Modulation of HSP47 expression

RELATED PATENT APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. Nos. 61/372,072, filed Aug. 9, 2010, 61/307,412, filed Feb. 23, 2010 and 61/285,149, filed Dec. 9, 2009 each entitled “Modulation of HSP47 Expression” and which are incorporated herein by reference in their entirety and for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which is entitled 220-PCT1_ST25_—07Dec-10.txt, said ASCII copy, created on Dec. 7, 2010 and is 533 kb in size, is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are compositions and methods for modulating expression of hsp47.

BACKGROUND OF THE INVENTION

Sato, Y., et al. disclose the administration of vitamin A-coupled liposomes to deliver small interfering RNA (siRNA) against gp46, the rat homolog of human heat shock protein 47, to liver cirrhosis rat animal models. Sato, Y., et al., Nature Biotechnology, vol. 26 (4), p. 431-442 (2008).

Chen, J-J., et al. disclose transfecting human keloid samples with HSP47-shRNA (small hairpin RNA) to examine proliferation of keloid fibroblast cells. Chen, J-J., et al., British Journal of Dermatology, vol. 156, p. 1188-1195 (2007).

PCT Patent Publication No. WO 2006/068232 discloses an astrocyte specific drug carrier which includes a retinoid derivative and/or a vitamin A analog.

SUMMARY OF THE INVENTION

Compositions, methods and kits for modulating expression of target genes are provided herein. In various aspects and embodiments, compositions, methods and kits provided herein modulate expression of heat shock protein 47 (hsp47), also known as SERPINH1. The compositions, methods and kits may involve use of nucleic acid molecules (for example, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA) or short hairpin RNA (shRNA)) that bind a nucleotide sequence (such as an mRNA sequence) encoding hsp47, for example, the mRNA coding sequence for human hsp47 exemplified by SEQ ID NO:1. In certain preferred embodiments, the compositions, methods and kits disclosed herein inhibit expression of hsp47. For example, siNA molecules (e.g., RISC length dsNA molecules or Dicer length dsNA molecules) are provided that reduce or inhibit hsp47 expression. Also provided are compositions, methods and kits for treating and/or preventing diseases, conditions or disorders associated with hsp47, such as liver fibrosis, cirrhosis, pulmonary fibrosis including lung fibrosis (including ILF), kidney fibrosis resulting from any condition (e.g., CKD including ESRD), peritoneal fibrosis, chronic hepatic damage, fibrillogenesis, fibrotic diseases in other organs, abnormal scarring (keloids) associated with all possible types of skin injury accidental and jatrogenic (operations); scleroderma; cardiofibrosis, failure of glaucoma filtering operation; and intestinal adhesions.

In one aspect, provided are nucleic acid molecules (e.g., siNA molecules) in which (a) the nucleic acid molecule includes a sense strand and an antisense strand; (b) each strand of the nucleic acid molecule is independently 15 to 49 nucleotides in length; (c) a 15 to 49 nucleotide sequence of the antisense strand is complementary to a sequence of an mRNA encoding human hsp47 (e.g., SEQ ID NO: 1); and (d) a 15 to 49 nucleotide sequence of the sense strand is complementary to the a sequence of the antisense strand and includes a 15 to 49 nucleotide sequence of an mRNA encoding human hsp47 (e.g., SEQ ID NO: 1).

In certain embodiments, the sequence of the antisense strand that is complementary to a sequence of an mRNA encoding human hsp47 includes a sequence complimentary to a sequence between nucleotides 600-800; or 801-899; or 900-1000; or 1001-1300 of SEQ ID NO: 1; or between nucleotides 650-730; or 900-975 of SEQ ID NO: 1. In some embodiments, the antisense strand includes a sequence that is complementary to a sequence of an mRNA encoding human hsp47 corresponding to nucleotides 674-693 of SEQ ID NO: 1 or a portion thereof; or nucleotides 698-716 of SEQ ID NO: 1 or a portion thereof; or nucleotides 698-722 of SEQ ID NO: 1 or a portion thereof; or nucleotides 701-720 of SEQ ID NO: 1 or a portion thereof; or nucleotides 920-939 of SEQ ID NO: 1 or a portion thereof; or nucleotides 963-982 of SEQ ID NO: 1 or a portion thereof; or nucleotides 947-972 of SEQ ID NO: 1 or a portion thereof; or nucleotides 948-966 of SEQ ID NO: 1 or a portion thereof; or nucleotides 945-969 of SEQ ID NO: 1 or a portion thereof; or nucleotides 945-963 of SEQ ID NO: 1 or a portion thereof.

In certain embodiments, the antisense strand of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes a sequence corresponding to SEQ ID NO: 4 or a portion thereof; or SEQ ID NO: 6 or a portion thereof; or SEQ ID NO: 8 or a portion thereof; or SEQ ID NO: 10 or a portion thereof; or SEQ ID NO: 12 or a portion thereof; or SEQ ID NO: 14 or a portion thereof; or SEQ ID NO: 16 or a portion thereof; or SEQ ID NO: 18 or a portion thereof; or SEQ ID NO: 20 or a portion thereof; or SEQ ID NO: 22 or a portion thereof; or SEQ ID NO: 24 or a portion thereof; or SEQ ID NO: 26 or a portion thereof; or SEQ ID NO: 28 or a portion thereof; or SEQ ID NO: 30 or a portion thereof; or SEQ ID NO: 32 or a portion thereof; or SEQ ID NO: 34 or a portion thereof; or SEQ ID NO: 36 or a portion thereof; or SEQ ID NO: 38 or a portion thereof; or SEQ ID NO: 40 or a portion thereof; or SEQ ID NO: 42 or a portion thereof; or SEQ ID NO: 44 or a portion thereof; or SEQ ID NO: 46 or a portion thereof; or SEQ ID NO: 48 or a portion thereof; or SEQ ID NO: 50 or a portion thereof; or SEQ ID NO: 52 or a portion thereof; or SEQ ID NO: 54 or a portion thereof; or SEQ ID NO: 56 or a portion thereof; or SEQ ID NO: 58 or a portion thereof. In certain embodiments, the sense strand of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes a sequence corresponding to SEQ ID NO: 3 or a portion thereof; or SEQ ID NO: 5 or a portion thereof; or SEQ ID NO: 7 or a portion thereof; or SEQ ID NO: 9 or a portion thereof; or SEQ ID NO: 11 or a portion thereof; or SEQ ID NO: 13 or a portion thereof; or SEQ ID NO: 15 or a portion thereof; or SEQ ID NO: 17 or a portion thereof; or SEQ ID NO: 19 or a portion thereof; or SEQ ID NO: 21 or a portion thereof; or SEQ ID NO: 23 or a portion thereof; or SEQ ID NO: 25 or a portion thereof; or SEQ ID NO: 27 or a portion thereof; or SEQ ID NO: 29 or a portion thereof; or SEQ ID NO: 31 or a portion thereof; or SEQ ID NO: 33 or a portion thereof; or SEQ ID NO: 35 or a portion thereof; or SEQ ID NO: 37 or a portion thereof; or SEQ ID NO: 39 or a portion thereof; or SEQ ID NO: 41 or a portion thereof; or SEQ ID NO: 43 or a portion thereof; or SEQ ID NO: 45 or a portion thereof; or SEQ ID NO: 47 or a portion thereof; or SEQ ID NO: 49 or a portion thereof; or SEQ ID NO: 51 or a portion thereof; or SEQ ID NO: 53 or a portion thereof; or SEQ ID NO: 55 or a portion thereof; or SEQ ID NO: 57 or a portion thereof.

In some embodiments the antisense and sense strands of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes the sequence pairs set forth in SERPINH1_—4 (SEQ ID NOS:195 and 220). In some embodiments of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes the antisense and sense strands of the sequence pairs set forth in SERPINH1_—12 (SEQ ID NOS:196 and 221). In some embodiments the antisense and sense strands of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes the sequence pairs set forth in SERPINH1_—30 (SEQ ID NOS:199 and 224). In some embodiments of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes the antisense and sense strands of the sequence pairs set forth in SERPINH1_—58 (SEQ ID NOS:208 and 233).

In certain embodiments, the antisense strand of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes a sequence corresponding to any one of the antisense sequences shown on any one of Tables B or C.

In certain preferred embodiments, the antisense strand of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes a sequence corresponding to any one of the antisense sequences shown on Table A-18. In certain preferred embodiments the antisense strand and the strand are selected from the sequence pairs shown in Table A-18. In some embodiments of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes the antisense and sense strands selected from the sequence pairs set forth in SERPINH1_—2 (SEQ ID NOS: 60 and 127), SERPINH1_—6 (SEQ ID NOS: 63 and 130), SERPINH1_—11 (SEQ ID NOS: 68 and 135), SERPINH1_—13 (SEQ ID NOS: 69 and 136), SERPINH1_—45 (SEQ ID NOS: 97 and 164), SERPINH1_—45a (SEQ ID NOS: 98 and 165), SERPINH1_—51 (SEQ ID NOS: 101 and 168), SERPINH1_—52 (SEQ ID NOS:102 and 169) or SERPINH1_—86 (SEQ ID NOS: 123 and 190). In some preferred embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—2 (SEQ ID NOS: 60 and 127), SERPINH1_—6 (SEQ ID NOS: 63 and 130). SERPINH1_—45a (SEQ ID NOS: 98 and 165), and SERPINH1_—51 (SEQ ID NOS: 101 and 168).

In some preferred embodiments of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes the antisense and sense strands selected from the sequence pairs set forth in SERPINH1_—2 (SEQ ID NOS: 60 and 127). In some embodiments the antisense and sense strands include the sequence pairs set forth in SERPINH1_—6 (SEQ ID NOS: 63 and 130). In some embodiments of a nucleic acid molecule (e.g., a siNA molecule) as disclosed herein includes the antisense and sense strands of the sequence pairs set forth in SERPINH1_—11 (SEQ ID NOS: 68 and 135). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—13 (SEQ ID NOS: 69 and 136). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—45 (SEQ ID NOS: 97 and 164). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—45a (SEQ ID NOS: 98 and 165). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—51 (SEQ ID NOS: 101 and 168).

In various embodiments of nucleic acid molecules (e.g., siNA molecules) as disclosed herein, the antisense strand may be 15 to 49 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleotides in length); or 17-35 nucleotides in length; or 17-30 nucleotides in length; or 15-25 nucleotides in length; or 18-25 nucleotides in length; or 18-23 nucleotides in length; or 19-21 nucleotides in length; or 25-30 nucleotides in length; or 26-28 nucleotides in length. In some embodiments of nucleic acid molecules (e.g., siNA molecules) as disclosed herein, the antisense strand may be 19 nucleotides in length Similarly the sense strand of nucleic acid molecules (e.g., siNA molecules) as disclosed herein may be 15 to 49 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleotides in length); or 17-35 nucleotides in length; or 17-30 nucleotides in length; or 15-25 nucleotides in length; or 18-25 nucleotides in length; or 18-23 nucleotides in length; or 19-21 nucleotides in length; or 25-30 nucleotides in length; or 26-28 nucleotides in length. In some embodiments of nucleic acid molecules (e.g., siNA molecules) as disclosed herein, the sense strand may be 19 nucleotides in length. In some embodiments of nucleic acid molecules (e.g., siNA molecules) as disclosed herein, the antisense strand and the sense strand may be 19 nucleotides in length. The duplex region of the nucleic acid molecules (e.g., siNA molecules) as disclosed herein may be 15-49 nucleotides in length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleotides in length), 15-35 nucleotides in length; or 15-30 nucleotides in length; or about 15-25 nucleotides in length; or 17-25 nucleotides in length; or 17-23 nucleotides in length; or 17-21 nucleotides in length; or 25-30 nucleotides in length; or 25-28 nucleotides in length. In various embodiments of nucleic acid molecules (e.g., siNA molecules) as disclosed herein, the duplex region may be 19 nucleotides in length.

In certain embodiments, the sense and antisense strands of a nucleic acid (e.g., an siNA nucleic acid molecule) as provided herein are separate polynucleotide strands. In some embodiments, the separate antisense and sense strands form a double stranded structure via hydrogen bonding, for example, Watson-Crick base pairing. In some embodiments the sense and antisense strands are two separate strands that are covalently linked to each other. In other embodiments, the sense and antisense strands are part of a single polynucleotide strand having both a sense and antisense region; in some preferred embodiments the polynucleotide strand has a hairpin structure.

In certain embodiments, the nucleic acid molecule (e.g., siNA molecule) is a double stranded nucleic acid (dsNA) molecule that is symmetrical with regard to overhangs, and has a blunt end on both ends. In other embodiments the nucleic acid molecule (e.g., siNA molecule) is a dsNA molecule that is symmetrical with regard to overhangs, and has an overhang on both ends of the dsNA molecule; preferably the molecule has overhangs of 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides; preferably the molecule has 2 nucleotide overhangs. In some embodiments the overhangs are 5′ overhangs; in alternative embodiments the overhangs are 3′ overhangs. In certain embodiments, the overhang nucleotides are modified with modifications as disclosed herein. In some embodiments the overhang nucleotides are 2′-deoxynucleotides.

In certain preferred embodiments, the nucleic acid molecule (e.g., siNA molecule) is a dsNA molecule that is asymmetrical with regard to overhangs, and has a blunt end on one end of the molecule and an overhang on the other end of the molecule. In certain embodiments the overhang is 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides; preferably the overhang is 2 nucleotides. In some preferred embodiments an asymmetrical dsNA molecule has a 3′-overhang (for example a two nucleotide 3′-overhang) on one side of a duplex occurring on the sense strand; and a blunt end on the other side of the molecule. In some preferred embodiments an asymmetrical dsNA molecule has a 5′-overhang (for example a two nucleotide 5′-overhang) on one side of a duplex occurring on the sense strand; and a blunt end on the other side of the molecule. In other preferred embodiments an asymmetrical dsNA molecule has a 3′-overhang (for example a two nucleotide 3′-overhang) on one side of a duplex occurring on the antisense strand; and a blunt end on the other side of the molecule. In some preferred embodiments an asymmetrical dsNA molecule has a 5′-overhang (for example a two nucleotide 5′-overhang) on one side of a duplex occurring on the antisense strand; and a blunt end on the other side of the molecule. In certain preferred embodiments, the overhangs are 2′-deoxynucleotides.

In some embodiments, the nucleic acid molecule (e.g., siNA molecule) has a hairpin structure (having the sense strand and antisense strand on one polynucleotide), with a loop structure on one end and a blunt end on the other end. In some embodiments, the nucleic acid molecule has a hairpin structure, with a loop structure on one end and an overhang end on the other end (for example a 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide overhang); in certain embodiments, the overhang is a 3′-overhang; in certain embodiments the overhang is a 5′-overhang; in certain embodiments the overhang is on the sense strand; in certain embodiments the overhang is on the antisense strand.

In some preferred embodiments, the nucleic acid molecule is selected from the nucleic acid molecules shown on Table I.

The nucleic acid molecules (e.g., siNA molecule) disclosed herein may include one or more modifications or modified nucleotides such as described herein. For example, a nucleic acid molecule (e.g., siNA molecule) as provided herein may include a modified nucleotide having a modified sugar; a modified nucleotide having a modified nucleobase; or a modified nucleotide having a modified phosphate group. Similarly, a nucleic acid molecule (e.g., siNA molecule) as provided herein may include a modified phosphodiester backbone and/or may include a modified terminal phosphate group.

Nucleic acid molecules (e.g., siNA molecules) as provided may have one or more nucleotides that include a modified sugar moiety, for example as described herein. In some preferred embodiments the modified sugar moiety is selected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-deoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-(CH₂)₂—O-2′-bridge, 2′-locked nucleic acid, and 2′-O—(N-methylcarbamate).

Nucleic acid molecules (e.g., siNA molecules) as provided may have one or more modified nucleobase(s) for example as described herein, which preferably may be one selected from the group consisting of xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methyl guanine, and acyclonucleotides.

Nucleic acid molecules (e.g., siNA molecules) as provided may have one or more modifications to the phosphodiester backbone, for example as described herein. In some preferred embodiments the phosphodiester bond is modified by substituting the phosphodiester bond with a phosphorothioate, 3′-(or -5′)deoxy-3′-(or -5′)thio-phosphorothioate, phosphorodithioate, phosphoroselenates, 3′-(or -5′)deoxy phosphinates, borano phosphates, 3′-(or -5′)deoxy-3′-(or 5′-)amino phosphoramidates, hydrogen phosphonates, borano phosphate esters, phosphoramidates, alkyl or aryl phosphonates and phosphotriester or phosphorus linkages.

In various embodiments, the provided nucleic acid molecules (e.g., siNA molecules) may include one or modifications in the sense strand but not the antisense strand. In some embodiments the provided nucleic acid molecules (e.g., siNA molecules) include one or more modifications in the antisense strand but not the sense strand. In some embodiments the provided nucleic acid molecules (e.g., siNA molecules) include one or more modifications in the both the sense strand and the antisense strand.

In some embodiments in which the provided nucleic acid molecules (e.g., siNA molecules) have modifications, the sense strand includes a pattern of alternating modified and unmodified nucleotides, and/or the antisense strand includes a pattern of alternating modified and unmodified nucleotides; in some preferred versions of such embodiments the modification is a 2′-O-methyl (2′ methoxy or 2′OMe) sugar moiety. The pattern of alternating modified and unmodified nucleotides may start with a modified nucleotide at the 5′ end or 3′ end of one of the strands; for example the pattern of alternating modified and unmodified nucleotides may start with a modified nucleotide at the 5′ end or 3′ end of the sense strand and/or the pattern of alternating modified and unmodified nucleotides may start with a modified nucleotide at the 5′ end or 3′ end of the antisense strand. When both the antisense and sense strand include a pattern of alternating modified nucleotides, the pattern of modified nucleotides may be configured such that modified nucleotides in the sense strand are opposite modified nucleotides in the antisense strand; or there may be a phase shift in the pattern such that modified nucleotides of the sense strand are opposite unmodified nucleotides in the antisense strand and vice-versa.

The nucleic acid molecules (e.g., siNA molecules) as provided herein may include 1-3 (i.e., 1, 2 or 3) deoxynucleotides at the 3′ end of the sense and/or antisense strand.

The nucleic acid molecules (e.g., siNA molecules) as provided herein may include a phosphate group at the 5′ end of the sense and/or antisense strand.

In one aspect, provided are double stranded nucleic acid molecules having the structure (A1):

(A1) 5′ (N)x-Z 3′ (antisense strand)

- 3′ Z′—(N′)y-z″ 5′ (sense strand)
  
  wherein each of N and N′ is a nucleotide which may be unmodified or modified, or an unconventional moiety;
  
  wherein each of (N)x and (N′)y is an oligonucleotide in which each consecutive N or N′ is joined to the next N or N′ by a covalent bond;
  
  wherein each of Z and Z′ is independently present or absent, but if present independently includes 1-5 consecutive nucleotides or non-nucleotide moieties or a combination thereof covalently attached at the 3′ terminus of the strand in which it is present;
  
  wherein z″ may be present or absent, but if present is a capping moiety covalently attached at the 5′ terminus of (N′)y;
  
  wherein each of x and y is independently an integer between 18 and 40;
  
  wherein the sequence of (N′)y has complementary to the sequence of (N)x; and wherein (N)x includes an antisense sequence to SEQ ID NO:1. In some embodiments (N)x includes an antisense oligonucleotide present in Table A-19. In other embodiments (N)x is selected from an antisense oligonucleotide present in Tables B or C.

In some embodiments the covalent bond joining each consecutive N or N′ is a phosphodiester bond.

In some embodiments x=y and each of x and y is 19, 20, 21, 22 or 23. In various embodiments x=y=19.

In some embodiments of nucleic acid molecules (e.g., siNA molecules) as disclosed herein, the double stranded nucleic acid molecule is a siRNA, siNA or a miRNA.

In some embodiments, the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—4 (SEQ ID NOS:195 and 220), SERPINH1_—12 (SEQ ID NOS:196 and 221), SERPINH1_—30 (SEQ ID NOS:199 and 224), and SERPINH1_—58 (SEQ ID NOS:208 and 233),

In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—4 (SEQ ID NOS:195 and 220). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—12 (SEQ ID NOS:196 and 221). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—30 (SEQ ID NOS:199 and 224), In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—58 (SEQ ID NOS:208 and 233).

In some embodiments the double stranded nucleic acid molecules comprise a DNA moiety or a mismatch to the target at position 1 of the antisense strand (5′ terminus). Such a structure is described herein. According to one embodiment provided are modified nucleic acid molecules having a structure (A2) set forth below:

(A2) 5′ N¹—(N)x-Z 3′ (antisense strand)

- 3′ Z′—N²—(N′)y-z″ 5′ (sense strand)
  
  wherein each of N², N and N′ is an unmodified or modified ribonucleotide, or an unconventional moiety;
  
  wherein each of (N)x and (N′)y is an oligonucleotide in which each consecutive N or N′ is joined to the adjacent N or N′ by a covalent bond;
  
  wherein each of x and y is independently an integer between 17 and 39;
  
  wherein the sequence of (N′)y has complementarity to the sequence of (N)x and (N)x has complementarity to a consecutive sequence in a target RNA;
  
  wherein N¹is covalently bound to (N)x and is mismatched to the target RNA or is a complementary DNA moiety to the target RNA;
  
  wherein N¹is a moiety selected from the group consisting of natural or modified uridine, deoxyribouridine, ribothymidine, deoxyribothymidine, adenosine or deoxyadenosine;
  
  wherein z″ may be present or absent, but if present is a capping moiety covalently attached at the 5′ terminus of N²—(N′)y; and
  
  wherein each of Z and Z′ is independently present or absent, but if present is independently 1-5 consecutive nucleotides, consecutive non-nucleotide moieties or a combination thereof covalently attached at the 3′ terminus of the strand in which it is present.

In some embodiments the sequence of (N′)y is fully complementary to the sequence of (N)x. In various embodiments sequence of N²—(N′)y is complementary to the sequence of N¹—(N)x. In some embodiments (N)x comprises an antisense that is fully complementary to about 17 to about 39 consecutive nucleotides in a target RNA. In other embodiments (N)x comprises an antisense that is substantially complementary to about 17 to about 39 consecutive nucleotides in a target RNA.

In some embodiments N¹and N²form a Watson-Crick base pair, In some embodiments N¹and N²form a non-Watson-Crick base pair. In some embodiments a base pair is formed between a ribonucleotide and a deoxyribonucleotide.

In some embodiments x=y=18, x=y=19 or x=y=20. In preferred embodiments x=y=18. When x=18 in N¹—(N)x, N¹refers to position land positions 2-19 are included in (N)₁₈. When y=18 in N²—(N′)y, N²refers to position 19 and positions 1-18 are included in (N′)₁₈.

In some embodiments N¹is covalently bound to (N)x and is mismatched to the target RNA. In various embodiments N¹is covalently bound to (N)x and is a DNA moiety complementary to the target RNA.

In some embodiments a uridine in position 1 of the antisense strand is substituted with an N¹selected from adenosine, deoxyadenosine, deoxyuridine (dU), ribothymidine or deoxythymidine. In various embodiments N¹selected from adenosine, deoxyadenosine or deoxyuridine.

In some embodiments guanosine in position 1 of the antisense strand is substituted with an N¹selected from adenosine, deoxyadenosine, uridine, deoxyuridine, ribothymidine or deoxythymidine. In various embodiments N¹is selected from adenosine, deoxyadenosine, uridine or deoxyuridine.

In some embodiments cytidine in position 1 of the antisense strand is substituted with an N¹selected from adenosine, deoxyadenosine, uridine, deoxyuridine, ribothymidine or deoxythymidine. In various embodiments N¹is selected from adenosine, deoxyadenosine, uridine or deoxyuridine.

In some embodiments adenosine in position 1 of the antisense strand is substituted with an N¹selected from deoxyadenosine, deoxyuridine, ribothymidine or deoxythymidine. In various embodiments N¹selected from deoxyadenosine or deoxyuridine.

In some embodiments N¹and N²form a base pair between uridine or deoxyuridine, and adenosine or deoxyadenosine. In other embodiments N¹and N²form a base pair between deoxyuridine and adenosine.

In some embodiments the double stranded nucleic acid molecule is a siRNA, siNA or a miRNA. The double stranded nucleic acid molecules as provided herein are also referred to as “duplexes”.

In some embodiments (N)x includes and antisense oligonucleotide present in Table A-18. In some embodiments x=y=18 and N1-(N)x includes an antisense oligonucleotide present in Table A-18. In some embodiments x=y=19 or x=y=20. In certain preferred embodiments x=y=−18. In some embodiments x=y=18 and the sequences of N1-(N)x and N2-(N′)y are selected from the pair of oligonucleotides set forth in Table A-18. In some embodiments x=y=18 and the sequences of N1-(N)x and N2-(N′)y are selected from the pair of oligonucleotides set forth in Tables D and E. In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—2 (SEQ ID NOS: 60 and 127), SERPINH1_—6 (SEQ ID NOS: 63 and 130), SERPINH1_—11 (SEQ ID NOS: 68 and 135), SERPINH1_—13 (SEQ ID NOS: 69 and 136), SERPINH1_—45 (SEQ ID NOS: 97 and 164), SERPINH1_—45a (SEQ ID NOS: 98 and 165), SERPINH1_—51 (SEQ ID NOS: 101 and 168), SERPINH1_—51a (SEQ ID NOS: 105 and 172), SERPINH1_—52 (SEQ ID NOS:102 and 169) or SERPINH1_—86 (SEQ ID NOS: 123 and 190). In some preferred embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—2 (SEQ ID NOS: 60 and 127), SERPINH1_—6 (SEQ ID NOS: 63 and 130), SERPINH1_—45a (SEQ ID NOS: 98 and 165), SERPINH1_—51 (SEQ ID NOS: 101 and 168) and SERPINH1_—51a (SEQ ID NOS: 105 and 172).

In some preferred embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—2 (SEQ ID NOS: 60 and 127). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—6 (SEQ ID NOS: 63 and 130). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—11 (SEQ ID NOS: 68 and 135). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—13 (SEQ ID NOS: 69 and 136). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—45 (SEQ ID NOS: 97 and 164). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—45a (SEQ ID NOS: 98 and 165). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—51 (SEQ ID NOS: 101 and 168). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—51a (SEQ ID NOS: 105 and 172). In some embodiments the antisense and sense strands are the sequence pairs set forth in SERPINH1_—52 (SEQ ID NOS:102 and 169). In some embodiments the antisense and sense strands are the sequence pairs set forth in (SEQ ID NOS: 123 and 190). In some preferred embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—2 (SEQ ID NOS: 60 and 127), SERPINH1_—6 (SEQ ID NOS: 63 and 130), SERPINH1_—45a (SEQ ID NOS: 98 and 165), SERPINH1_—51 (SEQ ID NOS: 101 and 168) and SERPINH1_—51a (SEQ ID NOS: 105 and 172).

In some embodiments N1 and N2 form a Watson-Crick base pair. In other embodiments N1 and N2 form a non-Watson-Crick base pair. In some embodiments N1 is a modified riboadenosine or a modified ribouridine.

In some embodiments N1 and N2 form a Watson-Crick base pair. In other embodiments N1 and N2 form a non-Watson-Crick base pair. In certain embodiments N1 is selected from the group consisting of riboadenosine, modified riboadenosine, deoxyriboadenosine, modified deoxyriboadenosine. In other embodiments N1 is selected from the group consisting of ribouridine, deoxyribouridine, modified ribouridine, and modified deoxyribouridine,

In certain embodiments position 1 in the antisense strand (5′ terminus) includes deoxyribouridine (dU) or adenosine. In some embodiments N1 is selected from the group consisting of riboadenosine, modified riboadenosine, deoxyriboadenosine, modified deoxyriboadenosine and N2 is selected from the group consisting of ribouridine, deoxyribouridine, modified ribouridine, and modified deoxyribouridine. In certain embodiments N1 is selected from the group consisting of riboadenosine and modified riboadenosine and N2 is selected from the group consisting of ribouridine and modified ribouridine.

In certain embodiments N1 is selected from the group consisting of ribouridine, deoxyribouridine, modified ribouridine, and modified deoxyribouridine and N2 is selected from the group consisting of riboadenosine, modified riboadenosine, deoxyriboadenosine, modified deoxyriboadenosine. In certain embodiments N1 is selected from the group consisting of ribouridine and deoxyribouridine and N2 is selected from the group consisting of riboadenosine and modified riboadenosine. In certain embodiments N1 is ribouridine and N2 is riboadenosine. In certain embodiments N1 is deoxyribouridine and N2 is riboadenosine.

In some embodiments of Structure (A2), N1 includes 2′OMe sugar-modified ribouracil or 2′OMe sugar-modified riboadenosine. In certain embodiments of structure (A), N2 includes a 2′OMe sugar modified ribonucleotide or deoxyribonucleotide.

In some embodiments of Structure (A2), N1 includes 2′OMe sugar-modified ribouracil or 2′OMe sugar-modified ribocytosine. In certain embodiments of structure (A), N2 includes a 2′OMe sugar modified ribonucleotide.

In some embodiments each of N and N′ is an unmodified nucleotide. In some embodiments at least one of N or N′ includes a chemically modified nucleotide or an unconventional moiety. In some embodiments the unconventional moiety is selected from a mirror nucleotide, an abasic ribose moiety and an abasic deoxyribose moiety. In some embodiments the unconventional moiety is a mirror nucleotide, preferably an L-DNA moiety. In some embodiments at least one of N or N′ includes a 2′OMe sugar-modified ribonucleotide.

In some embodiments the sequence of (N′)y is fully complementary to the sequence of (N)x. In other embodiments the sequence of (N′)y is substantially complementary to the sequence of (N)x.

In some embodiments (N)x includes an antisense sequence that is fully complementary to about 17 to about 39 consecutive nucleotides in a target mRNA. In other embodiments (N)x includes an antisense that is substantially complementary to about 17 to about 39 consecutive nucleotides in a target mRNA.

In some embodiments of Structure A1 and Structure A2 the compound is blunt ended, for example wherein both Z and Z′ are absent. In an alternative embodiment, at least one of Z or Z′ is present. Z and Z′ independently include one or more covalently linked modified and or unmodified nucleotides, including deoxyribonucleotides and ribonucleotides, or an unconventional moiety for example inverted abasic deoxyribose moiety or abasic ribose moiety; a non-nucleotide C3, C4 or C5 moiety, an amino-6 moiety, a mirror nucleotide and the like. In some embodiments each of Z and Z′ independently includes a C3 moiety or an amino-C6 moiety. In some embodiments Z′ is absent and Z is present and includes a non-nucleotide C3 moiety. In some embodiments Z is absent and Z′ is present and includes a non-nucleotide C3 moiety.

In some embodiments of Structure A1 and Structure A2, each N consists of an unmodified ribonucleotide. In some embodiments of Structure A1 and Structure A2, each N′ consists of an unmodified nucleotide. In preferred embodiments, at least one of N and N′ is a modified ribonucleotide or an unconventional moiety.

In other embodiments the compound of Structure A1 or Structure A2 includes at least one ribonucleotide modified in the sugar residue. In some embodiments the compound includes a modification at the 2′ position of the sugar residue. In some embodiments the modification in the 2′ position includes the presence of an amino, a fluoro, an alkoxy or an alkyl moiety. In certain embodiments the 2′ modification includes an alkoxy moiety. In preferred embodiments the alkoxy moiety is a methoxy moiety (also known as 2′-O-methyl; 2′OMe; 2′-OCH3). In some embodiments the nucleic acid compound includes 2′OMe sugar modified alternating ribonucleotides in one or both of the antisense and the sense strands. In other embodiments the compound includes 2′OMe sugar modified ribonucleotides in the antisense strand, (N)x or N1-(N)x, only. In certain embodiments the middle ribonucleotide of the antisense strand; e.g. ribonucleotide in position 10 in a 19-mer strand is unmodified. In various embodiments the nucleic acid compound includes at least 5 alternating 2′OMe sugar modified and unmodified ribonucleotides. In additional embodiments the compound of Structure A1 or Structure A2 includes modified ribonucleotides in alternating positions wherein each ribonucleotide at the 5′ and 3′ termini of (N)x or N1-(N)x are modified in their sugar residues, and each ribonucleotide at the 5′ and 3′ termini of (N′)y or N2-(N)y are unmodified in their sugar residues.

In some embodiments the double stranded molecule includes one or more of the following modifications

a) N in at least one of positions 5, 6, 7, 8, or 9 from the 5′ terminus of the antisense strand is selected from a 2′5′ nucleotide or a mirror nucleotide;

b) N′ in at least one of positions 9 or 10 from the 5′ terminus of the sense strand is selected from a 2′5′ nucleotide and a pseudoUridine; and

c) N′ in 4, 5, or 6 consecutive positions at the 3′ terminus positions of (N′)y comprises a 2′5′ nucleotide.

In some embodiments the double stranded molecule includes a combination of the following modifications

a) the antisense strand includes a 2′5′ nucleotide or a mirror nucleotide in at least one of positions 5, 6, 7, 8, or 9 from the 5′ terminus; and

b) the sense strand includes at least one of a 2′5′ nucleotide and a pseudoUridine in positions 9 or 10 from the 5′ terminus.

In some embodiments the double stranded molecule includes a combination of the following modifications

a) the antisense strand includes a 2′5′ nucleotide or a mirror nucleotide in at least one of positions 5, 6, 7, 8, or 9 from the 5′ terminus; and

c) the sense strand includes 4, 5, or 6 consecutive 2′5′ nucleotides at the 3′ penultimate or 3′ terminal positions.

In some embodiments, the sense strand [(N)x or N1-(N)x] includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 2′OMe sugar modified ribonucleotides. In some embodiments, the antisense strand includes 2′OMe modified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19. In other embodiments antisense strand includes 2′OMe modified ribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. In other embodiments the antisense strand includes 2′OMe modified ribonucleotides at positions 3, 5, 7, 9, 11, 13, 15, 17 and 19. In some embodiments the antisense strand includes one or more 2′OMe sugar modified pyrimidines. In some embodiments all the pyrimidine nucleotides in the antisense strand are 2′OMe sugar modified. In some embodiments the sense strand includes 2′OMe sugar modified pyrimidines.

In some embodiments of Structure A1 and Structure A2, neither the sense strand nor the antisense strand is phosphorylated at the 3′ and 5′ termini. In other embodiments one or both of the sense strand or the antisense strand are phosphorylated at the 3′ termini.

In some embodiments of Structure A1 and Structure A2 (N)y includes at least one unconventional moiety selected from a mirror nucleotide, a 2′5′ nucleotide and a TNA. In some embodiments the unconventional moiety is a mirror nucleotide. In various embodiments the mirror nucleotide is selected from an L-ribonucleotide (L-RNA) and an L-deoxyribonucleotide (L-DNA). In preferred embodiments the mirror nucleotide is L-DNA. In certain embodiments the sense strand comprises an unconventional moiety in position 9 or 10 (from the 5′ terminus). In preferred embodiments the sense strand includes an unconventional moiety in position 9 (from the 5′ terminus). In some embodiments the sense strand is 19 nucleotides in length and comprises 4, 5, or 6 consecutive unconventional moieties in positions 15, (from the 5′ terminus). In some embodiments the sense strand includes 4 consecutive 2′5′ ribonucleotides in positions 15, 16, 17, and 18. In some embodiments the sense strand includes 5 consecutive 2′5′ ribonucleotides in positions 15, 16, 17, 18 and 19. In various embodiments the sense strand further comprises Z′. In some embodiments Z′ includes a C3OH moiety or a C3Pi moiety.

In some embodiments of Structure A1 (N′)y includes at least one L-DNA moiety. In some embodiments x=y=19 and (N′)y, consists of unmodified ribonucleotides at positions 1-17 and 19 and one L-DNA at the 3′ penultimate position (position 18). In other embodiments x=y=19 and (N′)y consists of unmodified ribonucleotides at positions 1-16 and 19 and two consecutive L-DNA at the 3′ penultimate position (positions 17 and 18). In various embodiments the unconventional moiety is a nucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotide phosphate linkage. According to various embodiments (N′)y includes 2, 3, 4, 5, or 6 consecutive ribonucleotides at the 3′ terminus linked by 2′-5′ internucleotide linkages. In one embodiment, four consecutive nucleotides at the 3′ terminus of (N′)y are joined by three 2′-5′ phosphodiester bonds, wherein one or more of the 2′-5′ nucleotides which form the 2′-5′ phosphodiester bonds further includes a 3′-O-methyl (3′OMe) sugar modification. Preferably the 3′ terminal nucleotide of (N′)y includes a 2′OMe sugar modification. In certain embodiments x=y=19 and (N′)y includes two or more consecutive nucleotides at positions 15, 16, 17, 18 and 19 include a nucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotide bond (2′-5′ nucleotide). In various embodiments the nucleotide forming the 2′-5′ internucleotide bond includes a 3′ deoxyribose nucleotide or a 3′ methoxy nucleotide (3′ H or 3′OMe in place of a 3′ OH). In some embodiments x=y=19 and (N′)y includes 2′-5′ nucleotides at positions 15, 16 and 17 such that adjacent nucleotides are linked by a 2′-5′ internucleotide bond between positions 15-16, 16-17 and 17-18; or at positions, 15, 16, 17, 18, and 19 such that adjacent nucleotides are linked by a 2′-5′ internucleotide bond between positions 15-16, 16-17, 17-18 and 18-19 and a 3′OH is available at the 3′ terminal nucleotide or at positions 16, 17 and 18 such that adjacent nucleotides are linked by a 2′-5′ internucleotide bond between positions 16-17, 17-18 and 18-19. In some embodiments x=y=19 and (N′)y includes 2′-5′ nucleotides at positions 16 and 17 or at positions 17 and 18 or at positions 15 and 17 such that adjacent nucleotides are linked by a 2′-5′ internucleotide bond between positions 16-17 and 17-18 or between positions 17-18 and 18-19 or between positions 15-16 and 17-18, respectively. In other embodiments the pyrimidine ribonucleotides (rU, rC) in (N′)y are substituted with nucleotides joined to the adjacent nucleotide by a 2′-5′ internucleotide bond. In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—4, SERPINH1_—12, SERPINH1_—18, SERPINH1_—30, SERPINH1_—58 or SERPINH1_—88, and x=y=19 and (N′)y comprises five consecutive nucleotides at the 3′ terminus joined by four 2′-5′ linkages, specifically the linkages between the nucleotides position 15-16, 16-17, 17-18 and 18-19.

In some embodiments the linkages include phosphodiester bonds. In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—4, SERPINH1_—12, SERPINH1_—18, SERPINH1_—30, SERPINH1_—58 or SERPINH1_—88 and x=y=19 and (N′)y comprises five consecutive nucleotides at the 3′ terminus joined by four 2′-5′ linkages and optionally further includes Z′ and z′ independently selected from an inverted abasic moiety and a C3 alkyl [C3; 1,3-propanediol mono(dihydrogen phosphate)] cap. The C3 alkyl cap is covalently linked to the 3′ or 5′ terminal nucleotide. In some embodiments the 3′ C3 terminal cap further comprises a 3′ phosphate. In some embodiments the 3′ C3 terminal cap further comprises a 3′ terminal hydroxy group.

In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—4, SERPINH1_—12, SERPINH1_—18, SERPINH1_—30, SERPINH1_—58 or SERPINH1_—88 and x=y=19 and (N′)y includes an L-DNA position 18; and (N′)y optionally further includes Z′ and z′ independently selected from an inverted abasic moiety and a C3 alkyl [C3; 1,3-propanediol mono(dihydrogen phosphate)] cap.

