfaGfcAfaGfaAfcAfcUfgUfusUfsu 0.08 0.17 0.57 D1341 S1341 AfaCfaGfuguUfcUfuGfcUfcUfaUfasAf AS1341 uUfaUfaGfaGfcAfaGfaAfCfAfcUfgUfususu 0.08 0.22 0.63 D1342 S1342 Af Af CfaGf u Gfu Ufcu u GfcUfc Ufa Ufa sAf AS1342 uUfaUfaGfaGfcAfAfGfaAfcAfcUfguusUfsu 0.21 0.56 0.86 D1343 S1343 AfacaGfuGfUfUfcUfuGfcUfcUfaUfasAf AS1343 uUfaUfaGfaGfcAfaGfaacAfcUfGfUfusUfsu 0.14 0.37 0.73 D1344 S1344 AfaCfaGfuGfuucUfUfGfcUfcUfaUfasAf AS1344 uUfaUfaGfaGfcaaGfAfAfcAfcUfgUfusUfsu 0.08 0.20 0.66 D1345 S1345 Afa CfAf Gf u Gfu Ufcu u GfcUfc Ufa Ufa sAf AS1345 uUfaUfaGfaGfcAfAfGfaAfcAfcugUfusUfsu 0.12 0.34 0.73 D1346 S1346 AfaCfaGfuGfUfUfcUfuGfcUfcUfauasAf AS1346 uUfAfUfaGfaGfcAfaGfaacAfcUfgUfusUfsu 0.16 0.42 0.90 D1347 S1347 AfaCfaGfuGfUfUfcUfuGfcUfcUfaUfasAf AS1347 uUfaUfaGfaGfcAfaGfaacAfcUfgUfusUfsUf 0.17 0.43 0.85 D1348 S1348 AfaCfAfGfuGfuucUfuGfcUfcUfaUfasAf AS1348 uUfaUfaGfaGfcAfaGfAfAfcAfcugUfusUfsu 0.08 0.21 0.58 D1349 S1349 AfaCfaGfuGfUfUfcUfuGfcUfcuaUfasAf AS1349 uUfaUfAfGfaGfcAfaGfaacAfcUfgUfusUfsu 0.21 0.39 0.88 D135O S135O AfaCfaguGfuUfcUfUfGfcUfcUfaUfasAf AS135O uUfaUfaGfaGfcaaGfaAfcAfCfUfgUfusUfsu 0.06 0.13 0.52 D1351 S1351 AfaCfAfGfuguUfcUfuGfcUfcUfaUfasAf AS1351 uUfaUfaGfaGfcAfaGfaAfCfAfcugUfusUfsu 0.08 0.21 0.58 D1352 S1352 AfaCfaGfUfGfuUfcuuGfcUfcUfaUfasAf AS1352 uUfaUfaGfaGfcAfAfGfaAfcacUfgUfusUfsu 0.18 0.49 0.84 D1353 S1353 AfaCfaGfuGfUfUfcUfuGfcucUfaUfasAf AS1353 uUfaUfaGfAfGfcAfaGfaacAfcUfgUfusUfsu 0.11 0.25 0.68 D1354 S1354 AfacaGfuGfuUfcUfUfGfcUfcUfaUfasAf AS1354 uUfaUfaGfaGfcaaGfaAfcAfcUfGfUfusUfsu 0.07 0.15 0.52 D1355 S1355 AfaCfaGfUfGfuucUfuGfcUfcUfaUfasAf AS1355 uUfaUfaGfaGfcAfaGfAfAfcacUfgUfusUfsu 0.10 0.26 0.63 D1356 S1356 AfaCfaGfuGfUfUfcUfugcUfcUfaUfasAf AS1356 uUfaUfaGfaGfCfAfaGfaacAfcUfgUfusUfsu 0.16 0.33 0.79 D1357 S1357 AfaCfAfGfuGfuUfcUfuGfcUfcUfaUfasAf