In some embodiments (N′)y includes a 3′ terminal phosphate. In some embodiments (N′)y includes a 3′ terminal hydroxyl.

In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—4, SERPINH1_—12, SERPINH1_—18, SERPINH1_—30, SERPINH1_—58 or SERPINH1_—88 and x=y=19 and (N)x includes 2′OMe sugar modified ribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or at positions 2, 4, 6, 8, 11, 13, 15, 17, 19. In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—4, SERPINH1_—12, SERPINH1_—18, SERPINH1_—30, SERPINH1_—58 and SERPINH1_—88 and x=y=19 and (N)x includes 2′OMe sugar modified pyrimidines. In some embodiments all pyrimidines in (N)x include the 2′OMe sugar modification.

In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—2, SERPINH1_—6, SERPINH1_—11, SERPINH1_—13, SERPINH1_—45. SERPINH1_—45a, SERPINH1_—51. SERPINH51a, SERPINH1_—52 or SERPINH1_—86 and x=y=18, and N2-(N′)y includes five consecutive nucleotides at the 3′ terminus joined by four 2′-5′ linkages, specifically the linkages between the nucleotides position 15-16, 16-17, 17-18 and 18-19. In some embodiments the linkages include phosphodiester bonds.

In some embodiments N²—(N′)y comprises a 3′ terminal phosphate. In some embodiments N2-(N′)y comprises a 3′ terminal hydroxyl.

In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—2, SERPINH1_—6, SERPINH1_—11, SERPINH1_—13, SERPINH1_—45, SERPINH1_—45a, SERPINH1_—51, SERPINH1_—51a, SERPINH1_—52 or SERPINH1_—86 and x=y=18 and N¹—(N)x includes 2′OMe sugar modified ribonucleotides in positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or in positions 1, 3, 5, 9, 11, 13, 15, 17, 19, or in positions 3, 5, 9, 11, 13, 15, 17, or in positions 2, 4, 6, 8, 11, 13, 15, 17, 19. In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—2, SERPINH1_—6, SERPINH1_—11, SERPINH1_—13, SERPINH1_—45, SERPINH1_—45a, SERPINH1_—51, SERPINH1_—52 or SERPINH1_—86 and x=y=18 and N¹—(N)x includes 2′OMe sugar modified ribonucleotides at positions 11, 13, 15, 17 and 19 (from 5′ terminus). In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—2, SERPINH1_—6, SERPINH1_—11, SERPINH1_—13, SERPINH1-45. SERPINH1_—45a, SERPINH1_—51, SERPINH1_—51a, SERPINH1_—52 or SERPINH1_—86 and x=y=18 and N¹—(N)x includes 2′OMe sugar modified ribonucleotides in positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or in positions 3, 5, 7, 9, 11, 13, 15, 17, 19. In some embodiments the antisense and sense strands are selected from the sequence pairs set forth in SERPINH1_—2, SERPINH1_—6, SERPINH1_—11, SERPINH1_—13, SERPINH1_—45, SERPINH1_—45a, SERPINH1_—51, SERPINH1_—52 or SERPINH1_—86 and x=y=18 and N1-(N)x includes 2′OMe sugar modified ribonucleotides in positions 2, 4, 6, 8, 11, 13, 15, 17, 19.

In additional embodiments N1-(N)x further includes Z wherein Z includes a non-nucleotide overhang. In some embodiments the non-nucleotide overhang is C3-C3 [1,3-propanediol mono(dihydrogen phosphate)]2.

In some embodiments of Structure A2, (N)y includes at least one L-DNA moiety. In some embodiments x=y=18 and (N′)y consists of unmodified ribonucleotides at positions 1-16 and 18 and one L-DNA at the 3′ penultimate position (position 17). In other embodiments x=y=18 and (N′)y consists of unmodified ribonucleotides at position 1-15 and 18 and two consecutive L-DNA at the 3′ penultimate position (positions 16 and 17). In various embodiments the unconventional moiety is a nucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotide phosphate linkage. According to various embodiments (N′)y includes 2, 3, 4, 5, or 6 consecutive ribonucleotides at the 3′ terminus linked by 2′-5′ internucleotide linkages. In one embodiment, four consecutive nucleotides at the 3′ terminus of (N′)y are joined by three 2′-5′ phosphodiester bonds, wherein one or more of the 2′-5′ nucleotides which form the 2′-5′ phosphodiester bonds further includes a 3′-O-methyl (3′OMe) sugar modification. Preferably the 3′ terminal nucleotide of (N′)y includes a 2′OMe sugar modification. In certain embodiments x=y=18 and in (N′)y two or more consecutive nucleotides at positions 14, 15, 16, 17, and 18 include a nucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotide bond. In various embodiments the nucleotide forming the 2′-5′ internucleotide bond includes a 3′ deoxyribose nucleotide or a 3′ methoxy nucleotide. In some embodiments x=y=18 and (N′)y includes nucleotides joined to the adjacent nucleotide by a 2′-5′ internucleotide bond between positions 15-16, 16-17 and 17-18 or between positions 16-17 and 17-18. In some embodiments x=y=18 and (N′)y includes nucleotides joined to the adjacent nucleotide by a 2′-5′ internucleotide bond between positions 14-15, 15-16, 16-17, and 17-18 or between positions 15-16, 16-17, and 17-18 or between positions 16-17 and 17-18 or between positions 17-18 or between positions 15-16 and 17-18. In other embodiments the pyrimidine ribonucleotides (rU, rC) in (N′)y are substituted with nucleotides joined to the adjacent nucleotide by a 2′-5′ internucleotide bond.

In some embodiments the antisense and sense strands are selected from the oligonucleotide pairs set forth in Table A-18 and identified herein as SERPINH1_—2 (SEQ ID NOS: 60 and 127), SERPINH1_—6 (SEQ ID NOS: 63 and 130), SERPINH1_—45a (SEQ ID NOS: 98 and 165), SERPINH1_—51 (SEQ ID NOS: 101 and 168) and SERPINH1_—51a (SEQ ID NOS: 105 and 172).

In some embodiments the double stranded nucleic acid molecule includes the antisense strand set forth in SEQ ID NO:127 and sense strand set forth in SEQ ID NO:60; identified herein as SERPINH1_—2. In some embodiments the double stranded nucleic acid molecule has the structure

embedded image

wherein each “|” represents base pairing between the ribonucleotides;

wherein each of A, C, G, U is independently an unmodified or modified ribonucleotide, or an unconventional moiety;

wherein each of Z and Z′ is independently present or absent, but if present is independently 1-5 consecutive nucleotides or non-nucleotide moieties or a combination thereof covalently attached at the 3′ terminus of the strand in which it is present; and

wherein z″ may be present or absent, but if present is a capping moiety covalently attached at the 5′ terminus of N2-(N′)y.

In some embodiments provided is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:127) includes one or more 2′OMe sugar modified pyrimidines and or purines, a 2′-5′ ribonucleotide in position 5, 6, 7 or 8, and a 3′ terminal nucleotide or non-nucleotide overhang. In some embodiments the sense strand (SEQ ID NO:60) includes 4 or 5 consecutive 2′5′ nucleotides at the 3′ terminal or penultimate positions, a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus and a cap moiety covalently attached at the 5′ terminus. In other embodiments the sense strand (SEQ ID NO:60) includes one or more 2′OMe primidine, a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus and a cap moiety covalently attached at the 5′ terminus.

In some embodiments provided is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:127) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 1, 3, 5, 9, 11, 15, 17 and 19, a 2′-5′ ribonucleotide at position 7, and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:60) is selected from a sense strand which includes

a) 2′-5′ ribonucleotides at positions 15, 16, 17, 18 and 19, a C3OH 3′ terminal non-nucleotide overhang; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; or

b) 2′-5′ ribonucleotides at positions 15, 16, 17, 18 and 19, a 3′ terminal phosphate; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; or

c) 2′OMe sugar modified ribonucleotides at positions (5′>3′) 5, 7, 13, and 16; a 2′5′ ribonucleotide at position 18; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; or

d) 2′OMe sugar modified ribonucleotides at positions (5′>3′) 7, 13, 16 and 18; a 2′5′ ribonucleotide at position 9; a C3OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; or

e) 2′-5′ ribonucleotides at positions 15, 16, 17, 18, and 19: a C3-Pi moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:127) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 1, 3, 5, 9, 11, 15, 17, 19, a 2′-5′ ribonucleotide at position 7 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:60) includes 2′-5′ ribonucleotides at positions 15, 16, 17, 18, and 19: a C3 3′ terminal overhang; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:127) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 1, 3, 5, 9, 11, 15, 17, 19, a 2′-5′ ribonucleotide at position 7 and a C3Pi-C3OH, 3′ terminal overhang; and the sense strand (SEQ ID NO:60) includes 2′-5′ ribonucleotides at positions 15, 16, 17, 18, and 19: a 3′ terminal phosphate; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:127) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 1, 3, 5, 9, 11, 15, 17, 19, a 2′-5′ ribonucleotide at position 7 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:60) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 5, 7, 13, and 16; a 2′-5′ ribonucleotide at position 18; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:127) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 1, 3, 5, 9, 11, 15, 17, 19, a 2′-5′ ribonucleotide at position 7 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:60) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 7, 13, 16 and 18; a 2′-5′ ribonucleotide at position 9; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:127) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 1, 3, 5, 9, 11, 15, 17, 19, a 2′-5′ ribonucleotide at position 7 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:60) includes 2′-5′ ribonucleotides at positions 15, 16, 17, 18, and 19: a C3-Pi moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

In some embodiments provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:127) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 1, 3, 5, 9, 11, 13, 15, 17, 19 and a C3-C3 3′ terminal overhang; and the sense strand (SEQ ID NO:60) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 7, 9, 13, 16 and 18; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

In some embodiments provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:60) includes 2′-5′ ribonucleotides at positions 15, 16, 17, 18, and 19: a 3′ terminal phosphate and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus and the antisense strand (SEQ ID NO:127) includes an antisense strand selected from one of

a) 2′OMe sugar modified ribonucleotides at positions (5′>3′) 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; or

b) 2′OMe sugar modified ribonucleotides at positions (5′>3′) 1, 3, 6, 8, 10, 12, 14, 17, 18 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus.

In some embodiments provided herein is a double stranded nucleic acid molecule which includes the antisense strand set forth in SEQ ID NO:130 and the sense strand set forth in SEQ ID NO:63; identified herein as SERPINH1_—6. In some embodiments the duplex comprises the structure

In some embodiments provided is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:63) includes one or more 2′OMe sugar modified pyrimidines; a 3′ terminal nucleotide or non-nucleotide overhang; and cap moiety covalently attached at the 5′ terminus. In some embodiments the antisense strand (SEQ ID NO:130) includes one or more 2′OMe sugar modified pyrimidine, a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus and a cap moiety covalently attached at the 5′ terminus.

In some embodiments provided is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:63) includes 2′OMe sugar modified ribonucleotides at positions (5′>3′) 2, 14 and 18; a C3OH or C3Pi moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:130) is selected from an antisense strand which includes

a) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 9, 11, 13, 15 and 17; a 2′-5′ ribonucleotide at position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; or

b) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 7, 9, 12, 13 and 17; a 2′-5′ ribonucleotide at position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; or

c) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 9, 11, 13, 15 and 17; a 2′-5′ ribonucleotide at position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; or

d) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 9, 11, 13, 15 and 17; a dU in position 1; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus.

In some embodiments provided is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:63) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 2, 14 and 18; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:130) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 9, 11, 13, 15 and 17; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus.

In some embodiments provided is a duplex oligonucleotide molecule wherein the sense strand (SEQ ID NO:63) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 14 and 18 and optionally in position 2; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:130) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 7, 9, 12, 13, and 17; a 2′-5′ ribonucleotide at position 7; and a C3:Pi-C3OH moiety covalently attached to the 3′ terminus.

a) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 9, 11, 13, 15 and 17; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3Pi or C3Pi-C3OH moiety covalently attached to the 3′ terminus; or

b) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 7, 9, 12, 13, and 17; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3Pi or C3Pi-C3OH moiety covalently attached to the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:63) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 14 and 18; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:130) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 9, 11, 13, 15 and 17; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:63) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 14 and 18; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:130) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 7, 9, 12, 13, and 17; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3OH 3′ terminal overhang.

In some embodiments the duplex includes the antisense strand set forth in SEQ ID NO:165 and sense strand set forth in SEQ ID NO:98; identified herein as SERPINH1_—45a. In some embodiments the duplex comprises the structure

In some embodiments the sense strand (SEQ ID NO:98) includes 2′-5′ ribonucleotides in positions (5′>3′) 15, 16, 17, and 18 or 15, 16, 17, 18, and 19: a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus, and a cap moiety covalently attached at the 5′ terminus. In some embodiments the antisense strand (SEQ ID NO:165) includes 2′OMe sugar modified pyrimidine and or purines, a 2′-5′ nucleotide in position 5, 6, 7, or 8 (5′>3′); and a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus.

In some embodiments the sense strand (SEQ ID NO:98) includes 2′-5′ ribonucleotides in positions (5′>3′) 15, 16, 17, 18, and 19: a C3Pi or C3-OH 3′ terminal non-nucleotide moiety and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:165) includes an antisense strand selected from one of

a) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 2, 4, 6, 8, 11, 13, 15, 17, and 19; a 2′-5′ ribonucleotide in position 7 and a C3Pi-C3Pi or C3Pi-C3OH 3′ terminal overhang; or

b) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 2, 4, 6, 8, 11, 13, 15, 17 and 19 and a C3Pi-C3Pi or C3Pi-C3OH 3′ terminal overhang;

c) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′ ribonucleotide in position 7 and a C3Pi-C3Pi or C3Pi-C3OH 3′ terminal overhang; or

d) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 and a C3Pi-C3Pi or C3Pi-C3OH 3′ terminal overhang.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:98) includes 2′-5′ ribonucleotides in positions (5′>3′) 15, 16, 17, 18, and 19: a C3-OH 3′ terminal moiety and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:165) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 2, 4, 6, 8, 11, 13, 15, 17, and 19; a 2′-5′ ribonucleotide in position 7 and a C3Pi-COH 3′ terminal overhang.

In some embodiments the double stranded nucleic acid molecule includes the antisense strand set forth in SEQ ID NO:168 and sense strand set forth in SEQ ID NO:101; identified herein as SERPINH1_—51. In some embodiments the duplex comprises the structure

In some embodiments provided is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:101) includes 2′OMe sugar modified pyrimidines, optionally a 2′-5′ ribonucleotide in position 9 or 10; a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus and optionally a cap moiety covalently attached at the 5′ terminus. In some embodiments the antisense strand (SEQ ID NO:168) includes 2′OMe sugar modified pyrimidine and or purines, a 2′-5′ nucleotide in position 5, 6, 7, or 8 (5′>3′); and a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus.

In some embodiments provided is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:101) includes 2′OMe sugar modified pyrimidines in positions (5′>3′) 4, 11, 13, and 17, optionally a 2′-5′ ribonucleotide in position 9 or 10, a C3Pi or C3OH non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:168) is selected from an antisense strand which includes

a) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 8, and 15, a 2′5′ ribonucleotide in position 6 or 7; a C3Pi-C3OH overhang covalently attached at the 3′ terminus; or

b) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 4, 8, 13 and 15, a 2′5′ ribonucleotide in position 6 or 7; a C3Pi-C3OH overhang covalently attached at the 3′ terminus; or

c) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 4, 8, 11 and 15, a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH overhang covalently attached at the 3′ terminus; or

d) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 8, 12, 13, and 15; a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:101) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 11, 13, and 17, optionally a 2′-5′ ribonucleotide in position 9, a C3-OH non-nucleotide moiety covalently attached at the 3′ and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:168) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 8, and 15, a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:101) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 11, 13, and 17, optionally a 2′-5′ ribonucleotide in position 9, a C3-OH non-nucleotide moiety covalently attached at the 3′ and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:168) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 4, 8, 13 and 15, a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:101) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 11, 13, and 17, a 2′-5′ ribonucleotide in position 9, a C3OH non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:168) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 4, 8, 11 and 15, a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:101) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 11, 13, and 17, a 2′-5′ ribonucleotide in position 9, a C3OH non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:168) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 8, 12, 13, and 15; a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus.

In some embodiments the double stranded nucleic acid molecule includes the antisense strand set forth in SEQ ID NO:172 and sense strand set forth in SEQ ID NO:105; identified herein as SERPINH1_—51a. In some embodiments the duplex comprises the structure

embedded image

wherein each “|” represents base pairing between the ribonucleotides;

wherein each of A, C, G and U is independently an unmodified or modified ribonucleotide, or an unconventional moiety;

wherein each of Z and Z′ is independently present or absent, but if present is independently 1-5 consecutive nucleotides or non-nucleotide moieties or a combination thereof covalently attached at the 3′ terminus of the strand in which it is present; and

wherein z″ may be present or absent, but if present is a capping moiety covalently attached at the 5′ terminus of the sense strand.

In some embodiments provided is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:105) includes 2′OMe sugar modified pyrimidines, optionally a 2′-5′ ribonucleotide in position 9 or 10; a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus and optionally a cap moiety covalently attached at the 5′ terminus. In some embodiments the antisense strand (SEQ ID NO:172) includes 2′OMe sugar modified pyrimidine and or purines, a 2′-5′ nucleotide in position 5, 6, 7, or 8 (5′>3′); and a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus.

In some embodiments provided is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:105) includes 2′OMe sugar modified pyrimidines in positions (5′>3′) 4, 11, 13, and 17, optionally a 2′-5′ ribonucleotide in position 9 or 10, a C3Pi or C3OH non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:172) is selected from an antisense strand which includes

a) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 8, and 15, a 2′5′ ribonucleotide in position 6 or 7; a C3Pi-C3OH moiety covalently attached at the 3′ terminus; or

b) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 8, 13 and 15, a 2′5′ ribonucleotide in position 6 or 7; a C3Pi-C3OH moiety covalently attached at the 3′ terminus; or

c) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 8, 11 and 15, a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus; or

d) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 8, 12, 13, and 15; a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:105) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 11, 13, and 17, optionally a 2′-5′ ribonucleotide in position 9, a C3-OH non-nucleotide moiety covalently attached at the 3′ and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:172) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′)₈, and 15, a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:105) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 11, 13, and 17, optionally a 2′-5′ ribonucleotide in position 9, a C3-OH non-nucleotide moiety covalently attached at the 3′ and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:172) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 8, 13 and 15, a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:105) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 11, 13, and 17, a 2′-5′ ribonucleotide in position 9, a C3-OH non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:172) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 8, 11 and 15, a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:105) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 11, 13, and 17, a 2′-5′ ribonucleotide in position 9, a C3OH non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:172) includes 2′OMe sugar modified ribonucleotides in positions (5′>3″) 3, 8, 12, 13, and 15; a 2′5′ ribonucleotide in position 6; a C3Pi-C3OH moiety covalently attached at the 3′ terminus.

In some embodiments the antisense and sense strands are selected from the oligonucleotide pairs set forth in Table A-19 and identified herein as SERPINH1_—4 (SEQ ID NOS: 195 and 220) and SERPINH1_—12 (SEQ ID NOS: 196 and 221).

In some embodiments the double stranded nucleic acid molecule includes the antisense strand set forth in SEQ ID NO:220 and sense strand set forth in SEQ ID NO:194; identified herein as SERPINH1_—4. In some embodiments the double stranded nucleic acid molecule has the structure

In some embodiments provided is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:220) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 9, 11, 15, 17 and 19, a 2′-5′ ribonucleotide in position 7, and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:195) is selected from a sense strand which includes

a) 2′-5′ ribonucleotides in positions 15, 16, 17, 18 and 19, a C3OH moiety covalently attached to the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; or

b) 2′-5′ ribonucleotides in positions 15, 16, 17, 18 and 19, a 3′ terminal phosphate; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; or

c) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 5, 7, 13, and 16; a 2′5′ ribonucleotide in position 18; a C3OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; or

d) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 7, 13, 16 and 18; a 2′5′ ribonucleotide in position 9; a C3OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; or

e) 2′-5′ ribonucleotides in positions 15, 16, 17, 18, and 19: a C3Pi moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:220) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 9, 11, 15, 17, 19, a 2′-5′ ribonucleotide in position 7 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:195) includes 2′-5′ ribonucleotides in positions 15, 16, 17, 18, and 19: a C3 moiety covalently attached to the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:220) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 9, 11, 15, 17, 19, a 2′-5′ ribonucleotide in position 7 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:195) includes 2′-5′ ribonucleotides in positions 15, 16, 17, 18, and 19: a 3′ terminal phosphate; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:220) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 9, 11, 15, 17, 19, a 2′-5′ ribonucleotide in position 7 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:195) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 5, 7, 13, and 16; a 2′-5′ ribonucleotide in position 18; a C3OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:220) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 9, 11, 15, 17, 19, a 2′-5′ ribonucleotide in position 7 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:195) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 7, 13, 16 and 18; a 2′-5′ ribonucleotide in position 9; a C3OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:220) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 9, 11, 15, 17, 19, a ribonucleotide in position 7 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:195) includes 2′-5′ ribonucleotides in positions 15, 16, 17, 18, and 19: a C3Pi moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

In some embodiments provided herein is a double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:220) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 9, 11, 13, 15, 17, 19 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:195) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 7, 9, 13, 16 and 18; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus.

In some embodiments provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:195) includes 2′-5′ ribonucleotides in positions 15, 16, 17, 18, and 19: a 3′ terminal phosphate and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus and the antisense strand (SEQ ID NO:220) includes an antisense strand selected from one of

a) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 7, 9, 11, 13, 15, 17, 19 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; or

b) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 6, 8, 10, 12, 14, 17, 18 and a C3Pi-C3OH moiety covalently attached to the 3′ terminus.

In some embodiments provided herein is a double stranded nucleic acid molecule which includes the antisense strand set forth in SEQ ID NO:130 and the sense strand set forth in SEQ ID NO:63; identified herein as SERPINH1_—12. In some embodiments the duplex comprises the structure

embedded image

wherein each “|” represents base pairing between the ribonucleotides;

wherein each of A, C, G, U is independently an unmodified or modified ribonucleotide, or an unconventional moiety;

wherein each of Z and Z′ is independently present or absent, but if present is independently 1-consecutive nucleotides or non-nucleotide moieties or a combination thereof covalently attached at the 3′ terminus of the strand in which it is present; and

wherein z″ may be present or absent, but if present is a capping moiety covalently attached at the 5′ terminus of N2-(N′)y.

In some embodiments provided is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:196) includes one or more 2′OMe sugar modified pyrimidines; a 3′ terminal nucleotide or non-nucleotide overhang; and a cap moiety covalently attached at the 5′ terminus. In some embodiments the antisense strand (SEQ ID NO:221) includes one or more 2′OMe sugar modified pyrimidines, a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus, and a cap moiety covalently attached at the 5′ terminus.

In some embodiments provided is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:196) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 2, 14 and 18; a C3OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:221) is selected from an antisense strand which includes

a) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 9, 11, 13, 15 and 17; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; or

b) 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 7, 9, 12, 13 and 17; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus.

In some embodiments provided is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:196) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 2, 14 and 18; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:221) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 9, 11, 13, 15 and 17; a ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus.

In some embodiments provided is a duplex oligonucleotide molecule wherein the sense strand (SEQ ID NO:196) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 14 and 18 and optionally in position 2; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:221) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 3, 5, 7, 9, 12, 13, and 17; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus.

In some embodiments provided is a duplex oligonucleotide molecule wherein the sense strand (SEQ ID NO:196) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 14 and 18; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:221) is selected from an antisense strand which includes

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:196) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 14 and 18; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:220) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 9, 11, 13, 15 and 17; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus.

Provided herein is a double stranded nucleic acid molecule wherein the sense strand (SEQ ID NO:196) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 14 and 18; a C3-OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand (SEQ ID NO:220) includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 7, 9, 12, 13, and 17; a 2′-5′ ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached to the 3′ terminus.

In further embodiments of Structures A1 and A2 (N′)y includes 1-8 modified ribonucleotides wherein the modified ribonucleotide is a DNA nucleotide. In certain embodiments (N′)y includes 1, 2, 3, 4, 5, 6, 7, or up to 8 DNA moieties.

In some embodiments either Z or Z′ is present and independently includes two non-nucleotide moieties.

In additional embodiments Z and Z′ are present and each independently includes two non-nucleotide moieties.

In some embodiments each of Z and Z′ includes an abasic moiety, for example a deoxyriboabasic moiety (referred to herein as “dAb”) or riboabasic moiety (referred to herein as “rAb”). In some embodiments each of Z and/or Z′ includes two covalently linked abasic moieties and is for example dAb-dAb or rAb-rAb or dAb-rAb or rAb-dAb, wherein each moiety is covalently attached to an adjacent moiety, preferably via a phospho-based bond. In some embodiments the phospho-based bond includes a phosphorothioate, a phosphonoacetate or a phosphodiester bond. In preferred embodiments the phospho-based bond includes a phosphodiester bond.

In some embodiments each of Z and/or Z′ independently includes an alkyl moiety, optionally propane [(CH2)3] moiety (C3) or a derivative thereof including propanol (C3-OH) and phospho derivative of propanediol (“C3-3′Pi”). In some embodiments each of Z and/or Z′ includes two alkyl moieties covalently linked to the 3′ terminus of the antisense strand or sense strand via a phosphodiester or phosphorothioate linkage and covalently linked to one another via a phosphodiester or phosphorothioate linkage and in some examples is C3Pi-C3Pi or C3Pi-C3OH. The 3′ terminus of the antisense strand and/or the 3′ terminus of the sense strand is covalently attached to a C3 moiety via a phospho-based bond and the C3 moiety is covalently conjugated a C3-OH moiety via a phospho-based bond. In some embodiments the phospho-based bonds include a phosphorothioate, a phosphonoacetate or a phosphodiester bond. In preferred embodiments the phospho-based bond includes a phosphodiester bond.

In various embodiments of Structure A1 or Structure A2, Z and Z′ are absent. In other embodiments Z or Z′ is present. In some embodiments each of Z and/or Z′ independently includes a C2, C3, C4, C5 or C6 alkyl moiety, optionally a C3 [propane, —(CH2)₃-] moiety or a derivative thereof including propanol (C3-OH/C3OH), propanediol, and phosphodiester derivative of propanediol (“C3Pi”). In preferred embodiments each of Z and/or Z′ includes two hydrocarbon moieties and in some examples is C3Pi-C3OH or C3Pi-C3Pi, Each C3 is covalently conjugated to an adjacent C3 via a covalent bond, preferably a phospho-based bond. In some embodiments the phospho-based bond is a phosphorothioate, a phosphonoacetate or a phosphodiester bond.

In specific embodiments x=y=19 and Z comprises at least one C3 alkyl overhang. In some embodiments the C3-C3 overhang is covalently attached to the 3′ terminus of (N)x or (N′)y via a covalent linkage, preferably a phosphodiester linkage. In some embodiments the linkage between a first C3 and a second C3 is a phosphodiester linkage. In some embodiments the 3′ non-nucleotide overhang is C3Pi-C3Pi. In some embodiments the 3′ non-nucleotide overhang is C3Pi-C3Ps. In some embodiments the 3′ non-nucleotide overhang is C3Pi-C3OH (OH is hydroxy). In some embodiments the 3′ non-nucleotide overhang is C3Pi-C3OH.

In various embodiments the alkyl moiety comprises an alkyl derivative including a C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl moiety comprising a terminal hydroxyl, a terminal amino, or terminal phosphate group. In some embodiments the alkyl moiety is a C3 alkyl or C3 alkyl derivative moiety. In some embodiments the C3 alkyl moiety comprises propanol, propylphosphate, propylphosphorothioate or a combination thereof. The C3 alkyl moiety is covalently linked to the 3′ terminus of (N′)y and/or the 3′ terminus of (N)x via a phosphodiester bond. In some embodiments the alkyl moiety comprises propanol, propyl phosphate or propyl phosphorothioate. In some embodiments each of Z and Z′ is independently selected from propanol, propyl phosphate propyl phosphorothioate, combinations thereof or multiples thereof in particular 2 or 3 covalently linked propanol, propyl phosphate, propyl phosphorothioate or combinations thereof. In some embodiments each of Z and Z′ is independently selected from propyl phosphate, propyl phosphorothioate, propyl phospho-propanol; propyl phospho-propyl phosphorothioate; propylphospho-propyl phosphate; (propyl phosphate)₃, (propyl phosphate)₂-propanol, (propyl phosphate)₂-propyl phosphorothioate. Any propane or propanol conjugated moiety can be included in Z or Z′.

The structures of exemplary 3′ terminal non-nucleotide moieties are as follows:

embedded image

In some embodiments each of Z and Z′ is independently selected from propanol, propyl phosphate, propyl phosphorothioate, combinations thereof or multiples thereof.

In some embodiments each of Z and Z′ is independently selected from propyl phosphate, propyl phosphorothioate, propyl phospho-propanol; propyl phospho-propyl phosphorothioate; propylphospho-propyl phosphate; (propyl phosphate)3, (propyl phosphate)2-propanol, (propyl phosphate)2-propyl phosphorothioate. Any propane or propanol conjugated moiety can be included in Z or Z′.

In additional embodiments each of Z and/or Z′ includes a combination of an abasic moiety and an unmodified deoxyribonucleotide or ribonucleotide or a combination of a hydrocarbon moiety and an unmodified deoxyribonucleotide or ribonucleotide or a combination of an abasic moiety (deoxyribo or ribo) and a hydrocarbon moiety. In such embodiments, each of Z and/or Z′ includes C3-rAb or C3-dAb wherein each moiety is covalently bond to the adjacent moiety vi a phospho-based bond, preferably a phosphodiester, phosphorothioate or phosphonoacetate bond.

In certain embodiments nucleic acid molecules as disclosed herein include a sense oligonucleotide sequence selected from any one of Oligo #s 2-67 or 68-92, shown infra in Tables A-18 and A-19, respectfully.

In certain preferred embodiments compounds provided include Compound_—1, Compound_—2, Compound_—3, Compound_—4, Compound_—5, Compound_—6, Compound_—7, Compound_—8 and Compound_—9 as described herein.

In some embodiments (such as, for example, Compound_—1, Compound_—5 and Compound_—6 as described herein) provided are 19 mer double stranded nucleic acid molecules wherein the antisense strand is SEQ ID NO:127 and the sense strand is SEQ ID NO:60. In certain embodiments, provided are 19 mer double stranded nucleic acid molecules wherein the antisense strand is SEQ ID NO:127 and includes 2′OMe sugar modified ribonucleotides, a ribonucleotide in at least one of positions 1, 5, 6, or 7, and a 3′ terminal non-nucleotide moiety covalently attached to the 3′ terminus; and the sense strand is SEQ ID NO:60 and includes at least one 2′5′ ribonucleotide or 2′OMe modified ribonucleotide, a non-nucleotide moiety covalently attached at the 3′ terminus and a cap moiety covalently attached at the 5′ terminus. In some embodiments, provided are 19 mer double stranded nucleic acid molecule wherein the antisense strand is SEQ ID NO:127 and includes 2′OMe sugar modified ribonucleotides at positions 3, 5, 9, 11, 13, 15, 17, and 19 (5′>3′), a 2′-5′ ribonucleotide in position 7, and a 3′ terminal C3OH non-nucleotide moiety covalently attached at the 3′ terminus; and the sense strand is SEQ ID NO:60 and includes 5 consecutive 2′5′ ribonucleotides in the 3′ terminal positions 15, 16, 17, 18, and 19 (5′>3′), a C3Pi non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic moiety covalently attached at the 5′ terminus.

In one embodiment provided is Compound_—1 that is a 19 mer double stranded nucleic acid molecule wherein the antisense strand is SEQ ID NO:127 and includes 2′OMe sugar modified ribonucleotides at positions 3, 5, 9, 11, 13, 15, 17, and 19 (5′>3′), a 2′-5′ ribonucleotide in position 7, and a C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′ terminus; and the sense strand is SEQ ID NO:60 and includes 5 consecutive 2′5′ ribonucleotides in the 3′ terminal positions 15, 16, 17, 18, and 19 (5′>3′), a C3Pi non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic moiety covalently attached at the 5′ terminus; and that further includes a 2′OMe sugar modified ribonucleotide at position 1 of the antisense strand.

In one embodiment, provided is Compound_—6 that is a 19 mer double stranded nucleic acid molecule wherein the antisense strand is SEQ ID NO:127 and includes 2′OMe sugar modified ribonucleotides at positions 3, 5, 9, 11, 13, 15, 17, and 19 (5′>3′), a 2′-5′ ribonucleotide in position 7, and a C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′ terminus; and the sense strand is SEQ ID NO:60 and includes 5 consecutive 2′5′ ribonucleotides in the 3′ terminal positions 15, 16, 17, 18, and 19 (5′>3′), a C3Pi non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic moiety covalently attached at the 5′ terminus; and that further includes a 2′5′ ribonucleotide at position 1 of the antisense strand.

In one embodiment, provided is Compound_—5 that is a 19 mer double stranded nucleic acid molecule wherein the antisense strand is SEQ ID NO:127 and includes 2′OMe sugar modified ribonucleotides in positions 1, 3, 5, 9, 11, 13, 15, 17, and 19 (5′>3′), a 2′-5′ ribonucleotide in position 7, and a C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′ terminus; and the sense strand is SEQ ID NO:60 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 7, 13, 16 and 18, a 2′5′ ribonucleotide at position 9, a C3OH non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic moiety covalently attached at the 5′ terminus.

In some embodiments (such as, for example, Compound_—2, and Compound_—7 as described herein) provided are 19 mer double stranded nucleic acid molecules wherein the sense strand is SEQ ID NO:63 and the antisense strand is SEQ ID NO:130. In some embodiments provided are 19-mer double stranded nucleic acid molecules wherein the sense strand is SEQ ID NO:63 and includes 2′OMe sugar modified pyrimidine ribonucleotides; a non-nucleotide moiety covalently attached at the 3′ terminus; and a cap moiety covalently attached at the 5′ terminus; and the antisense strand is SEQ ID NO:130 and includes 2′OMe sugar modified ribonucleotides; a 2′-5′ ribonucleotide at position 7; and a non-nucleotide moiety covalently attached at the 3′ terminus. In some embodiments provided are 19-mer double stranded nucleic acid molecules wherein the sense strand is SEQ ID NO:63 and includes 2′OMe sugar modified ribonucleotides, a non-nucleotide moiety covalently attached at the 3′ terminus, and a cap moiety covalently attached at the 5′ terminus; and the antisense strand is SEQ ID NO:130 and includes 2′OMe sugar modified ribonucleotides; a ribonucleotide in at least one of positions 5, 6 or 7; and a non-nucleotide moiety covalently attached at the 3′ terminus.

In one embodiment, provided is Compound_—2 that is a 19-mer double stranded nucleic acid molecule wherein the sense strand is SEQ ID NO:63 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 2, 14 and 18; a C3OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand is SEQ ID NO:130 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 9, 12, 13, and 17; a 2′-5′ ribonucleotide in at least one of positions 5, 6 or 7; and C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′ terminus.

In one embodiment, provided is Compound_—7 that is a 19-mer double stranded nucleic acid molecule wherein the sense strand is SEQ ID NO:63 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 2, 14 and 18; a C3OH moiety covalently attached at the 3′ terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand is SEQ ID NO:130 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 3, 5, 9, 11, 13, and 17; a 2′-5′ ribonucleotide at position 7; and a C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′ terminus.

In some embodiments (such as, for example, Compound_—3 as described herein) provided are 19 mer double stranded nucleic acid molecules wherein the sense strand is SEQ ID NO:98 and the antisense strand is SEQ ID NO:165. In some embodiments, provided are 19-mer double stranded nucleic acid molecules wherein the sense strand is SEQ ID NO:98 and includes 2′-5′ ribonucleotides in positions at the 3′ terminus: a non-nucleotide moiety covalently attached at the 3′ terminus and a cap moiety covalently attached at the 5′ terminus; and the antisense strand is SEQ ID NO:165 and includes 2′OMe sugar modified ribonucleotides; a 2′-5′ ribonucleotide in at least one of positions 5, 6 or 7 and a non-nucleotide moiety covalently attached at the 3′ terminus. In one embodiment, provided is Compound_—3 that is a 19-mer double stranded nucleic acid molecule wherein the sense strand is SEQ ID NO:98 and includes 2′-5′ ribonucleotides in positions (5′>3′) 15, 16, 17, 18, and 19: a C3-OH 3′ moiety covalently attached at the 3′ terminus and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand is SEQ ID NO:165 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 2, 4, 6, 8, 11, 13, 15, 17, and 19; a 2′-5′ ribonucleotide in position 7 and a C3Pi-C3OH covalently attached at the 3′ terminus.

In some embodiments (such as, for example, Compound_—4, Compound_—8 and Compound_—9 described herein) provided are 19-mer double stranded nucleic acid molecules wherein the sense strand is SEQ ID NO:101 and the antisense strand is SEQ ID NO:168. In some embodiments provided are 19-mer double stranded nucleic acid molecules wherein the sense strand is SEQ ID NO:101 and includes 2′OMe sugar modified pyrimidine ribonucleotides, an optional ribonucleotide in one of position 9 or 10, a non-nucleotide moiety covalently attached at the 3′ terminus and a cap moiety covalently attached at the 5′ terminus; and the antisense strand is SEQ ID NO:168 and includes 2′OMe sugar modified ribonucleotides, a 2′5′ ribonucleotide in at least one of positions 5, 6, or 7; and a non-nucleotide moiety covalently attached at the 3′ terminus.

In one embodiment, provided is Compound 4 that is a 19-mer double stranded nucleic acid molecule wherein sense strand is SEQ ID NO:101 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 11, 13, and 17, a 2′-5′ ribonucleotide in position 9, a C3OH non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand is SEQ ID NO:168 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 4, 8, 11 and 15, a 2′5′ ribonucleotide in position 6; a 3′ C3Pi-C3OH overhang covalently attached at the 3′ terminus.

In one embodiment, provided is Compound_—8 that is a 19-mer double stranded nucleic acid molecule wherein sense strand is SEQ ID NO:101 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 4, 11, 13, and 17, a C3OH non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand is SEQ ID NO:168 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 4, 8, 13 and 15, a 2′5′ ribonucleotide in position 6; and a 3′ C3Pi-C3OH overhang covalently attached at the 3′ terminus.

In one embodiment, provided is Compound_—9 that is a 19-mer double stranded nucleic acid molecule wherein the sense strand is SEQ ID NO:101 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 2, 4, 11, 13, and 17, a C3OH non-nucleotide moiety covalently attached at the 3′ terminus and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′ terminus; and the antisense strand is SEQ ID NO:168 and includes 2′OMe sugar modified ribonucleotides in positions (5′>3′) 1, 4, 8, 11 and 15, a 2′5′ ribonucleotide in position 6; a 3′ C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′ terminus.

In another aspect, provided are methods for reducing the expression of hsp47 in a cell by introducing into a cell a nucleic acid molecule as provided herein in an amount sufficient to reduce expression of hsp47. In one embodiment, the cell is hepatocellular stellate cell. In another embodiment, the cell is a stellate cell in renal or pulmonary tissue. In certain embodiments, the method is performed in vitro, in other embodiments, the method is performed in vivo.

In yet another aspect, provided are methods for treating an individual suffering from a disease associated with hsp47. The methods include administering to the individual a nucleic acid molecule such as provided herein in an amount sufficient to reduce expression of hsp47. In certain embodiments the disease associated with hsp47 is a disease selected from the group consisting of liver fibrosis, cirrhosis, pulmonary fibrosis including lung fibrosis (including ILF), any condition causing kidney fibrosis (e.g., CKD including ESRD), peritoneal fibrosis, chronic hepatic damage, fibrillogenesis, fibrotic diseases in other organs, abnormal scarring (keloids) associated with all possible types of skin injury accidental and jatrogenic (operations); scleroderma; cardiofibrosis, failure of glaucoma filtering operation; and intestinal adhesions. In some embodiments, the compounds may be useful in treating organ-specific indications, for example indications including those shown in Table 2 below:

TABLE 2

Organ
Indication

Skin
Pathologic scarring as keloid and hypertrophic scar

Surgical scarring

Injury scarring

keloid, or nephrogenic fibrosing dermatopathy

Peritoneum
Peritoneal fibrosis

Adhesions

Peritoneal Sclerosis associated with continual ambulatory peritoneal

dialysis (CAPD)

Liver
Cirrhosis including post-hepatitis C cirrhosis, primary biliary cirrhosis

Liver fibrosis, e.g. Prevention of Liver Fibrosis in Hepatitis C carriers

schistomasomiasis

cholangitis

Liver cirrhosis due to Hepatitis C post liver transplant or Non-Alcoholic

Steatohepatitis (NASH)

Pancreas
inter(peri)lobular fibrosis (as in alcoholic chronic pancreatitis), periductal

fibrosis (as in hereditary pancreatitis), periductal and interlobular fibrosis

(as in autoimmune pancreatitis), diffuse inter- and intralobular fibrosis (as

in obstructive chronic pancreatitis)

Kidney
Chronic Kidney Disease (CKD) of any etiology. Treatment of early stage

CKD (elevated SCr) in diabetic patients (“prevent” further deterioration in

renal function)

kidney fibrosis associated with lupus glomeruloschelerosis

Diabetic Nephropathy

Heart
Congestive heart failure,

Endomyocardial fibrosis,

cardiofibrosis

fibrosis associated with myocardial infarction

Lung
Asthma, Idiopathic pulmonary fibrosis (IPF);

Interstitial lung fibrosis (ILF)

Radiation Pneumonitis leading to Pulmonary Fibrosis (e.g. due to cancer

treating radiation)

Bone marrow
Myeloproliferative disorders: Myelofibrosis (MF), Polycythemia vera

(PV), Essential thrombocythemia (ET)

idiopathic myelofibrosis

drug induced myelofibrosis.

Eye
Anterior segment: Corneal opacification e,g, following inherited

dystrophies, herpetic keratitis or pterygia; Glaucoma

Posterior segment fibrosis and traction retinal detachment, a complication

of advanced diabetic retinopathy (DR); Fibrovascular scarring and gliosis

in the retina;

Under the retina fibrosis for example subsequent to subretinal hemorrhage

associated with neovascular AMD

Retro-orbital fibrosis, postcataract surgery, proliferative vitreoretinopathy.

Ocular cicatricial pemphigoid

Intestine
Intestinal fibrosis, Crohn's disease

Vocal cord
Vocal cord scarring, vocal cord mucosal fibrosis, laryngeal fibrosis

Vasculature
Atherosclerosis, postangioplasty arterial restenosis

Multisystemic
Scleroderma systemic sclerosis; multifocal fibrosclerosis; sclerodermatous

graft-versus-host disease in bone marrow transplant recipients, and

nephrogenic systemic fibrosis (exposure to gadolinium-based contrast

agents (GBCAs), 30% of MRIs)

Malignancies of
Metastatic and invasive cancer by inhibiting function of activated tumor

various origin
associated myofibroblasts

In some embodiments the preferred indications include, Liver cirrhosis due to Hepatitis C post liver transplant; Liver cirrhosis due to\ Non-Alcoholic Steatohepatitis (NASH); Idiopathic Pulmonary Fibrosis; Radiation Pneumonitis leading to Pulmonary Fibrosis; Diabetic Nephropathy; Peritoneal Sclerosis associated with continual ambulatory peritoneal dialysis (CAPD) and Ocular cicatricial pemphigoid.

Fibrotic Liver indications include Alcoholic Cirrhosis, Hepatitis B cirrhosis, Hepatitis C cirrhosis, Hepatitis C (Hep C) cirrhosis post orthotopic liver transplant (OLTX), NASH/NAFLD, Primary biliary cirrhosis (PBC), Primary sclerosing cholangitis (PSC), Biliary atresia, alpha1 antitrypsin deficiency (A1AD), Copper storage diseases (Wilson's disease), Fructosemia, Galactosemia, Glycogen storage diseases (especially types III, IV, VI, IX, and X), Iron-overload syndromes (hemochromatosis), Lipid abnormalities (e.g., Gaucher's disease). Peroxisomal disorders (eg, Zellweger syndrome), Tyrosinemia, Congenital hepatic fibrosis, Bacterial Infections (eg, brucellosis), Parasitic (eg, echinococcosis), Budd-Chiari syndrome (hepatic veno-occlusive disease).

Pulmonary Indications indications include Idiopathic Pulmonary Fibrosis, Silicosis, Pneumoconiosis, Bronchopulmonary Dysplasia in newborn following neonatal respiratory distress syndrome, Bleomycin/chemo lung injury, Brochiolitis Obliterans (BOS) post lung transplant, Chronic obstructive pulmonary disorder (COPD), Cystic Fibrosis, Asthma.

Cardiac indications include Cardiomyopathy, Atherosclerosis (Bergers disease, etc), Endomyocardial fibrosis, Atrial Fibrillation, Scarring post Myocardial Infarction (MI)

Other Thoracic indications include Radiation-induced capsule tissue reactions around textured breast implants and Oral submucosal fibrosis.

Renal indications include Autosomal Dominant Polycystic Kidney Disease (ADPKD), Diabetic nephropathy (diabetic glomerulosclerosis), FSGS (collapsing vs, other histologic variants), IgA Nephropathy (Berger Disease), Lupus Nephritis, Wegner's, Scleroderma, Goodpasture Syndrome, tubulointerstitial fibrosis: drug induced (protective) pencillins, cephalosporins, analgesic nephropathy, Membranoproliferative glomerulonephritis (MPGN), Henoch-Schonlein Purpura, Congenital nephropathies: Medullary Cystic Disease, Nail-Patella Syndrome and Alport Syndrome.

Bone Marrow indications include lympangiolyomyositosis (LAM), Chronic graft vs. host disease, Polycythemia vera, Essential thrombocythemia, Myelofibrosis.

Ocular indications include Retinopathy of Prematurity (RoP), Ocular cicatricial pemphigoid, Lacrimal gland fibrosis, Retinal attachment surgery, Corneal opacity, Herpetic keratitis, Pterygia, Glaucoma, Age-related macular degeneration (AMD/ARMD), Retinal fibrosis associated Diabetes mellitus (DM) retinopathy

Gynecological indications include Endometriosis add on to hormonal therapy for prevention of scarring, post STD fibrosis/salphingitis,

Systemic indications include Dupuytren's disease, palmar fibromatosis, Peyronie's disease, Ledderhose disease, keloids, multifocal fibrosclerosis, nephrogenic systemic fibrosis, nephrogenic myelofibrosis (anemia).

Injury Associated Fibrotic Diseases include Burn (chemical included) induced skin & soft tissue scarring and contraction, Radiation induce skin & organ scarring post cancer therapeutic radiation treatment, Keloid (skin).

Surgical indications include peritoneal fibrosis post peritoneal dialysis catheter, corneal implant, cochlear implant, other implants, silicone implants in breasts, chronic sinusitis; adhesions, pseudointimal hyperplasia of dialysis grafts.

Other indications include Chronic Pancreatitis.

In some embodiments provided is a method for treatment of a subject suffering from liver fibrosis comprising administering to the subject an effective amount of a nucleic acid molecule disclosed herein, thereby treating liver fibrosis. In some embodiments the subject is suffering from cirrhosis of the liver due to hepatitis. In some embodiments the subject is suffering from cirrhosis of the liver due to NASH.

In some embodiments provided is the use of a nucleic acid molecule disclosed herein for the manufacture of a medicament to treat liver fibrosis. In some embodiments the liver fibrosis is due to hepatitis. In some embodiments the liver fibrosis is due to NASH.

In some embodiments provided is a method for remodeling of scar tissue comprising administering to a subject in need thereof an effective amount of a nucleic acid molecule disclosed herein, thereby effecting scar tissue remodeling. In some embodiments the scar tissue is in the liver. In some embodiments the subject is suffering from cirrhosis of the liver due to hepatitis. In some embodiments the subject is suffering from cirrhosis of the liver due to NASH.

In some embodiments provided is a method for effecting fibrosis regression comprising administering to a subject in need thereof an effective amount of a nucleic acid molecule disclosed herein, thereby effecting fibrosis regression.

In some embodiments provided is a method for reduction of scar tissue in a subject comprising the step of administering to the subject an effective amount of a nucleic acid molecule disclosed herein to reduce the scar tissue. In some embodiments provided is a method for reducing scar tissue in a subject comprising the step of topically applying to scar tissue an effective amount of a nucleic acid molecule disclosed herein to reduce scar tissue.

In some embodiments provided is a method for improving the appearance of scar tissue comprising the step of topically applying to scar tissue an effective amount of a nucleic acid molecule disclosed herein to improve the appearance of the scar tissue.

In some embodiments provided is a method for treatment of a subject suffering from lung fibrosis comprising administering to the subject an effective amount of a nucleic acid molecule disclosed herein, thereby treating the lung fibrosis. In some embodiments the subject is suffering from interstitial lung fibrosis (ILF). In some embodiments the subject is suffering from Radiation Pneumonitis leading to Pulmonary Fibrosis. In some embodiments the subject is suffering from drug induced lung fibrosis.

In some embodiments provided is the use of a nucleic acid molecule disclosed herein for the manufacture of a medicament to treat lung fibrosis. In some embodiments the lung fibrosis is ILF. In some embodiments the lung fibrosis drug- or radiatio-induced lung fibrosis.

In one aspect, provided are pharmaceutical compositions that include a nucleic acid molecule (e.g., an siNA molecule) as described herein in a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical formulation includes, or involves, a delivery system suitable for delivering nucleic acid molecules (e.g., siNA molecules) to an individual such as a patient; for example delivery systems described in more detail below.

In a related aspect, provided are compositions or kits that include a nucleic acid molecule (e.g., an siNA molecule) packaged for use by a patient. The package may be labeled or include a package label or insert that indicates the content of the package and provides certain information regarding how the nucleic acid molecule (e.g., an siNA molecule) should be or can be used by a patient, for example the label may include dosing information and/or indications for use. In certain embodiments the contents of the label will bear a notice in a form prescribed by a government agency, for example the United States Food and Drug administration. In certain embodiments, the label may indicate that the nucleic acid molecule (e.g., an siNA molecule) is suitable for use in treating a patient suffering from a disease associated with hsp47; for example, the label may indicate that the nucleic acid molecule (e.g., an siNA molecule) is suitable for use in treating fibroids; or for example the label may indicate that the nucleic acid molecule (e.g., an siNA molecule) is suitable for use in treating a disease selected from the group consisting of fibrosis, liver fibrosis, cirrhosis, pulmonary fibrosis, kidney fibrosis, peritoneal fibrosis, chronic hepatic damage, and fibrillogenesis,

As used herein, the term “heat shock protein 47” or “hsp47” or “HSP47” are used interchangeably and refer to any heat shock protein 47, peptide, or polypeptide having any hsp47 protein activity. Heat shock protein 47 is a serine proteinase inhibitor (serpin) also known, for example, as serpin peptidase inhibitor, Glade H, member 1 (SERPINH1), SERPINH2, collagen binding protein 1 (CBP1), CBP2, gp46; arsenic-transactivated protein 3 (AsTP3); HSP47; proliferation-inducing gene 14 (PIG14); PPROM; rheumatoid arthritis antigen A-47 (RA-A47); colligin-1; and colligin-2. In certain preferred embodiments, “hsp47” refers to human hsp47. Heat shock protein 47 (or more particularly human hsp47) may have an amino acid sequence that is the same, or substantially the same, as SEQ ID NO. 2 (FIG. 7).

As used herein the term “nucleotide sequence encoding hsp47” means a nucleotide sequence that codes for an hsp47 protein or portion thereof. The term “nucleotide sequence encoding hsp47” is also meant to include hsp47 coding sequences such as hsp47 isoforms, mutant hsp47 genes, splice variants of hsp47 genes, and hsp47 gene polymorphisms. A nucleic acid sequence encoding hsp47 includes mRNA sequences encoding hsp47, which can also be referred to as “hsp47 mRNA.” An exemplary sequence of human hsp47 mRNA is SEQ ID. NO. 1.

As used herein, the term “nucleic acid molecule” or “nucleic acid” are used interchangeably and refer to an oligonucleotide, nucleotide or polynucleotide. Variations of “nucleic acid molecule” are described in more detail herein. A nucleic acid molecule encompasses both modified nucleic acid molecules and unmodified nucleic acid molecules as described herein. A nucleic acid molecule may include deoxyribonucleotides, ribonucleotides, modified nucleotides or nucleotide analogs in any combination.

As used herein, the term “nucleotide” refers to a chemical moiety having a sugar (or an analog thereof, or a modified sugar), a nucleotide base (or an analog thereof, or a modified base), and a phosphate group (or analog thereof, or a modified phosphate group). A nucleotide encompasses both modified nucleotides or unmodified nucleotides as described herein. As used herein, nucleotides may include deoxyribonucleotides (e.g., unmodified deoxyribonucleotides), ribonucleotides (e.g., unmodified ribonucleotides), and modified nucleotide analogs including, inter alia, locked nucleic acids and unlocked nucleic acids, peptide nucleic acids, L-nucleotides (also referred to as mirror nucleotides), ethylene-bridged nucleic acid (ENA), arabinoside, PACE, nucleotides with a 6 carbon sugar, as well as nucleotide analogs (including abasic nucleotides) often considered to be non-nucleotides. In some embodiments, nucleotides may be modified in the sugar, nucleotide base and/or in the phosphate group with any modification known in the art, such as modifications described herein. A “polynucleotide” or “oligonucleotide” as used herein refer to a chain of linked nucleotides; polynucleotides and oligonucleotides may likewise have modifications in the nucleotide sugar, nucleotide bases and phosphate backbones as are well known in the art and/or are disclosed herein.

As used herein, the term “short interfering nucleic acid”, “siNA”, or “short interfering nucleic acid molecule” refers to any nucleic acid molecule capable of modulating gene expression or viral replication. Preferably siNA inhibits or down regulates gene expression or viral replication. siNA includes without limitation nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. As used herein, “short interfering nucleic acid”, “siNA”, or “short interfering nucleic acid molecule” has the meaning described in more detail elsewhere herein.

As used herein, the term “complementary” means that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules disclosed herein, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). “Fully complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, a nucleic acid molecule disclosed herein includes about 15 to about 35 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.

As used herein, the term “sense region” refers to a nucleotide sequence of a siNA molecule complementary (partially or fully) to an antisense region of the siNA molecule. The sense strand of a siNA molecule can include a nucleic acid sequence having homology with a target nucleic acid sequence. As used herein, “sense strand” refers to nucleic acid molecule that includes a sense region and may also include additional nucleotides.

As used herein, the term “antisense region” refers to a nucleotide sequence of a siNA molecule complementary (partially or fully) to a target nucleic acid sequence. The antisense strand of a siNA molecule can optionally include a nucleic acid sequence complementary to a sense region of the siNA molecule. As used herein, “antisense strand” refers to nucleic acid molecule that includes an antisense region and may also include additional nucleotides.

As used herein, the term “RNA” refers to a molecule that includes at least one ribonucleotide residue.

As used herein, the term “duplex region” refers to the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the “duplex region” consists of 19 base pairs. The remaining base pairs may, for example, exist as 5′ and 3′ overhangs. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well know in the art. Alternatively, two strands can be synthesized and added together under biological conditions to determine if they anneal to one another.

As used herein, the terms “non-pairing nucleotide analog” means a nucleotide analog which includes a non-base pairing moiety including but not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In some embodiments the non-base pairing nucleotide analog is a ribonucleotide. In other embodiments it is a deoxyribonucleotide.

As used herein, the term, “terminal functional group” includes without limitation a halogen, alcohol, amine, carboxylic, ester, amide, aldehyde, ketone, ether groups.

An “abasic nucleotide” or “abasic nucleotide analog” is as used herein may also be often referred to herein and in the art as a pseudo-nucleotide or an unconventional moiety. While a nucleotide is a monomeric unit of nucleic acid, generally consisting of a ribose or deoxyribose sugar, a phosphate, and a base (adenine, guanine, thymine, or cytosine in DNA; adenine, guanine, uracil, or cytosine in RNA), an abasic or pseudo-nucleotide lacks a base, and thus is not strictly a nucleotide as the term is generally used in the art. Abasic deoxyribose moieties include for example, abasic deoxyribose-3′-phosphate; 1,2-dideoxy-D-ribofuranose-3-phosphate; 1,4-anhydro-2-deoxy-D-ribitol-3-phosphate. Inverted abasic deoxyribose moieties include inverted deoxyriboabasic; 3′,5′ inverted deoxyabasic 5′-phosphate.

The term “capping moiety” (z″) as used herein includes a moiety which can be covalently linked to the 5′ terminus of (N′)y and includes abasic ribose moiety, abasic deoxyribose moiety, modifications abasic ribose and abasic deoxyribose moieties including 2′O alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and modifications thereof; C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5′OMe nucleotide; and nucleotide analogs including 4′,5′-methylene nucleotide; 1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate; 5′-amino; and bridging or non bridging methylphosphonate and 5′-mercapto moieties.

Certain capping moieties may be abasic ribose or abasic deoxyribose moieties; inverted abasic ribose or abasic deoxyribose moieties; C6-amino-Pi; a mirror nucleotide including L-DNA and L-RNA. The nucleic acid molecules as disclosed herein may be synthesized using one or more inverted nucleotides, for example inverted thymidine or inverted adenine (for example see Takei, et al., 2002. JBC 277 (26)23800-06).

The term “unconventional moiety” as used herein refers to non-nucleotide moieties including an abasic moiety, an inverted abasic moiety, a hydrocarbon (alkyl) moiety and derivatives thereof, and further includes a deoxyribonucleotide, a modified deoxyribonucleotide, a mirror nucleotide (L-DNA or L-RNA), a non-base pairing nucleotide analog and a nucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotide phosphate bond; bridged nucleic acids including LNA and ethylene bridged nucleic acids, linkage modified (e.g. PACE) and base modified nucleotides as well as additional moieties explicitly disclosed herein as unconventional moieties.

As used herein, the term “inhibit”, “down-regulate”, or “reduce” with respect to gene expression means the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits (e.g., mRNA), or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of an inhibitory factor (such as a nucleic acid molecule, e.g., an siNA, for example having structural features as described herein); for example the expression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less than that observed in the absence of an inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph showing the effect of GFP siNA on various reporter cell lines. Cell lines were established by lenti-viral induction of human HSP47 cDNA-GFP or rat GP46 cDNA-GFP construct into HEK293, human fibrosarcoma cell line HT1080, human HSC line hTERT or NRK cell line. Negative control siNA or siNA against GFP was introduced into the cells and GFP fluorescence was measured. The results showed that siNA against GFP knocks down the fluorescence to different extent in different cell lines. 293 HSP47-GFP and 293_GP46-GFP cell lines were selected for siHsp47 screening due to their easiness of being transfected and sensitivity to fluorescence knockdown.

FIG. 2 is a series of bar graphs showing the cytotoxicity and knockdown efficiency of various siHsp47s in 293_HSP47-GFP and 293_GP46-GFP cell lines. The result showed that siHsp47-C, siHsp47-2 and siHsp47-2d efficiently knockdown both human HSP47 and rat GP46 (the human hsp47 homolog) without substantial cytotoxicity. siGp46A against GP46 does not knock down human HSP47. Additionally, the newly designed siHsp47s outperformed siGp46A in knocking down rat GP46.

FIG. 3 is a bar graph showing the knock down effect of various siHsp47s on hsp47 mRNA, measured by TaqMan® qPCR using the human HSC cell line hTERT. The Y axis represents the remaining mRNA level of hsp47. HSP47-C was most effective among all the hsp47 siNAs tested.

FIG. 4 is a bar graph showing the effect of different hsp47 siNAs on collagen I expression in hTERT cells. The level of collagen I mRNA levels were measured by real-time quantitative PCR using TaqMan® probe. The Y axis represents the remaining mRNA expression level of collagen I. The result showed that collagen I mRNA level is significantly reduced in the cells treated with some of the candidates (siHsp47-2, siHsp47-2d, and their combination with siHsp47-1).

FIG. 5 shows the decrease in fibrotic areas of the liver in animals treated with siHSP47.

FIG. 6 is an exemplary nucleic acid sequence of human hsp47 mRNA cDNA (SEQ ID NO: 1; based on the cDNA disclosed in GenBank accession number: NM 001235).

FIG. 7 is an exemplary amino acid sequence of human hsp47 (SEQ ID NO:2).

FIG. 8 is protein coding nucleic acid sequence of human hsp47 cDNA (SEQ ID NO:59), which corresponds to nucleotides 230-1486 of SEQ ID NO: 1.

FIGS. 9A-91 show plasma stability of Compound_—1, Compound_—2, Compound 3, Compound_—4, Compound_—5, Compound_—6, Compound_—7, Compound_—8 and Compound_—9, respectively, as detected by ethidium bromide staining.

FIGS. 10A-10I show ontarget/off-target activity of Compound_—1, Compound_—2, Compound_—3, Compound_—4, Compound_—5, Compound_—6, Compound_—7, Compound_—8 and Compound_—9, respectively. AS_CM shows activity of antisense strand of compound to a plasmid comprising a full match insert; AS_SM shows activity of antisense strand of compound to a plasmid comprising seed sequence insert; S_CM shows activity of sense strand of compound to a plasmid comprising a full match insert. All assays were performed in human cells, except for data shown in FIG. 10F which was performed in rat REF52 cells.

DETAILED DESCRIPTION OF THE INVENTION
RNA Interference and siNA Nucleic Acid Molecules

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

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

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283 and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., International PCT Publication No. WO 01/75164, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity.

Nucleic acid molecules (for example having structural features as disclosed herein) may inhibit or down regulate gene expression or viral replication by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner; see e.g., Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).

An siNA nucleic acid molecule can be assembled from two separate polynucleotide strands, where one strand is the sense strand and the other is the antisense strand in which the antisense and sense strands are self-complementary (i.e. each strand includes nucleotide sequence that is complementary to nucleotide sequence in the other strand); such as where the antisense strand and sense strand form a duplex or double stranded structure having any length and structure as described herein for nucleic acid molecules as provided, for example wherein the double stranded region (duplex region) is about 15 to about 49 (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 base pairs); the antisense strand includes nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule (i.e., hsp47 mRNA) or a portion thereof and the sense strand includes nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 17 to about 49 or more nucleotides of the nucleic acid molecules herein are complementary to the target nucleic acid or a portion thereof).

In certain aspects and embodiments a nucleic acid molecule (e.g., a siNA molecule) provided herein may be a “RISC length” molecule or may be a Dicer substrate as described in more detail below.

An siNA nucleic acid molecule may include separate sense and antisense sequences or regions, where the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and/or stacking interactions. Nucleic acid molecules may include a nucleotide sequence that is complementary to nucleotide sequence of a target gene. Nucleic acid molecules may interact with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.

Alternatively, an siNA nucleic acid molecule is assembled from a single polynucleotide, where the self-complementary sense and antisense regions of the nucleic acid molecules are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), i.e., the antisense strand and the sense strand are part of one single polynucleotide that having an antisense region and sense region that fold to form a duplex region (for example to form a “hairpin” structure as is well known in the art). Such siNA nucleic acid molecules can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region includes nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence (e.g., a sequence of hsp47 mRNA). Such siNA nucleic acid molecules can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region includes nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active nucleic acid molecule capable of mediating RNAi.

The following nomenclature is often used in the art to describe lengths and overhangs of siNA molecules and may be used throughout the specification and Examples. Names given to duplexes indicate the length of the oligomers and the presence or absence of overhangs. For example, a “21+2” duplex contains two nucleic acid strands both of which are 21 nucleotides in length, also termed a 21-mer siRNA duplex or a 21-mer nucleic acid and having a 2 nucleotides 3′-overhang. A “21−2” design refers to a 21-mer nucleic acid duplex with a 2 nucleotides 5′-overhang. A 21−0 design is a 21-mer nucleic acid duplex with no overhangs (blunt). A “21+2UU” is a 21-mer duplex with 2-nucleotides 3′-overhang and the terminal 2 nucleotides at the 3′-ends are both U residues (which may result in mismatch with target sequence). The aforementioned nomenclature can be applied to siNA molecules of various lengths of strands, duplexes and overhangs (such as 19−0, 21+2, 27+2, and the like). In an alternative but similar nomenclature, a “25/27” is an asymmetric duplex having a 25 base sense strand and a 27 base antisense strand with a 2-nucleotides 3′-overhang. A “27/25” is an asymmetric duplex having a 27 base sense strand and a 25 base antisense strand.

Chemical Modifications

In certain aspects and embodiments, nucleic acid molecules (e.g., siNA molecules) as provided herein include one or more modifications (or chemical modifications). In certain embodiments, such modifications include any changes to a nucleic acid molecule or polynucleotide that would make the molecule different than a standard ribonucleotide or RNA molecule (i.e., that includes standard adenosine, cytosine, uracil, or guanosine moieties); which may be referred to as an “unmodified” ribonucleotide or unmodified ribonucleic acid. Traditional DNA bases and polynucleotides having a 2′-deoxy sugar represented by adenosine, cytosine, thymine, or guanosine moieties may be referred to as an “unmodified deoxyribonucleotide” or “unmodified deoxyribonucleic acid”; accordingly, the term “unmodified nucleotide” or “unmodified nucleic acid” as used herein refers to an “unmodified ribonucleotide” or “unmodified ribonucleic acid” unless there is a clear indication to the contrary. Such modifications can be in the nucleotide sugar, nucleotide base, nucleotide phosphate group and/or the phosphate backbone of a polynucleotide.

In certain embodiments modifications as disclosed herein may be used to increase RNAi activity of a molecule and/or to increase the in vivo stability of the molecules, particularly the stability in serum, and/or to increase bioavailability of the molecules. Non-limiting examples of modifications include without limitation internucleotide or internucleoside linkages; deoxynucleotides or dideoxyribonucleotides at any position and strand of the nucleic acid molecule; nucleic acid (e.g., ribonucleic acid) with a modification at the 2′-position preferably selected from an amino, fluoro, methoxy, alkoxy and alkyl; 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, biotin group, and terminal glyceryl and/or inverted deoxy abasic residue incorporation, sterically hindered molecules, such as fluorescent molecules and the like. Other nucleotides modifiers could include 3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphate nucleotides of 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxy-3′-thiacytidine (3TC) and 2′,3′-didehydro-2′,3′-dide-oxythymidine (d4T). Further details on various modifications are described in more detail below.

Modified nucleotides include those having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methyl nucleotides. Locked nucleic acids, or LNA's are described, for example, in Elman et al., 2005; Kurreck et al., 2002; Crinelli et al., 2002; Braasch and Corey, 2001; Bondensgaard et al., 2000; Wahlestedt et al., 2000; and International Patent Publication Nos. WO 00/47599, WO 99/14226, and WO 98/39352 and WO 2004/083430. In one embodiment, an LNA is incorporated at the 5′ terminus of the sense strand.

Chemical modifications also include unlocked nucleic acids, or UNAs, which are non-nucleotide, acyclic analogues, in which the C2′-C3′ bond is not present (although UNAs are not truly nucleotides, they are expressly included in the scope of “modified” nucleotides or modified nucleic acids as contemplated herein). In particular embodiments, nucleic acid molecules with an overhang may be modified to have UNAs at the overhang positions (i.e., 2 nucleotide overhand). In other embodiments, UNAs are included at the 3′- or 5′-ends. A UNA may be located anywhere along a nucleic acid strand, i.e. in position 7. Nucleic acid molecules may contain one or more than UNA. Exemplary UNAs are disclosed in Nucleic Acids Symposium Series No. 52 p. 133-134 (2008). In certain embodiments a nucleic acid molecule (e.g., a siNA molecule) as described herein include one or more UNAs; or one UNA. In some embodiments, a nucleic acid molecule (e.g., a siNA molecule) as described herein that has a 3′-overhang include one or two UNAs in the 3′ overhang. In some embodiments a nucleic acid molecule (e.g., a siNA molecule) as described herein includes a UNA (for example one UNA) in the antisense strand; for example in position 6 or position 7 of the antisense strand. Chemical modifications also include non-pairing nucleotide analogs, for example as disclosed herein. Chemical modifications further include unconventional moieties as disclosed herein.

Chemical modifications also include terminal modifications on the 5′ and/or 3′ part of the oligonucleotides and are also known as capping moieties. Such terminal modifications are selected from a nucleotide, a modified nucleotide, a lipid, a peptide, and a sugar.

Chemical modifications also include six membered “six membered ring nucleotide analogs.” Examples of six-membered ring nucleotide analogs are disclosed in Allart, et al (Nucleosides & Nucleotides, 1998, 17:1523-1526; and Perez-Perez, et al., 1996, Bioorg. and Medicinal Chem Letters 6:1457-1460) Oligonucleotides including 6-membered ring nucleotide analogs including hexitol and altritol nucleotide monomers are disclosed in International patent application publication No. WO 2006/047842.

Chemical modifications also include “mirror” nucleotides which have a reversed chirality as compared to normal naturally occurring nucleotide; that is a mirror nucleotide may be an “L-nucleotide” analogue of naturally occurring D-nucleotide (see U.S. Pat. No. 6,602,858). Mirror nucleotides may further include at least one sugar or base modification and/or a backbone modification, for example, as described herein, such as a phosphorothioate or phosphonate moiety. U.S. Pat. No. 6,602,858 discloses nucleic acid catalysts including at least one L-nucleotide substitution. Mirror nucleotides include for example L-DNA (L-deoxyriboadenosine-3′-phosphate (mirror dA); L-deoxyribocytidine-3′-phosphate (mirror dC); L-deoxyriboguanosine-3′-phosphate (mirror dG); L-deoxyribothymidine-3′-phosphate (mirror image dT)) and L-RNA (L-riboadenosine-3′-phosphate (mirror rA); L-ribocytidine-3-phosphate (mirror rC); L-riboguanosine-3′-phosphate (mirror rG); L-ribouracil-3′-phosphate (mirror dU).

In some embodiments, modified ribonucleotides include modified deoxyribonucleotides, for example 5′OMe DNA (5-methyl-deoxyriboguanosine-3′-phosphate) which may be useful as a nucleotide in the 5′ terminal position (position number 1); PACE (deoxyriboadenosine 3′ phosphonoacetate, deoxyribocytidine 3′ phosphonoacetate, deoxyriboguanosine 3′ phosphonoacetate, deoxyribothymidine 3′ phosphonoacetate.

Modifications may be present in one or more strands of a nucleic acid molecule disclosed herein, e.g., in the sense strand, the antisense strand, or both strands. In certain embodiments, the antisense strand may include modifications and the sense strand my only include unmodified RNA.

Nucleobases

Nucleobases of the nucleic acid disclosed herein may include unmodified ribonucleotides (purines and pyrimidines) such as adenine, guanine, cytosine, uracil. The nucleobases in one or both strands can be modified with natural and synthetic nucleobases such as, thymine, xanthine, hypoxanthine, inosine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, any “universal base” nucleotides; 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, deazapurines, heterocyclic substituted analogs of purines and pyrimidines, e.g., aminoethyoxy phenoxazine, derivatives of purines and pyrimidines (e.g., 1-alkyl-, 1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) and tautomers thereof, 8-oxo-N-6-methyladenine, 7-diazaxanthine, 5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl) cytosine and 4,4-ethanocytosine). Other examples of suitable bases include non-purinyl and non-pyrimidinyl bases such as 2-aminopyridine and triazines.

Sugar Moieties

Sugar moieties in nucleic acid disclosed herein may include 2′-hydroxyl-pentofuranosyl sugar moiety without any modification. Alternatively, sugar moieties can be modified such as, 2′-deoxy-pentofuranosyl sugar moiety, D-ribose, hexose, modification at the 2′ position of the pentofuranosyl sugar moiety such as 2′-O-alkyl (including 2′-O-methyl and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino, 2′-O-allyl, 2′-S-alkyl, 2′-halogen (including 2′-fluoro, chloro, and bromo), 2′-methoxyethoxy, 2′-O-methoxyethyl, 2′-O-2-methoxyethyl, 2′-allyloxy (—OCH2CH═CH2), 2′-propargyl, 2′-propyl, ethynyl, ethenyl, propenyl, CF, cyano, imidazole, carboxylate, thioate, C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF3, OCN, O-, S-, or N-alkyl; O-, S, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2, N3; heterozycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl, as, among others, for example as described in European patents EP 0 586 520 B1 or EP 0 618 925 B1.

Alkyl group includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl(alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and more preferably 4 or fewer. Likewise, preferred cycloalkyls may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C1-C6 includes alkyl groups containing 1 to 6 carbon atoms. The alkyl group can be substituted alkyl group such as alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Alkoxy group includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkyl aryl amino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

In some embodiments, the pentafuronosyl ring may be replaced with acyclic derivatives lacking the C2′-C3′-bond of the pentafuronosyl ring. For example, acyclonucleotides may substitute a 2-hydroxyethoxymethyl group for-the 2′-deoxyribofuranosyl sugar normally present in dNMPs.

Halogens include fluorine, bromine, chlorine, iodine.

Backbone

The nucleoside subunits of the nucleic acid disclosed herein may be linked to each other by phosphodiester bond. The phosphodiester bond may be optionally substituted with other linkages. For example, phosphorothioate, thiophosphate-D-ribose entities, triester, thioate, 2′-5′ bridged backbone (may also be referred to as 5′-2′ or 2′5′ nucleotide or 2′5′ ribonucleotide), PACE, 3′-(or -5′)deoxy-3′-(or -5′)thio-phosphorothioate, phosphorodithioate, phosphoroselenates, 3′-(or -5′)deoxy phosphinates, borano phosphates, 3′-(or -5′)deoxy-3′-(or 5′-)amino phosphoramidates, hydrogen phosphonates, phosphonates, borano phosphate esters, phosphoramidates, alkyl or aryl phosphonates and phosphotriester modifications such as alkylphosphotriesters, phosphotriester phosphorus linkages, 5′-ethoxyphosphodiester, P-alkyl oxyphosphotriester, methylphosphonate, and nonphosphorus containing linkages for example, carbonate, carbamate, silyl, sulfur, sulfonate, sulfonamide, formacetal, thioformacetyl, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino linkages.

Nucleic acid molecules disclosed herein may include a peptide nucleic acid (PNA) backbone. The PNA backbone is includes repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various bases such as purine, pyrimidine, natural and synthetic bases are linked to the backbone by methylene carbonyl bonds.

Terminal Phosphates

Modifications can be made at terminal phosphate groups. Non-limiting examples of different stabilization chemistries can be used, e.g., to stabilize the 3′-end of nucleic acid sequences, including (1) [3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3) [5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5) [5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7) [3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9) [5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. In addition to unmodified backbone chemistries can be combined with one or more different backbone modifications described herein.

Exemplary chemically modified terminal phosphate groups include those shown below:

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Conjugates

Modified nucleotides and nucleic acid molecules (e.g., siNA molecules) as provided herein may include conjugates, for example, a conjugate covalently attached to the chemically-modified nucleic acid molecule. Non-limiting examples of conjugates include conjugates and ligands described in Vargeese et al., U.S. Ser. No. 10/427,160. The conjugate may be covalently attached to a nucleic acid molecule (such as an siNA molecule) via a biodegradable linker. The conjugate molecule may be attached at the 3′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified nucleic acid molecule. The conjugate molecule may be attached at the 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified nucleic acid molecule. The conjugate molecule may be attached both the 3′-end and 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified nucleic acid molecule, or any combination thereof. In one embodiment, a conjugate molecule may include a molecule that facilitates delivery of a chemically-modified nucleic acid molecule into a biological system, such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified nucleic acid molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified nucleic acid molecules are described in Vargeese et al., U.S. Ser. No. 10/201,394.

Linkers

A nucleic acid molecule provided herein (e.g., an siNA) molecule may include a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the nucleic acid to the antisense region of the nucleic acid. A nucleotide linker can be a linker of ≧2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. The nucleotide linker can be a nucleic acid aptamer. By “aptamer” or “nucleic acid aptamer” as used herein refers to a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that includes a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule (such as hsp47 mRNA) where the target molecule does not naturally bind to a nucleic acid. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. See e.g., Gold et al.; 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.

A non-nucleotide linker may include an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000.

5′ Ends, 3′ Ends and Overhangs

Nucleic acid molecules disclosed herein (e.g., siNA molecules) may be blunt-ended on both sides, have overhangs on both sides or a combination of blunt and overhang ends. Overhangs may occur on either the 5′- or 3′-end of the sense or antisense strand.

5′- and/or 3′-ends of double stranded nucleic acid molecules (e.g., siNA) may be blunt ended or have an overhang. The 5′-end may be blunt ended and the 3′-end has an overhang in either the sense strand or the antisense strand. In other embodiments, the 3′-end may be blunt ended and the 5′-end has an overhang in either the sense strand or the antisense strand. In yet other embodiments, both the 5′- and 3′-end are blunt ended or both the 5′- and 3′-ends have overhangs.

The 5′- and/or 3′-end of one or both strands of the nucleic acid may include a free hydroxyl group. The 5′- and/or 3′-end of any nucleic acid molecule strand may be modified to include a chemical modification. Such modification may stabilize nucleic acid molecules, e.g., the 3′-end may have increased stability due to the presence of the nucleic acid molecule modification. Examples of end modifications (e.g., terminal caps) include, but are not limited to, abasic, deoxy abasic, inverted (deoxy) abasic, glyceryl, dinucleotide, acyclic nucleotide, amino, fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate, C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF3, OCN, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2, N3; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl, as, among others, described in European patents EP 586,520 and EP 618,925 and other modifications disclosed herein.

Nucleic acid molecules include those with blunt ends, i.e., ends that do not include any overhanging nucleotides. A nucleic acid molecule can include one or more blunt ends. The blunt ended nucleic acid molecule has a number of base pairs equal to the number of nucleotides present in each strand of the nucleic acid molecule. The nucleic acid molecule can include one blunt end, for example where the 5′-end of the antisense strand and the 3′-end of the sense strand do not have any overhanging nucleotides. Nucleic acid molecule may include one blunt end, for example where the 3′-end of the antisense strand and the 5′-end of the sense strand do not have any overhanging nucleotides. A nucleic acid molecule may include two blunt ends, for example where the 3′-end of the antisense strand and the 5′-end of the sense strand as well as the 5′-end of the antisense strand and 3′-end of the sense strand do not have any overhanging nucleotides. Other nucleotides present in a blunt ended nucleic acid molecule can include, for example, mismatches, bulges, loops, or wobble base pairs to modulate the activity of the nucleic acid molecule to mediate RNA interference.