AS1357 uUfaUfaGfaGfcAfaGfaAfcAfcugUfusUfsUf 0.09 0.19 0.51 D1358 S1358 AfaCfaGfuGfUfUfcuuGfcUfcUfaUfasAf AS1358 uUfaUfaGfaGfcAfAfGfaacAfcUfgUfusUfsu 0.22 0.48 0.71 D1359 S1359 AfaCfaGfuGfuUfcUfUfGfcUfcUfaUfasAf AS1359 uUfaUfaGfaGfcaaGfaAfcAfcUfgUfusUfsUf 0.10 0.17 0.61 D1360 S1360 AfaCfaguGfUfUfcUfuGfcUfcUfaUfasAf AS1360 uUfaUfaGfaGfcAfaGfaacAfCfUfgUfusUfsu 0.14 0.40 0.87 D1361 S1361 AfaCfaGfuGfuUfcUfUfGfcUfcuaUfasAf AS1361 uUfaUfAfGfaGfcaaGfaAfcAfcUfgUfusUfsu 0.07 0.14 0.52 D1362 S1362 aaCfaGfuGfuUfcUfuGfCfUfcUfaUfasAf AS1362 u Ufa Ufa Gf agcAf a Gf a Af cAfc UfgUf Uf sUfsu 0.10 0.28 0.81 D1363 S1363 AfaCfaGfuGfuucUfuGfcUfcUfAfUfasAf AS1363 u Ufa u a GfaGfc Af a Gf Af Afc Af c UfgUf u sUfsu 0.06 0.16 0.68 D1364 S1364 AfaCfaGfuGfuUfcUfugcUfcUfaUfAfsAf AS1364 uuaUfaGfaGfCfAfaGfaAfcAfcUfgUfusUfsu 0.09 0.26 0.67 D1365 S1365 aacaguguucuugcucuauasa AS1365 uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu 0.20 0.59 0.95 D1366 S1366 AfaCfaGfuGfuUfcUfuGfCfUfcUfauasAf AS1366 uUfAfUfaGfagcAfaGfaAfcAfcUfgUfusUfsu 0.06 0.13 0.53 D1367 S1367 AfaCfaGfuGfuUfcUfuGfCfUfcUfaUfasAf AS1367 u Ufa Ufa Gf age Afa Gf a Af cAf c UfgUf u s UfsUf 0.08 0.16 0.53 D1368 S1368 AfaCfaGfuguUfcUfuGfcUfcUfAfUfasAf AS1368 uUfauaGfaGfcAfaGfaAfCfAfcUfgUfusUfsu 0.07 0.15 0.54 D1369 S1369 AfaCfaGfuGfuUfcuuGfcUfcUfaUfAfsAf AS1369 uuaUfaGfaGfcAfAfGfaAfcAfcUfgUfusUfsu 0.23 0.56 0.89 D137O S137O AfaCfaGfuGfuUfcUfuGfCfUfcuaUfasAf AS137O uUfaUfAfGfagcAfaGfaAfcAfcUfgUfusUfsu 0.06 0.12 0.55 D1371 S1371 AfaCfaGfuGfuUfcUfuGfCfUfcuaUfasAf AS1371 uUfaUfAfGfagcAfaGfaAfcAfcUfgUfusUfsu 0.07 0.18 0.58 D1372 S1372 AfaCfaguGfuUfcUfuGfcUfcUfAfUfasAf AS1372 uUfauaGfaGfcAfaGfaAfcAfCfUfgUfusUfsu 0.06 0.15 0.56 D1373 S1373 AfaCfaGfuGfuucUfuGfcUfcUfaUfAfsAf AS1373 uuaUfaGfaGfcAfaGfAfAfcAfcUfgUfusUfsu 0.21 0.51 0.89