In certain embodiments of the nucleic acid molecules (e.g., siNA molecules) provided herein, at least one end of the molecule has an overhang of at least one nucleotide (for example 1 to 8 overhang nucleotides). For example, one or both strands of a double stranded nucleic acid molecule disclosed herein may have an overhang at the 5′-end or at the 3′-end or both. An overhang may be present at either or both the sense strand and antisense strand of the nucleic acid molecule. The length of the overhang may be as little as one nucleotide and as long as 1 to 8 or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides; in some preferred embodiments an overhang is 2, 3, 4, 5, 6, 7 or 8 nucleotides; for example an overhang may be 2 nucleotides. The nucleotide(s) forming the overhang may be include deoxyribonucleotide(s), ribonucleotide(s), natural and non-natural nucleobases or any nucleotide modified in the sugar, base or phosphate group such as disclosed herein. A double stranded nucleic acid molecule may have both 5′- and 3′-overhangs. The overhangs at the 5′- and 3′-end may be of different lengths. An overhang may include at least one nucleic acid modification which may be deoxyribonucleotide. One or more deoxyribonucleotides may be at the 5′-terminal. The 3′-end of the respective counter-strand of the nucleic acid molecule may not have an overhang, more preferably not a deoxyribonucleotide overhang. The one or more deoxyribonucleotide may be at the 3′-terminal. The 5′-end of the respective counter-strand of the dsRNA may not have an overhang, more preferably not a deoxyribonucleotide overhang. The overhang in either the 5′- or the 3′-end of a strand may be 1 to 8 (e.g., about 1, 2, 3, 4, 5, 6, 7 or 8) unpaired nucleotides, preferably, the overhang is 2-3 unpaired nucleotides; more preferably 2 unpaired nucleotides. Nucleic acid molecules may include duplex nucleic acid molecules with overhanging ends of about 1 to about 20 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1, 15, 16, 17, 18, 19 or 20); preferably 1-8 (e.g., about 1, 2, 3, 4, 5, 6, 7 or 8) nucleotides, for example, about 21-nucleotide duplexes with about 19 base pairs and 3′-terminal mononucleotide, dinucleotide, or trinucleotide overhangs. Nucleic acid molecules herein may include duplex nucleic acid molecules with blunt ends, where both ends are blunt, or alternatively, where one of the ends is blunt. Nucleic acid molecules disclosed herein can include one or more blunt ends, i.e. where a blunt end does not have any overhanging nucleotides. In one embodiment, the blunt ended nucleic acid molecule has a number of base pairs equal to the number of nucleotides present in each strand of the nucleic acid molecule. The nucleic acid molecule may include one blunt end, for example where the 5′-end of the antisense strand and the 3′-end of the sense strand do not have any overhanging nucleotides. The nucleic acid molecule may include one blunt end, for example where the 3′-end of the antisense strand and the 5′-end of the sense strand do not have any overhanging nucleotides. A nucleic acid molecule may include two blunt ends, for example where the 3′-end of the antisense strand and the 5′-end of the sense strand as well as the 5′-end of the antisense strand and 3′-end of the sense strand do not have any overhanging nucleotides. In certain preferred embodiments the nucleic acid compounds are blunt ended. Other nucleotides present in a blunt ended siNA molecule can include, for example, mismatches, bulges, loops, or wobble base pairs to modulate the activity of the nucleic acid molecule to mediate RNA interference.

In many embodiments one or more, or all, of the overhang nucleotides of a nucleic acid molecule (e.g., a siNA molecule) as described herein includes are modified such as described herein; for example one or more, or all, of the nucleotides may be 2′-deoxynucleotides.

Amount, Location and Patterns of Modifications.

Nucleic acid molecules (e.g., siNA molecules) disclosed herein may include modified nucleotides as a percentage of the total number of nucleotides present in the nucleic acid molecule. As such, a nucleic acid molecule may include about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given nucleic acid molecule will depend on the total number of nucleotides present in the nucleic acid. If the nucleic acid molecule is single stranded, the percent modification can be based upon the total number of nucleotides present in the single stranded nucleic acid molecule. Likewise, if the nucleic acid molecule is double stranded, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.

Nucleic acid molecules disclosed herein may include unmodified RNA as a percentage of the total nucleotides in the nucleic acid molecule. As such, a nucleic acid molecule may include about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of total nucleotides present in a nucleic acid molecule.

A nucleic acid molecule (e.g., an siNA molecule) may include a sense strand that includes about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand includes about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. A nucleic acid molecule may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense nucleic acid strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

A nucleic acid molecule may include about 1 to about 5 or more (specifically about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages in each strand of the nucleic acid molecule.

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

A chemically-modified short interfering nucleic acid (siNA) molecule may include an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).

A chemically-modified short interfering nucleic acid (siNA) molecule may include an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

A chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against hsp47 inside a cell or reconstituted in vitro system may include a sense region, wherein one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and an antisense region, wherein one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides). The sense region and/or the antisense region can have a terminal cap modification, such as any modification, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/or antisense sequence. The sense and/or antisense region can optionally further include a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhang nucleotides can further include one or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages. The purine nucleotides in the sense region may alternatively be 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides) and one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides). One or more purine nucleotides in the sense region may alternatively be purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides) and any purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides). One or more purine nucleotides in the sense region and/or present in the antisense region may alternatively selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides).

In some embodiments, a nucleic acid molecule (e.g., a siNA molecule) as described herein includes a modified nucleotide (for example one modified nucleotide) in the antisense strand; for example in position 6 or position 7 of the antisense strand.

Modification Patterns and Alternating Modifications

Nucleic acid molecules (e.g., siNA molecules) provided herein may have patterns of modified and unmodified nucleic acids. A pattern of modification of the nucleotides in a contiguous stretch of nucleotides may be a modification contained within a single nucleotide or group of nucleotides that are covalently linked to each other via standard phosphodiester bonds or, at least partially, through phosphorothioate bonds. Accordingly, a “pattern” as contemplated herein, does not necessarily need to involve repeating units, although it may. Examples of modification patterns that may be used in conjunction with the nucleic acid molecules (e.g., siNA molecules) provided herein include those disclosed in Giese, U.S. Pat. No. 7,452,987. For example, nucleic acid molecules (e.g., siNA molecules) provided herein include those having modification patters such as, similar to, or the same as, the patterns shown diagrammatically in FIG. 2 of the Giese U.S. Pat. No. 7,452,987.

A modified nucleotide or group of modified nucleotides may be at the 5′-end or 3′-end of the sense or antisense strand, a flanking nucleotide or group of nucleotides is arrayed on both sides of the modified nucleotide or group, where the flanking nucleotide or group either is unmodified or does not have the same modification of the preceding nucleotide or group of nucleotides. The flanking nucleotide or group of nucleotides may, however, have a different modification. This sequence of modified nucleotide or group of modified nucleotides, respectively, and unmodified or differently modified nucleotide or group of unmodified or differently modified nucleotides may be repeated one or more times.

In some patterns, the 5′-terminal nucleotide of a strand is a modified nucleotide while in other patterns the 5′-terminal nucleotide of a strand is an unmodified nucleotide. In some patterns, the 5′-end of a strand starts with a group of modified nucleotides while in other patterns, the 5′-terminal end is an unmodified group of nucleotides. This pattern may be either on the first stretch or the second stretch of the nucleic acid molecule or on both.

Modified nucleotides of one strand of the nucleic acid molecule may be complementary in position to the modified or unmodified nucleotides or groups of nucleotides of the other strand.

There may be a phase shift between modifications or patterns of modifications on one strand relative to the pattern of modification of the other strand such that the modification groups do not overlap. In one instance, the shift is such that the modified group of nucleotides of the sense strand corresponds to the unmodified group of nucleotides of the antisense strand and vice versa.

There may be a partial shift of the pattern of modification such that the modified groups overlap. The groups of modified nucleotides in any given strand may optionally be the same length, but may be of different lengths. Similarly, groups of unmodified nucleotides in any given strand may optionally be the same length, or of different lengths.

In some patterns, the second (penultimate) nucleotide at the terminus of the strand, is an unmodified nucleotide or the beginning of group of unmodified nucleotides. Preferably, this unmodified nucleotide or unmodified group of nucleotides is located at the 5′-end of the either or both the sense and antisense strands and even more preferably at the terminus of the sense strand. An unmodified nucleotide or unmodified group of nucleotide may be located at the 5′-end of the sense strand. In a preferred embodiment the pattern consists of alternating single modified and unmodified nucleotides.

In some double stranded nucleic acid molecules include a 2′-O-methyl modified nucleotide and a non-modified nucleotide, preferably a nucleotide which is not 2′-O-methyl modified, are incorporated on both strands in an alternating fashion, resulting in a pattern of alternating 2′-O-methyl modified nucleotides and nucleotides that are either unmodified or at least do not include a 2′-O-methyl modification. In certain embodiments, the same sequence of 2′-O-methyl modification and non-modification exists on the second strand; in other embodiments the alternating 2′-O-methyl modified nucleotides are only present in the sense strand and are not present in the antisense strand; and in yet other embodiments the alternating 2′-O-methyl modified nucleotides are only present in the sense strand and are not present in the antisense strand. In certain embodiments, there is a phase shift between the two strands such that the 2′-O-methyl modified nucleotide on the first strand base pairs with a non-modified nucleotide(s) on the second strand and vice versa. This particular arrangement, i.e. base pairing of 2′-O-methyl modified and non-modified nucleotide(s) on both strands is particularly preferred in certain embodiments. In certain embodiments, the pattern of alternating 2′-O-methyl modified nucleotides exists throughout the entire nucleic acid molecule; or the entire duplex region. In other embodiments the pattern of alternating 2′-O-methyl modified nucleotides exists only in a portion of the nucleic acid; or the entire duplex region.

In “phase shift” patterns, it may be preferred if the antisense strand starts with a 2′-O-methyl modified nucleotide at the 5′ end whereby consequently the second nucleotide is non-modified, the third, fifth, seventh and so on nucleotides are thus again 2′-O-methyl modified whereas the second, fourth, sixth, eighth and the like nucleotides are non-modified nucleotides.

Exemplary Modification Locations and Patterns

While exemplary patterns are provided in more detail below, all permutations of patterns with of all possible characteristics of the nucleic acid molecules disclosed herein and those known in the art are contemplated (e.g., characteristics include, but are not limited to, length of sense strand, length of antisense strand, length of duplex region, length of hangover, whether one or both ends of a double stranded nucleic acid molecule is blunt or has an overhang, location of modified nucleic acid, number of modified nucleic acids, types of modifications, whether a double overhang nucleic acid molecule has the same or different number of nucleotides on the overhang of each side, whether a one or more than one type of modification is used in a nucleic acid molecule, and number of contiguous modified/unmodified nucleotides). With respect to all detailed examples provided below, while the duplex region is shown to be 19 nucleotides, the nucleic acid molecules provided herein can have a duplex region ranging from 1 to 49 nucleotides in length as each strand of a duplex region can independently be 17-49 nucleotides in length Exemplary patterns are provided herein.

Nucleic acid molecules may have a blunt end (when n is 0) on both ends that include a single or contiguous set of modified nucleic acids. The modified nucleic acid may be located at any position along either the sense or antisense strand. Nucleic acid molecules may include a group of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 contiguous modified nucleotides. Modified nucleic acids may make up 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 100% of a nucleic acid strand. Modified nucleic acids of the examples immediately below may be in the sense strand only, the antisense strand only, or in both the sense and antisense strand.

General nucleic acid patters are shown below where X=sense strand nucleotide in the duplex region; X_a=5′-overhang nucleotide in the sense strand; X_b=3′-overhang nucleotide in the sense strand; Y=antisense strand nucleotide in the duplex region; Y_a=3′-overhang nucleotide in the antisense strand; Y_b=5′-overhang nucleotide in the antisense strand; and M=a modified nucleotide in the duplex region. Each a and b are independently 0 to 8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7 or 8). Each X, Y, a and b are independently modified or unmodified. The sense and antisense strands can are each independently 17-49 nucleotides in length. The examples provided below have a duplex region of 19 nucleotides; however, nucleic acid molecules disclosed herein can have a duplex region anywhere between 17 and 49 nucleotides and where each strand is independently between 17 and 49 nucleotides in length.

5′
X_aXXXXXXXXXXXXXXXXXXXX_b

3′
Y_bYYYYYYYYYYYYYYYYYYYY_a

Further exemplary nucleic acid molecule patterns are shown below where X=unmodified sense strand nucleotides; x=an unmodified overhang nucleotide in the sense strand; Y=unmodified antisense strand nucleotides; y=an unmodified overhang nucleotide in the antisense strand; and M=a modified nucleotide. The sense and antisense strands can are each independently 17-49 nucleotides in length. The examples provided below have a duplex region of 19 nucleotides; however, nucleic acid molecules disclosed herein can have a duplex region anywhere between 17 and 49 nucleotides and where each strand is independently between 17 and 49 nucleotides in length.

5′

M
_nXXXXXXXXXMXXXXXXXXXM_n

3′

M
_nYYYYYYYYYYYYYYYYYYYM_n

5′
XXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYMYYYYYYYYY

5′
XXXXXXXXMMXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYMMYYYYYYYYY

5′
XXXXXXXXXMXXXXXXXXX

3′
YYYYYYYYYMYYYYYYYYY

5′
XXXXXMXXXXXXXXXXXXX

3′
YYYYYYYYYMYYYYYYYYY

5′

MXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYMYYYYYY

5′
XXXXXXXXXXXXXXXXXXM

3′
YYYYYMYYYYYYYYYYYYY

5′
XXXXXXXXXMXXXXXXXX

3′

MYYYYYYYYYYYYYYYYY

5′
XXXXXXXMXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYM

5′
XXXXXXXXXXXXXMXXXX

3′

MYYYYYYYYYYYYYYYYY

5′

MMMMMMMMMMMMMMMMMM

3′

MMMMMMMMMMMMMMMMMM

Nucleic acid molecules may have blunt ends on both ends with alternating modified nucleic acids. The modified nucleic acids may be located at any position along either the sense or antisense strand.

5′

MXMXMXMXMXMXMXMXMXM

3′
YMYMYMYMYMYMYMYMYMY

5′
XMXMXMXMXMXMXMXMXMX

3′

MYMYMYMYMYMYMYMYMYM

5′

MMXMMXMMXMMXMMXMMXM

3′
YMMYMMYMMYMMYMMYMMY

5′
XMMXMMXMMXMMXMMXMMX

3′

MMYMMYMMYMMYMMYMMYM

5′

MMMXMMMXMMMXMMMXMMM

3′
YMMMYMMMYMMMYMMMYMM

5′
XMMMXMMMXMMMXMMMXMM

3′

MMMYMMMYMMMYMMMYMMM

Nucleic acid molecules with a blunt 5′-end and 3′-end overhang end with a single modified nucleic acid.

Nucleic acid molecules with a 5′-end overhang and a blunt 3′-end with a single modified nucleic acid.

Nucleic acid molecules with overhangs on both ends and all overhangs are modified nucleic acids. In the pattern immediately below, M is n number of modified nucleic acids, where n is an integer from 0 to 8 (i.e., 0, 1, 2, 3, 4, 5, 6, 7 and 8).

5′
XXXXXXXXXXXXXXXXXXXM

3′

MYYYYYYYYYYYYYYYYYYY

Nucleic acid molecules with overhangs on both ends and some overhang nucleotides are modified nucleotides. In the patterns immediately below, M is n number of modified nucleotides, x is n number of unmodified overhang nucleotides in the sense strand, y is n number of unmodified overhang nucleotides in the antisense strand, where each n is independently an integer from 0 to 8 (i.e., 0, 1, 2, 3, 4, 5, 6, 7 and 8), and where each overhang is maximum of 20 nucleotides; preferably a maximum of 8 nucleotides (modified and/or unmodified).

5′
XXXXXXXXXXXXXXXXXXXM

3′
yYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMx

3′
yYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMxM

3′
yYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMxMx

3′
yYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMxMxM

3′
yYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMxMxMx

3′
yYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMxMxMxM

3′
yYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMxMxMxMx

3′
yYYYYYYYYYYYYYYYYYYY

5′
MXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYy

5′
xMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYy

5′
MxMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYy

5′
xMxMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYy

5′
MxMxMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYy

5′
xMxMxMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYy

5′
MxMxMxMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYy

5′
xMxMxMxMXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYy

5′
xXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYM

5′
xXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYMy

5′
xXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYMyM

5′
xXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYMyMy

5′
xXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYMyMyM

5′
xXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYMyMyMy

5′
xXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYMyMyMyM

5′
xXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYYMyMyMyMy

5′
XXXXXXXXXXXXXXXXXXXx

3′
MYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXx

3′
yMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXx

3′
MyMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXx

3′
yMyMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXx

3′
MyMyMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXx

3′
yMyMyMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXx

3′
MyMyMyMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXx

3′
yMyMyMyMYYYYYYYYYYYYYYYYYYY

Modified nucleotides at the 3′ end of the sense region.

5′
XXXXXXXXXXXXXXXXXXXM

3′
YYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMM

3′
YYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMMM

3′
YYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMMMM

3′
YYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMMMMM

3′
YYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMMMMMM

3′
YYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMMMMMMMM

3′
YYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMMMMMMMM

3′
YYYYYYYYYYYYYYYYYYY

Overhang at the 5′ end of the sense region.

5′
MXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYY

5′
MMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYY

5′
MMMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYY

5′
MMMMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYY

5′
MMMMMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYY

5′
MMMMMMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYY

5′
MMMMMMMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYY

5′

MMMMMMMMXXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYY

Overhang at the 3′ end of the antisense region.

5′
XXXXXXXXXXXXXXXXXXX

3′
MYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXX

3′
MMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXX

3′
MMMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXX

3′
MMMMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXX

3′
MMMMMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXX

3′
MMMMMMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXX

3′
MMMMMMMYYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXX

3′

MMMMMMMMYYYYYYYYYYYYYYYYYYY

Modified nucleotide(s) within the sense region

5′
XXXXXXXXXMXXXXXXXXX

3′
YYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXX

3′
YYYYYYYYYMYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXXMM

3′
YYYYYYYYYYYYYYYYYYY

5′
XXXXXXXXXXXXXXXXXXX

3′

MMYYYYYYYYYYYYYYYYYYY

Exemplary nucleic acid molecules are provided below along with the equivalent general structure in line with the symbols used above:

siHSP47-C siRNA to human and rat hsp47 having a 19 nucleotide (i.e., 19mer) duplex region and modified 2 nucleotide (i.e., deoxynucleotide) overhangs at the 3′-ends of the sense and antisense strands.

5′
GGACAGGCCUCUACAACUAdTdT
3′

3′

dTdTCCUGUCCGGAGAUGUUGAU
5′

5′
XXXXXXXXXXXXXXXXXXXMM

3′

MMYYYYYYYYYYYYYYYYYYY

siHSP47-Cd siRNA to human and rat hsp47 having a 25-mer duplex region, a 2 nucleotide overhang at the 3′-end of the antisense strand and 2 modified nucleotides at the 5′-terminal and penultimate positions of the sense strand.

5′
GGACAGGCCUCUACAACUACUACdGdA
3′

3′
UUCCUGUCCGGAGAUGUUGAUGAUGCU
5′

5′
XXXXXXXXXXXXXXXXXXXXXXXMM
3′

3′
yyYYYYYYYYYYYYYYYYYYYYYYYYY
5′

siHSP47-1 siRNA to human and rat hsp47 cDNA 719-737 having a 19-mer duplex region, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs at the 3′-ends of the sense and antisense strands.

5′
CAGGCCUCUACAACUACUAdTdT
3′

3′

dTdTGUCCGGAGAUGUUGAUGAU
5′

5′
XXXXXXXXXXXXXXXXXXXMM
3′

3′
MMYYYYYYYYYYYYYYYYYYY
5′

siHSP47-1d siRNA to human hsp47 cDNA 719-743 having a 25-mer with a blunt end at the 3′-end of the sense strand and a 2 nucleotide overhang at the 3′-end of the antisense strand, and 2 modified nucleotides at the 5′-terminal and penultimate positions of the sense strand.

5′
CAGGCCUCUACAACUACUACGACdGdA
3′

3′
UUGUCCGGAGAUGUUGAUGAUGCUGCU
5′

5′
XXXXXXXXXXXXXXXXXXXXXXXMM
3′

3′
yyYYYYYYYYYYYYYYYYYYYYYYYYY
5′

siHSP47-2 siRNA to human hsp47 cDNA 469-487 having a 19-mer duplex region, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs at the 3′-ends of the sense and antisense strands.

5′
GAGCACUCCAAGAUCAACUdTdT
3′

3′

dTdTCUCGUGAGGUUCUAGUUGA
5′

5′
XXXXXXXXXXXXXXXXXXXMM
3′

3′
MMYYYYYYYYYYYYYYYYYYY
5′

siHSP47-2d siRNA to human hsp47 cDNA 469-493 having a 25-mer duplex region with a blunt end at the 3′-end of the sense strand and a 2 nucleotide overhang at the 3′-end of the antisense strand, and 2 modified nucleotides at the 5′-terminal and penultimate positions of the sense strand.

5′
GAGCACUCCAAGAUCAACUUCCGdCdG
3′

3′
UUCUCGUGAGGUUCUAGUUGAAGGCGC
5′

5′
XXXXXXXXXXXXXXXXXXXXXXXMM
3′

3′
yyYYYYYYYYYYYYYYYYYYYYYYYYY
5′

siHSP47-2d rat siRNA to rat Gp46 cDNA 466-490 having a 25-mer duplex region with a blunt end at the 3′-end of the sense strand and a 2 nucleotide overhang at the 3′-end of the antisense strand, and 2 modified nucleotides at the 5′-terminal and penultimate positions of the sense strand.

5′
GAACACUCCAAGAUCAACUUCCGdAdG
3′

3′
UUCUUGUGAGGUUCUAGUUGAAGGCUC
5′

5′
XXXXXXXXXXXXXXXXXXXXXXXMM
3′

3′
yyYYYYYYYYYYYYYYYYYYYYYYYYY
5′

siHSP47-3 siRNA to human hsp47 cDNA 980-998 having a 19-mer duplex region, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs at the 3′-ends of the sense and antisense strands.

5′
CTGAGGCCATTGACAAGAAdTdT
3′

3′

dTdTGACUCCGGUAACUGUUCUU
5′

5′
XXXXXXXXXXXXXXXXXXXMM
3′

3′
MMYYYYYYYYYYYYYYYYYYY
5′

siHSP47-3d siRNA to human hsp47 cDNA 980-1004 having a 25-mer duplex region with a blunt end at the 3′-end of the sense strand and a 2 nucleotide overhang at the 3′-end of the antisense strand, and 2 modified nucleotides at the 5′-terminal and penultimate positions of the sense strand.

5′
CTGAGGCCATTGACAAGAACAAGdGdC
3′

3′
UUGACUCCGGUAACUGUUCUUGUUCCG
5′

5′
XXXXXXXXXXXXXXXXXXXXXXXMM
3′

3′
yyYYYYYYYYYYYYYYYYYYYYYYYYY
5′

siHSP47-4 siRNA to human hsp47 cDNA 735-753 having a 19-mer duplex region, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs at the 3′-ends of the sense and antisense strands.

5′
CUACGACGACGAGAAGGAAdTdT
3′

3′

dTdTGAUGCUGCUGCUCUUCCUU
5′

5′
XXXXXXXXXXXXXXXXXXXMM
3′

3′
MMYYYYYYYYYYYYYYYYYYY
5′

siHSP47-4-d siRNA to human hsp47 cDNA 735-759 having a 25-mer duplex region with a blunt end at the 3′-end of the sense strand and a 2 nucleotide overhang at the 3′-end of the antisense strand, and 2 modified nucleotides at the 5′-terminal and penultimate positions of the sense strand.

5′
CUACGACGACGAGAAGGAAAAGCdTdG
3′

3′
UUGAUGCUGCUGCUCUUCCUUUUCGAC
5′

5′
XXXXXXXXXXXXXXXXXXXXXXXMM
3′

3′
yyYYYYYYYYYYYYYYYYYYYYYYYYY
5′

siHSP47-5 siRNA to human hsp47 cDNA 621-639 having a 19-mer duplex region, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs at the 3′-ends of the sense and antisense strands.

5′
GCCACACUGGGAUGAGAAAdTdT
3′

3′

dTdTCGGUGUGACCCUACUCUUU
5′

5′
XXXXXXXXXXXXXXXXXXXMM
3′

3′
MMYYYYYYYYYYYYYYYYYYY
5′

siHSP47-6 siRNA to human hsp47 cDNA 446-464 having a 19-mer duplex region, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs at the 3′-ends of the sense and antisense strands.

5′
GCAGCAAGCAGCACUACAAdTdT
3′

3′

dTdTCGUCGUUCGUCGUGAUGUU
5′

5′
XXXXXXXXXXXXXXXXXXXMM
3′

3′
MMYYYYYYYYYYYYYYYYYYY
5′

siHSP47-7 siRNA to human hsp47 cDNA 692-710 having a 19-mer duplex region, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs at the 3′-ends of the sense and antisense strands.

5′
CCGUGGGUGUCAUGAUGAUdTdT
3′

3′

dTdTGGCACCCACAGUACUACUA
5′

5′
XXXXXXXXXXXXXXXXXXXMM
3′

3′
MMYYYYYYYYYYYYYYYYYYY
5′

Nicks and Gaps in Nucleic Acid Strands

Nucleic acid molecules (e.g., siNA molecules) provided herein may have a strand, preferably the sense strand, that is nicked or gapped. As such, nucleic acid molecules may have three or more strand, for example, such as a meroduplex RNA (mdRNA) disclosed in International Patent Application No. PCT/US07/081,836. Nucleic acid molecules with a nicked or gapped strand may be between about 1-49 nucleotides, or may be RISC length (e.g., about 15 to 25 nucleotides) or Dicer substrate length (e.g., about 25 to 30 nucleotides) such as disclosed herein.

Nucleic acid molecules with three or more strands include, for example, an ‘A’ (antisense) strand, ‘S1’ (second) strand, and ‘S2’ (third) strand in which the ‘S1’ and ‘S2’ strands are complementary to and form base pairs with non-overlapping regions of the ‘A’ strand (e.g., an mdRNA can have the form of A:S1S2). The S1, S2, or more strands together form what is substantially similar to a sense strand to the ‘A’ antisense strand. The double-stranded region formed by the annealing of the ‘S1’ and ‘A’ strands is distinct from and non-overlapping with the double-stranded region formed by the annealing of the ‘S2’ and ‘A’ strands. An nucleic acid molecule (e.g., an siNA molecule) may be a “gapped” molecule, meaning a “gap” ranging from 0 nucleotides up to about 10 nucleotides (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides). Preferably, the sense strand is gapped. In some embodiments, the A:S1 duplex is separated from the A:S2 duplex by a gap resulting from at least one unpaired nucleotide (up to about 10 unpaired nucleotides) in the ‘A’ strand that is positioned between the A:S1 duplex and the A:S2 duplex and that is distinct from any one or more unpaired nucleotide at the 3′-end of one or more of the ‘A’, ‘S1’, or ‘S2 strands. The A:S1 duplex may be separated from the A:B2 duplex by a gap of zero nucleotides (i.e., a nick in which only a phosphodiester bond between two nucleotides is broken or missing in the polynucleotide molecule) between the A:S1 duplex and the A:S2 duplex-which can also be referred to as nicked dsRNA (ndsRNA). For example, A:S1S2 may be include a dsRNA having at least two double-stranded regions that combined total about 14 base pairs to about 40 base pairs and the double-stranded regions are separated by a gap of about 0 to about 10 nucleotides, optionally having blunt ends, or A:S1S2 may include a dsRNA having at least two double-stranded regions separated by a gap of up to 10 nucleotides wherein at least one of the double-stranded regions includes between about 5 base pairs and 13 base pairs.

Dicer Substrates

In certain embodiments, the nucleic acid molecules (e.g., siNA molecules) provided herein may be a precursor “Dicer substrate” molecule, e.g., double stranded nucleic acid, processed in vivo to produce an active nucleic acid molecules, for example as described in Rossi, US Patent App. No. 20050244858. In certain conditions and situations, it has been found that these relatively longer dsRNA siNA species, e.g., of from about 25 to about 30 nucleotides, can give unexpectedly effective results in terms of potency and duration of action. Without wishing to be bound by any particular theory, it is thought that the longer dsRNA species serve as a substrate for the enzyme Dicer in the cytoplasm of a cell. In addition to cleaving double stranded nucleic acid into shorter segments, Dicer may facilitate the incorporation of a single-stranded cleavage product derived from the cleaved dsRNA into the RNA-induced silencing complex (RISC complex) that is responsible for the destruction of the cytoplasmic RNA derived from the target gene.

Dicer substrates may have certain properties which enhance its processing by Dicer. Dicer substrates are of a length sufficient such that it is processed by Dicer to produce an active nucleic acid molecule and may further include one or more of the following properties: (i) the dsRNA is asymmetric, e.g., has a 3′ overhang on the first strand (antisense strand) and (ii) the dsRNA has a modified 3′ end on the antisense strand (sense strand) to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA. In certain embodiments, the longest strand in the Dicer substrate may be 24-30 nucleotides.

Dicer substrates may be symmetric or asymmetric. The Dicer substrate may have a sense strand includes 22-28 nucleotides and the antisense strand may include 24-30 nucleotides; thus, in some embodiments the resulting Dicer substrate may have an overhang on the 3′ end of the antisense strand. Dicer substrate may have a sense strand 25 nucleotides in length, and the antisense strand having 27 nucleotides in length with a 2 base 3′-overhang. The overhang may be 1-3 nucleotides, for example 2 nucleotides. The sense strand may also have a 5′ phosphate.

An asymmetric Dicer substrate may further contain two deoxynucleotides at the 3′-end of the sense strand in place of two of the ribonucleotides. Some exemplary Dicer substrates lengths and structures are 21+0, 21+2, 21−2, 22+0, 22+1, 22−1, 23+0, 23+2, 23−2, 24+0, 24+2, 24−2, 25+0, 25+2, 25−2, 26+0, 26+2, 26−2, 27+0, 27+2, and 27−2.

The sense strand of a Dicer substrate may be between about 22 to about 30 (e.g., about 22, 23, 24, 25, 26, 27, 28, 29 or 30); about 22 to about 28; about 24 to about 30; about 25 to about 30; about 26 to about 30; about 26 and 29; or about 27 to about 28 nucleotides in length. In certain preferred embodiments Dicer substrates contain sense and antisense strands, that are at least about 25 nucleotides in length and no longer than about 30 nucleotides; between about 26 and 29 nucleotides; or 27 nucleotides in length. The sense and antisense strands may be the same length (blunt ended), different lengths (have overhangs), or a combination. The sense and antisense strands may exist on the same polynucleotide or on different polynucleotides. A Dicer substrate may have a duplex region of about 19, 20, 21, 22, 23, 24, 25 or 27 nucleotides.

Like other siNA molecules provided herein, the antisense strand of a Dicer substrate may have any sequence that anneals to the antisense strand under biological conditions, such as within the cytoplasm of a eukaryotic cell.

Dicer substrates may have any modifications to the nucleotide base, sugar or phosphate backbone as known in the art and/or as described herein for other nucleic acid molecules (such as siNA molecules). In certain embodiments, Dicer substrates may have a sense strand is modified for Dicer processing by suitable modifiers located at the 3′ end of the sense strand, i.e., the dsRNA is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for-the 2′-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotides modifiers that could be used in Dicer substrate siNA molecules include 3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphate nucleotides of 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxy-3′-thiacytidine (3TC) and 2′,3′-didehydro-2′,3′-dide-oxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, they may replace ribonucleotides (e.g., 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3′ end of the sense strand) such that the length of the Dicer substrate does not change. When sterically hindered molecules are utilized, they may be attached to the ribonucleotide at the 3′ end of the antisense strand. Thus, in certain embodiments the length of the strand does not change with the incorporation of the modifiers. In certain embodiments, two DNA bases in the dsRNA are substituted to direct the orientation of Dicer processing of the antisense strand. In a further embodiment of two terminal DNA bases are substituted for two ribonucleotides on the 3′-end of the sense strand forming a blunt end of the duplex on the 3′ end of the sense strand and the 5′ end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3′-end of the antisense strand. This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

In certain embodiments modifications are included in the Dicer substrate such that the modification does not prevent the nucleic acid molecule from serving as a substrate for Dicer. In one embodiment, one or more modifications are made that enhance Dicer processing of the Dicer substrate. One or more modifications may be made that result in more effective RNAi generation. One or more modifications may be made that support a greater RNAi effect. One or more modifications are made that result in greater potency per each Dicer substrate to be delivered to the cell. Modifications may be incorporated in the 3′-terminal region, the 5′-terminal region, in both the 3′-terminal and 5′-terminal region or at various positions within the sequence. Any number and combination of modifications can be incorporated into the Dicer substrate so long as the modification does not prevent the nucleic acid molecule from serving as a substrate for Dicer. Where multiple modifications are present, they may be the same or different. Modifications to bases, sugar moieties, the phosphate backbone, and their combinations are contemplated. Either 5′-terminus can be phosphorylated.

Examples of Dicer substrate phosphate backbone modifications include phosphonates, including methylphosphonate, phosphorothioate, and phosphotriester modifications such as alkylphosphotriesters, and the like. Examples of Dicer substrate sugar moiety modifications include 2′-alkyl pyrimidine, such as 2′-O-methyl, 2′-fluoro, amino, and deoxy modifications and the like (see, e.g., Amarzguioui et al., 2003). Examples of Dicer substrate base group modifications include abasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like. Locked nucleic acids, or LNA's, could also be incorporated.

The sense strand may be modified for Dicer processing by suitable modifiers located at the 3′ end of the sense strand, i.e., the Dicer substrate is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for-the 2′-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotides modifiers could include 3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphate nucleotides of 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxy-3′-thiacytidine (3TC) and 2′,3′-didehydro-2′,3′-dide-oxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3′ end of the sense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3′ end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the Dicer substrate to direct the orientation of Dicer processing of the antisense strand. In a further embodiment of the present invention, two terminal DNA bases are substituted for two ribonucleotides on the 3′-end of the sense strand forming a blunt end of the duplex on the 3′ end of the sense strand and the 5′ end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3′-end of the antisense strand. This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

The antisense strand may be modified for Dicer processing by suitable modifiers located at the 3′ end of the antisense strand, i.e., the dsRNA is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2′-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphate nucleotides of 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxy-3′-thiacytidine (3TC) and 2′,3′-didehydro-2′,3′-dide-oxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3′ end of the antisense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3′ end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the dsRNA to direct the orientation of Dicer processing. In a further invention, two terminal DNA bases are located on the 3′ end of the antisense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5′ end of the sense strand and the 3′ end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3′-end of the sense strand. This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

Dicer substrates with a sense and an antisense strand can be linked by a third structure. The third structure will not block Dicer activity on the Dicer substrate and will not interfere with the directed destruction of the RNA transcribed from the target gene. The third structure may be a chemical linking group. Suitable chemical linking groups are known in the art and can be used. Alternatively, the third structure may be an oligonucleotide that links the two oligonucleotides of the dsRNA is a manner such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the Dicer substrate. The hairpin structure preferably does not block Dicer activity on the Dicer substrate or interfere with the directed destruction of the RNA transcribed from the target gene.

The sense and antisense strands of the Dicer substrate are not required to be completely complementary. They only need to be substantially complementary to anneal under biological conditions and to provide a substrate for Dicer that produces an siRNA sufficiently complementary to the target sequence.

Dicer substrate can have certain properties that enhance its processing by Dicer. The Dicer substrate can have a length sufficient such that it is processed by Dicer to produce an active nucleic acid molecules (e.g., siRNA) and may have one or more of the following properties: (i) the Dicer substrate is asymmetric, e.g., has a 3′ overhang on the first strand (antisense strand) and (ii) the Dicer substrate has a modified 3′ end on the second strand (sense strand) to direct orientation of Dicer binding and processing of the Dicer substrate to an active siRNA. The Dicer substrate can be asymmetric such that the sense strand includes 22-28 nucleotides and the antisense strand includes 24-30 nucleotides. Thus, the resulting Dicer substrate has an overhang on the 3′ end of the antisense strand. The overhang is 1-3 nucleotides, for example 2 nucleotides. The sense strand may also have a 5′ phosphate.

A Dicer substrate may have an overhang on the 3′ end of the antisense strand and the sense strand is modified for Dicer processing. The 5′ end of the sense strand may have a phosphate. The sense and antisense strands may anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. A region of one of the strands, particularly the antisense strand, of the Dicer substrate may have a sequence length of at least 19 nucleotides, wherein these nucleotides are in the 21-nucleotide region adjacent to the 3′ end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene. A Dicer substrate may also have one or more of the following additional properties: (a) the antisense strand has a right shift from a corresponding 21-mer (i.e., the antisense strand includes nucleotides on the right side of the molecule when compared to the corresponding 21-mer), (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatch pairings and (c) base modifications such as locked nucleic acid(s) may be included in the 5′ end of the sense strand.

An antisense strand of a Dicer substrate nucleic acid molecule may be modified to include 1-9 ribonucleotides on the 5′-end to give a length of 22-28 nucleotides. When the antisense strand has a length of 21 nucleotides, then 1-7 ribonucleotides, or 2-5 ribonucleotides and or 4 ribonucleotides may be added on the 3′-end. The added ribonucleotides may have any sequence. Although the added ribonucleotides may be complementary to the target gene sequence, full complementarity between the target sequence and the antisense strands is not required. That is, the resultant antisense strand is sufficiently complementary with the target sequence. A sense strand may then have 24-30 nucleotides. The sense strand may be substantially complementary with the antisense strand to anneal to the antisense strand under biological conditions. In one embodiment, the antisense strand may be synthesized to contain a modified 3′-end to direct Dicer processing. The sense strand may have a 3′ overhang. The antisense strand may be synthesized to contain a modified 3′-end for Dicer binding and processing and the sense strand has a 3′ overhang.

Heat Shock Protein 47

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and resides in the endoplasmic reticulum. It interacts with procollagen during the process of folding, assembling and transporting from the endoplasmic reticulum (Nagata Trends Biochem Sci 1996; 21:22-6; Razzaque et al. 2005; Contrib Nephrol 2005; 148: 57-69; Koide et al. 2006 J. Biol. Chem.; 281: 3432-38; Leivo et al. Dev. Biol. 1980; 76:100-114; Masuda et al. J. Clin. Invest. 1994; 94:2481-2488; Masuda et al. Cell Stress Chaperones 1998; 3:256-264). HSP47 has been reported to have an upregulated expression in various tissue fibrosis (Koide et al. J Biol Chem 1999; 274: 34523-26), such as liver cirrhosis (Masuda et al. J Clin Invest 1994; 94:2481-8), pulmonary fibrosis (Razzaque et al. Virchows Arch 1998; 432:455-60; Kakugawa et al. Eur Respir J 2004; 24: 57-65), and glomerulosclerosis (Moriyama et al. Kidney Int 1998; 54: 110-19). Exemplary nucleic acid sequence of target human hsp47 cDNA is disclosed in GenBank accession number: NM_—001235 and the corresponding mRNA sequence, for example as listed as SEQ ID NO: 1. One of ordinary skill in the art would understand that a given sequence may change over time and to incorporate any changes needed in the nucleic acid molecules herein accordingly.

The specific association of HSP47 with a diverse range of collagen types makes HSP47 a potential target for the treatment of fibrosis. Inhibition of hsp47 expression may prevent extracellular collagen I secretion. Sato et al. (Nat Biotechnol 2008; 26:431-442) explored this possibility by using siRNA for the inhibition hsp47 expression and preventing the progression of hepatic fibrosis in rats. Similarly, Chen et al. (Br J Dermatol 2007; 156: 1188-1195) and Wang et al. (Plast. Reconstr Surg 2003; 111: 1980-7) investigated the inhibition hsp47 expression by RNA interference technology.

Methods and Compositions for Inhibiting hsp47

Provided are compositions and methods for inhibition of hsp47 expression by using small nucleic acid molecules, such as short interfering nucleic acid (siNA), interfering RNA (RNAi), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating or that mediate RNA interference against hsp47 gene expression. The composition and methods disclosed herein are also useful in treating various fibrosis such as liver fibrosis, lung fibrosis, and kidney fibrosis.

Nucleic acid molecule(s) and/or methods of the invention are used to down regulate the expression of gene(s) that encode RNA referred to, by example, Genbank Accession NM_—001235.

Compositions, methods and kits provided herein may include one or more nucleic acid molecules (e.g., siNA) and methods that independently or in combination modulate (e.g., downregulate) the expression of hsp47 protein and/or genes encoding hsp47 proteins, proteins and/or genes encoding hsp47 associated with the maintenance and/or development of diseases, conditions or disorders associated with hsp47, such as liver fibrosis, cirrhosis, pulmonary fibrosis, kidney fibrosis, peritoneal fibrosis, chronic hepatic damage, and fibrillogenesis (e.g., genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. NM_—001235), or a hsp47 gene family member where the genes or gene family sequences share sequence homology. The description of the various aspects and embodiments is provided with reference to exemplary gene hsp47. However, the various aspects and embodiments are also directed to other related hsp47 genes, such as homolog genes and transcript variants, and polymorphisms (e.g., single nucleotide polymorphism, (SNPs)) associated with certain hsp47 genes. As such, the various aspects and embodiments are also directed to other genes that are involved in hsp47 mediated pathways of signal transduction or gene expression that are involved, for example, in the maintenance or development of diseases, traits, or conditions described herein. These additional genes can be analyzed for target sites using the methods described for the hsp47 gene herein. Thus, the modulation of other genes and the effects of such modulation of the other genes can be performed, determined, and measured as described herein.

In one embodiment, compositions and methods provided herein include a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a hsp47 gene (e.g., human hsp47 exemplified by SEQ ID NO:1), where the nucleic acid molecule includes about 15 to about 49 base pairs.

In one embodiment, a nucleic acid disclosed may be used to inhibit the expression of the hsp47 gene or a hsp47 gene family where the genes or gene family sequences share sequence homology. Such homologous sequences can be identified as is known in the art, for example using sequence alignments. Nucleic acid molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences. In instances where mismatches are identified, non-canonical base pairs (for example, mismatches and/or wobble bases) can be used to generate nucleic acid molecules that target more than one gene sequence. In a non-limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate nucleic acid molecules that are capable of targeting sequences for differing hsp47 targets that share sequence homology. As such, one advantage of using siNAs disclosed herein is that a single nucleic acid can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the homologous genes. In this approach, a single nucleic acid can be used to inhibit expression of more than one gene instead of using more than one nucleic acid molecule to target the different genes.

Nucleic acid molecules may be used to target conserved sequences corresponding to a gene family or gene families such as hsp47 family genes. As such, nucleic acid molecules targeting multiple hsp47 targets can provide increased therapeutic effect. In addition, nucleic acid can be used to characterize pathways of gene function in a variety of applications. For example, nucleic acid molecules can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The nucleic acid molecules can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The nucleic acid molecules can be used to understand pathways of gene expression involved in, for example fibroses such as liver, kidney or pulmonary fibrosis, and/or inflammatory and proliferative traits, diseases, disorders, and/or conditions.

In one embodiment, the compositions and methods provided herein include a nucleic acid molecule having RNAi activity against hsp47 RNA, where the nucleic acid molecule includes a sequence complementary to any RNA having hsp47 encoding sequence, such as those sequences having sequences as shown in Table I. In another embodiment, a nucleic acid molecule may have RNAi activity against hsp47 RNA, where the nucleic acid molecule includes a sequence complementary to an RNA having variant hsp47 encoding sequence, for example other mutant hsp47 genes not shown in Table I but known in the art to be associated with the maintenance and/or development of fibrosis. Chemical modifications as shown in Table I or otherwise described herein can be applied to any nucleic acid construct disclosed herein. In another embodiment, a nucleic acid molecule disclosed herein includes a nucleotide sequence that can interact with nucleotide sequence of a hsp47 gene and thereby mediate silencing of hsp47 gene expression, for example, wherein the nucleic acid molecule mediates regulation of hsp47 gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the hsp47 gene and prevent transcription of the hsp47 gene.

Nucleic acid molecules disclosed herein may have RNAi activity against hsp47 RNA, where the nucleic acid molecule includes a sequence complementary to any RNA having hsp47 encoding sequence, such as those sequences having GenBank Accession Nos. NM_—001235. Nucleic acid molecules may have RNAi activity against hsp47 RNA, where the nucleic acid molecule includes a sequence complementary to an RNA having variant hsp47 encoding sequence, for example other mutant hsp47 genes known in the art to be associated with the maintenance and/or development of fibrosis. Nucleic acid molecules disclosed herein include a nucleotide sequence that can interact with nucleotide sequence of a hsp47 gene and thereby mediate silencing of hsp47 gene expression, e.g., where the nucleic acid molecule mediates regulation of hsp47 gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the hsp47 gene and prevent transcription of the hsp47 gene.

Methods of Treatment

The specific association of HSP47 with a diverse range of collagen types makes hsp47 a target for the treatment of fibrosis. Inhibition of hsp47 expression may prevent extracellular collagen I secretion. Sato et al. (Nat Biotechnol 2008; 26:431-442) explored this possibility by using siRNA for the inhibition hsp47 expression and preventing the progression of hepatic fibrosis in rats. Similarly, Chen et al. (Br J Dermatol 2007; 156: 1188-1195) and Wang et al. (Plast. Reconstr Surg 2003; 111: 1980-7) investigated the inhibition hsp47 expression by RNA interference technology.

In one embodiment, nucleic acid molecules may be used to down regulate or inhibit the expression of hsp47 and/or hsp47 proteins arising from hsp47 and/or hsp47 haplotype polymorphisms that are associated with a disease or condition, (e.g., fibrosis). Analysis of hsp47 and/or hsp47 genes, or hsp47 and/or hsp47 protein or RNA levels can be used to identify subjects with such polymorphisms or those subjects who are at risk of developing traits, conditions, or diseases described herein. These subjects are amenable to treatment, for example, treatment with nucleic acid molecules disclosed herein and any other composition useful in treating diseases related to hsp47 and/or hsp47 gene expression. As such, analysis of hsp47 and/or hsp47 protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of hsp47 and/or hsp47 protein or RNA levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of certain hsp47 and/or hsp47 proteins associated with a trait, condition, or disease.

Provided are compositions and methods for inhibition of hsp47 expression by using small nucleic acid molecules as provided herein, such as short interfering nucleic acid (siNA), interfering RNA (RNAi), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating or that mediate RNA interference against hsp47 gene expression. The composition and methods disclosed herein are also useful in treating various fibrosis such as liver fibrosis, lung fibrosis, and kidney fibrosis.

The nucleic acid molecules disclosed herein individually, or in combination or in conjunction with other drugs, can be use for preventing or treating diseases, traits, conditions and/or disorders associated with hsp47, such as liver fibrosis, cirrhosis, pulmonary fibrosis, kidney fibrosis, peritoneal fibrosis, chronic hepatic damage, and fibrillogenesis.

The nucleic acid molecules disclosed herein are able to inhibit the expression of hsp47 in a sequence specific manner. The nucleic acid molecules may include a sense strand and an antisense strand which include contiguous nucleotides that are at least partially complementary (antisense) to a hsp47 mRNA.

In some embodiments, dsRNA specific for hsp47 can be used in conjunction with other dsRNA specific for other molecular chaperones that assist in the folding of newly synthesized proteins such as, calnexin, calreticulin, BiP (Bergeron et al. Trends Biochem. Sci. 1994; 19:124-128; Herbert et al. 1995; Cold Spring Harb. Symp. Quant. Biol. 60:405-415)

Fibrosis can be treated by RNA interference using nucleic acid molecules as disclosed herein. Exemplary fibrosis include liver fibrosis, peritoneal fibrosis, lung fibrosis, kidney fibrosis. The nucleic acid molecules disclosed herein may inhibit the expression of hsp47 in a sequence specific manner.

Treatment of fibrosis can be monitored by determining the level of extracellular collagen using suitable techniques known in the art such as, using anti-collagen I antibodies. Treatment can also be monitored by determining the level of hsp47 mRNA or the level of HSP47 protein in the cells of the affected tissue. Treatment can also be monitored by non-invasive scanning of the affected organ or tissue such as by computer assisted tomography scan, magnetic resonance elastography scans.

A method for treating or preventing hsp47 associated disease or condition in a subject or organism may include contacting the subject or organism with a nucleic acid molecule as provided herein under conditions suitable to modulate the expression of the hsp47 gene in the subject or organism.

A method for treating or preventing fibrosis in a subject or organism may include contacting the subject or organism with a nucleic acid molecule under conditions suitable to modulate the expression of the hsp47 gene in the subject or organism.

A method for treating or preventing one or more fibroses selected from the group consisting of liver fibrosis, kidney fibrosis, and pulmonary fibrosis in a subject or organism may include contacting the subject or organism with a nucleic acid molecule under conditions suitable to modulate the expression of the hsp47 gene in the subject or organism.

Fibrotic Diseases

Fibrotic diseases are generally characterized by the excess deposition of a fibrous material within the extracellular matrix, which contributes to abnormal changes in tissue architecture and interferes with normal organ function.

All tissues damaged by trauma respond by the initiation of a wound-healing program. Fibrosis, a type of disorder characterized by excessive scarring, occurs when the normal self-limiting process of wound healing response is disturbed, and causes excessive production and deposition of collagen. As a result, normal organ tissue is replaced with scar tissue, which eventually leads to the functional failure of the organ.

Fibrosis may be initiated by diverse causes and in various organs. Liver cirrhosis, pulmonary fibrosis, sarcoidosis, keloids and kidney fibrosis are all chronic conditions associated with progressive fibrosis, thereby causing a continuous loss of normal tissue function.

Acute fibrosis (usually with a sudden and severe onset and of short duration) occurs as a common response to various forms of trauma including accidental injuries (particularly injuries to the spine and central nervous system), infections, surgery, ischemic illness (e.g. cardiac scarring following heart attack), burns, environmental pollutants, alcohol and other types of toxins, acute respiratory distress syndrome, radiation and chemotherapy treatments).

Fibrosis, a fibrosis related pathology or a pathology related to aberrant crosslinking of cellular proteins may all be treated by the siRNAs disclosed herein. Fibrotic diseases or diseases in which fibrosis is evident (fibrosis related pathology) include both acute and chronic forms of fibrosis of organs, including all etiological variants of the following: pulmonary fibrosis, including interstitial lung disease and fibrotic lung disease, liver fibrosis, cardiac fibrosis including myocardial fibrosis, kidney fibrosis including chronic renal failure, skin fibrosis including scleroderma, keloids and hypertrophic scars; myelofibrosis (bone marrow fibrosis); all types of ocular scarring including proliferative vitreoretinopathy (PVR) and scarring resulting from surgery to treat cataract or glaucoma; inflammatory bowel disease of variable etiology, macular degeneration, Grave's ophthalmopathy, drug induced ergotism, keloid scars, scleroderma, psoriasis, glioblastoma in Li-Fraumeni syndrome, sporadic glioblastoma, myleoid leukemia, acute myelogenous leukemia, myelodysplastic syndrome, myeloproferative syndrome, gynecological cancer, Kaposi's sarcoma, Hansen's disease, and collagenous colitis.

In various embodiments, the compounds (nucleic acid molecules) as disclosed herein may be used to treat fibrotic diseases, for example as disclosed herein, as well as many other diseases and conditions apart from fibrotic diseases, for example such as disclosed herein. Other conditions to be treated include fibrotic diseases in other organs—kidney fibrosis for any reason (CKD including ESRD); lung fibrosis (including ILF); myelofibrosis, abnormal scarring (keloids) associated with all possible types of skin injury accidental and jatrogenic (operations); scleroderma; cardiofibrosis, failure of glaucoma filtering operation; intestinal adhesions.

Ocular Surgery and Fibrotic Complications

Contracture of scar tissue resulting from eye surgery may often occur. Glaucoma surgery to create new drainage channels often fails due to scarring and contraction of tissues and the generated drainage system may be blocked requiring additional surgical intervention. Current anti-scarring regimens (Mitomycin C or 5FU) are limited due to the complications involved (e.g. blindness) e.g. see Cordeiro M F, et al., Human anti-transforming growth factor-beta2 antibody: a new glaucoma anti-scarring agent Invest Ophthalmol V is Sci. 1999 September; 40 (10):2225-34. There may also be contraction of scar tissue formed after corneal trauma or corneal surgery, for example laser or surgical treatment for myopia or refractive error in which contraction of tissues may lead to inaccurate results. Scar tissue may be formed on/in the vitreous humor or the retina, for example, and may eventually causes blindness in some diabetics, and may be formed after detachment surgery, called proliferative vitreoretinopathy (PVR). PVR is the most common complication following retinal detachment and is associated with a retinal hole or break. PVR refers to the growth of cellular membranes within the vitreous cavity and on the front and back surfaces of the retina containing retinal pigment epithelial (RPE) cells. These membranes, which are essentially scar tissues, exert traction on the retina and may result in recurrences of retinal detachment, even after an initially successful retinal detachment procedure.

Scar tissue may be formed in the orbit or on eye and eyelid muscles after squint, orbital or eyelid surgery, or thyroid eye disease, and where scarring of the conjunctiva occurs as may happen after glaucoma surgery or in cicatricial disease, inflammatory disease, for example, pemphigoid, or infective disease, for example, trachoma. A further eye problem associated with the contraction of collagen-including tissues is the opacification and contracture of the lens capsule after cataract extraction. Important role for MMPs has been recognized in ocular diseases including wound healing, dry eye, sterile corneal ulceration, recurrent epithelial erosion, corneal neovascularization, pterygium, conjuctivochalasis, glaucoma, PVR, and ocular fibrosis.

Liver Fibrosis

Liver fibrosis (LF) is a generally irreversible consequence of hepatic damage of several etiologies. In the Western world, the main etiologic categories are: alcoholic liver disease (30-50%), viral hepatitis (30%), biliary disease (5-10%), primary hemochromatosis (5%), and drug-related and cryptogenic cirrhosis of, unknown etiology, (10-15%). Wilson's disease, α₁-antitrypsin deficiency and other rare diseases also have liver fibrosis as one of the symptoms. Liver cirrhosis, the end stage of liver fibrosis, frequently requires liver transplantation and is among the top ten causes of death in the Western world.

Kidney Fibrosis and Related Conditions.

Chronic Renal Failure (CRF)

Chronic renal failure is a gradual and progressive loss of the ability of the kidneys to excrete wastes, concentrate urine, and conserve electrolytes. CRF is slowly progressive. It most often results from any disease that causes gradual loss of kidney function, and fibrosis is the main pathology that produces CRF.

Diabetic Nephropathy

Diabetic nephropathy, hallmarks of which are glomerulosclerosis and tubulointerstitial fibrosis, is the single most prevalent cause of end-stage renal disease in the modern world, and diabetic patients constitute the largest population on dialysis. Such therapy is costly and far from optimal. Transplantation offers a better outcome but suffers from a severe shortage of donors.

Chronic Kidney Disease

Chronic kidney disease (CKD) is a worldwide public health problem and is recognized as a common condition that is associated with an increased risk of cardiovascular disease and chronic renal failure (CRF).

The Kidney Disease Outcomes Quality Initiative (K/DOQI) of the National Kidney Foundation (NKF) defines chronic kidney disease as either kidney damage or a decreased kidney glomerular filtration rate (GFR) for three or more months. Other markers of CKD are also known and used for diagnosis. In general, the destruction of renal mass with irreversible sclerosis and loss of nephrons leads to a progressive decline in GFR. Recently, the K/DOQI published a classification of the stages of CKD, as follows:

Stage 1: Kidney damage with normal or increased GFR (>90 mL/min/1.73 m2)

Stage 2: Mild reduction in GFR (60-89 mL/min/1.73 m2)

Stage 3: Moderate reduction in GFR (30-59 mL/min./1.73 m2)

Stage 4: Severe reduction in GFR (15-29 mL/min/1.73 m2)

Stage 5: Kidney failure (GFR<15 mL/min/1.73 m2 or dialysis)

In stages 1 and 2 CKD, GFR alone does not confirm the diagnosis. Other markers of kidney damage, including abnormalities in the composition of blood or urine or abnormalities in imaging tests, may be relied upon.

Pathophysiology of CKD

Approximately 1 million nephrons are present in each kidney, each contributing to the total GFR. Irrespective of the etiology of renal injury, with progressive destruction of nephrons, the kidney is able to maintain GFR by hyperfiltration and compensatory hypertrophy of the remaining healthy nephrons. This nephron adaptability allows for continued normal clearance of plasma solutes so that substances such as urea and creatinine start to show significant increases in plasma levels only after total GFR has decreased to 50%, when the renal reserve has been exhausted. The plasma creatinine value will approximately double with a 50% reduction in GFR. Therefore, a doubling in plasma creatinine from a baseline value of 0.6 mg/dL to 1.2 mg/dL in a patient actually represents a loss of 50% of functioning nephron mass.

The residual nephron hyperfiltration and hypertrophy, although beneficial for the reasons noted, is thought to represent a major cause of progressive renal dysfunction. This is believed to occur because of increased glomerular capillary pressure, which damages the capillaries and leads initially to focal and segmental glomerulosclerosis and eventually to global glomerulosclerosis. This hypothesis has been based on studies of five-sixths nephrectomized rats, which develop lesions that are identical to those observed in humans with CKD.

The two most common causes of chronic kidney disease are diabetes and hypertension. Other factors include acute insults from nephrotoxins, including contrasting agents, or decreased perfusion; Proteinuria; Increased renal ammoniagenesis with interstitial injury; Hyperlipidemia; Hyperphosphatemia with calcium phosphate deposition; Decreased levels of nitrous oxide and smoking.

In the United States, the incidence and prevalence of CKD is rising, with poor outcomes and high cost to the health system. Kidney disease is the ninth leading cause of death in the US. The high rate of mortality has led the US Surgeon General's mandate for America's citizenry, Healthy People 2010, to contain a chapter focused on CKD. The objectives of this chapter are to articulate goals and to provide strategies to reduce the incidence, morbidity, mortality, and health costs of chronic kidney disease in the United States.

The incidence rates of end-stage renal disease (ESRD) have also increased steadily internationally since 1989. The United States has the highest incident rate of ESRD, followed by Japan. Japan has the highest prevalence per million population, followed by the US.

The mortality rates associated with hemodialysis are striking and indicate that the life expectancy of patients entering into hemodialysis is markedly shortened. At every age, patients with ESRD on dialysis have significantly increased mortality when compared with nondialysis patients and individuals without kidney disease. At age 60 years, a healthy person can expect to live for more than 20 years, whereas the life expectancy of a 60-year-old patient starting hemodialysis is closer to 4 years (Aurora and Verelli, May 21, 2009. Chronic Renal Failure: Treatment & Medication. Emedicine. http://emedicine.medscape.com/article/238798-treatment).

Pulmonary Fibrosis

Interstitial pulmonary fibrosis (IPF) is scarring of the lung caused by a variety of inhaled agents including mineral particles, organic dusts, and oxidant gases, or by unknown reasons (idiopathic lung fibrosis). The disease afflicts millions of individuals worldwide, and there are no effective therapeutic approaches. A major reason for the lack of useful treatments is that few of the molecular mechanisms of disease have been defined sufficiently to design appropriate targets for therapy (Lasky J A., Brody A R. (2000), “Interstitial fibrosis and growth factors”, Environ Health Perspect.; 108 Suppl 4:751-62).

Cardiac Fibrosis

Heart failure is unique among the major cardiovascular disorders in that it alone is increasing in prevalence while there has been a striking decrease in other conditions. Some of this can be attributed to the aging of the populations of the United States and Europe. The ability to salvage patients with myocardial damage is also a major factor, as these patients may develop progression of left ventricular dysfunction due to deleterious remodelling of the heart.

The normal myocardium is composed of a variety of cells, cardiac myocytes and noncardiomyocytes, which include endothelial and vascular smooth muscle cells and fibroblasts.

Structural remodeling of the ventricular wall is a key determinant of clinical outcome in heart disease. Such remodeling involves the production and destruction of extracellular matrix proteins, cell proliferation and migration, and apoptotic and necrotic cell death. Cardiac fibroblasts are crucially involved in these processes, producing growth factors and cytokines that act as autocrine and paracrine factors, as well as extracellular matrix proteins and proteinases. Recent studies have shown that the interactions between cardiac fibroblasts and cardiomyocytes are essential for the progression of cardiac remodeling of which the net effect is deterioration in cardiac function and the onset of heart failure (Manabe I, et al., (2002), “Gene expression in fibroblasts and fibrosis: involvement in cardiac hypertrophy”, Circ Res. 13; 91 (12):1103-13).

Burns and Scars

A particular problem which may arise, particularly in fibrotic disease, is contraction of tissues, for example contraction of scars. Contraction of tissues including extracellular matrix components, especially of collagen-including tissues, may occur in connection with many different pathological conditions and with surgical or cosmetic procedures. Contracture, for example, of scars, may cause physical problems, which may lead to the need for medical treatment, or it may cause problems of a purely cosmetic nature. Collagen is the major component of scar and other contracted tissue and as such is the most important structural component to consider. Nevertheless, scar and other contracted tissue also includes other structural components, especially other extracellular matrix components, for example, elastin, which may also contribute to contraction of the tissue.

Contraction of collagen-including tissue, which may also include other extracellular matrix components, frequently occurs in the healing of burns. The burns may be chemical, thermal or radiation burns and may be of the eye, the surface of the skin or the skin and the underlying tissues. It may also be the case that there are burns on internal tissues, for example, caused by radiation treatment. Contraction of burnt tissues is often a problem and may lead to physical and/or cosmetic problems, for example, loss of movement and/or disfigurement.

Skin grafts may be applied for a variety of reasons and may often undergo contraction after application. As with the healing of burnt tissues the contraction may lead to both physical and cosmetic problems. It is a particularly serious problem where many skin grafts are needed as, for example, in a serious burns case.

Contraction is also a problem in production of artificial skin. To make a true artificial skin it is necessary to have an epidermis made of epithelial cells (keratinocytes) and a dermis made of collagen populated with fibroblasts. It is important to have both types of cells because they signal and stimulate each other using growth factors. The collagen component of the artificial skin often contracts to less than one tenth of its original area when populated by fibroblasts.

Cicatricial contraction, contraction due to shrinkage of the fibrous tissue of a scar, is common. In some cases the scar may become a vicious cicatrix, a scar in which the contraction causes serious deformity. A patient's stomach may be effectively separated into two separate chambers in an hour-glass contracture by the contraction of scar tissue formed when a stomach ulcer heals. Obstruction of passages and ducts, cicatricial stenosis, may occur due to the contraction of scar tissue. Contraction of blood vessels may be due to primary obstruction or surgical trauma, for example, after surgery or angioplasty. Stenosis of other hollow visci, for examples, ureters, may also occur. Problems may occur where any form of scarring takes place, whether resulting from accidental wounds or from surgery. Conditions of the skin and tendons which involve contraction of collagen-including tissues include post-trauma conditions resulting from surgery or accidents, for example, hand or foot tendon injuries, post-graft conditions and pathological conditions, such as scleroderma, Dupuytren's contracture and epidermolysis bullosa. Scarring and contraction of tissues in the eye may occur in various conditions, for example, the sequelae of retinal detachment or diabetic eye disease (as mentioned above). Contraction of the sockets found in the skull for the eyeballs and associated structures, including extra-ocular muscles and eyelids, may occur if there is trauma or inflammatory damage. The tissues contract within the sockets causing a variety of problems including double vision and an unsightly appearance.

For further information on different types of fibrosis see: Molina V, et al., (2002), “Fibrotic diseases”, Harefuah, 141 (11): 973-8, 1009; Yu L, et al., (2002), “Therapeutic strategies to halt renal fibrosis”, Curr Opin Pharmacol. 2 (2):177-81; Keane W F and Lyle P A. (2003), “Recent advances in management of type 2 diabetes and nephropathy: lessons from the RENAAL study”, Am J Kidney Dis. 41 (3 Suppl 2): S22-5; Bohle A, et al., (1989), “The pathogenesis of chronic renal failure”, Pathol Res Pract. 185 (4):421-40; Kikkawa R, et al., (1997), “Mechanism of the progression of diabetic nephropathy to renal failure”, Kidney Int Suppl. 62:S39-40; Bataller R, and Brenner D A. (2001), “Hepatic stellate cells as a target for the treatment of liver fibrosis”, Semin Liver Dis. 21 (3):437-51; Gross T J and Hunninghake G W, (2001) “Idiopathic pulmonary fibrosis”, N Engl J. Med. 345 (7):517-25; Frohlich E D. (2001) “Fibrosis and ischemia: the real risks in hypertensive heart disease”, Am J Hypertens; 14 (6 Pt 2):194S-199S; Friedman S L. (2003), “Liver fibrosis—from bench to bedside”, J. Hepatol. 38 Suppl 1:S38-53; Albanis E, et al., (2003), “Treatment of hepatic fibrosis: almost there”, Curr Gastroenterol Rep. 5 (1):48-56; (Weber K T. (2000), “Fibrosis and hypertensive heart disease”, Curr Opin Cardiol. 15 (4):264-72).

Delivery of Nucleic Acid Molecules and Pharmaceutical Formulations

Nucleic acid molecules may be adapted for use to prevent or treat fibroses (e.g., liver, kidney, peritoneal, and pulmonary) diseases, traits, conditions and/or disorders, and/or any other trait, disease, disorder or condition that is related to or will respond to the levels of hsp47 in a cell or tissue, alone or in combination with other therapies. A nucleic acid molecule may include a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations.

Nucleic acid molecules disclosed herein may be delivered or administered directly with a carrier or diluent but not any delivery vehicle that acts to assist, promote or facilitate entry to the cell, including viral vectors, viral particles, liposome formulations, lipofectin or precipitating agents and the like.

Nucleic acid molecules may be delivered or administered to a subject by direct application of the nucleic acid molecules with a carrier or diluent or any other delivery vehicle that acts to assist, promote or facilitate entry into a cell, including viral sequences, viral particular, liposome formulations, lipofectin or precipitating agents and the like. Polypeptides that facilitate introduction of nucleic acid into a desired subject such as those described in US. Application Publication No. 20070155658 (e.g., a melamine derivative such as 2,4,6-Triguanidino Traizine and 2,4,6-Tramidosarcocyl Melamine, a polyarginine polypeptide, and a polypeptide including alternating glutamine and asparagine residues).

Methods for the delivery of nucleic acid molecules are described in Akhtar et al., Trends Cell Bio., 2: 139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, (1995), Maurer et al., Mol. Membr. Biol., 16: 129-140 (1999); Hofland and Huang, Handb. Exp. Pharmacol., 137: 165-192 (1999); and Lee et al., ACS Symp. Ser., 752: 184-192 (2000); U.S. Pat. Nos. 6,395,713; 6,235,310; 5,225,182; 5,169,383; 5,167,616; 4,959217; 4,925,678; 4,487,603; and 4,486,194 and Sullivan et al., PCT WO 94/02595; PCT WO 00/03683 and PCT WO 02/08754; and U.S. Patent Application Publication No. 2003077829. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see e.g., Gonzalez et al., Bioconjugate Chem., 10: 1068-1074 (1999); Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and U.S. Application Publication No. 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., Clin. Cancer Res., 5: 2330-2337 (1999) and Barry et al., International PCT Publication No. WO 99/31262. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.

Nucleic acid molecules may be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers. The nucleic acid molecules of the invention may include sequences shown in Tables I. Examples of such nucleic acid molecules consist essentially of sequences provided in Table I.

Delivery systems include surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).

Nucleic acid molecules may be formulated or complexed with polyethylenimine (e.g., linear or branched PEI) and/or polyethylenimine derivatives, including for example polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives, grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, Pharmaceutical Research, 19, 810-817; Choi et al, 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999, PNAS USA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release, 60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274, 19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; Sagara, U.S. Pat. No. 6,586,524 and United States Patent Application Publication No. 20030077829.

Nucleic acid molecules may be complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666. The membrane disruptive agent or agents and the nucleic acid molecule may also be complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Pat. No. 6,235,310.

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

Aerosols of liquid particles may include a nucleic acid molecules disclosed herein and can be produced by any suitable means, such as with a nebulizer (see e.g., U.S. Pat. No. 4,501,729). Nebulizers are commercially available devices which transform solutions or suspensions of an active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers include the active ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, e.g., sodium chloride or other suitable salts. Optional additives include preservatives if the formulation is not prepared sterile, e.g., methyl hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants. The aerosols of solid particles including the active composition and surfactant can likewise be produced with any solid particulate aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a therapeutic composition at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which can be delivered by means of an insufflator. In the insufflator, the powder, e.g., a metered dose thereof effective to carry out the treatments described herein, is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically includes from 0.1 to 100 w/w of the formulation. A second type of illustrative aerosol generator includes a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquefied propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation can additionally contain one or more co-solvents, for example, ethanol, emulsifiers and other formulation surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents. Other methods for pulmonary delivery are described in, e.g., US Patent Application No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728; 6,565,885. PCT Patent Publication No. WO2008/132723 relates to aerosol delivery of oligonucleotides in general, and of siRNA in particular, to the respiratory system.

Nucleic acid molecules may be administered to the central nervous system (CNS) or peripheral nervous system (PNS). Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. See e.g., Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75; Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469; Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J. Pharmocol., 340 (2/3), 153; Bannai et al., 1998, Brain Research, 784 (1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12 (1), 32; Bannai et al., 1998, Brain Res. Protoc., 3 (1), 83; and Simantov et al., 1996, Neuroscience, 74 (1), 39. Nucleic acid molecules are therefore amenable to delivery to and uptake by cells in the CNS and/or PNS.

Delivery of nucleic acid molecules to the CNS is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, e.g., as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.

Delivery systems may include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. Examples of liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA, the neutral lipid DOPE (GIBCO BRL) and Di-Alkylated Amino Acid (DiLA2).

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

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

Nucleic acid molecules may include a bioconjugate, for example a nucleic acid conjugate as described in Vargeese et al., U.S. Ser. No. 10/427,160; U.S. Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886; U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,138,045.

Compositions, methods and kits disclosed herein may include an expression vector that includes a nucleic acid sequence encoding at least one nucleic acid molecule of the invention in a manner that allows expression of the nucleic acid molecule. Methods of introducing nucleic acid molecules or one or more vectors capable of expressing the strands of dsRNA into the environment of the cell will depend on the type of cell and the make up of its environment. The nucleic acid molecule or the vector construct may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing an organism or a cell in a solution containing dsRNA. The cell is preferably a mammalian cell; more preferably a human cell. The nucleic acid molecule of the expression vector can include a sense region and an antisense region. The antisense region can include a sequence complementary to a RNA or DNA sequence encoding hsp47 and the sense region can include a sequence complementary to the antisense region. The nucleic acid molecule can include two distinct strands having complementary sense and antisense regions. The nucleic acid molecule can include a single strand having complementary sense and antisense regions.

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

Expression vectors may include a nucleic acid sequence encoding at least one nucleic acid molecule disclosed herein, in a manner which allows expression of the nucleic acid molecule. For example, the vector may contain sequence(s) encoding both strands of a nucleic acid molecule that include a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a nucleic acid molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725. Expression vectors may also be included in a mammalian (e.g., human) cell.

An expression vector may include a nucleic acid sequence encoding two or more nucleic acid molecules, which can be the same or different. Expression vectors may include a sequence for a nucleic acid molecule complementary to a nucleic acid molecule referred to by a Genbank Accession number NM_—001235, for example those shown in Table I.

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

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

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

Nucleic acid molecule may be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res. 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

A viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of dsRNA construct encoded by the expression construct.

Methods for oral introduction include direct mixing of RNA with food of the organism, as well as engineered approaches in which a species that is used as food is engineered to express an RNA, then fed to the organism to be affected. Physical methods may be employed to introduce a nucleic acid molecule solution into the cell. Physical methods of introducing nucleic acids include injection of a solution containing the nucleic acid molecule, bombardment by particles covered by the nucleic acid molecule, soaking the cell or organism in a solution of the RNA, or electroporation of cell membranes in the presence of the nucleic acid molecule.

Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like. Thus the nucleic acid molecules may be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or other-wise increase inhibition of the target gene.

The nucleic acid molecules or the vector construct can be introduced into the cell using suitable formulations. One preferable formulation is with a lipid formulation such as in Lipofectamine™ 2000 (Invitrogen, CA, USA), vitamin A coupled liposomes (Sato et al. Nat Biotechnol 2008; 26:431-442, PCT Patent Publication No. WO 2006/068232). Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art. When the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable. Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used. In some instances, it may be preferable to formulate dsRNA in a buffer or saline solution and directly inject the formulated dsRNA into cells, as in studies with oocytes. The direct injection of dsRNA duplexes may also be done. For suitable methods of introducing dsRNA see U.S. published patent application No. 2004/0203145, 20070265220 which are incorporated herein by reference.

Polymeric nanocapsules or microcapsules facilitate transport and release of the encapsulated or bound dsRNA into the cell. They include polymeric and monomeric materials, especially including polybutylcyanoacrylate. A summary of materials and fabrication methods has been published (see Kreuter, 1991). The polymeric materials which are formed from monomeric and/or oligomeric precursors in the polymerization/nanoparticle generation step, are per se known from the prior art, as are the molecular weights and molecular weight distribution of the polymeric material which a person skilled in the field of manufacturing nanoparticles may suitably select in accordance with the usual skill.

Nucleic acid moles may be formulated as a microemulsion. A microemulsion is a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution. Typically microemulsions are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a 4th component, generally an intermediate chain-length alcohol to form a transparent system.

Surfactants that may be used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.

Water Soluble Crosslinked Polymers

Delivery formulations can include water soluble degradable crosslinked polymers that include one or more degradable crosslinking lipid moiety, one or more PEI moiety, and/or one or more mPEG (methyl ether derivative of PEG (methoxypoly(ethylene glycol)).

Degradable lipid moieties preferably include compounds having the following structural motif:

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In the above formula, ester linkages are biodegradable groups, R represents a relatively hydrophobic “lipo” group, and the structural motif shown occurs m times where m is in the range of about 1 to about 30. For example, in certain embodiments R is selected from the group consisting of C2-C50 alkyl, C2-C50 heteroalkyl, C2-C50 alkenyl, C2-C50 heteroalkenyl, C5-C50 aryl; C2-C50 heteroaryl; C2-C50 alkynyl, C2-C50 heteroalkynyl, C2-C50 carboxyalkenyl, and C2-C50 carboxyheteroalkenyl. In preferred embodiments. R is a saturated or unsaturated alkyl having 4 to 30 carbons, more preferably 8 to 24 carbons or a sterol, preferably a cholesteryl moiety. In preferred embodiments, R is oleic, lauric, myristic, palmitic margaric, stearic, arachidic, behenic, or lignoceric. In a most preferred embodiment, R is oleic.

The N in formula (B) may have an electron pair or a bond to a hydrogen atom. When N has an electron pair, the recurring unit may be cationic at low pH.

The degradable crosslinking lipid moiety may be reacted with a polyethyleneimine (PEI) as shown in Scheme A below:

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In formula (A), R has the same meanings as described above. The PEI may contain recurring units of formula (B) in which x is an integer in the range of about 1 to about 100 and y is an integer in the range of about 1 to about 100.

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The reaction illustrated in Scheme A may be carried out by intermixing the PEI and the diacrylate (I) in a mutual solvent such as ethanol, methanol or dichloromethane with stirring, preferably at room temperature for several hours, then evaporating the solvent to recover the resulting polymer. While not wishing to be bound to any particular theory, it is believed that the reaction between the PEI and diacrylate (I) involves a Michael reaction between one or more amines of the PEI with double bond(s) of the diacrylate (see J. March, Advanced Organic Chemistry 3rd Ed, pp. 711-712 (1985)). The di acrylate shown in Scheme A may be prepared in the manner as described in U.S. application Ser. No. 11/216,986 (US Publication No. 2006/0258751).

The molecular weight of the PEI is preferably in the range of about 200 to 25,000 Daltons more preferably 400 to 5,000 Daltons, yet more preferably 600 to 2000 Daltons. PEI may be either branched or linear.

The molar ratio of PEI to diacrylate is preferably in the range of about 1:2 to about 1:20. The weight average molecular weight of the cationic lipopolymer may be in the range of about 500 Daltons to about 1,000,000 Daltons preferably in the range of about 2,000 Daltons to about 200.000 Daltons. Molecular weights may be determined by size exclusion chromatography using PEG standards or by agarose gel electrophoresis.

The cationic lipopolymer is preferably degradable, more preferably biodegradable, e.g., degradable by a mechanism selected from the group consisting of hydrolysis, enzyme cleavage, reduction, photo-cleavage, and sonication. While not wishing to be bound to any particular theory, but it is believed that degradation of the cationic lipopolymer of formula (II) within the cell proceeds by enzymatic cleavage and/or hydrolysis of the ester linkages.

Synthesis may be carried out by reacting the degradable lipid moiety with the PEI moiety as described above. Then the mPEG (methyl ether derivative of PEG (methoxypoly (ethylene glycol)), is added to form the degradable crosslinked polymer. In preferred embodiments, the reaction is carried out at room temperature. The reaction products may be isolated by any means known in the art including chromatographic techniques. In a preferred embodiment, the reaction product may be removed by precipitation followed by centrifugation.

Dosages

The useful dosage to be administered and the particular mode of administration will vary depending upon such factors as the cell type, or for in vivo use, the age, weight and the particular animal and region thereof to be treated, the particular nucleic acid and delivery method used, the therapeutic or diagnostic use contemplated, and the form of the formulation, for example, suspension, emulsion, micelle or liposome, as will be readily apparent to those skilled in the art. Typically, dosage is administered at lower levels and increased until the desired effect is achieved.

When lipids are used to deliver the nucleic acid, the amount of lipid compound that is administered can vary and generally depends upon the amount of nucleic acid being administered. For example, the weight ratio of lipid compound to nucleic acid is preferably from about 1:1 to about 30:1, with a weight ratio of about 5:1 to about 10:1 being more preferred.

A suitable dosage unit of nucleic acid molecules may be in the range of 0.001 to 0.25 milligrams per kilogram body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kilogram body weight per day, or in the range of 0.01 to 10 micrograms per kilogram body weight per day, or in the range of 0.10 to 5 micrograms per kilogram body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram body weight per day.