- 42 042669

D1374 S1374 AfacaGfuguUfcUfuGfcUfcUfaUfasAf AS1374 u Ufa UfaGfaGfcAfaGfa AfCfAfc UfGf Uf usUfsu 0.08 0.21 0.64 D1375 S1375 AfaCfaGfuGfuUfcuuGfCfUfcUfaUfasAf AS1375 uUfaUfaGfagcAfAfGfaAfcAfcUfgUfusUfsu 0.15 0.40 0.94 D1376 S1376 AfaCfaGfuGfuUfcuuGfCfUfcUfaUfasAf AS1376 uUfaUfaGfagcAfAfGfaAfcAfcUfgUfusUfsu 0.13 0.40 0.96 D1377 S1377 AfaCfaGfuGfuUfcUfuGfcUfCfUfauasAf AS1377 uUfAfUfagaGfcAfaGfaAfcAfcUfgUfusUfsu 0.08 0.17 0.64 D1378 S1378 AfaCfaGfuguUfcUfuGfcUfcUfaUfAfsAf AS1378 uuaUfaGfaGfcAfaGfaAfCfAfcUfgUfusUfsu 0.18 0.50 0.97 D1379 S1379 AfaCfaGfuGfuucUfuGfCfUfcUfaUfasAf AS1379 uUfaUfaGfagcAfaGfAfAfcAfcUfgUfusUfsu 0.08 0.24 0.79 D1380 S1380 aaCfaGfuGfuUfcUfuGfcUfcUfAfUfasAf AS1380 uUfauaGfaGfcAfaGfaAfcAfcUfgUfUfsUfsu 0.07 0.14 0.58 D1381 S1381 AfaCfaguGfuUfcUfuGfcUfcUfaUfAfsAf AS1381 uuaUfaGfaGfcAfaGfaAfcAfCfUfgUfusUfsu 0.11 0.34 0.96 D1382 S1382 AfaCfaGfuguUfcUfuGfCfUfcUfaUfasAf AS1382 uUfaUfaGfagcAfaGfaAfCfAfcUfgUfusUfsu 0.08 0.18 0.69 D1383 S1383 AfaCfaGfuGfuUfcuuGfcUfCfUfaUfasAf AS1383 uUfaUfagaGfcAfAfGfaAfcAfcUfgUfusUfsu 0.14 0.38 0.85 D1384 S1384 AfaCfaGfuGfuUfcUfuGfcUfcUfAfUfasAf AS1384 uUfauaGfaGfcAfaGfaAfcAfcUfgUfusUfsUf 0.07 0.16 0.54 D1385 S1385 AfacaGfuGfuUfcUfuGfcUfcUfaUfAfsAf AS1385 uuaUfaGfaGfcAfaGfaAfcAfcUfGfUfusUfsu 0.08 0.20 0.75 D1386 S1386 aacaguguucUfuGfcUcUaudAsa AS1386 uUfdAUdAGfaGfcAfaGfaadCadCudGdTdTsusu 0.25 0.56 0.90 D1387 S1387 AfaCfaguGfuUfcUfuGfCfUfcUfaUfasAf AS1387 uUfaUfaGfagcAfaGfaAfcAfCfUfgUfusUfsu 0.08 0.19 0.70 D1388 S1388 AfaCfaGfuGfuucUfuGfcUfCfUfaUfasAf AS1388 uUfaUfagaGfcAfaGfAfAfcAfcUfgUfusUfsu 0.08 0.14 0.60 D1389 S1389 AfaCfaGfuGfuUfcUfuGfcUfcuaUfAfsAf AS1389 uuaUfAfGfaGfcAfaGfaAfcAfcUfgUfusUfsu 0.08 0.19 0.62 D1390 S1390 aaCfaGfuGfuUfcUfuGfcUfcUfaUfAfsAf AS1390 uuaUfaGfaGfcAfaGfaAfcAfcUfgUfUfsUfsu 0.08 0.27 0.76 D1391 S1391 aacaguguucdTudGcdTcdTadTasa AS1391 uUfdAUdAGfaGfcAfaGfaadCadCudGudTsusu 0.18 0.36 0.81 D1392 S1392 AfacaGfuGfuUfcUfuGfCfUfcUfaUfasAf AS1392 uUfaUfaGfagcAfaGfaAfcAfcUfGfUfusUfsu 0.07 0.17 0.55 D1393 S1393 AfaCfaGfuguUfcUfuGfcUfCfUfaUfasAf AS1393 uUfaUfagaGfcAfaGfaAfCfAfcUfgUfusUfsu 0.07 0.15 0.57 D1394 S1394 AfaCfaGfuGfuUfcuuGfcUfcUfAfUfasAf AS1394 uUfauaGfaGfcAfAfGfaAfcAfcUfgUfusUfsu 0.26 0.68 1.06 D1395 S1395 AfaCfaGfuGfuUfcUfuGfcucUfaUfAfsAf AS1395 uuaUfaGfAfGfcAfaGfaAfcAfcUfgUfusUfsu 0.06 0.18 0.58 D1396 S1396 AfaCfaGfuGfuUfcUfuGfcUfcUfaUfAfsAf AS1396 uuaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsUf 0.09 