Suitable amounts of nucleic acid molecules may be introduced and these amounts can be empirically determined using standard methods. Effective concentrations of individual nucleic acid molecule species in the environment of a cell may be about 1 femtomolar, about 50 femtomolar, 100 femtomolar, 1 picomolar, 1.5 picomolar, 2.5 picomolar, 5 picomolar, 10 picomolar, 25 picomolar, 50 picomolar, 100 picomolar, 500 picomolar, 1 nanomolar, 2.5 nanomolar, 5 nanomolar, 10 nanomolar, 25 nanomolar, 50 nanomolar, 100 nanomolar, 500 nanomolar, 1 micromolar, 2.5 micromolar, 5 micromolar, 10 micromolar, 100 micromolar or more.

Dosage may be from 0.01 μg to 1 g per kg of body weight (e.g., 0.1 μg, 0.25 μg, 0.5 μg, 0.75 μg, 1 μg, 2.5 μg, 5 μg, 10 μg, 25 μg, 50 μg, 100 μg, 250 μg, 500 μg, 1 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, or 500 mg per kg).

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

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

Pharmaceutical compositions that include the nucleic acid molecule disclosed herein may be administered once daily, qid, tid, bid, QD, or at any interval and for any duration that is medically appropriate. However, the therapeutic agent may also be dosed in dosage units containing two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In that case, the nucleic acid molecules contained in each sub-dose may be correspondingly smaller in order to achieve the total daily dosage unit. The dosage unit can also be compounded for a single dose over several days, e.g., using a conventional sustained release formulation which provides sustained and consistent release of the dsRNA over a several day period. Sustained release formulations are well known in the art. The dosage unit may contain a corresponding multiple of the daily dose. The composition can be compounded in such a way that the sum of the multiple units of a nucleic acid together contain a sufficient dose.

Pharmaceutical Compositions, Kits, and Containers

Also provided are compositions, kits, containers and formulations that include a nucleic acid molecule (e.g., an siNA molecule) as provided herein for reducing expression of hsp47 for administering or distributing the nucleic acid molecule to a patient. A kit may include at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass, metal or plastic. The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), cell population(s) and/or antibody(s). In one embodiment, the container holds a polynucleotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose. In another embodiment a container includes an antibody, binding fragment thereof or specific binding protein for use in evaluating hsp47 protein expression cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or directions for such uses can be included on or with such container, as can reagents and other compositions or tools used for these purposes. Kits may further include associated indications and/or directions; reagents and other compositions or tools used for such purpose can also be included.

The container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be a nucleic acid molecule capable of specifically binding hsp47 and/or modulating the function of hsp47.

A kit may further include a second container that includes a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.

The units dosage ampoules or multidose containers, in which the nucleic acid molecules are packaged prior to use, may include an hermetically sealed container enclosing an amount of polynucleotide or solution containing a polynucleotide suitable for a pharmaceutically effective dose thereof, or multiples of an effective dose. The polynucleotide is packaged as a sterile formulation, and the hermetically sealed container is designed to preserve sterility of the formulation until use.

The container in which the polynucleotide including a sequence encoding a cellular immune response element or fragment thereof may include a package that is labeled, and the label may bear a notice in the form prescribed by a governmental agency, for example the Food and Drug Administration, which notice is reflective of approval by the agency under Federal law, of the manufacture, use, or sale of the polynucleotide material therein for human administration.

Federal law requires that the use of pharmaceutical compositions in the therapy of humans be approved by an agency of the Federal government. In the United States, enforcement is the responsibility of the Food and Drug Administration, which issues appropriate regulations for securing such approval, detailed in 21 U.S.C. §301-392. Regulation for biologic material, including products made from the tissues of animals is provided under 42 U.S.C. §262. Similar approval is required by most foreign countries. Regulations vary from country to country, but individual procedures are well known to those in the art and the compositions and methods provided herein preferably comply accordingly.

The dosage to be administered depends to a large extent on the condition and size of the subject being treated as well as the frequency of treatment and the route of administration. Regimens for continuing therapy, including dose and frequency may be guided by the initial response and clinical judgment. The parenteral route of injection into the interstitial space of tissues is preferred, although other parenteral routes, such as inhalation of an aerosol formulation, may be required in specific administration, as for example to the mucous membranes of the nose, throat, bronchial tissues or lungs.

As such, provided herein is a pharmaceutical product which may include a polynucleotide including a sequence encoding a cellular immune response element or fragment thereof in solution in a pharmaceutically acceptable injectable carrier and suitable for introduction interstitially into a tissue to cause cells of the tissue to express a cellular immune response element or fragment thereof, a container enclosing the solution, and a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of manufacture, use, or sale of the solution of polynucleotide for human administration.

Indications

The nucleic acid molecules disclosed herein can be used to treat diseases, conditions or disorders associated with hsp47, such as liver fibrosis, cirrhosis, pulmonary fibrosis, kidney fibrosis, peritoneal fibrosis, chronic hepatic damage, and fibrillogenesis and any other disease or conditions that are related to or will respond to the levels of hsp47 in a cell or tissue, alone or in combination with other therapies. As such, compositions, kits and methods disclosed herein may include packaging a nucleic acid molecule disclosed herein that includes a label or package insert. The label may include indications for use of the nucleic acid molecules such as use for treatment or prevention of liver fibrosis, peritoneal fibrosis, kidney fibrosis and pulmonary fibrosis, and any other disease or conditions that are related to or will respond to the levels of hsp47 in a cell or tissue, alone or in combination with other therapies. A label may include an indication for use in reducing expression of hsp47. A “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products, etc.

Those skilled in the art will recognize that other anti-fibrosis treatments, drugs and therapies known in the art can be readily combined with the nucleic acid molecules herein (e.g. siNA molecules) and are hence contemplated herein.

The methods and compositions provided herein will now be described in greater detail by reference to the following non-limiting examples.

EXAMPLE 1
Selecting hsp47 Nucleic Acid Molecule Sequences

Nucleic acid molecules (e.g., siNA≦25 nucleotides) against Hsp47 were designed using several computer programs including siRNA at Whitehead (Whitehead Institute for Biomedical Research), IDT siRNA Design (Integrated DNA Technologies), BLOCK-iT RNAi Designer (Invitrogen), siDESIGN Center (Dharmacon), and BIOPREDsi (Friedrich Miescher Institute for Biomedical Research, part of the Novartis Research Foundation, available at http://www.biopredsi.orgistart.html). The sequences of top scored siRNAs from these programs were compared and selected (see Table 1) based on the algorithms as well as the sequence homology between human and rat. Candidate sequences were validated by in vitro knocking down assays.

Several parameters were considered for selecting a nucleic acid molecule (e.g., a 21-mer siRNA) sequence. Exemplary parameters include:

- 1) thermodynamic stability (RISC favors the strand with less stable 5′ end)
- 2) 30-52% GC content
- 3) positional nucleotide preference:
- (C/G)NNNNNNNN(A/U)10NNNNNNNN(A/U), where N is any nucleotide
- 4) devoid of putative immunostimulatory motifs
- 5) 2-nucleotide 3′ overhang
- 6) position of siRNA within the transcript (preferably within cDNA region)
- 7) sequence specificity (checked by using BLAST)
- 8) variations in single nucleotide by checking SNP database

siRNA sequences having ≦25 nucleotides were designed based on the foregoing methods. Corresponding Dicer substrate siRNA (e.g., ≧26 nucleotides) were designed based on the smaller sequences and extend the target site of the siNA≦2.5 nucleotide by adding 4 bases to the 3′-end of the sense strand and 6 bases to the 5′-end of the antisense strand. The Dicer substrates that were made generally have a 25 base sense strand a 27 base antisense strand with an asymmetric blunt ended and 3′-overhang molecule. The sequences of the sense and the anti-sense strand without base modification (base sequence) and with modifications (experimental sequence) are provided in Table 1.

EXAMPLE 2

In order to screen for the potent of various siNA molecules against both the human and rat hsp47 genes, various reporter cell lines were established by lenti-viral induction of human HSP47 cDNA-green fluorescent protein (GFP) or rat GP46 cDNA-GFP construct into 293, HT1080, human HSC line hTERT, or NRK cell lines. These cell lines were further evaluated by siRNA against GFP. The remaining fluorescence signal was measured and normalized to scrambled siRNA (Ambion) and subsequently normalized to cell viability. The results showed that siRNA against GFP knocks down the fluorescence to different extent in different cell lines (FIG. 1). 293_HSP47-GFP and 293_GP46-GFP cell lines were selected for siHsp47 screening due to their ease of transfection and sensitivity to fluorescence knockdown.

siRNA Transfection:

Cells were transfected with 1.5 pmol per well of siNA against GFP in 96-well tissue culture plates using Lipofectamine RNAiMAX (Invitrogen) in a reverse transfection manner. Cells were seeded at 6,000 cells per well and mixed with the siNA complexs. Fluorescence readings were taken after 72 hours incubation on a Synergy 2 Multi-Mode Microplate Reader (BioTek).

Cell Viability Assay:

Cells treated with or without siNA were measured for viability after 72 hours incubation using CellTiter-Glo Luminescent Cell Viability Assay Kit according to the manual (Promega). The readings were normalized to samples treated with scrambled siNA molecules.

EXAMPLE 3
Evaluation of Inhibitory Efficiency of siHsp47 on hsp47 Expression in Reporter Cell Lines

siNAs against hsp47 were evaluated for their inhibitory efficiency in 293 HSP47-GFP and 293_GP46-GFP cell lines by evaluating the change in fluorescent signal from the reporter GFP. The experiments were carried out as described in Example 2. The fluorescent signals were normalized to fluorescent signals from cells treated with scrambled siRNA (Ambion) which served as a control. The results indicate that the tested hsp47 siNA molecules were effective in inhibiting hsp47 mRNA in both cell lines. However, siNA against GP46 mRNA (as published in the 2008 Sato et al paper) was effective only in the 293 GP46-GFP cell line. The results are shown in FIG. 2 A-B.

The 293_HSP47-GFP and 293_GP46-GFP cell lines treated with siRNA against hsp47 and gp46 were evaluated for viability using the methods described in Example 2. The cell viability was normalized to cells treated with scrambled siRNA (Ambion). The results indicate that the cell viability was not affected significantly by the treatment with siNA molecules. However, the cell viability of 293_HSP47-GFP cell lines treated with different hsp47 siNA molecules varied depending on the siNA molecules used, while the viability of 293_GP46-GFP cell lines were similar. Viability for 293 HSP47-GFP cells were lower for siHsp47-6 and Hsp47-7 treated cells than the rest. The results are shown in FIG. 2C-D.

EXAMPLE 4
Evaluation of siHsp47 Inhibitory Effect on hsp47 mRNA by TaqMan® qPCR

In Example 3, the knock down efficiency of siHsp47s in reporter cell lines was evaluated by change in fluorescent signal. To validate the results at the mRNA level, siRNAs targeting endogenous hsp47 were transfected into cells of the human HSC cell line hTERT using Lipofectamine RNAiMAX (Invitrogen) in a reverse transfection manner as described in Example 2.

The hsp47 mRNA level was evaluated for knock down efficiency of the various tested siHsp47 siNA molecules. Briefly, mRNA were isolated from hTERT cells after 72 hours after transfection using an RNeasy mini kit (Qiagen). The level of hsp47 mRNA was determined by reverse transcription coupled with quantitative PCR using TaqMan® probes. Briefly, cDNA synthesis was carried out using High-Capacity cDNA Reverse Transcription Kit (ABI) according to the manufacturer's instruction, and subjected to TaqMan Gene Expression Assay (ABI, hsp47 assay ID Hs01060395_g1). The level of hsp47 mRNA was normalized to the level of GAPDH mRNA according to the manufacturer's instruction (ABI). The results indicate that siHsp47-C was the most effective among all the hsp47 siRNAs, siHsp47-2 and siHsp47-2d were the next most effective. The combinations of siHsp47-1 with siHsp47-2 or siHsp47-1 with siHsp47-2d were more effective than siHsp47-1 alone. The results are shown in FIG. 3.

EXAMPLE 5
Validation of siHsp47 Knock Down Effect at the Protein Level

The inhibitory effect of different Hsp47 siNA molecules (siHsp47) on hsp47 mRNA expression were validated at the protein level by measuring the HSP47 in hTERT cells transfected with different siHsp47. Transfection of hTERT cells with different siHsp47 were performed as described in Example 2. Transfected hTERT cells were lysed and the cell lysate were clarified by centrifugation. Proteins in the clarified cell lysate were resolved by SDS polyacrylamide gel electrophoresis. The level of HSP47 protein in the cell lysate were determined using an anti-HSP47 antibody (Assay Designs) as the primary antibody, Goat anti-mouse IgG conjugated with HRP (Millipore) as the secondary antibody, and subsequently detected by Supersignal West Pico Chemiluminescence kit (Pierce). Anti-actin antibody (Abeam) was used as a protein loading control. The result showed significant decrease in the level of Hsp47 protein in cells treated with siHsp47-C, siHsp47-2d, alone or combination of siHsp47-1 with siHsp47-2d.

EXAMPLE 6
Downregulation of Collagen I expression by hsp47 siRNA

To determine the effect of siHsp47 on collagen I expression level, collagen I mRNA level in hTERT cells treated with different siRNA against hsp47 was measured. Briefly, hTERT cells were transfected with different siHsp47 as described in Example 2. The cells were lysed after 72 hours and mRNA were isolated using RNeasy mini kit according to the manual (Qiagen). The level of collagen 1 mRNA was determined by reverse transcription coupled with quantitative PCR using TaqMan® probes. Briefly, cDNA synthesis was carried out using High-Capacity cDNA Reverse Transcription Kit (ABI) according to the manual, and subjected to TaqMan Gene Expression Assay (ABI, COL1A1 assay ID Hs01076780_g1). The level of collagen I mRNA was normalized to the level of GAPDH mRNA according to the manufacturer's instruction (ABI). The signals were normalized to the signal obtained from cells transfected with scrambled siNA. The result indicated that collagen I mRNA level is significantly reduced in the cells treated with some of the candidates siHsp47-2, siHsp47-2d, and their combination with siHsp47-1 and shown in FIG. 4.

EXAMPLE 7
Immunofluorescence Staining of hsp47 siRNA Treated hTERT Cells

To visualize the expression of two fibrosis markers, collagen 1 and alpha-smooth muscle actin (SMA), in hTERT cells transfected with or without siHsp47, the cells were stained with rabbit anti-collagen I antibody (Abeam) and mouse anti-alpha-SMA antibody (Sigma). Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen (Molecular Probes)) were used as secondary antibodies to visualize collagen I (green) and alpha-SMA (red). Hoescht was used to visualize nucleus (blue). The results indicate correlation between siRNA knocking down of some of the target genes and collagen/SMA expression.

EXAMPLE 8
In Vivo Testing of siHSP47 in Animal Models of Liver Fibrosis

siRNA for Rat Liver Cirrhosis Treatment

The siRNA duplex sequence for HSP47 (siHSP47C) is as listed below.

Sense (5′->3′)
ggacaggccucuacaacuaTT

Antisense (5′->3′)
uaguuguagaggccuguccTT

10 mg/ml siRNA stock solution was prepared by dissolving in nuclease free water (Ambion). For treatment of cirrhotic rats, siRNA was formulated with vitamin A-coupled liposome as described by Sato et al (Sato Y. et al. Nature Biotechnology 2008. Vol. 26, p 431) in order to target activated hepatic stellate cells that produce collagen. The vitamin A (VA)-liposome-siRNA formulation consists of 0.33 μmol/ml of VA, 0.33 μmol/ml of liposome (Coatsome EL-01-D, NOF Corporation) and 0.5 μg/μl of siRNA in 5% glucose solution.

The liver cirrhosis animal model was reported by Sato et al (Sato Y. et al. Nature Biotechnology 2008. Vol. 26, p 431). 4 week-old male SD rats were induced with liver cirrhosis with 0.5% dimethylnitrosoamine (DMN) (Wako Chemicals, Japan) in phosphate-buffered saline (PBS). A dose of 2 ml/kg per body weight was administered intraperitoneally for 3 consecutive days per week on days 0, 2, 4, 7, 9, 11, 14, 16, 18, 21, 23, 25, 28, 30, 32, 34, 36, 38 and 40

siRNA treatment: siRNA treatment was carried out from day 32 and for 5 intravenous injections. In detail, rats were treated with siRNA on day-32, 34, 36, 38 and 40. Then rats were sacrificed on day-42 or 43. 3 different siRNA doses (1.5 mg siRNA per kg body weight, 2.25 mg siRNA per kg body weight, 3.0 mg siRNA per kg body weight) were tested. Detail of tested groups and number of animals in each group are as follows:

1) Cirrhosis was induced by DMN injection, then 5% glucose was injected instead of siRNA) (n=10)
2) VA-Lip-siHSP47C1.5 mg/kg (n=10)
3) VA-Lip-siHSP47C 2.25 mg/kg (n=10)
4) VA-Lip-siHSP47C 3.0 mg/Kg (n=10)
5) Sham (PBS was injected instead of DMN. 5% Glucose was injected instead of siRNA) (n=6)
6) No treatment control (Intact) (n=6)

VA-Lip Refers to Vitamin A—Liposome Complex.

Evaluation of therapeutic efficacy: On day 43, 2 out of 10 animals in the “diseased rat” group and 1 out of 10 animals in “VA-Lip-siHSP47C siRNA 1.5 mg/kg” died due to development of liver cirrhosis. The remainder of the animals survived. After rats were sacrificed, liver tissues were fixed in 10% formalin. Then, the left lobule of each liver was embedded in paraffin for histology. Tissue slides were stained with Sirius red, and hematoxylin and eosin (HE). Sirius red staining was employed to visualize collagen-deposits and to determine the level of cirrhosis. HE staining was for nuclei and cytoplasm as counter-staining. Each slide was observed under microscope (BZ-8000, Keyence Corp. Japan) and percentage of Sirius red-stained area per slide was determined by image analysis software attached to the microscope. At least 4 slides per each liver were prepared for image analysis, and whole area of each slide (slice of liver) was captured by camera and analyzed. Statistical analysis was carried out by Student's t-test.

Results: FIG. 5 shows the fibrotic areas. The area of fibrosis in “diseased rats” was higher than in the “sham” or “no treatment control” groups. Therefore, DMN treatment induced collagen deposition in the liver, which was a typical observation of liver fibrosis. However, the area of fibrosis was significantly reduced by the treatment of siRNA targeting HSP47 gene, compared with “disease rat” group (FIG. 5). This result indicates that siRNA as disclosed herein has a therapeutic efficacy in actual disease.

Additional siRNA compounds are tested in the liver fibrosis animal model, and were shown to reduce the fibrotic area in the liver.

EXAMPLE 9
Generation of Sequences for Active Double Stranded RNA Compounds to HSP47/SERPINH1 and Production of the siRNAs Shown in Tables A-18, A-19, and B-E

Using proprietary algorithms and the known sequence of the target genes, the sequences of many potential siRNAs were generated. The sequences that have been generated using this method are complementary to the corresponding mRNA sequence.

Duplexes are generated by annealing complementary single stranded oligonucleotides. In a laminar flow hood, a ˜500 μM Stock Solution of single stranded oligonucleotide is prepared by diluting in WFI (water for injection, Norbrook). Actual ssRNA concentrations are determined by diluting each 500 μM ssRNA 1:200 using WFI, and measuring the OD using Nano prop. The procedure is repeated 3 times and the average concentration is calculated. The Stock Solution was then diluted to a final concentration of 250 μM. Complementary single strands were annealed by heating to 85° C. and allowing to cool to room temperature over at least 45 minutes. Duplexes were tested for complete annealing by testing 5 μl on a 20% polyacrylamide gel and staining. Samples were stored at −80° C.

Tables A-18, A-19 and B-E provide siRNAs for HSP47/SERPINH1. For each gene there is a separate list of 19-mer siRNA sequences, which are prioritized based on their score in the proprietary algorithm as the best sequences for targeting the human gene expression.

The following abbreviations are used in the Tables A-18, A-19 and B-E herein: “other spec or Sp.” refers to cross species identity with other animals: D-dog, Rt-rat, Rb-Rabbit, Rh-rhesus monkey, P-Pig, M-Mouse; ORF: open reading frame. 19-mers, and 18+1-mers refer to oligomers of 19 and 18+1 (U in position 1 of Antisense, A in position 19 of sense strand) ribonucleic acids in length, respectively.

In Vitro Testing of the siRNA Compounds for the Target Genes

Low-Throughput-Screen (LTS) for siRNA Oligos Directed to Human and Rat SERPINH1 Gene.

Cell Lines: Human prostate adenocarcinoma PC3 cells (ATCC, Cat# CRL-1435) were grown in RPMI medium supplemented with 10% FBS and 2 mM L-Glutamine and human epithelial cervical cancer HeLa cells (ATCC, Cat#CCL-2) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine. Cells were maintained at 37° C. in 5% CO₂.

About 2×10⁵human PC-3 cells endogenously expressing SERPINH1 gene, were inoculated in 1.5 mL growth medium in order to reach 30-50% confluence after 24 hours. Cells were transfected with Lipofectamine™2000 reagent to a final concentration of 0.01-5 nM per transfected cells. Cells were incubated at 37±1° C., 5% CO₂for 48 hours. siRNA transfected cells were harvested and RNA was isolated using EZ-RNA kit [Biological Industries (#20-410-100)].

Reverse transcription was performed as follows: Synthesis of cDNA was performed and human SERPINH1 mRNA levels were determined by Real Time qPCR and normalized to those of the Cyclophilin A (CYNA, PPIA) mRNA for each sample. siRNA activity was determined based on the ratio of the SERPINH1 mRNA quantity in siRNA-treated samples versus non-transfected control samples.

The most active sequences were selected from further assays. From Table A-18 siRNA compounds SERPINH1_—2, SERPINH1_—6, SERPINH1_—13, SERPINH1_—45 SERPINH1_—45a, SERPINH1_—51, SERPINH1_—51a, SERPINH1_—52 and SERPINH1_—86 were selected as preferred compounds. From Table A-19 siRNA compounds SERPINH1_—4, SERPINH1_—12, SERPINH1_—18, SERPINH1_—30, SERPINH1_—58 and SERPINH1_—88 were selected as preferred compounds.

Other preferred compounds include SERPINH1_—50, SERPINH1_—67, SERPINH1_—73, SERPINH1_—74.

IC50 Values for the LTS Selected SERPINH1 siRNA Oligos

About 2×10⁵human PC-3 or 0.9×10⁵rat REF52 cells endogenously expressing SERPINH1 gene, were inoculated in 1.5 mL growth medium in order to reach 30-50% confluence. Cells were transfected with SERPINH1 double stranded RNA compounds (I.e. SERPINH1_—2, 4, 6, 12, 13, 18, 45, 51, 58, 88) with Lipofectamine™2000 reagent to reach final transfection concentrations ranging between 0.0029-100 nM. As negative control cells are treated with Lipofectamine™2000 reagent or with Synthetic randomized-sequence, non-targeting siRNA at final concentrations of 20-100 nM. Cy3-labeled siRNA transfected cells were used as positive control for transfection efficiency.

Cells were incubated at 37±1° C., 5% CO2 for 48 hours. siRNA transfected cells were harvested and RNA was isolated using EZ-RNA kit [Biological Industries (#20-410-100) Reverse transcription: Synthesis of cDNA is performed and human SERPINH1 mRNA levels were determined by Real Time qPCR and normalized to those of the Cyclophilin A (CYNA, PPIA) mRNA for each sample.

The IC₅₀value of the tested RNAi activity was determined by constructing a dose-response curve using the activity results obtained with the various final siRNA concentrations. The dose response curve was constructed by plotting the relative amount of residual SERPINH1 mRNA versus the logarithm of transfected siRNA concentration. The curve is calculated by fitting the best sigmoid curve to the measured data. The method for the sigmoid fit is also known as a 3-point curve fit.

$Y = Bot + \frac{100 - Bot}{1 + 10^{(Log IC 50 - X) \times HillSlope}}$

where Y is the residual SERPINH1 mRNA response, X is the logarithm of transfected siRNA concentration, Bot is the Y value at the bottom plateau, LogIC50 is the X value when Y is halfway between bottom and top plateaus and HillSlope is the steepness of the curve.

The percent of inhibition of gene expression using specific siRNAs was determined using qPCR analysis of target gene in cells expressing the endogenous gene. Other siRNA compounds according to Tables A-18, A-19 and B-E are tested in vitro where it is shown that these siRNA compounds inhibit gene expression. Activity is shown as percent residual mRNA; accordingly, a lower value reflects better activity.

In order to test the stability of the siRNA compounds in serum, specific siRNA molecules were incubated in four different batches of human serum (100% concentration) at 37° C. for up to 24 hours. Samples are collected at 0.5, 1, 3, 6, 8, 10, 16 and 24 hours. The migration patterns as an indication of were determined at each collection time by polyacrylamide gel electrophoresis (PAGE).

Table 3 shows 1050 (or activity where 1050 not calculated) in human cell line for unmodified double stranded nucleic acid compounds (sense and antisense strand unmodified, dTdT 3′ terminal overhangs) selected from Tables A-18 and A-19.

TABLE 3

AntiSense
struc-

0.1
0.5

siRNA
1^stposition
ture
IC50
nM
nM
5 nM

SERPINH1_6_S709
U
A2
0.019

SERPINH1_12_S709
A
A1
0.065

SERPINH1_23_S709
U
A2
0.377

SERPINH1_54_S709
A
A1
0.522

SERPINH1_37_S709
U
A2
0.11

SERPINH1_73_S709
A
A1
0.189

SERPINH1_24_S709
U
A2
0.271

SERPINH1_55_S709
A
A1
0.268

SERPINH1_60_S709
U
A2
0.163

SERPINH1_88_S709
A
A1
0.135

SERPINH1_11_S709
U
A2
0.079

SERPINH1_30_S709
A
A1
0.093

SERPINH1_25_S709
U
A2
0.229

SERPINH1_56_S709
A
A1
0.469

SERPINH1_5_S709
U
A2
0.178

SERPINH1_81_S709
G
A1
1.404

SERPINH1_52_S709
U
A2
0.06

SERPINH1_58_S709
A
A1
0.304

SERPINH1_2_S709
U
A2
0.008

SERPINH1_4_S709
A
A1
0.006

SERPINH1_43_S709
U
A2
1.403

SERPINH1_67_S709
A
A1
2.39

SERPINH1_16_S709
U
A2

134
95
16

SERPINH1_46_S709
A
A1

112
84
28

SERPINH1_8_S709
U
A2

103
90
39

SERPINH1_85_S709
C
A1

166
109
59

SERPINH1_45_S709
U
A2
0.029

SERPINH1_45a_S1354
A
A2
0.051

siRNA Knock Down Activity:

About 2×10⁵human PC-3 cells endogenously expressing SERPINH1 gene were seeded per well in 6 well plates and allowed to grow for about 24 hr to 30-70% confluency. Cells were transfected with the siRNAs being tested at different concentrations using the Lipofectamine™2000 reagent (Invitrogen). The cells were incubated at 37° C. in a 5% CO₂incubator for either 48 h or 72 h. At 48-72 h after transfection cells were harvested and cell RNA was extracted. Cy3-labeled siRNA duplexes were used as a positive control for transfection efficiency. Mock cells treated with Lipofectamine™2000 reagent defined as “Control not active samples” (negative control) and cells treated with a known active siRNA (HSP47-C) at final concentration of 5 nM defined as “Control active samples” (positive control). Z′ and controls fold {Fold=mean (Negative)/mean (Positive)} are the means to describe the assay efficiency.

The percent inhibition of target gene expression by each siRNA tested was determined by Qpcr analysis of a target mRNA from cells. Reverse transcription was performed by synthesizing cDNA from the cells and determining target gene mRNA levels by Real Time qPCR. Measured cell mRNA levels were normalized to those of the Cyclophilin A (CYNA, PPIA) mRNA for each sample. siRNA activity was determined based on the ratio of the target gene mRNA quantity siRNA-treated samples versus non-transfected control samples. Z′ and controls fold {Fold=mean (Negative)/mean(Positive)} are the means to describe the assay efficiency.

The qPCR results are those that passed QC standards, i.e. the value of the standard curve slope was in the interval [−4, −3], R2>0.99, no primer dimers. Results that did not pass the QC requirements were disqualified from analysis.

IC50 value of the tested RNAi activity was determined by constructing a dose-response curve using the activity results obtained with the various final siRNA concentrations. The dose response curve was constructed by plotting the relative amount of residual SERPINH1 mRNA versus the logarithm of transfected siRNA concentration, as described above.

On-Target and Off-Target Testing of Double Stranded RNA Molecules:

The psiCHECK system enables evaluation of the guide strand (GS) (antisense) and the passenger strand (PS) (sense strand) to elicit targeted (on-target) and off-targeted effects, by monitoring the changes in expression levels of their target sequences. Four psiCHECK™-2-based (Promega) constructs were prepared for the evaluation of target activity and potential off-target activity of each test molecule GS and PS strands. In each of the constructs one copy or three copies of either the full target or the seed-target sequence, of test molecule PS or GS, was cloned into the multiple cloning site located downstream of the Renilla luciferase translational stop codon in the 3′-UTR region.

The resulting vectors were termed:

1—GS-CM (guide strand, complete-match) vector containing one copy of the full target sequence (nucleotide sequence fully complementary to the whole 19-base sequence of the GS of the test molecule);

2—PS-CM (passenger strand, complete-match) vector containing one copy of the full target sequence (nucleotide sequence fully complementary to the whole 19-base sequence of the PS of the test molecule);

3—GS-SM (guide strand, seed-match) vector containing one copy or three copies of the seed region target sequence (sequence complementary to nucleotides 1-8 of the GS of the test molecule);

4—PS-SM (passenger strand, seed-match) vector containing one copy of the seed region target sequence (sequence complementary to nucleotides 1-8 of the PS of the test molecule).

Nomenclature:

guide strand: strand of siRNA that enters the RISC complex and guides cleavage/silencing of the complementary RNA sequence

seed sequence: Nucleotides 2-8 from the 5′ end of the guide strand.

cm (complete match): DNA fragment fully complementary to the guide strand of siRNA. This DNA fragment is cloned in 3′UTR of a reporter gene and serves as a target for the straightforward RNA silencing.

sm (seed match): 19-mer DNA fragment with nucleotides ns 12-18 fully complementary to the ns 2-8 of the guide strand of siRNA. This DNA fragment is cloned in 3′UTR of a reporter gene and serves as a target for the “off-target” silencing.

X1: A single copy of cm or sm cloned in 3′UTR of a reporter gene.

X3 Three copies of cm or sm cloned in 3′UTR of a reporter gene, separated with 4 nucleotides one from another.

TABLE 4

non-limiting examples of psiCHECK cloning targets

Nomenclature
Description
structure

S2a_cm_X1
SERPINH1_2, antisense

CTCGAGGAGACACATGGGTGCTATAG

clone name
complete match = fully

CGGCCGC SEQ_ID_NO: 2724

complimentary to the
XhoI SERPINH1_2 sense strand NotI

SERPINH1_2 antisense strand,

a single copy.

S2acmS_X1
The sense strand = the strand of
5′-

the S2a_cm_X1 clone to be
TCGAGGAGACACATGGGTGCTATAGC

expressed in the vector, with
SEQ_ID_NO: 2725

XhoI and NotI sticky ends.

S2acmA_X1
The complimentary (antisense)
5′-

strand of the S2a_cm_X1
GGCCGCTATAGCACCCATGTGTCTCC

clone, with XhoI and NotI
SEQ_ID_NO: 2726

sticky ends.

S2a_sm_X1
SERPINH1_2, antisense seed

CTCGAGTCTCAAACGTTGTGCTATCG

clone name
match, a single copy,

CGGCCGC SEQ_ID_NO: 2727

nucleotides 12-18 are
AS(3′-CTCTGTGTACCCACGATAT)

complimentary to the
SEQ_ID_NO: 2728

nucleotides 2-8 of the
seed

SERPINH1_2 antisense strand.

S2s_cm_X1
SERPINH1_2, sense complete

CTCGAGTATAGCACCCATGTGTCTCG

clone name
match = fully complimentary to

CGGCCGC SEQ_ID_NO: 2729

the SERPINH1_2 sense strand ≡
XhoI SERPINH1_2 antisense strand NotI

antisense strand, a single

copy.

S2s_sm_X1
SERPINH1_2, sense seed

CTCGAGGCGATACAAACTGTGTCTAG

clone name
match, a single copy,

CGGCCGC SEQ_ID_NO: 2730

nucleotides 12-18 are
S(3′-ATATCGTGGGTACACAGAG)

complimentary to the
SEQ_ID_NO: 2731

nucleotides 2-8 of the
seed

SERPINH1_2 sense strand.

S2a_sm_X3
SERPINH1_2, antisense seed

CTCGAGTCTCAAACGTTGTGCTATCttc

clone name
match, a triple copy
cTCTCAAACGTTGTGCTATCttccTCTCA

AACGTTGTGCTATCGCGGCCGC

SEQ_ID_NO: 2732

(ttcc - a spacer)

Sts_sm_X3
SERPINH1_2, sense seed

CTCGAGGCGATACAAACTGTGTCTAtt

clone name
match, a triple copy
ccGCGATACAAACTGTGTCTAttccGCG

ATACAAACTGTGTCTAGCGGCCGC

SEQ_ID_NO: 2733

(ttcc - a spacer)

The target sequences are cloned using the XhoI and NotI compatible restriction enzyme sites. Annealing mixtures are prepared in tightly closed 0.5 ml Eppendorf tubes, heated in a water bath to 850 C, submerged into the boiling water bath and finally gradually cooled to room temperature.

Ligation: The double stranded oligonucleotide generated by the annealing procedure is ligated to the linearized (by XhoI and NotI) psiCHECK™-2, and transfected into cells using standard techniques. Positive colonies were identified and sequenced for verification of insert sequence. Table 5 shows nucleotide sequences of inserted oligonucleotides.

TABLE 5

Clone Full
SEQ ID

siRNA
name
NO:
Oligonucleotide sequence (5′>3′)

SERPINH1_11
S11s_cm_X1
2734
GGCCGCCGGACAGGCCTCTACAACAC

2735
TCGAGTGTTGTAGAGGCCTGTCCGGC

S11a_cm_X1
2736
GGCCGCTGTTGTAGAGGCCTGTCCGC

2737
TCGAGCGGACAGGCCTCTACAACAGC

S11s_sm_X1
2738
GGCCGCAGGACAGGAAGAGCACCACC

2739
TCGAGGTGGTGCTCTTCCTGTCCTGC

S11a_sm_X1
2740
GGCCGCGGTTGTAGCTTAAGGGAATC

2741
TCGAGATTCCCTTAAGCTACAACCGC

S11s_sm_X3
2742
GGCCGCAGGACAGGAAGAGCACCACGGAAAGGACAG

GAAGAGCACCACGGAAAGGACAGGAAGAGCACCACC

2743
TCGAGGTGGTGCTCTTCCTGTCCTTTCCGTGGTGCTCT

TCCTGTCCTTTCCGTGGTGCTCTTCCTGTCCTGC

S11a_sm_X3
2744
GGCCGCGGTTGTAGCTTAAGGGAATGGAAGGTTGTAG

CTTAAGGGAATGGAAGGTTGTAGCTTAAGGGAATC

2745
TCGAGATTCCCTTAAGCTACAACCTTCCATTCCCTTAA

GCTACAACCTTCCATTCCCTTAAGCTACAACCGC

SERPINH1_30
S30s_cm_X1
2746
GGCCGCCGGACAGGCCTCTACAACTC

2747
TCGAGAGTTGTAGAGGCCTGTCCGGC

S30a_cm_X1
2748
GGCCGCAGTTGTAGAGGCCTGTCCGC

2749
TCGAGCGGACAGGCCTCTACAACTGC

SERPINH1_2
S2s_cm_X1
2750
GGCCGCGAGACACATGGGTGCTATAC

2751
TCGAGTATAGCACCCATGTGTCTCGC

S2a_cm_X1
2752
GGCCGCTATAGCACCCATGTGTCTCC

2753
TCGAGGAGACACATGGGTGCTATAGC

S2s_sm_X1
2754
GGCCGCTAGACACAGTTTGTATCGCC

2755
TCGAGGCGATACAAACTGTGTCTAGC

S2a_sm_X1
2756
GGCCGCGATAGCACAACGTTTGAGAC

2757
TCGAGTCTCAAACGTTGTGCTATCGC

S2s_sm_X3
2758
GGCCGCTAGACACAGTTTGTATCGCGGAATAGACACA

GTTTGTATCGCGGAATAGACACAGTTTGTATCGCC

2759
TCGAGGCGATACAAACTGTGTCTATTCCGCGATACAA

ACTGTGTCTATTCCGCGATACAAACTGTGTCTAGC

S2a_sm_X3
2760
GGCCGCGATAGCACAACGTTTGAGAGGAAGATAGCA

CAACGTTTGAGAGGAAGATAGCACAACGTTTGAGAC

2761
TCGAGTCTCAAACGTTGTGCTATCTTCCTCTCAAACGT

TGTGCTATCTTCCTCTCAAACGTTGTGCTATCGC

SERPINH1_4
S4s_cm_X1
2762
GGCCGCGAGACACATGGGTGCTATTC

2763
TCGAGAATAGCACCCATGTGTCTCGC

S4a_cm_X1
2764
GGCCGCAATAGCACCCATGTGTCTCC

2765
TCGAGGAGACACATGGGTGCTATTGC

SERPINH1_6
S6s_cm X1
2766
GGCCGCACAAGATGCGAGACGAGTAC

2767
TCGAGTACTCGTCTCGCATCTTGTGC

S6a_cm_X1
2768
GGCCGCTACTCGTCTCGCATCTTGTC

2769
TCGAGACAAGATGCGAGACGAGTAGC

S6s_sm_X1
2770
GGCCGCCCAAGATGATCTAATCTGCC

2771
TCGAGGCAGATTAGATCATCTTGGGC

S6a_sm_X1
2772
GGCCGCGACTCGTCGATACTAGGTGC

2773
TCGAGCACCTAGTATCGACGAGTCGC

S6s_sm_X3
2774
TCGAGGCAGATTAGATCATCTTGGTTCCGCAGATTAG

ATCATCTTGGTTCCGCAGATTAGATCATCTTGGGC

2775
GGCCGCCCAAGATGATCTAATCTGCGGAACCAAGATG

ATCTAATCTGCGGAACCAAGATGATCTAATCTGCC

S6a_sm_X3
2776
GGCCGCGACTCGTCGATACTAGGTGGGAAGACTCGTC

GATACTAGGTGGGAAGACTCGTCGATACTAGGTGC

2777
TCGAGCACCTAGTATCGACGAGTCTTCCCACCTAGTAT

CGACGAGTCTTCCCACCTAGTATCGACGAGTCGC

SERPINH1_12
S12s_cm_X1
2778
GGCCGCACAAGATGCGAGACGAGTTC

2779
TCGAGAACTCGTCTCGCATCTTGTGC

S12a_cm_X1
2780
GGCCGCAACTCGTCTCGCATCTTGTC

2781
TCGAGACAAGATGCGAGACGAGTTGC

SERPINH1_45a
S450s_cmX1
2782
GGCCGCACTCCAAGATCAACTTCCTC

2783
TCGAGAGGAAGTTGATCTTGGAGTGC

(SERPINH1_45_S450)
S450a_cmX1
2784
GGCCGCAGGAAGTTGATCTTGGAGTC

2785
TCGAGACTCCAAGATCAACTTCCTGC

S450s_smX1
2786
GGCCGCCCTCCAAGCGACCATGAAGC

2787
TCGAGCTTCATGGTCGCTTGGAGGGC

S450a_smX1
2788
GGCCGCCGGAAGTTTCGATGTTCTGC

2789
TCGAGCAGAACATCGAAACTTCCGGC

S450s_smX3
2790
GGCCGCCCTCCAAGCGACCATGAAGGGAACCTCCAAG

CGACCATGAAGGGAACCTCCAAGCGACCATGAAGC

2791
TCGAGCTTCATGGTCGCTTGGAGGTTCCCTTCATGGTC

GCTTGGAGGTTCCCTTCATGGTCGCTTGGAGGGC

S450a_smX3
2792
GGCCGCCGGAAGTTTCGATGTTCTGGGAACGGAAGTT

TCGATGTTCTGGGAACGGAAGTTTCGATGTTCTGC

2793
TCGAGCAGAACATCGAAACTTCCGTTCCCAGAACATC

GAAACTTCCGTTCCCAGAACATCGAAACTTCCGGC

SERPINH1_51
S51s_cm_X1
2794
GGCCGCTCCTGAGACACATGGGTGAC

2795
TCGAGTCACCCATGTGTCTCAGGAGC

S51a_cm_X1
2796
GGCCGCTCACCCATGTGTCTCAGGAC

2797
TCGAGTCCTGAGACACATGGGTGAGC

S51s_sm_X1
2798
GGCCGCGCCTGAGAACACGTGTGTCC

2799
TCGAGGACACACGTGTTCTCAGGCGC

S51a_sm_X1
2800
GGCCGCGCACCCATTGTGATACTTCC

2801
TCGAGGAAGTATCACAATGGGTGCGC

S51s_sm_X3
2802
GGCCGCGCCTGAGAACACGTGTGTCGGAAGCCTGAGA

ACACGTGTGTCGGAAGCCTGAGAACACGTGTGTCC

2803
TCGAGGACACACGTGTTCTCAGGCTTCCGACACACGT

GTTCTCAGGCTTCCGACACACGTGTTCTCAGGCGC

S51a_sm_X3
2804
GGCCGCGCACCCATTGTGATACTTCGGAAGCACCCAT

TGTGATACTTCGGAAGCACCCATTGTGATACTTCC

2805
TCGAGGAAGTATCACAATGGGTGCTTCCGAAGTATCA

CAATGGGTGCTTCCGAAGTATCACAATGGGTGCGC

SERPINH1_86
S86s_cm_X1
2806
GGCCGCACAGGCCTCTACAACTACAC

2807
TCGAGTGTAGTTGTAGAGGCCTGTGC

S86a_cm_X1
2808
GGCCGCTGTAGTTGTAGAGGCCTGTC

2809
TCGAGACAGGCCTCTACAACTACAGC

S86s_sm_X1
2810
GGCCGCACAGGCCTAGCACAAGCACC

2811
TCGAGGTGCTTGTGCTAGGCCTGTGC

S86a_sm_X1
2812
GGCCGCGGTAGTTGGCTCTGAAGTGC

2813
TCGAGCACTTCAGAGCCAACTACCGC

S86s_sm_X3
2814
GGCCGCACAGGCCTAGCACAAGCACGGAAACAGGCC

TAGCACAAGCACGGAAACAGGCCTAGCACAAGCACC

2815
TCGAGGTGCTTGTGCTAGGCCTGTTTCCGTGCTTGTGC

TAGGCCTGTTTCCGTGCTTGTGCTAGGCCTGTGC

S86a_sm_X3
2816
GGCCGCGGTAGTTGGCTCTGAAGTGGGAAGGTAGTTG

GCTCTGAAGTGGGAAGGTAGTTGGCTCTGAAGTGC

2817
TCGAGCACTTCAGAGCCAACTACCTTCCCACTTCAGA

GCCAACTACCTTCCCACTTCAGAGCCAACTACCGC

SERPINH1_52
S52s_cm_X1
2818
GGCCGCGACAAGATGCGAGACGAGAC

2819
TCGAGTCTCGTCTCGCATCTTGTCGC

S52a_cm_X1
2820
GGCCGCTCTCGTCTCGCATCTTGTCC

2821
TCGAGGACAAGATGCGAGACGAGAGC

S52s_sm_X1
2822
GGCCGCTACAAGATTATCTCATCTCC

2823
TCGAGGAGATGAGATAATCTTGTAGC

S52a_sm_X1
2824
GGCCGCGCTCGTCTATACTAGGTGAC

2825
TCGAGTCACCTAGTATAGACGAGCGC

S52s_sm_X3
2826
GGCCGCTACAAGATTATCTCATCTCGGAATACAAGAT

TATCTCATCTCGGAATACAAGATTATCTCATCTCC

2827
TCGAGGAGATGAGATAATCTTGTATTCCGAGATGAGA

TAATCTTGTATTCCGAGATGAGATAATCTTGTAGC

S52a_sm_X3
2828
GGCCGCGCTCGTCTATACTAGGTGAGGAAGCTCGTCT

ATACTAGGTGAGGAAGCTCGTCTATACTAGGTGAC

2829
TCGAGTCACCTAGTATAGACGAGCTTCCTCACCTAGT

ATAGACGAGCTTCCTCACCTAGTATAGACGAGCGC

SERPINH1_58
S58s_cm_X1
2830
GGCCGCGACAAGATGCGAGACGAGTC

2831
TCGAGACTCGTCTCGCATCTTGTCGC

S58a_cm_X1
2832
GGCCGCACTCGTCTCGCATCTTGTCC

2833
TCGAGGACAAGATGCGAGACGAGTGC

SERPINH1_95
S95s_cm_X1
2834
GGCCGCACTCCAAGATCAACTTCCGC

2835
TCGAGCGGAAGTTGATCTTGGAGTGC

S95a_cm_X1
2836
GGCCGCCGGAAGTTGATCTTGGAGTC

2837
TCGAGACTCCAAGATCAACTTCCGGC

SERPINH1_96
S96s_cm_X1
2838
GGCCGCTCCTGAGACACATGGGTGCC

2839
TCGAGGCACCCATGTGTCTCAGGAGC

S96a_cm_X1
2840
GGCCGCGCACCCATGTGTCTCAGGAC

2841
TCGAGTCCTGAGACACATGGGTGCGC

SERPINH1_97
S97s_cm_X1
2842
GGCCGCACAGGCCTCTACAACTACTC

2843
TCGAGAGTAGTTGTAGAGGCCTGTGC

S97a_cm_X1
2844
GGCCGCAGTAGTTGTAGAGGCCTGTC

2845
TCGAGACAGGCCTCTACAACTACTGC

Relevant strands, as described above, were cloned in the 3′UTR of the reporter mRNA, Renilla Luciferase in the psiCHECK™-2 (Promega) vector. XhoI and NotI were used as cloning sites using standard molecular biology techniques. Each strand was chemically synthesized and annealed by heating to 100° C. and cooled to room temperature. Ligation was carried out for 3 hours using standard molecular biology techniques and transformed into E. coli DH5a cells. Resulting colonies were screened for presence of plasmid constructs by colony-PCR using relevant primers. Each of the plasmids (vectors) was purified from one positive colony and its sequence was verified.

About 1.3×10⁶human HeLa cells were inoculated in 10 cm dish. Cells were then incubated in 37±1° C., 5% CO2 incubator for 24 hours. Growth medium was replaced one day post inoculation by 8 mL fresh growth medium and each plate was transfected with one of the plasmids mentioned above, using Lipofectamine™2000 reagent according manufacturing protocol and incubated for 5 hours at 37±1° C. and 5% CO2. Following incubation, cells were re-plated in a 96-well plate at final concentration of 5×103 cells per well in 80 μL growth medium. 16 hours later, cells were transfected with SERPINH1 siRNA molecules using Lipofectamine™2000 reagent at different concentrations ranging from 0.001 nM to 5 nM in a 1000 μL final volume. Mock cells treated with Lipofectamine™2000 reagent with the corresponding psiCHECK™-2 plasmid defined as “Control not active samples” (negative control) and cells treated with a known active siRNA (HSP47-C) at final concentration of 5 nM defined as “Control active samples” (positive control). Z′ and controls fold {Fold=mean (Negative)/mean(Positive)} are the means to describe the assay efficiency.

Cells were then incubated for 48 hours at 37±1° C. and Renilla and FireFly Luciferase activities were measured in each of the siRNA transfected samples, using Dual-Luciferase® Assay kit (Promega, Cat#E1960) according to manufacturer procedure. The activity of a synthetic siRNA toward this target sequence results either in cleavage and subsequent degradation of the fused mRNA or in translation inhibition of the encoded protein. Measuring the decrease in Renilla luciferase activity thus provides a convenient way of monitoring siRNA effect while Firefly luciferase, allows normalization of Renilla luciferase expression. Renilla Luciferase activity value was divided by Firefly Luciferase activity value for each sample (normalization). Renilla luciferase activity is finally expressed as the percentage of the normalized activity value in tested sample relative to “Control not active samples”.

Results of TNFα and IL-6 levels in peripheral blood mononuclear cells (PMNC) exposed to unmodified or modified siRNA/Lipofectamine™2000. Results are provided in pg/ml values calculated based on standard curve. “Control Lipofec2000” relates to level of cytokine secretion induced by the transfection reagent, Lipofectamine™2000. None of the modified compounds levels of cytokines TNFa or IL6 above those of the control transfection reagent.

Donor II

TNFa
IL-6

control

162 +/− 280

Control Lipofec2000

308 +/− 75
1303 +/− 440

dsRNA SEQ ID
860 nM
610
2915

NOS: 101 and 168)
287 nM
6963
4021

unmodified
96 nM
641
2278

32 nM
1095
4126

Compound_4
860 nM
660 +/− 227
1166 +/− 280

287 nM
484 +/− 84
1844 +/− 1072

96 nM
571 +/− 170
2015 +/− 1667

32 nM
865 +/− 90
2201 +/− 952

Donor I
Donor II

TNFa
IL-6
TNFa
IL-6

control

115 +/− 64
162 +/− 280

control Lipofec2000

427 +/− 87
1848 +/− 194
308 +/− 75
1303 +/− 440

dsRNA SEQ ID
860 nM
326
1014
873
4015

NOS: 60 and 127)
287 nM
305
638
909
3046

unmodified
96 nM
546
1007
690
2451

32 nM
707
1331
637
2159

Compound_1
860 nM
491
1480
1017
4492

287 nM
363
956
981
3126

96 nM
294
840
952
2491

32 nM
355
848
902
2779

Donor I

TNFa
IL-6

control

115 +/− 64

control Lipofec2000

427 +/− 87
1848 +/− 194

dsRNA SEQ ID
860 nM
228
553

NOS: 63 and 130)
287 nM
395
569

unmodified
96 nM
561
966

32 nM
737
1021

Compound_2
860 nM
598
1560

287 nM
621
1440

96 nM
570
1825

32 nM
517
1510

Donor I
Donor II

TNFa
IL-6
TNFa
IL-6

control

115 +/− 64
162 +/− 280

control Lipofec2000

427 +/− 87
1848 +/− 194
308 +/− 75
1303 +/− 440

dsRNA SEQ ID
860 nM
137
225
521
4223

NOS: 98 and 165)
287 nM
750
105
463
3755

unmodified
96 nM
504
180
627
2784

32 nM
312
442
711
3084

Compound_3
860 nM
540
2170
1474
3896

287 nM
698
2428
1000
1864

96 nM
582
1876
1089
1760

32 nM
614
1341
724
1044

Ctrl cells

115 +/− 64
162 +/− 280

CL075 (ug/ml)
2
13878

26464

0.67
8115
28471
17013

0.22
1575
10873
7589
22111

0.074
219
906
1389
7072

Results are pg/ml

Data for induction of interferon (IFN) responsive genes, MX1 and IFIT1, unmodified and modified double stranded nucleic acid compounds. Results shown are residual (fold of Ctrl Lipofectamine-2000 treated cells) human IFIT1 and MX1 genes as tested in human PMNC. Data shows that all modified compounds induced negligible levels of IFN downstream genes, compared to unmodified (_S709) compounds.

Donor II

IFIT1
MX1

Ctrl Lipo2000
1
1

dsRNA SEQ ID NOS: 101 and 168)

unmodified

32 nM
5.5
3.3

96 nM
7.5
4.3

297 nM
3.9
3.8

860 nM
0.8
0.8

Compound_4

32 nM
1.2 +/− 0.5
1.7 +/− 0.35

96 nM
1.1 +/− 0.3
1.5 +/− 0.06

297 nM
0.7 +/− 0.3
0.9 +/− 0.7

860 nM
0.6 +/− 0.1
0.9 +/− 0.5

Donor I
Donor II

IFIT1
MX1
IFIT1
MX1

Ctrl Lipo2000
1
1
1
1

dsRNA SEQ ID NOS: 60 and 127)

unmodified

32 nM
27.9
—
2.2
2.7

96 nM
42.1
18.3
4.0
5.0

297 nM
53.8
18.0
3.4
2.8

860 nM
39.4
16.3
3.3
3.6

Compound_1

32 nM
1.2
0.2
0.8
1.3

96 nM
1.3
0.8
1.5
1.1

297 nM
1.1
0.3
1.2
1.6

860 nM
1.0
0.3
0.3
0.3

Ctrl Lipo2000
1
1.00
1
1

dsRNA SEQ ID NOS: 98 and 165)

unmodified

32 nM
29.7
18.5
4.3
4.1

96 nM
39.1
19.2
3.5

297 nM
25.1
9.3
4.8
5.2

860 nM

3.8
3.7

Compound_3

32 nM
1.4
0.4
1.0
1.4

96 nM
1.7
1.3
1.3
1.1

297 nM
1.9
1.4
1.1
1.4

860 nM
5.2
2.5
1.1
1.4

Donor I

IFIT1
MX1

Ctrl Lipo2000
1
1

dsRNA SEQ ID NOS: 63 and 130)

unmodified

32 nM
29.6
17.8

96 nM
31.5
16.1

297 nM

860 nM
36.6
11.4

Compound_2

32 nM
1.6
0.7

96 nM
1.1
1.0

297 nM
2.1
0.2

860 nM
1.8
1.4

Donor I

Donor II

IFIT1
MX1
IFIT1
MX1

Ctrl cells
1
1
1
1

0.125
18

5.4
3.7

0.56
26
11
4.9
4.7

1.7
41
14
4.5
5.1

5
24
7
0.9
0.8

0.075
4
2
1.8
1.8

0.12
27
10
4.5
3.6

0.67
21

4.6
4.2

2
26
12
4.1
3.7

The tables below show activity of Compound_—1, Compound_—2, Compound 3 and Compound_—4 compared to unmodified (_S709) compounds in rat cells. Results are shown as residual target (% of control Lipofectamine™2000 treated cells) rat SERPINH1 gene in REF52 cells. Results of two separate experiments are shown. Knockdown to target gene in rat cells is relevant to testing compounds in animal models of human disease.

Study_1
Study_2

Ctrl Lipo2000
100
100

dsRNA SEQ ID NOS: 60
0.8 nM
52
36

and 127) unmodified

2 nM
25
31

10 nM
16
28

50 nM
8
4

Compound_1
0.8 nM
53
14

2 nM
39
14

10 nM
19
24

50 nM
7
4

dsRNA SEQ ID NOS: 63
0.8 nM
45
15

and 130) unmodified

2 nM
28
18

10 nM
13
12

50 nM
12
8

Compound_2
0.8 nM
76
78

2 nM
61
68

10 nM
37
28

50 nM

4

dsRNA SEQ ID NOS: 98
0.8 nM
72
65

and 165) unmodified

2 nM
43
41

10 nM
32
42

50 nM
28
27

Compound_3
0.8 nM
88
30

2 nM
39
24

10 nM
24
23

50 nM
6
23

Study_3
Study_4

Ctrl Lipo2000
100
100

Compound_4
0.8 nM
66
106

2 nM
35
32

10 nM
10
12

50 nM
6
9

Serum Stability Assay

The modified compounds according to the present invention are tested for duplex stability in human serum or human tissue extract, as follows:

siRNA molecules at final concentration of 7 uM are incubated at 370 C in 100% human serum (Sigma Cat# H4522). (siRNA stock 100 uM diluted in human serum 1:14.29 or human tissue extract from various tissue types). Five ul (5 ul) are added to 15 ul 1.5×TBE-loading buffer at different time points (for example 0, 30 min, 1 h, 3 h, 6 h, 8 h, 10 h, 16 h and 24 h). Samples were immediately frozen in liquid nitrogen and kept at −20° C.

Each sample is loaded onto a non-denaturing 20% acrylamide gel, prepared according to methods known in the art. The oligos were visualized with ethidium bromide under UV light.

Exonuclease Stability Assay

To study the stabilization effect of 3′ non-nucleotide moieties on a nucleic acid molecule the sense strand, the antisense strand and the annealed siRNA duplex are incubated in cytosolic extracts prepared from different cell types.

Extract: HCT116 cytosolic extract (12 mg/ml).

Extract buffer: 25 mM Hepes pH-7.3 at 37° C.; 8 mM MgCl; 150 mM NaCl with 1 mM DTT was added fresh immediately before use.

Method: 3.5 ml of test siRNA (100 mM), were mixed with 46.5 ml contain 120 mg of HCT116 cytosolic extract. The 46.5 ml consists of 12 ml of HCT116 extract, and 34.5 ml of the extract buffer supplemented with DTT and protease inhibitors cocktail/100 (Calbiochem, setIII-539134). The final concentration of the siRNA in the incubation tube is 7 mM. The sample was incubated at 37° C., and at the indicated time point 5 ml were moved to fresh tube, mixed with 15 ml of 1×TBE-50% Glycerol loading buffer, and snap frozen in Liquid N2. The final concentration of the siRNA in the loading buffer is 1.75 mM (21 ng siRNA/ml). For Analyses by native PAGE and EtBr staining 50 ng are loaded per lane. For Northern analyses 1 ng of tested siRNA was loaded per lane.

Innate Immune Response to SERPINH1 siRNA Molecules:

Fresh human blood (at RT) was mixed at 1:1 ratio with sterile 0.9% NaCl at RT, and gently loaded (1:2 ratio) on Ficoll (Lymphoprep, Axis-Shield cat#1114547). Samples were centrifuged at RT (22° C., 800 g) in a swinging centrifuge for 30 minutes, washed with RPMI1640 medium and centrifuged (RT, 250 g) for 10 minutes. Cells were counted and seeded at final concentration of 1.5×10⁶cell/ml in growth medium (RPMI1640+10% FBS+2 mM L-glutamine+1% Pen-Strep) and incubated for 1 hours at 37° C. before siRNA treatment.

Cells were then treated with the siRNAs being tested at different concentrations using the Lipofectamine™2000 reagent (Invitrogen) according manufacturer's instructions and incubated at 37° C. in a 5% CO2 incubator for 24 hours.

As a positive control for IFN response, cells were treated with either poly(I:C), a synthetic analog of double strand RNA (dsRNA) which is a TLR3 ligand (InvivoGen Cat# tlr1-pic) at final concentrations of 0.25-5.0 μg/mL or to Thiazolaquinolone (CLO75), a TLR 7/8 ligand (InvivoGen Cat# tlr1-c75) at final concentrations of 0.075-2 μg/mL. Cell treated with Lipofectamine™2000 reagent were used as negative (reference) control for IFN response.

At about 24 hours following incubation, cells were collected and supernatant was transferred to new tubes. Samples were frozen immediately in liquid nitrogen and secretion of IL-6 and TNF-α cytokines was tested using IL-6, DuoSet ELISA kit (R&D System DY2060), and TNF-α, DuoSet ELISA kit (R&D System DY210), according to manufacturer's instructions. RNA was extracted from the cell pellets and mRNA levels of human genes IFIT1 (interferon-induced protein with tetratricopeptide repeats 1) and MX1 (myxovirus (influenza virus) resistance 1, interferon-inducible protein p78) were measured by qPCR. Measured mRNA quantities were normalized to the mRNA quantity of the reference gene peptidylprolyl isomerase A (cyclophilin A; CycloA). Induction of IFN-signaling was evaluated by comparing the quantity of mRNA from IFIT1 and MX1 genes from treated cells, relative to their quantities non-treated cells. The qPCR results are those that passed QC standards, i.e. the value of the standard curve slope was in the interval [−4, −3], R2>0.99, no primer dimers. Results that did not pass the QC requirements were disqualified from analysis.

Table 6 shows siSERPINH1 compounds. Activity and stability data for some of the compounds is presented in Table 6. The code for the sense and antisense strand structures is presented in Table 7, infra.

TABLE 6

%
%

residual
residual

1.
Stability in
5 nM
25 nM
Sense strand 5->3
AntiSense strand 5->3

Name
plasma (h)
001
002
003
004
006
code
code

SERPINH1_2_S1356
10
16
10
9

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; mA; rU; mG; rU; mG;

rU2p; rA2p; rU2p; rA2p
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1357

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; mC; rA; mU; rG; mU; rG;

rU2p; rA2p; rU2p; rA2p
mU; mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1358
16
52
41

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; rA; mU; rG; mU; rG;

rU2p; rA2p; rU2p; rA2p
mU; mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1359

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; rA; mU; rG; mU; rG; rU;

rU2p; rA2p; rU2p; rA2p
mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1360
10
47
31
8
20

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
rC; mC; rA; mU; rG; mU; rG; rU;

rU2p; rA2p; rU2p; rA2p
mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1361
8
31
34

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; rC2p; rA;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; mC; rC; rA; mU; rG; mU; rG;

rU2p; rA2p; rU2p; rA2p
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1362

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; LdC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; rA; mU; rG; mU; rG; rU;

rU2p; rA2p; rU2p; rA2p
mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1363
17
10
15
25

zc3p; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; mA; rU; mG; rU; mG;

rU2p; rA2p; rU2p; rA2p
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1364

zc3p; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; mC; rA; mU; rG; mU; rG;

rU2p; rA2p; rU2p; rA2p
mU; mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1365
16
41
52

zc3p; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; rA; mU; rG; mU; rG;

rU2p; rA2p; rU2p; rA2p
mU; mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1366

zc3p; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; rA; mU; rG; mU; rG; rU;

rU2p; rA2p; rU2p; rA2p
mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1367
16
51
39

zc3p; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
rC; mC; rA; mU; rG; mU; rG; rU;

rU2p; rA2p; rU2p; rA2p
mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1368

zc3p; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; rC2p; rA;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; mC; rC; rA; mU; rG; mU; rG;

rU2p; rA2p; rU2p; rA2p
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1369

zc3p; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; LdC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; rA; mU; rG; mU; rG; rU;

rU2p; rA2p; rU2p; rA2p
mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1370
17
15

61
20

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; mA; rU; mG; rU; mG;

rU2p; rA2p; rU2p; rA2p; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1371

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; mC; rA; mU; rG; mU; rG;

rU2p; rA2p; rU2p; rA2p; zc3p$
mU; mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1372
16
74
66

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; rA; mU; rG; mU; rG;

rU2p; rA2p; rU2p; rA2p; zc3p$
mU; mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1373
8
48
65

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; rA; mU; rG; mU; rG; rU;

rU2p; rA2p; rU2p; rA2p; zc3p$
mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1374
16
39
110
6

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
rC; mC; rA; mU; rG; mU; rG; rU;

rU2p; rA2p; rU2p; rA2p; zc3p$
mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1375

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; rC2p; rA;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; mC; rC; rA; mU; rG; mU; rG;

rU2p; rA2p; rU2p; rA2p; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1376

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; rG; LdC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; rA; mU; rG; mU; rG; rU;

rU2p; rA2p; rU2p; rA2p; zc3p$
mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1377
3

25
5

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; mA; rC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; mA; rU; mG; rU; mG;

mC; mU; rA; LdT; rA$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1378

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; mC; rA; mC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; mC; rA; mU; rG; mU; rG;

mC; mU; rA; LdT; rA$
mU; mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1379

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; mC; rA; mC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; rA; mU; rG; mU; rG;

mC; mU; rA; LdT; rA$
mU; mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1380
8
23
33

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; mC; rA; mC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; rA; mU; rG; mU; rG; rU;

mC; mU; rA; LdT; rA$
mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1381
16
25
56
12

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; mC; rA; mC;

mC; rA; rU; rG; rG; rG; mU; rG;
rC; mC; rA; mU; rG; mU; rG; rU;

mC; mU; rA; LdT; rA$
mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1382
8
22
31
11

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; rC2p; rA;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; mC; rC; rA; mU; rG; mU; rG;

mC; mU; rA; LdT; rA$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1383

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; LdC; rA; mC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; rA; mU; rG; mU; rG; rU;

mC; mU; rA; LdT; rA$
mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1384
16
7
20
7
4

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; mA; rC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; mU; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1385
16
55
37

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; mC; rA; mC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; mC; rA; mU; rG; mU; rG;

rC; mU; rA; mU; rA; zc3p$
mU; mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1386
16
42
45

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; mC; rA; mC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; rA; mU; rG; mU; rG;

rC; mU; rA; mU; rA; zc3p$
mU; mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1387

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; mC; rA; mC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; rA; mU; rG; mU; rG; rU;

rC; mU; rA; mU; rA; zc3p$
mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1388
16
21
39

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; mC; rA; mC;

mC; rA; rU; rG; rG; rG; mU; rG;
rC; mC; rA; mU; rG; mU; rG; rU;

rC; mU; rA; mU; rA; zc3p$
mC; mU; rC; zc3p; zc3p$

SERPINH1_2_S1389
16
20
27

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; rC2p; rA;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; mC; rC; rA; mU; rG; mU; rG;

rC; mU; rA; mU; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1390

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; rG; LdC; rA; mC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; rA; mU; rG; mU; rG; rU;

rC; mU; rA; mU; rA; zc3p$
mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1687

zidB; rG; rA; rG; rA; rC; rA; mC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU; rG; rG; rG; mU; rG; rC;
mC; rC; mA; rU; mG; rU; mG;

mU; rA; mU; rA; zc3p$
rU; mC; rU; rC; zc3p; zc3p$

SERPINH1_2_S1694
24

zidB; rG; rA; rG; rA; rC; rA; mC;
mU; rA; mU; rA; mG; rC; rA2p;

rA; rU2p; rG; rG; rG; mU; rG;
rC; mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; mU; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1700
16

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; rA2p;

mC; rA; rU; rG; rG; rG; mU; rG;
rC; mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; rU2p; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1705
10

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; mG; rC; rA2p;

rA; rU; rG; rG; rG; rU; rG; rC2p;
rC; mC; rC; mA; rU; mG; rU; mG;

rU2p; rA2p; rU2p; rA2p
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1707
10

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; mG; rC; rA2p;

rA; rU; rG; rG; rG; rU; rG; rC2p;
rC; mC; rC; mA; rU; mG; rU; mG;

rU2p; rA2p; rU2p; rA2p; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1754
24

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; mG; rC; rA2p;

rA; rU; rG; rG; rG; rU; rG; rC2p;
rC; mC; rC; mA; rU; mG; rU; mG;

rU2p; rA2p; rU2p; rA2p; zc3p
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1755
24

zidB; rG; rA; rG; rA; rC; rA; rC;
rU2p; rA; mU; rA; mG; rC; rA2p;

rA; rU; rG; rG; rG; rU; rG; rC2p;
rC; mC; rC; mA; rU; mG; rU;

rU2p; rA2p; rU2p; rA2p; zc3p
mG; rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1756

zidB; rG; rA; rG; rA; rC; rA; rC;
rU2p; rA; mU; rA; mG; rC; rA2p;

rA; rU; rG; rG; rG; rU; rG; rC2p;
rC; mC; rC; mA; rU; mG; rU;

rU2p; rA2p; rU2p; rA2p
mG; rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1787

zidB; rG; rA; rG; rA; rC; rA; rC;
dU; rA; mU; rA; mG; rC; rA2p; rC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; mA; rU; mG; rU; mG;

rU2p; rA2p; rU2p; rA2p; zc3p
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_4_S1391
0

58

zidB; rG; rA; rG; rA; rC; rA; rC;
rA; mA; rU; mA; rG; mC; rA; mC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
rC; rC; mA; rU; mG; rU; mG; rU;

rU2p; rA2p; rU2p; rU2p
mC; rU; mC; zc3p; zc3p$

SERPINH1_4_S1782

zidB; rG; rA; rG; rA; rC; rA; rC;
rA; rA; mU; rA; mG; rC; rA2p; rC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; mA; rU; mG; rU; mG;

rU2p; rA2p; rU2p; rU2p; zc3p
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_6_S1356

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; mU; rC;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mU; rC; mG; rC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1363

zc3p; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; mU; rC;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mU; rC; mG; rC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1370

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; mU; rC;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mU; rC; mG; rC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1414

zc3p; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; mU; mC; rG; mU;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mC; mU; mC; rG; mC; rA; mU;

rA2p; rG2p; rU2p; rA2p
mC; mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1415

zc3p; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; mU; mC; rG; mU;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mC; mU; rC; rG; mC; rA; mU; mC;

rA2p; rG2p; rU2p; rA2p
mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1416

zc3p; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; mU; mC; rG; mU;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mC; mU; rC; rG; mC; rA; rU; mC;

rA2p; rG2p; rU2p; rA2p
mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1417

zc3p; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; mU; mC;

rG; rC; rG; rA; rG; rA; rC; rG2p;
rU; mC; rG; mC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1418

zc3p; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; LdT; rC;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mU; rC; rG; mC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1419

zc3p; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; rC; rG2p;
rC; mU; rC; rG; mC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1420

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; mU; mC; rG; mU;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mC; mU; mC; rG; mC; rA; mU;

rA2p; rG2p; rU2p; rA2p
mC; mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1421

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; mU; mC; rG; mU;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mC; mU; rC; rG; mC; rA; mU; mC;

rA2p; rG2p; rU2p; rA2p
mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1422

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; mU; mC; rG; mU;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mC; mU; rC; rG; mC; rA; rU; mC;

rA2p; rG2p; rU2p; rA2p
mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1423

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; mU; mC;

rG; rC; rG; rA; rG; rA; rC; rG2p;
rU; mC; rG; mC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1424

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; LdT; rC;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mU; rC; rG; mC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1425

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; rC; rG2p;
rC; mU; rC; rG; mC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1426

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; mU; mC; rG; mU;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mC; mU; mC; rG; mC; rA; mU;

rA2p; rG2p; rU2p; rA2p; zc3p$
mC; mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1427

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; mU; mC; rG; mU;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mC; mU; rC; rG; mC; rA; mU; mC;

rA2p; rG2p; rU2p; rA2p; zc3p$
mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1428

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; mU; mC; rG; mU;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mC; mU; rC; rG; mC; rA; rU; mC;

rA2p; rG2p; rU2p; rA2p; zc3p$
mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1429

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; mU; mC;

rG; rC; rG; rA; rG; rA; rC; rG2p;
rU; mC; rG; mC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1430

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; LdT; rC;

rG; rC; rG; rA; rG; rA; rC; rG2p;
mU; rC; rG; mC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1431

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; rC; rG2p;
rC; mU; rC; rG; mC; mA; rU; mC;

rA2p; rG2p; rU2p; rA2p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1432
0
6
19
15

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; rC;

mU; rG; rC; rG; rA; rG; rA; mC;
mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; LdT; rA$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1435
6
37
46

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; mU; mC; rG; mU;

mU; rG; rC; rG; rA; rG; rA; mC;
mC; mU; rC; rG; mC; rA; rU; mC;

rG; rA; rG; LdT; rA$
mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1436
3
10
17
5

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; mC;

mU; rG; rC; rG; rA; rG; rA; mC;
rU; mC; rG; mC; mA; rU; mC;

rG; rA; rG; LdT; rA$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1437

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; LdT; rC;

mU; rG; rC; rG; rA; rG; rA; mC;
mU; rC; rG; mC; mA; rU; mC;

rG; rA; rG; LdT; rA$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1438
3
15
17

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; mC;

rG; rA; rG; LdT; rA$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1439
24
12
23
11

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; rC;

mU; rG; rC; rG; rA; rG; rA; mC;
mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; mU; rA; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1442
24
29

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; mU; mC; rG; mU;

mU; rG; rC; rG; rA; rG; rA; mC;
mC; mU; rC; rG; mC; rA; rU; mC;

rG; rA; rG; mU; rA; zc3p$
mU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1443
24
9
22
7

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; mC;

mU; rG; rC; rG; rA; rG; rA; mC;
rU; mC; rG; mC; mA; rU; mC;

rG; rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1444

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; LdT; rC;

mU; rG; rC; rG; rA; rG; rA; mC;
mU; rC; rG; mC; mA; rU; mC;

rG; rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1445
24

19
18

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; mC;

rG; rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1739
24

11
zidB; rA; mC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; mC; rG;
rC; mU; rC; mG; rC; mA; rU; mC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1741
24

12
zidB; rA; mC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; mC; rG;
rC; mU; rC; rG; mC; mA; rU; rC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1744
0

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; mC; rG;
rC; mU; rC; rG; mC; mA; rU; mC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1746
0

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; mC; rG;
rC; mU; rC; mG; rC; mA; rU; mC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1785

zidB; rA; mC; rA; rA; rG; rA; rU;
dU; rA; mC; rU; mC; rG; rU2p; rC;

rG; rC; rG; rA; rG; rA; mC; rG;
mU; rC; rG; mC; mA; rU; rC; rU;

rA; rG; mU; rA; zc3p$
mU; rG; rU; zc3p; zc3p$

SERPINH1_11_S1356

zidB; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; rU; mG; rU; mA; rG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
mA; rG; mG; rC; mC; rU; mG;

rA2p; rA2p; rC2p; rA2p
rU; mC; rC; mG; zc3p; zc3p$

SERPINH1_11_S1363

zc3p; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; rU; mG; rU; mA; rG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
mA; rG; mG; rC; mC; rU; mG;

rA2p; rA2p; rC2p; rA2p
rU; mC; rC; mG; zc3p; zc3p$

SERPINH1_11_S1370

zidB; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; rU; mG; rU; mA; rG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
mA; rG; mG; rC; mC; rU; mG;

rA2p; rA2p; rC2p; rA2p; zc3p$
rU; mC; rC; mG; zc3p; zc3p$

SERPINH1_11_S1446

zc3p; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; mU; rG; mU; rA; rG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rA; rG; rG; mC; mC; mU; rG;

rA2p; rA2p; rC2p; rA2p
mU; mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1449

zc3p; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; mU; rG; rU2p; rA;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rG; rA; rG; rG; mC; rC; mU; rG; rU;

rA2p; rA2p; rC2p; rA2p
mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1450

zc3p; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; mU; rG; LdT; rA; rG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rA; rG; rG; mC; rC; mU; rG; rU;

rA2p; rA2p; rC2p; rA2p
mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1451

zidB; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; mU; rG; mU; rA; rG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rA; rG; rG; mC; mC; mU; rG;

rA2p; rA2p; rC2p; rA2p
mU; mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1454

zidB; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; mU; rG; rU2p; rA;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rG; rA; rG; rG; mC; rC; mU; rG; rU;

rA2p; rA2p; rC2p; rA2p
mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1455

zidB; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; mU; rG; LdT; rA; rG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rA; rG; rG; mC; rC; mU; rG; rU;

rA2p; rA2p; rC2p; rA2p
mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1456

zidB; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; mU; rG; mU; rA; rG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rA; rG; rG; mC; mC; mU; rG;

rA2p; rA2p; rC2p; rA2p; zc3p$
mU; mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1457

zidB; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; mU; rG; mU; rA; rG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rA; rG; rG; rC; mC; mU; rG;

rA2p; rA2p; rC2p; rA2p; zc3p$
mU; mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1459

zidB; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; mU; rG; rU2p; rA;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rG; rA; rG; rG; mC; rC; mU; rG; rU;

rA2p; rA2p; rC2p; rA2p; zc3p$
mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1460

zidB; rC; rG; rG; rA; rC; rA; rG;
mU; rG; mU; mU; rG; LdT; rA; rG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rA; rG; rG; mC; rC; mU; rG; rU;

rA2p; rA2p; rC2p; rA2p; zc3p$
mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1461

zidB; mC; rG; rG; rA; mC; rA;
mU; rG; mU; rU; mG; rU; mA; rG;

rG; rG; rC; mC; rU; rC; mU; rA;
mA; rG; mG; rC; mC; rU; mG;

mC; rA; rA; LdC; rA$
rU; mC; rC; mG; zc3p; zc3p$

SERPINH1_11_S1462

zidB; mC; rG; rG; rA; mC; rA;
mU; rG; mU; mU; rG; mU; rA; rG;

rG; rG; rC; mC; rU; rC; mU; rA;
rA; rG; rG; mC; mC; mU; rG;

mC; rA; rA; LdC; rA$
mU; mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1464

45
43

zidB; mC; rG; rG; rA; mC; rA;
mU; rG; mU; mU; rG; mU; rA; rG;

rG; rG; rC; mC; rU; rC; mU; rA;
rA; rG; rG; mC; rC; mU; rG; rU;

mC; rA; rA; LdC; rA$
mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1467

zidB; mC; rG; rG; rA; mC; rA;
mU; rG; mU; rU; mG; rU; mA; rG;

rG; rG; rC; rC; rU; rC; mU; rA;
mA; rG; mG; rC; mC; rU; mG;

mC; rA; rA; mC; rA; zc3p$
rU; mC; rC; mG; zc3p; zc3p$

SERPINH1_11_S1468

zidB; mC; rG; rG; rA; mC; rA;
mU; rG; mU; mU; rG; mU; rA; rG;

rG; rG; rC; rC; rU; rC; mU; rA;
rA; rG; rG; mC; mC; mU; rG;

mC; rA; rA; mC; rA; zc3p$
mU; mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1469

zidB; mC; rG; rG; rA; mC; rA;
mU; rG; mU; mU; rG; mU; rA; rG;

rG; rG; rC; rC; rU; rC; mU; rA;
rA; rG; rG; rC; mC; mU; rG;

mC; rA; rA; mC; rA; zc3p$
mU; mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1470

zidB; mC; rG; rG; rA; mC; rA;
mU; rG; mU; mU; rG; mU; rA; rG;

rG; rG; rC; rC; rU; rC; mU; rA;
rA; rG; rG; mC; rC; mU; rG; rU;

mC; rA; rA; mC; rA; zc3p$
mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1471

zidB; mC; rG; rG; rA; mC; rA;
mU; rG; mU; mU; rG; rU2p; rA;

rG; rG; rC; rC; rU; rC; mU; rA;
rG; rA; rG; rG; mC; rC; mU; rG; rU;

mC; rA; rA; mC; rA; zc3p$
mC; mC; rG; zc3p; zc3p$

SERPINH1_11_S1472

zidB; mC; rG; rG; rA; mC; rA;
mU; rG; mU; mU; rG; LdT; rA; rG;

rG; rG; rC; rC; rU; rC; mU; rA;
rA; rG; rG; mC; rC; mU; rG; rU;

mC; rA; rA; mC; rA; zc3p$
mC; mC; rG; zc3p; zc3p$

SERPINH1_12_S1391

zidB; rA; rC; rA; rA; rG; rA; rU;
rA; mA; rC; mU; rC; mG; rU; mC;

rG; rC; rG; rA; rG; rA; rC; rG2p;
rU; rC; mG; rC; mA; rU; mC; rU;

rA2p; rG2p; rU2p; rU2p
mU; rG; mU; zc3p; zc3p$

SERPINH1_12_S1780

zidB; rA; mC; rA; mA; rG; rA;
rA; rA; mC; rU; mC; rG; rU2p; rC;

rU; rG; rC; rG; rA; rG; rA; mC;
mU; rC; rG; mC; mA; rU; rC; rU;

rG; rA; rG; mU; rU; zc3p$
mU; rG; rU; zc3p; zc3p$

SERPINH1_30_S1391

zidB; rC; rG; rG; rA; rC; rA; rG;
rA; mG; rU; mU; rG; mU; rA; mG;

rG; rC; rC; rU; rC; rU; rA; rC2p;
rA; rG; mG; rC; mC; rU; mG;

rA2p; rA2p; rC2p; rU2p
rU; mC; rC; mG; zc3p; zc3p$

SERPINH1_45_S1354

174
40

rA; rC; rU; rC; rC; rA; rA; rG; rA;
yrA; rG; rG; rA; rA; rG; rU; rU; rG;

rU; rC; rA; rA; rC; rU; rU; rC;
rA; rU; rC; rU; rU; rG; rG; rA;

rC; yrU; zdT; zdT$
rG; rU; zdT; zdT$

SERPINH1_45_S1500
16
96
54

zidB; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; rG; rA; rA; rG; mU; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
rG; rA; mU; mC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p
rG; rA; rG; rU; zc3p; zc3p$