0.27 0.73 D1397 S1397 AfaCfaAfuGfuUfcUfuGfcdAdCdTdAUfasAf AS1397 uUfadTdAdGdAGfcAfaGfaGfcAfcAfgUfusUfsu 0.20 0.51 0.73 D1398 S1398 AfacaGfuguUfcuuGfcucUfauasAf AS1398 uUfAfUfaGfAfGfcAfAfGfaAfCfAfcUfGfUfusUfsu 0.13 0.34 0.86 D1399 S1399 dAacadGugudTcuudGcucdTauasdA AS1399 udTdAdTadGdAdGcdAdAdGadAdCdAcdTdGdTusdTsu 0.24 0.42 0.82 D1400 S1400 AfaCfaAfuGfuUfcUfuGfdCdAdCdTaUfasAf AS1400 uUfaUfdAdGdAdGcAfaGfaGfcAfcAfgUfusUfsu 0.49 0.85 0.78 D1401 S1401 AfaCfaAfuGfuUfcUfudGdCdAdCUfaUfasAf AS1401 uUfaUfadGdAdGdCAfaGfaGfcAfcAfgUfusUfsu 0.67 0.83 0.85 D1402 S1402 aaCfAfguGfUfucUfUfgcUfCfuaUfAfsa AS1402 uUfaUfAfgaGfCfaaGfAfacAfCfugUfUfsusu 0.18 0.47 0.80 D1403 S1403 AfaCfaAfuGfuUfcUfuGfcdAdCUfadTdAsAf AS 1403 udTdAUfadGdAGfcAfaGfaGfcAfcAfgUfusUfsu 0.73 0.89 0.77 D1404 S1404 aacAgugUucuUgcuCuauAsa AS1404 uUaUAgAGCaAGAaCACuGUUsusu 0.12 0.39 0.79 D1405 S1405 AacaGuguUcuuGcucUauasA AS 1405 uUAUaGAGcAAGaACAcUGUusUsu 0.12 0.37 0.77 D1406 S1406 AfaCfaAfuGfuUfcUfudGdCAfcUfadTdAsAf AS 1406 udTdAUfaGfadGdCAfaGfaGfcAfcAfgUfusUfsu 0.59 0.93 0.89 D1407 S1407 aACagUGuuCUugCUcuAUasa AS1407 UUauAGagCAagAAcaCUguUsUsu 0.09 0.16 0.55 D1408 S1408 AfaCfaAfuGfuUfcUfuGfcAfcdTdAdTdAsAf AS1408 udTdAdTdAGfaGfcAfaGfaAfcAfcAfgUfusUfsu 0.22 0.64 0.86 D1409 S1409 aaCAguGUucUUgcUCuaUAsa AS1409 uUaUAgaGCaaGAacACugUUsusu 0.13 0.31 0.76 D1410 S1410 AfaCfaAfuGfuUfcUfuGfcAfdCdTdAdTdAsAf AS1410 udTdAdTdAdGaGfcAfaGfaGfcAfcAfgUfusUfsu 0.77 0.94 0.93 D1411 S1411 aacAfgugUfucuUfgcuCfuauAfsa AS1411 uUfaUfAfgAfGfCfaAfGfAfaCfAfCfuGfUfUfsusu 0.23 0.53 1.04 D1412 S1412 aacdAgugdTucudTgcudCuaudAsa AS1412 udTadTdAgdAdGdCadAdGdAadCdAdCudGdTdTsusu 0.30 0.64 0.90 D1413 S1413 Afa Cf a Gf u Gf u Uf c Uf u Gf c Ufc Ufa Uf a sa AS1413 UfUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu 0.09 0.19 0.63 D1414 S1414 AfaCfaGfuGfUfUfcUfuGfcUfcUfaUfasa AS1414 UfUfaUfaGfaGfcAfaGfaacAfcUfgUfusUfsu 0.11 0.28 0.66 D1415 S1415 AfaCfaGfuGfuUfcUfuGfCfUfcUfaUfasa AS1415 UfUfaUfaGfagcAfaGfaAfcAfcUfgUfusUfsu 0.06 0.13 0.53 D1416 S1416 aacaguguucuugcucuauasa AS1416 UfUfAfUfAfGfAfGfCfAfAfGfAfAfCfAfCfUfGfUfUfsusu 0.20 0.53 0.99 D1417 S1417 Afa Cf a Gf u Gf u Uf c Uf u Gfc Ufc UfAf Uf a sa AS1417 UfUfauaGfaGfcAfaGfaAfcAfcUfgUfusUfsu 0.07 0.17 0.53 D1418 S1418 aAfCfagUfGfuuCfUfugCfUfcuAfUfasa AS1418 UfUfauAfGfagCfAfagAfAfcaCfUfguUfsUfsu 0.08 0.20 0.70 D1419 S1419 Afa Cf Af Gf u Gf u Uf c Uf u Gf c Uf c Ufa Uf a sAf AS1419 uUfaUfaGfaGfcAfaGfaAfcAfcugUfusUfsUf 0.08 0.20 0.70

Example 3 In vitro silencing activity with various chemical modifications on siRNA TTR.

The IC ₅₀ for each modified siRNA is determined on Hep3B cells by standard reversible transfection using Lipofectamine RNAiMAX. Briefly, reversible transfection is performed by adding 5 µl of Opti-MEM to 5 µl of siRNA duplex per well in a 96-well plate along with 10 µl of Opti-MEM plus 0.5 µl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad, CA, cat. 13778-150) and incubating at room temperature for 15-20 minutes. Then, after incubation, 100 µl of complete growth medium without antibiotics containing 12,000-15,000 Hep3B cells is added to each well. Cells are incubated for 24 hours at 37° C. in a 5% CO ₂ atmosphere, then lysed and ApoB and GAPDH mRNA analyzed with bDNA (Quantigene). Analyze seven different concentrations of siRNA, ranging from 10 nm to 0.6 pM, to determine the IC ₅₀ and ApoB/GAPDH for ApoB-transfected cells normalized to cells transfected with 10 nm siRNA Luc.

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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'-0-methyladenosine c 2'-O-methylcytidine g 2'-0-methylguanosine u 2'-O-methyluridine dT 2'-deoxythymidine s phosphorothioate bond

Modified duplex ANGPTL3

table 2

Duplex ID semantic ID SS sequence AS ID AS sequence RNAimax, Ner3 IOihM 0.1 nM 0.025 nM D2000 S2000 UfcAfcAfa Uf u Af AfGfcUfcCf u UfcUfu Uf A2OOO aAfaGfaAfgGfaGfcuuAfaUfuGfuGfasAfsc 0.036 0.274 0.233 D2001 S2001 UfuAfuUfgUfuCfCfUfcUfaGfuUfaUfuUf A2OO1 aAfaUfaAfcUfaGfaggAfaCfaAfuAfasAfsa 0.044 0.278 0.247 D2002 S2002 GfcUfaUfgUfuAfGfAfcGfaUfgUfaAfaAf A2OO2 uUfuUfaCfaUfcGfucuAfaCfaUfaGfcsAfsa 0.062 0.474 0.449 D2003 S2003 GfgAfcAfuGfgUfCfUfuAfaAfgAfcUfuUf A2OO3 a Af a Gf u Cf u Uf u Af aga Cf cAf u Gf u Cf csCf sa 0.303 1.042 0.912 D2004 S2004 CfaAfaAfaCfuCfAfAfcAfuAfuUfuGfaUf A2004 a UfcAfa Afu AfuGf u ugAfgUfu Uf u UfgsAfsa 0.102 0.623 0.499 D2005 S2005 AfcCfaGfuGfaAfAfUfcAfaAfgAfaGfaAf A2OO5 uUfcUfuCfuUfuGfauuUfcAfcUfgGfusUfsu 0.124 0.901 0.756 D2006 S2006 CfaCfaAfuUfaAfGfCfuCfcUfuCfuUfuUf A2OO6 aAfaAfgAfaGfgAfgcuUfaAfuUfgUfgsAfsa 0.069 0.269 0.244 D2007 S2007 CfuAfuGfuUfaGfAfCfgAfuGfuAfaAfaAf A2OO7 uUfuUfuAfcAfuCfgucUfaAfcAfuAfgsCfsa 0.052 0.622 0.589 D2008 S2008 UfcAfaCfaUfaUfUfUfgAfuCfaGfuCfuUf A2008 aAfgAfcUfgAfuCfaaaUfaUfgUfuGfasGfsu 0.133 0.798 0.785 D2009 S2009 AfaCfuGfaGfaAfGfAfaCfuAfcAfuAfuAf A2OO9 uAfuAfuGfuAfgUfucuUfcUfcAfgUfusCfsc 0.097 0.671 0.528 D2010 S2010 AfcAfa Uf u AfaGfCfUfcCfu UfcUfu Ufu Uf A2O1O aAfaAfaGfaAfgGfagcUfuAfaUfuGfusGfsa 0.145 0.308 0.293 D2011 S2011 CfuCfcAfgAfgCfCfAfaAfaUfcAfaGfaUf A2O11 aUfcUfuGfaUfuUfuggCfuCfuGfgAfgsAfsu 0.122 0.882 0.938 D2012 S2012 CfgAfuGfuAfaAfAfAfuUfuUfaGfcCfaAf A2O12 uUfgGfcUfaAfaAfuuuUfuAfcAfuCfgsUfsc 0.102 0.843 0.733 D2013 S2013 GfuCfuUfaAfaGfAfCfuUfuGfuCfcAfuAf A2O13 uAfuGfgAfcAfaAfgucUfuUfaAfgAfcsCfsa 1.133 1.105 1.022 D2014 S2014 CfaAfcAfuAfuUfUfGfaUfcAfgUfcUfuUf A2014 aAfaGfaCfuGfaUfcaaAfuAfuGfuUfgsAfsg 0.077 0.413 0.450 D2015 S2015 Af c Uf g Af gAf a G fAf Af c UfaCfaUfaUfaAf A2O15 uUfaUfaUfgUfaGfuucUfuCfuCfaGfusUfsc 0.055 0.293 0.364 D2016 S2016 CfcAfgAfgCfcAf Af Afa UfcAfaGfa Ufu Uf A2O16 a Af a Ufc Uf u Gfa Uf u u u GfgCf u Cf u GfgsAf sg 0.080 0.650 0.499 D2017 S2017 GfaUfgUfaAfaAfAfUfuUfuAfgCfcAfaUf A2O17 aUfuGfgCfuAfaAfauuUfuUfaCfaUfcsGfsu 0.076 0.605 0.579 D2018 S2018 UfcUfuAfaAfgAfCfUfuUfgUfcCfaUfaAf A2018 uUfaUfgGfaCfaAfaguCfuUfuAfaGfasCfsc 1.326 1.098 0.927 D2019 S2019 AfaCfaUfaUfuUfGfAfuCfaGfuCfuUfuUf A2O19 aAfaAfgAfcUfgAfucaAfaUfaUfgUfusGfsa 0.047 0.560 0.477 D2020 S2020 CfuGfaGfaAfgAfAfCfuAfcAfuAfuAfaAf A2O2O uUfuAfuAfuGfuAfguuCfuUfcUfcAfgsUfsu 0.066 0.690 0.681 D2021 S2021 Afa Uf uAfaGfcUfCfCf u UfcUfu Ufu Ufa Uf A2O21 aUfaAfaAfaGfaAfggaGfcUfuAfaUfusGfsu 0.041 0.611 0.251 D2022 S2022 AfaAfuCfaAfgAfUfUfuGfcUfaUfgUfuAf A2O22 uAfaCfaUfaGfcAfaauCfuUfgAfuUfusUfsg 0.053 0.555 0.516 D2023 S2023 UfuCfaGfuUfgGfGfAfcAfuGfgUfcUfuAf A2O23 uAfaGfaCfcAfuGfuccCfaAfcUfgAfasGfsg 0.779 1.045 0.963

- 44 042669

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 Af u Ufa AfgCf uCfCf Uf uCf u Uf u Ufu Afu Uf 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 Gfa UfaAfu AfgCf AfUfcAfa AfgAfcCfu Uf 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 u Af u Uf u Cf a Cf u Uf u u u Gf u UfgAf a Gf u sAf sg 0.198 0.969 0.865 D2059 S2059 GfaGfcAfaCfuAfAfCfuAfaCfuUfaAfuUf A2059 aAfuUfaAfgUfuAfguuAfgUfuGfcUfcsUfsu 0.031 0.253 0.210 D2060 S2060 Afa Cf cAfa Cf a Gf Cf Af u AfgU f cAfa Af u Af A2060 uAfuUfuGfaCfuAfugcUfgUfuGfgUfusUfsa 0.035 0.561 0.569 D2061 S2061 AfgUfcAfaAfuAfAfAfaGfaAfaUfaGfaAf A2061 uUfcUfaUfuUfcUfuuuAfuUfuGfaCfusAfsu 0.057 0.668 0.386 D2062 S2062 AfgUfcAfaAfaAfUfGfaAfgAfgGfuAfaAf A2062 u Ufu AfcCf uCfu Ufca u Ufu UfuGfaCf usUfsg 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 0.520 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 u Ufu UfgAfa Ufu Afagu UfaGf u UfaGf usUfsg 0.020 0.312 0.316

- 45 042669

D2078 S2078 CfaAfcAfgCfaUfAfGfuCfaAfaUfaAfaAf A2078 u Ufu Ufa Ufu UfgAfcua UfgCf uGf u UfgsGfsu 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 u Uf uCf u Uf u Ufa Ufu ugAfcUfa UfgCfusGfsu 0.038 0.606 0.589 D2091 S2091 AfgAfcCfcAfgCfAfAfcUfcUfcAfaGfuUf A2091 aAfcUfuGfaGfaGfuugCfuGfgGfuCfusGfsa 0.301 0.850 0.753 D2092 S2092 AfgUfcCfaUfgGfAfCfaUfuAfaUfuCfaAf A2092 u UfgAfa Ufu Afa UfgucCfa UfgGfaCf usAfsc 0.407 0.791 0.726 D2093 S2093 Gfa UfgGfa UfcAfCf Afa AfaCfu UfcAfa Uf A2093 aUfuGfaAfgUfuUfuguGfaUfcCfaUfcsUfsa 0.120 0.658 0.654 D2094 S2094 CfuAfgAfgAfaGfAfUfaUfaCfuCfcAfuAf A2094 uAfuGfgAfgUfaUfaucUfuCfuCfuAfgsGfsc 0.071 0.610 0.645 D2095 S2095 AfaAfgAfcAfaCfAfAfaCfaUfuAfuAfuUf A2095 a Afu AfuAfa UfgUfu ugUfuGfuCfu Uf usCfsc 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 GfgAf u Cf a Cfa AfAfAf c Uf u Cf a Af u Gf a Af A2098 uUfcAfuUfgAfaGfuuuUfgUfgAfuCfcsAfsu 0.130 0.831 0.951 D2099 S2099 Gfa AfgAf uAfu AfCf UfcCfa UfaGf u Gfa Af A2099 uUfcAfcUfaUfgGfaguAfuAfuCfuUfcsUfsc 0.058 0.828 0.938 D21OO S21OO GfaCfaAfcAfaAfCfAfuUfaUfaUfuGfaAf A2100 uUfcAfaUfaUfaAfuguUfuGfuUfgUfcsUfsu 0.026 0.564 0.856 D21O1 S21O1 GfgGfaAfaUfcAfCfGfaAfaCfcAfaCfuAf A2101 uAfgUfuGfgUfuUfcguGfaUfuUfcCfcsAfsa 0.314 0.948 1.033 D21O2 S21O2 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 D21O5 S21O5 AfcUfcCfaUfaGfUfGfaAfgCfaAfuCfuAf A21O5 uAfgAfuUfgCfuUfcacUfaUfgGfaGfusAfsu 0.100 0.823 0.925 D2106 S2106 AfcAfaCfaAfaCfAfUfuAfuAfuUfgAfaUf A2106 aUfuCfaAfuAfuAfaugUfuUfgUfuGfusCfsu 0.035 0.565 0.843 D2107 S2107 GfgAfaAfuCfaCfGfAfaAfcCfaAfcUfaUf A2107 a UfaGfu UfgGfu Uf ucgUfgAf u UfuCfcsCfsa 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 D211O S211O AfaCfgUfgGfgAfGfAfaCfuAfcAfaAfuAf A211O 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 u Af u UfcGfa UfgUfuga Af u Ufa AfuGf usCfsc 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 D212O S212O UfcAfgGfuAfgUfCfCfaUfgGfaCfaUfuAf A212O 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 AfcAfa AfcAfu UfAfUfa Uf uGfa Af u Afu Uf A2124 aAfuAfuUfcAfaUfauaAfuGfuUfuGfusUfsg 0.044 0.680 0.828 D2125 S2125 AfaUfgCfaAfuCfCfCfgGfaAfaAfcAfaAf A2125 u UfuGfu Uf u UfcCfgggAfu UfgCfa Uf usGfsg 0.349 0.971 1.005 D2126 S2126 Cf a GfgUf a Gf u Cf Cf Af u GfgAf c Afu Ufa Af 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 D213O S213O AfaCfaUfuAfuAfUfUfgAfaUfaUfuCfuUf A213O aAfgAfaUfaUfuCfaauAfuAfaUfgUfusUfsg 0.249 0.916 0.981 D2131 S2131 GfcAfaUfcCfcGfGfAfaAfaCfaAfaGfaUf A2131 aUfcUfuUfgUfuUfuccGfgGfaUfuGfcsAfsu 0.435 1.002 1.019

- 46 042669

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 AfcAf u Ufa Ufa Uf UfGfa Af u Af u UfcUfu Uf 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 uCf u Uf u UfaAfa Uf u ugCfcUfcAfgUf usCfsa 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 u Ufu UfgCfu Uf uGf uga UfcCfcAfaGf usAfsg 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 a UfuGfcCf u Ufu Ufaaa Uf u UfgCfcUfcsAfsg 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 AfaGfa Ufu UfgGfUfGf u Uf u UfcUfaCfu Uf A2171 aAfgUfaGfaAfaAfcacCfaAfaUfcUfusUfsg 0.303 1.065 0.977 D2172 S2172 AfaAfcAfaCfcUfAfAfaUfgGfuAfaAfuAf A2172 uAfuUfuAfcCfaUfuuaGfgUfuGfuUfusUfsc 0.101 0.855 0.880 D2173 S2173 AfuAfcUfcUfaUfAfAfaAfuCfaAfcCfaAf A2173 u UfgGf u UfgAf u Ufu ua UfaGfaGf u Af usAfsa 0.107 0.961 0.960 D2174 S2174 UfgAfaCfuGfaGfGfCfaAfaUfuUfaAfaAf A2174 uUfuUfaAfaUfuUfgccUfcAfgUfuCfasUfsu 0.078 0.714 0.878 D2175 S2175 GfgCfa Afa Uf u Uf AfAfa AfgGfcAfa Ufa Af 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) are grown to near confluence at 37° C. in 5% CO2 in RPMI (ATCC) supplemented with 10% FBS, streptomycin and glutamine (ATCC) and then removed from the dish by trypsinization. Transfection is performed by adding 14.8 µl of Opti-MEM plus 0.2 µl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad, CA, cat. no. 13788-150) to 5 µl of siRNA duplexes per well in a 96-well plate, and incubated at room temperature for 15 min. Then, 80 µl of complete growth medium without antibiotics containing ~2x10 ⁴ Hep3B cells is added to the siRNA mixture. Cells are incubated for either 24 or 120 hours and then the RNA is purified. Single dose experiments are performed at a final concentration of duplexes of 10 and 0.1 nM, and dose-response experiments are performed using a final concentration of duplexes of 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005 , 0.001, 0.0005, 0.0001, 0.00005, and 0.00001 nM unless otherwise noted.

Synthesis of cDNA using ABI High Throughput cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat # 4368813).

To 10 µl total RNA is added a master mix of 2 µl 10X buffer, 0.8 µl 25X dNTP, 2 µl random primers, 1 µl reverse transcriptase, 1 µl RNase inhibitor and 3.2 µl H ₂ O per reaction.

Claims (22)

tion. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cell (Hercules, CA) through the following steps: 25°C 10 min, 37°C 120 min, 85°C 5 s, 4°C hold. PCR in real time. To a master mix containing 0.5 µl of GAPDH TaqMan probe (Applied Biosystems, cat. no. 4236317E), 0.5 µl of ANGPTL TaqMan probe (Applied Biosystems, cat. no. Hs00205581 ml), and 5 µl of Lightcycler 480 probe master mix (Roche, 04887301001) per well in 50 384-well plates (Roche, cat. no. 04887301001), add 2 µl of cDNA. Real-time PCR was carried out on an ABI 7900HT real-time PCR system (Applied Biosystems) using AACt(RQ) assay. Each duplex was tested in two independent transfections and each transfection was analyzed in duplicate unless otherwise noted in the summary tables. In order to calculate the relative fold change, real-time data are analyzed using the AACt method and normalized to analyzes performed with cells transfected with 10 nM AD-1955 or sham-transfected cells. IC50 is calculated using a 4-parameter fit model using XLFit and normalized to cells transfected with 10 nM AD-1955 or naive cells at the same dose range or their own lowest dose. The AD-1955 sequence used as a negative control targets luciferase and has the following sequence: sense: cuuAcGcuGAGuAcuucGAdTsdT, antisense: UCGAAGuACUcAGCGuAAGdT sdT The various embodiments described above can be combined to produce other embodiments. All US patents, US patent application publications, patents of other countries, patent applications of other countries and non-patent publications mentioned in the present description are incorporated herein by reference. Aspects of the embodiments may be modified as the concepts of various patents, applications, and publications need to be used to provide additional embodiments. Such and other changes may 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 as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be construed as including all possible embodiments along with the full scope of equivalents to which such claims entitle. Accordingly, the claims are not limited to the disclosure. CLAIM 1. Double-stranded RNAi agent capable of inhibiting the expression of a target gene, containing a sense strand and an antisense strand, where each strand has from 14 to 30 nucleotides, where the duplex is represented by formula (III) sense: 5' n _p -N _a -(XXX) _i -N _b -YYY-N _b -(ZZZ) _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') _i -N _a '-n _q '5' (III), where i, j, k and l are each independently 0 or 1; p and q are each independently 0-6; each Na and Na' is independently an oligonucleotide sequence containing 2-20 modified nucleotides, where each sequence contains at least two differently modified nucleotides, each Nb and Nb' is independently an oligonucleotide sequence containing 1-10 modified nucleotides; each Dp, n _p ', nq and n _q ' independently represents an overhanging nucleotide sequence containing 0-6 nucleotides; and each XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' independently represents one motif of three identical modifications on three consecutive nucleotides; and where the modification in Nb is different from the modification in Y, and the modification in Nb' is different from the modification in Y'; where motif ΎΎΎ' is in positions 11, 12 and 13, counting from the first pair of nucleotides in the duplex region from the 5' end of the antisense strand; and where e Ύ' represents 2'-F and Ύ' represents 2'-OMe, where each nucleotide Ύ forms a base pair with the nucleotide Ύ'; where the antisense strand contains two blocks of two phosphorothioate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide bonds connections. 2. The double stranded RNAi agent of claim 1, wherein i is 1, j is 1, or both i and j are 1. 3. The double-stranded RNAi agent of claim 1, wherein k is 1, l is 1, or both k and l are 1. 4. Double-stranded RNAi agent according to claim 1, where the length of the duplex region is 17-30 nuk pairs - 48 042669 leotids. 5. The double-stranded RNAi agent according to claim 4, wherein the length of the duplex region is 17-19 base pairs. 6. The double-stranded RNAi agent according to claim 4, wherein the length of the duplex region is 27-30 base pairs. 7. The double stranded RNAi agent of claim 1, wherein each strand has 17-30 nucleotides. 8. The double-stranded RNAi agent of claim 1, wherein the nucleotide modifications are selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C- allyl, 2'-fluoro, 2'-deoxy, and combinations thereof. 9. The double-stranded RNAi agent of claim 8, wherein the nucleotides are modified with either 2'-OCH ₃ or 2'-F. 10. The double-stranded RNAi agent of claim 1 further comprising at least one ligand. 11. The double-stranded RNAi agent of claim 10 wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. 12. The double-stranded RNAi agent of claim 1, wherein the nucleotide modifications are selected from the group consisting of a 2'-O-methyl nucleotide, a 2'-deoxyfluoronucleotide, a 2'-O-N-methylacetamido (2'-O-NMA) nucleotide, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, 2'-O-aminopropyl (2'-O-AP) nucleotide, 2'-ara-F and combinations thereof. 13. Double-stranded RNAi agent at π. 11 where the ligand is attached to the 3' end of the sense strand. 14. The double-stranded RNAi agent of claim 1, wherein the base pair at position 1 of the 5' end of the duplex is an AU base pair. 15. The double stranded RNAi agent of claim 1, wherein each strand has 17-23 nucleotides. 16. Double-stranded RNAi agent according to claim 1, wherein formula (III) is represented as sense strand: 5' N _a -(XXX),-N _b -YYY-N _b - (ZZZ) _r N _a 3' antisense strand: 3 ' n _p '-N _a '-(X'X'X') _k -N _b '-YYY'-N _b '-(Z'Z'Z')i-Na'5' 17. The double stranded RNAi agent of claim 16 wherein each nucleotide modification is independently selected from the group consisting of 2'-fluoro and 2'-O-methyl. 18. The double-stranded RNAi agent of claim 16 further comprising at least one ligand. 19. The double-stranded RNAi agent of claim 18, wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. 20. The double stranded RNAi agent of claim 19 wherein the ligand is attached to the 3' end of the sense strand. 21. The double stranded RNAi agent of claim 16, wherein each strand has 17-30 nucleotides. 22. The double stranded RNAi agent of claim 16, wherein each strand has 17-23 nucleotides. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2