SERPINH1_45_S1501

zidB; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; mG; rA; mA; rG; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1502

zidB; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; mG; rA; mA; rG; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; rU; mC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1505
16
22
17

zidB; rA; rC; rU; rC; rC; rA; rA;
yrA; mG; rG; mA; rA; mG; rU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; rU; mG;

rU2p; rC2p; rC2p; yrU2p
rG; mA; rG; mU; zc3p; zc3p$

SERPINH1_45_S1506

zc3p; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; rG; rA; rA; rG; mU; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
rG; rA; mU; mC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p
rG; rA; rG; rU; zc3p; zc3p$

SERPINH1_45_S1507

zc3p; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; mG; rA; mA; rG; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1508

zc3p; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; mG; rA; mA; rG; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; rU; mC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1509
16

zc3p; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; rG; rA; rA; rG; rU2p;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1510

zc3p; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; rG; rA; rA; rG; LdT; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
rG; rA; mU; rC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1511
8

27

zc3p; rA; rC; rU; rC; rC; rA; rA;
yrA; mG; rG; mA; rA; mG; rU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; rU; mG;

rU2p; rC2p; rC2p; yrU2p
rG; mA; rG; mU; zc3p; zc3p$

SERPINH1_45_S1512

zidB; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; rG; rA; rA; rG; mU; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
rG; rA; mU; mC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p; zc3p$
rG; rA; rG; rU; zc3p; zc3p$

SERPINH1_45_S1513

zidB; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; mG; rA; mA; rG; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p; zc3p$
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1514

zidB; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; mG; rA; mA; rG; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; rU; mC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p; zc3p$
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1515

zidB; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; rG; rA; rA; rG; rU2p;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p; zc3p$
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1516

zidB; rA; rC; rU; rC; rC; rA; rA;
ymA; rG; rG; rA; rA; rG; LdT; mU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
rG; rA; mU; rC; mU; mU; rG;

rU2p; rC2p; rC2p; yrU2p; zc3p$
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1517
24
22
31
7
11
14
zidB; rA; rC; rU; rC; rC; rA; rA;
yrA; mG; rG; mA; rA; mG; rU;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; rU; mG;

rU2p; rC2p; rC2p; yrU2p; zc3p$
rG; mA; rG; mU; zc3p; zc3p$

SERPINH1_45_S1518
8
90
47

zidB; rA; rC; rU; mC; mC; rA;
ymA; rG; rG; rA; rA; rG; mU; mU;

rA; rG; rA; rU; mC; rA; rA; mC;
rG; rA; mU; mC; mU; mU; rG;

mU; rU; rC; LdC; yrU$
rG; rA; rG; rU; zc3p; zc3p$

SERPINH1_45_S1523
3
17
30

16
zidB; rA; rC; rU; mC; mC; rA;
yrA; mG; rG; mA; rA; mG; rU;

rA; rG; rA; rU; mC; rA; rA; mC;
mU; rG; rA; mU; rC; mU; rU; mG;

mU; rU; rC; LdC; yrU$
rG; mA; rG; mU; zc3p; zc3p$

SERPINH1_45_S1524

zidB; rA; rC; rU; rC; mC; rA; rA;
ymA; rG; rG; rA; rA; rG; mU; mU;

rG; rA; rU; mC; rA; rA; rC;
rG; rA; mU; mC; mU; mU; rG;

mU; rU; mC; mC; yrU; zc3p$
rG; rA; rG; rU; zc3p; zc3p$

SERPINH1_45_S1525

zidB; rA; rC; rU; rC; mC; rA; rA;
ymA; rG; mG; rA; mA; rG; mU;

rG; rA; rU; mC; rA; rA; rC;
mU; rG; rA; mU; rC; mU; mU; rG;

mU; rU; mC; mC; yrU; zc3p$
mG; rA; mG; rU; zc3p; zc3p$

SERPINH1_45_S1529
24
17
33

zidB; rA; rC; rU; rC; mC; rA; rA;
yrA; mG; rG; mA; rA; mG; rU;

rG; rA; rU; mC; rA; rA; rC;
mU; rG; rA; mU; rC; mU; rU; mG;

mU; rU; mC; mC; yrU; zc3p$
rG; mA; rG; mU; zc3p; zc3p$

SERPINH1_45_S1684
24

14
zidB; rA; rC; rU; rC; rC; rA; rA;
yrA; mG; rG; mA; rA; mG; rU2p;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; rU;

rU2p; rC2p; rC2p; yrU2p; zc3p$
mG; rG; mA; rG; mU; zc3p; zc3p$

SERPINH1_45_S1685
8

15
zidB; rA; rC; rU; mC; mC; rA;
yrA; mG; rG; mA; rA; mG; rU2p;

rA; rG; rA; rU; mC; rA; rA; mC;
mU; rG; rA; mU; rC; mU; rU;

mU; rU; rC; LdC; yrU$
mG; rG; mA; rG; mU; zc3p; zc3p$

SERPINH1_45_S1781

zidB; rA; rC; rU; rC; rC; rA; rA;
rU; rG; rG; mA; rA; mG; rU2p;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; rU; mG;

rU2p; rC2p; rC2p; yrU2p; zc3p$
rG; mA; rG; mU; zc3p; zc3p$

SERPINH1_45_S1786

zidB; rA; rC; rU; rC; rC; rA; rA;
dU; rG; rG; mA; rA; mG; rU2p;

rG; rA; rU; rC; rA; rA; rC; rU2p;
mU; rG; rA; mU; rC; mU; rU; mG;

rU2p; rC2p; rC2p; rA2p; zc3p$
rG; mA; rG; mU; zc3p; zc3p$

SERPINH1_51_S1356

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA; rU;

rA; rC; rA; rC; rA; rU; rG; rG2p;
mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1363

zc3p; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p;
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1370

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p; zc3p$
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1473

zc3p; rU; rC; rC; rU; rG; rA; rG;
mU; mC; rA; mC; mC; mC; rA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
mU; rG; mU; rG; mU; mC; mU;

rG2p; rU2p; rG2p; rA2p;
mC; rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1474

zc3p; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p;
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1475

zc3p; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p;
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1476

zc3p; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p;
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1477

zc3p; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p;
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1478

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; mC; rA; mC; mC; mC; rA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
mU; rG; mU; rG; mU; mC; mU;

rG2p; rU2p; rG2p; rA2p
mC; rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1479

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1480

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1481

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1482

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1483

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; mC; rA; mC; mC; mC; rA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
mU; rG; mU; rG; mU; mC; mU;

rG2p; rU2p; rG2p; rA2p; zc3p$
mC; rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1484

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p; zc3p$
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1485

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; rC; mA; rC; mC; rC; mA;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rU; mG; rU; mG; rU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p; zc3p$
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1486

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rG; rU; rG; mU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1487

zidB; rU; rC; rC; rU; rG; rA; rG;
mU; rC; rA; mC; rC; LdC; rA; mU;

rA; rC; rA; rC; rA; rU; rG; rG2p;
rG; rU; rG; mU; mC; rU; mC;

rG2p; rU2p; rG2p; rA2p; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1488

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; mA; rC; mC; rC; mA; rU;

rG; rA; rC; rA; mC; rA; mU; rG;
mG; rU; mG; rU; mC; rU; mC;

rG; rG; rU; LdG; rA$
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1489
8

25

zidB; rU; mC; rC; mU; rG; rA;
mU; mC; rA; mC; mC; mC; rA;

rG; rA; rC; rA; mC; rA; mU; rG;
mU; rG; mU; rG; mU; mC; mU;

rG; rG; rU; LdG; rA$
mC; rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1490

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; rA; mC; mC; mC; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; mU; mC;

rG; rG; rU; LdG; rA$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1491

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; rA; mC; mC; mC; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; rU; LdG; rA$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1492

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; rU; LdG; rA$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1493

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; rA; mC; rC; LdC; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; rU; LdG; rA$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1494

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; mA; rC; mC; rC; mA; rU;

rG; rA; rC; rA; mC; rA; mU; rG;
mG; rU; mG; rU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; mG; rG; mA; zc3p; zc3p$

SERPINH1_51_S1495

zidB; rU; mC; rC; mU; rG; rA;
mU; mC; rA; mC; mC; mC; rA;

rG; rA; rC; rA; mC; rA; mU; rG;
mU; rG; mU; rG; mU; mC; mU;

rG; rG; mU; rG; rA; zc3p$
mC; rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1496

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; rA; mC; mC; mC; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; mU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1497

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; rA; mC; mC; mC; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1498
24
22

10
7

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1499
24
25
31
18
28

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; rA; mC; rC; LdC; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1666

zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC2p; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1667

zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; LdC; rA; mU;

rA; rC2p; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1668
16

14
zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC2p; rA; mC; rA; mU; rG;
rG; rU; rG; rU; mC; rU; mC; rA;

rG; rG; mU; rG; rA; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1669
24

18
zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC2p; rA; mC; rA; mU; rG;
rG; rU; mG; rU; rC; rU; mC; rA;

rG; rG; mU; rG; rA; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1670
16

13
zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; mA; rC; rC; rC2p; rA; mU;

rA; rC2p; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1673
24

22
zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; rU; mC; rU; mC; rA;

rG; rG; mU; rG; rA; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1674
16

zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC; rA; mC; rA; mU; rG;
rG; rU; mG; rU; rC; rU; mC; rA;

rG; rG; mU; rG; rA; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1675
16

35
zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; mA; rC; rC; rC2p; rA; mU;

rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1676
10

zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; rU2p; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1677
10

zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; LdC; rA; mU;

rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; rU2p; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1678
10

zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; rU; mC; rU; mC; rA;

rG; rG; rU2p; rG; rA; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1679
10

zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC; rA; mC; rA; mU; rG;
rG; rU; mG; rU; rC; rU; mC; rA;

rG; rG; rU2p; rG; rA; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1680
10

zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; mA; rC; rC; rC2p; rA; mU;

rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; rU2p; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1682
8

16
zidB; rU; mC; rC; mU; rG; rA;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; mG; rU; rC; rU; mC; rA;

rG; rG; mU; rG; rA; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1778

zidB; rU; rC; rC; mU; rG; rA; rG;
yrA; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC2p; rA; mC; rA; mU; rG;
rG; rU; mG; rU; rC; rU; mC; rA;

rG; rG; mU; rG; yrU; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1779

zidB; rU; mC; rC; mU; rG; rA;
yrA; rC; rA; mC; rC; rC2p; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; mG; rU; rC; rU; mC; rA;

rG; rG; mU; rG; yrU; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1783

zidB; rU; rC; rC; mU; rG; rA; rG;
dU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC2p; rA; mC; rA; mU; rG;
rG; rU; mG; rU; rC; rU; mC; rA;

rG; rG; mU; rG; rA; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1784

zidB; rU; mC; rC; mU; rG; rA;
dU; rC; rA; mC; rC; rC2p; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; mG; rU; rC; rU; mC; rA;

rG; rG; mU; rG; rA; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_52_S1356

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; rC; mG; rU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; mU; rC; mU;

rG2p; rA2p; rG2p; rA2p
rU; mG; rU; mC; zc3p; zc3p$

SERPINH1_52_S1363

zc3p; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; rC; mG; rU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; mU; rC; mU;

rG2p; rA2p; rG2p; rA2p
rU; mG; rU; mC; zc3p; zc3p$

SERPINH1_52_S1370

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; rC; mG; rU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; mU; rC; mU;

rG2p; rA2p; rG2p; rA2p; zc3p$
rU; mG; rU; mC; zc3p; zc3p$

SERPINH1_52_S1552

zc3p; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mU; mC; rG; mC; rA; mU; mC;

rG2p; rA2p; rG2p; rA2p
mU; mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1553

zc3p; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; mU; mC; rU;

rG2p; rA2p; rG2p; rA2p;
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1554

zc3p; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; rU; mC; mU;

rG2p; rA2p; rG2p; rA2p
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1555

zc3p; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; mU; rC; mU;

rG2p; rA2p; rG2p; rA2p
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1556

zc3p; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; rU; LdC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; rU; mC; mU;

rG2p; rA2p; rG2p; rA2p
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1557

zc3p; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; rU; rC2p; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; rU; mC; mU;

rG2p; rA2p; rG2p; rA2p
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1558

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mU; mC; rG; mC; rA; mU; mC;

rG2p; rA2p; rG2p; rA2p
mU; mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1559

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; mU; mC; rU;

rG2p; rA2p; rG2p; rA2p
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1560

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; rU; mC; mU;

rG2p; rA2p; rG2p; rA2p
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1561

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; mU; rC; mU;

rG2p; rA2p; rG2p; rA2p
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1562

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; rU; LdC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; rU; mC; mU;

rG2p; rA2p; rG2p; rA2p
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1563

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; rU; rC2p; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; rU; mC; mU;

rG2p; rA2p; rG2p; rA2p
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1564
16
94

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mU; mC; rG; mC; rA; mU; mC;

rG2p; rA2p; rG2p; rA2p; zc3p$
mU; mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1565

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; mU; mC; rU;

rG2p; rA2p; rG2p; rA2p; zc3p$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1566

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; rU; mC; mU;

rG2p; rA2p; rG2p; rA2p; zc3p$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1567

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; mU; rC; mU;

rG2p; rA2p; rG2p; rA2p; zc3p$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1568

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; rU; LdC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; rU; mC; mU;

rG2p; rA2p; rG2p; rA2p; zc3p$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1569

zidB; rG; rA; rC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; rU; rC2p; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; rU; mC; mU;

rG2p; rA2p; rG2p; rA2p; zc3p$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1570

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; rC; mG; rU; mC; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; mU; rC; mU;

mC; rG; rA; LdG; rA$
rU; mG; rU; mC; zc3p; zc3p$

SERPINH1_52_S1571

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC;

mU; rG; mC; rG; rA; rG; rA;
mU; mC; rG; mC; rA; mU; mC;

mC; rG; rA; LdG; rA$
mU; mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1572

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; mU; mC; rU;

mC; rG; rA; LdG; rA$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1573

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; rU; mC; mU;

mC; rG; rA; LdG; rA$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1574

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; mU; rC; mU;

mC; rG; rA; LdG; rA$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1575

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; rU; LdC; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; rU; mC; mU;

mC; rG; rA; LdG; rA$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1576

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; rU; rC2p; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; rU; mC; mU;

mC; rG; rA; LdG; rA$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1577

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; rC; mG; rU; mC; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; mU; rC; mU;

mC; rG; rA; rG; rA; zc3p$
rU; mG; rU; mC; zc3p; zc3p$

SERPINH1_52_S1578

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC;

mU; rG; mC; rG; rA; rG; rA;
mU; mC; rG; mC; rA; mU; mC;

mC; rG; rA; rG; rA; zc3p$
mU; mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1579

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; mU; mC; rU;

mC; rG; rA; rG; rA; zc3p$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1580

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; rU; mC; mU;

mC; rG; rA; rG; rA; zc3p$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1581

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; mU; mC; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; mU; rC; mU;

mC; rG; rA; rG; rA; zc3p$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1582

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; rU; LdC; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; rU; mC; mU;

mC; rG; rA; rG; rA; zc3p$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_52_S1583

zidB; rG; rA; mC; rA; rA; rG; rA;
mU; rC; mU; mC; rG; rU; rC2p; rU;

mU; rG; mC; rG; rA; rG; rA;
mC; rG; mC; rA; rU; mC; mU;

mC; rG; rA; rG; rA; zc3p$
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_58_S1391

zidB; rG; rA; rC; rA; rA; rG; rA;
rA; mC; rU; mC; rG; mU; rC; mU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
rC; rG; mC; rA; mU; rC; mU; rU;

rG2p; rA2p; rG2p; rU2p
mG; rU; mC; zc3p; zc3p$

SERPINH1_58_S1584

zidB; rG; rA; rC; rA; rA; rG; rA;
rA; mC; rU; mC; rG; mU; mC; rU;

rU; rG; rC; rG; rA; rG; rA; rC2p;
mC; rG; mC; rA; rU; mC; mU;

rG2p; rA2p; rG2p; rU2p
mU; rG; mU; rC; zc3p; zc3p$

SERPINH1_86_S1356
16
68
65

zidB; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; mG; rU; mU; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; mG; rA; mG; rG; mC;

rU2p; rA2p; rC2p; rA2p
rC; mU; rG; mU; zc3p; zc3p$

SERPINH1_86_S1363

zc3p; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; mG; rU; mU; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; mG; rA; mG; rG; mC;

rU2p; rA2p; rC2p; rA2p
rC; mU; rG; mU; zc3p; zc3p$

SERPINH1_86_S1370

zidB; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; mG; rU; mU; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; mG; rA; mG; rG; mC;

rU2p; rA2p; rC2p; rA2p; zc3p$
rC; mU; rG; mU; zc3p; zc3p$

SERPINH1_86_S1530

zc3p; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; mU; mU; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC;

rU2p; rA2p; rC2p; rA2p
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1531

52
31

zc3p; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; mU; mU; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC; rC;

rU2p; rA2p; rC2p; rA2p
mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1532

zc3p; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; rU; LdT; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC;

rU2p; rA2p; rC2p; rA2p
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1533
8
70
74

zc3p; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; rU; rU2p; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC;

rU2p; rA2p; rC2p; rA2p
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1534

zidB; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; mU; mU; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC;

rU2p; rA2p; rC2p; rA2p
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1535

zidB; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; mU; mU; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC; rC;

rU2p; rA2p; rC2p; rA2p
mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1536

zidB; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; rU; LdT; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC;

rU2p; rA2p; rC2p; rA2p
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1537

zidB; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; rU; rU2p; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC;

rU2p; rA2p; rC2p; rA2p
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1538

zidB; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; mU; mU; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC;

rU2p; rA2p; rC2p; rA2p; zc3p$
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1539

zidB; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; mU; mU; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC; rC;

rU2p; rA2p; rC2p; rA2p; zc3p$
mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1540

zidB; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; rU; LdT; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC;

rU2p; rA2p; rC2p; rA2p; zc3p$
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1541

zidB; rA; rC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; rU; rU2p; rG;

rU; rC; rU; rA; rC; rA; rA; rC2p;
mU; rA; rG; rA; rG; rG; mC;

rU2p; rA2p; rC2p; rA2p; zc3p$
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1542

zidB; rA; mC; rA; rG; rG; mC;
mU; rG; mU; rA; mG; rU; mU; rG;

rC; rU; rC; mU; rA; mC; rA; rA;
mU; rA; mG; rA; mG; rG; mC;

rC; mU; rA; LdC; rA$
rC; mU; rG; mU; zc3p; zc3p$

SERPINH1_86_S1543
8
44
42

zidB; rA; mC; rA; rG; rG; mC;
mU; rG; mU; rA; rG; mU; mU; rG;

rC; rU; rC; mU; rA; mC; rA; rA;
mU; rA; rG; rA; rG; rG; mC;

rC; mU; rA; LdC; rA$
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1544
8
29
36

zidB; rA; mC; rA; rG; rG; mC;
mU; rG; mU; rA; rG; mU; mU; rG;

rC; rU; rC; mU; rA; mC; rA; rA;
mU; rA; rG; rA; rG; rG; mC; rC;

rC; mU; rA; LdC; rA$
mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1545

zidB; rA; mC; rA; rG; rG; mC;
mU; rG; mU; rA; rG; rU; LdT; rG;

rC; rU; rC; mU; rA; mC; rA; rA;
mU; rA; rG; rA; rG; rG; mC;

rC; mU; rA; LdC; rA$
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1546
16
67
63

zidB; rA; mC; rA; rG; rG; mC;
mU; rG; mU; rA; rG; rU; rU2p; rG;

rC; rU; rC; mU; rA; mC; rA; rA;
mU; rA; rG; rA; rG; rG; mC;

rC; mU; rA; LdC; rA$
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1547
16
24
63

zidB; rA; mC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; mG; rU; mU; rG;

rU; rC; mU; rA; mC; rA; rA;
mU; rA; mG; rA; mG; rG; mC;

rC; mU; rA; mC; rA; zc3p$
rC; mU; rG; mU; zc3p; zc3p$

SERPINH1_86_S1548
16
39
67

zidB; rA; mC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; mU; mU; rG;

rU; rC; mU; rA; mC; rA; rA;
mU; rA; rG; rA; rG; rG; mC;

rC; mU; rA; mC; rA; zc3p$
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1549
16
20
68

zidB; rA; mC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; mU; mU; rG;

rU; rC; mU; rA; mC; rA; rA;
mU; rA; rG; rA; rG; rG; mC; rC;

rC; mU; rA; mC; rA; zc3p$
mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1550
16
96
92

zidB; rA; mC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; rU; LdT; rG;

rU; rC; mU; rA; mC; rA; rA;
mU; rA; rG; rA; rG; rG; mC;

rC; mU; rA; mC; rA; zc3p$
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_86_S1551
16
70
51

zidB; rA; mC; rA; rG; rG; rC; rC;
mU; rG; mU; rA; rG; rU; rU2p; rG;

rU; rC; mU; rA; mC; rA; rA;
mU; rA; rG; rA; rG; rG; mC;

rC; mU; rA; mC; rA; zc3p$
mC; mU; rG; rU; zc3p; zc3p$

SERPINH1_2_S1686

zidB; rG; rA; rG; rA; rC; rA; mC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU; rG; rG; rG; mU; rG; rC;
mC; rC; mA; rU; mG; rU; mG;

mU; rA; mU; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1688

zidB; rG; rA; rG; rA; rC; rA; mC;
mU; rA; mU; rA; mG; rC; rA2p;

rA; rU; rG; rG; rG; mU; rG; rC;
rC; mC; rC; mA; rU; mG; rU; mG;

mU; rA; mU; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1689

zidB; rG; rA; rG; rA; rC; rA; mC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU; rG; rG; rG; mU; rG; rC;
mC; rC; mA; rU; mG; rU; mG;

mU; rA; mU; rA; zc3p; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1690

zidB; rG; rA; rG; rA; rC; rA; mC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU; rG; rG; rG; mU; rG; rC;
mC; rC; mA; rU; mG; rU; mG;

mU; rA; mU; rA; zc3p; zc3p$
rU; mC; rU; rC; zc3p; zc3p$

SERPINH1_2_S1691

zidB; rG; rA; rG; rA; rC; rA; mC;
mU; rA; mU; rA; mG; rC; rA2p;

rA; rU; rG; rG; rG; mU; rG; rC;
rC; mC; rC; mA; rU; mG; rU; mG;

mU; rA; mU; rA; zc3p; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1692

zidB; rG; rA; rG; rA; rC; rA; mC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU2p; rG; rG; rG; mU; rG;
mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; mU; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1693

zidB; rG; rA; rG; rA; rC; rA; mC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU2p; rG; rG; rG; mU; rG;
mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; mU; rA; zc3p$
rU; mC; rU; rC; zc3p; zc3p$

SERPINH1_2_S1695

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; mA; rC;

mC; rA; rU; rG; rG; rG; rU; rG;
mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; mU; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1696

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; mA; rC;

mC; rA; rU; rG; rG; rG; rU; rG;
mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; mU; rA; zc3p$
rU; mC; rU; rC; zc3p; zc3p$

SERPINH1_2_S1697

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; rA2p;

mC; rA; rU; rG; rG; rG; rU; rG;
rC; mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; mU; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1698

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; mA; rC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; rU2p; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1699

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; mA; rC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; rU2p; rA; zc3p$
rU; mC; rU; rC; zc3p; zc3p$

SERPINH1_2_S1701

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; mA; rC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; LdT; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1702

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; mA; rC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; LdT; rA; zc3p$
rU; mC; rU; rC; zc3p; zc3p$

SERPINH1_2_S1703

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; rA2p;

mC; rA; rU; rG; rG; rG; mU; rG;
rC; mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; LdT; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1704

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; mA; rU; mG; rU; mG;

rU2p; rA2p; rU2p; rA2p
rU; mC; rU; rC; zc3p; zc3p$

SERPINH1_2_S1706

zidB; rG; rA; rG; rA; rC; rA; rC;
mU; rA; mU; rA; mG; rC; mA; rC;

rA; rU; rG; rG; rG; rU; rG; rC2p;
mC; rC; mA; rU; mG; rU; mG;

rU2p; rA2p; rU2p; rA2p; zc3p$
rU; mC; rU; rC; zc3p; zc3p$

SERPINH1_2_S1708

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; mA; rC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; mA; rU; mG; rU; mG;

mC; mU; rA; LdT; rA$
rU; mC; rU; rC; zc3p; zc3p$

SERPINH1_2_S1709

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; rA2p;

mC; rA; rU; rG; rG; rG; mU; rG;
rC; mC; rC; mA; rU; mG; rU; mG;

mC; mU; rA; LdT; rA$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_2_S1710

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; mA; rC;

mC; rA; rU; rG; rG; rG; mU; rG;
mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; mU; rA; zc3p$
rU; mC; rU; rC; zc3p; zc3p$

SERPINH1_2_S1711

zidB; rG; rA; rG; rA; mC; rA;
mU; rA; mU; rA; mG; rC; rA2p;

mC; rA; rU; rG; rG; rG; mU; rG;
rC; mC; rC; mA; rU; mG; rU; mG;

rC; mU; rA; mU; rA; zc3p$
rU; mC; rU; mC; zc3p; zc3p$

SERPINH1_6_S1712

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; rC;

mU; rG; rC; rG; rA; rG; rA; mC;
mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; LdT; rA; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1713

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; mC;

mU; rG; rC; rG; rA; rG; rA; mC;
rU; mC; rG; mC; mA; rU; mC;

rG; rA; rG; LdT; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1714

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; mC;

rG; rA; rG; LdT; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1715

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; LdT; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1716

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; LdT; rA; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1717

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; rC;

rG; rA; rG; LdT; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1718

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; rC;

mU; rG; rC; rG; rA; rG; rA; mC;
mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; LdT; rA; zc3p; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1719

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; mC;

mU; rG; rC; rG; rA; rG; rA; mC;
rU; mC; rG; mC; mA; rU; mC;

rG; rA; rG; LdT; rA; zc3p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1720

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; mC;

rG; rA; rG; LdT; rA; zc3p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1721

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; LdT; rA; zc3p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1722

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; LdT; rA; zc3p; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1723

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; rC;

rG; rA; rG; LdT; rA; zc3p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1724

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; rC;

mU; rG; rC; rG; rA; rG; rA; mC;
mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; rU2p; rA; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1725

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; mC;

mU; rG; rC; rG; rA; rG; rA; mC;
rU; mC; rG; mC; mA; rU; mC;

rG; rA; rG; rU2p; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1726

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; mC;

rG; rA; rG; rU2p; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1727

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; rU2p; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1728

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; rU2p; rA; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1729

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; rC;

rG; rA; rG; rU2p; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1730

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; rC;

mU; rG; rC; rG; rA; rG; rA; mC;
mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; rU2p; rA; zc3p; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1731

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; mU; mC;

mU; rG; rC; rG; rA; rG; rA; mC;
rU; mC; rG; mC; mA; rU; mC;

rG; rA; rG; rU2p; rA; zc3p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1732

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; mC;

rG; rA; rG; rU2p; rA; zc3p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1733

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; rU2p; rA; zc3p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1734

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; rU2p; rA; zc3p; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1735

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; rC;

rG; rA; rG; rU2p; rA; zc3p; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1736

zidB; rA; mC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; mU; rC;

rG; rC; rG; rA; rG; rA; mC; rG;
mU; rC; mG; rC; mA; rU; mC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1737

zidB; rA; mC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; mU; mC;

rG; rC; rG; rA; rG; rA; mC; rG;
rU; mC; rG; mC; mA; rU; mC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1738

zidB; rA; mC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; mC; rG;
rC; mU; rC; rG; mC; mA; rU; mC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1740

zidB; rA; mC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; mC; rG;
rC; mU; rC; mG; rC; mA; rU; mC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1742

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; mU; rC;

rG; rC; rG; rA; rG; rA; mC; rG;
mU; rC; mG; rC; mA; rU; mC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1743

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; mU; mC;

rG; rC; rG; rA; rG; rA; mC; rG;
rU; mC; rG; mC; mA; rU; mC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1745

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; mC; rG;
rC; mU; rC; mG; rC; mA; rU; mC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1747

zidB; rA; rC; rA; rA; rG; rA; rU;
mU; rA; mC; rU; mC; rG; rU2p;

rG; rC; rG; rA; rG; rA; mC; rG;
rC; mU; rC; rG; mC; mA; rU; rC;

rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1748

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; LdT; rA$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1749

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; LdT; rA$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1750

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; rC;

rG; rA; rG; LdT; rA$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1751

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_6_S1752

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; mG; rC; mA; rU; mC;

rG; rA; rG; mU; rA; zc3p$
rU; mU; rG; mU; zc3p; zc3p$

SERPINH1_6_S1753

zidB; rA; mC; rA; rA; rG; rA;
mU; rA; mC; rU; mC; rG; rU2p;

mU; rG; rC; rG; rA; rG; rA; mC;
rC; mU; rC; rG; mC; mA; rU; rC;

rG; rA; rG; mU; rA; zc3p$
rU; mU; rG; rU; zc3p; zc3p$

SERPINH1_42_S1354

rG; rA; rC; rA; rG; rG; rC; rC; rU;
yrA; rU; rA; rG; rU; rU; rG; rU; rA;

rC; rU; rA; rC; rA; rA; rC; rU;
rG; rA; rG; rG; rC; rC; rU; rG;

rA; yrU; zdT; zdT$
rU; rC; zdT; zdT$

SERPINH1_51_S1671

zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1672

zidB; rU; rC; rC; mU; rG; rA; rG;
mU; rC; rA; mC; rC; LdC; rA; mU;

rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1681

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; rA; mC; rC; rC2p; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; rU; mC; rU; mC; rA;

rG; rG; mU; rG; rA; zc3p$
rG; rG; rA; zc3p; zc3p$

SERPINH1_51_S1683

zidB; rU; mC; rC; mU; rG; rA;
mU; rC; mA; rC; rC; rC2p; rA; mU;

rG; rA; rC; rA; mC; rA; mU; rG;
rG; rU; rG; mU; mC; rU; mC;

rG; rG; mU; rG; rA; zc3p$
rA; rG; rG; rA; zc3p; zc3p$

TABLE 7

Code of the modified nucleotides/unconventional moieties

as used in the Tables herein.

Code
Description

rA
riboadenosine-3′-phosphate; 3′-adenylic acid

rC
ribocytidine-3′-phosphate; 3′-cytidylic acid

rG
riboguanosine-3′-phosphate; 3′-guanylic acid

rU
ribouridine-3′-phosphate; 3′-uridylic acid

mA
2′-O-methyladenosine-3′-phosphate; 2′-O-methyl-3′-adenylic acid

mC
2′-O-methylcytidine-3′-phosphate; 2′-O-methyl-3′-cytidylic acid

mG
2′-O-methylguanosine-3′-phosphate; 2′-O-methyl-3′-guanylic acid

mU
2′-O-methyluridine-3′-phosphate; 2′-O-methyl-3′-uridylic acid

dA
deoxyriboadenosine-3′-phosphate; 2′-deoxyribo-3′-adenylic acid

dC
deoxyribocytidine-3′-phosphate; 2′-deoxyribo-3′-cytidylic acid

dG
deoxyriboguanosine-3′-phosphate; 2′-deoxyribo-3′-guanylic acid

dT
thymidine-3′-phosphate; 3′-thymidylic acid

rA2p
riboadenosine-2′-phosphate; 2′-adenylic acid (2′5′ A)

rC2p
ribocytidine-2′-phosphate; 2′-cytidylic acid (2′5′ C)

rG2p
riboguanosine-2′-phosphate; 2′-guanylic acid (2′5′ G)

rU2p
ribouridine-2′-phosphate; 2′-uridylic acid (2′5′U)

LdA
L-deoxyriboadenosine-3′-phosphate (mirror image dA)

LdC
L-deoxyribocytidine-3′-phosphate (mirror image dC)

LdG
L-deoxyriboguanosine-3′-phosphate (mirror image dG)

LdT
L-deoxyribothymidine-3′-phosphate (mirror image dT)

dB
abasic deoxyribose-3′-phosphate;

1,2-dideoxy-D-ribofuranose-3-phosphate;

1,4-2-deoxy-D-ribitol-3-phosphate

zidB
Inverted abasic deoxyribose-5′-phosphate; At 5′ = 5′-5′ idAb;

At 3′ = 3′-3′ idAb

z
Prefix to indicate moiety covalently attached to 3′ terminus or

5′ terminus

psiU
pseudouridne

p
5′ phosphate

s
5′ phosphorothioate

C3
C3 non-nucleotide

$
lacking a 3′ linker (used together with above nucleotides at the

3′ end of the sequence)

siRNA oligonucleotides useful in generating double stranded RNA molecules are disclosed in Tables A-18, A-19 and B-E below.

SERPINH1 Oligonucleotide Sequence Useful in the Preparation of siRNA Compounds.

TABLE A-18

SEQ

SEQ

ID

ID

NO

NO

Cross
Ident Human

Name
SEN
Sense (5′ > 3′)
AS
Antisense (5′ > 3′)
Species
gi″32454740

SERPINH1_2
60
GAGACACAUGGGUGCUAUA
127
UAUAGCACCCAUGUGUCUC
H, Rt, Rh,
[1533-1551]

M, D
(18/19)

SERPINH1_3
61
GGGAAGAUGCAGAAGAAGA
128
UCUUCUUCUGCAUCUUCCC
H, Rt, Rh,
[1112-1130]

Rb
(18/19)

SERPINH1_5
62
GAAGAAGGCUGUUGCCAUA
129
UAUGGCAACAGCCUUCUUC
H, Rt
[1123-1141]

(18/19)

SERPINH1_6
63
ACAAGAUGCGAGACGAGUA
130
UACUCGUCUCGCAUCUUGU
H, Rt, Rh,
[1464-1482]

(18/19)

SERPINH1_7
64
GGACAACCGUGGCUUCAUA
131
UAUGAAGCCACGGUUGUCC
H, Rh, M
[886-904]

(18/19)

SERPINH1_8
65
UGCAGUCCAUCAACGAGUA
132
UACUCGUUGAUGGACUGCA
H, Rt, Rh, M
[738-756]

(18/19)

SERPINH1_9
66
GCCUCAUCAUCCUCAUGCA
133
UGCAUGAGGAUGAUGAGGC
H, Rt, Rh,
[1026-1044]

M, D
(18/19)

SERPINH1_10
67
CGCGCUGCAGUCCAUCAAA
134
UUUGAUGGACUGCAGCGCG
H, Rt, Rh
[733-751]

(18/19)

SERPINH1_11
68
CGGACAGGCCUCUACAACA
135
UGUUGUAGAGGCCUGUCCG
H, Rt, Rh, P
[944-962]

(18/19)

SERPINH1_13
69
UGACAAGAUGCGAGACGAA
136
UUCGUCUCGCAUCUUGUCA
H, Rh
[1462-1480]

(18/19)

SERPINH1_14
70
CCAGCCUCAUCAUCCUCAA
137
UUGAGGAUGAUGAGGCUGG
H, M, Rt,
[1023-1041]

Rh, D-
(18/19)

SERPINH1_15
71
GCUGCAGUCCAUCAACGAA
138
UUCGUUGAUGGACUGCAGC
H, Rt, Rh
[736-754]

(18/19)

SERPINH1_16
72
GCAGCGCGCUGCAGUCCAA
139
UUGGACUGCAGCGCGCUGC
H, Rt, Rh
[729-747]

(18/19)

SERPINH1_17
73
UGAGACACAUGGGUGCUAA
140
UUAGCACCCAUGUGUCUCA
H, Rt, Rh
[1532-1550]

M, D
(18/19)

SERPINH1_19
74
GGUGGAGGUGACCCAUGAA
141
UUCAUGGGUCACCUCCACC
H, Rt, Rh, M
[1159-1177]

(18/19)

SERPINH1_20
75
CUUUGACCAGGACAUCUAA
142
UUAGAUGUCCUGGUCAAAG
H, Rt, Rh
[1324-1342]

(18/19)

SERPINH1_21
76
GGAGGUGACCCAUGACCUA
143
UAGGUCAUGGGUCACCUCC
H, Rt, Rh,
[1162-1180]

M, D
(18/19)

SERPINH1_22
77
CUCCUGAGACACAUGGGUA
144
UACCCAUGUGUCUCAGGAG
H, D
[1528-1546]

(18/19)

SERPINH1_23
78
AGAAGAAGGCUGUUGCCAA
145
UUGGCAACAGCCUUCUUCU
H, Rt
[1122-1140]

(18/19)

SERPINH1_24
79
AGCUCUCCAGCCUCAUCAA
146
UUGAUGAGGCUGGAGAGCU
H, Rt, D,
[1017-1035]

M, P, Rh
(18/19)

SERPINH1_25
80
CUGCAGUCCAUCAACGAGA
147
UCUCGUUGAUGGACUGCAG
H, Rt, Rh M
[737-755]

(18/19)

SERPINH1_26
81
CCGGACAGGCCUCUACAAA
148
UUUGUAGAGGCCUGUCCGG
H, Rt, Rh,
[943-961]

Rb, P
(18/19)

SERPINH1_27
82
GCACCGGACAGGCCUCUAA
149
UUAGAGGCCUGUCCGGUGC
H, Rt, Rh,
[940-958]

Rb, P
(18/19)

SERPINH1_28
83
GCAGAAGAAGGCUGUUGCA
150
UGCAACAGCCUUCUUCUGC
H, Rt
[1120-1138]

(18/19)

SERPINH1_31
84
AGAAGGCUGUUGCCAUCUA
151
UAGAUGGCAACAGCCUUCU
H, Rt
[1125-1143]

(18/19)

SERPINH1_32
85
AGCGCAGCGCGCUGCAGUA
152
UACUGCAGCGCGCUGCGCU
H, Rt, Rh,
[726-744]

(18/19)

SERPINH1_33
86
GACACAUGGGUGCUAUUGA
153
UCAAUAGCACCCAUGUGUC
H, Rt, Rh, M
[1535-1553]

(18/19)

SERPINH1_34
87
GGGCCUGACUGAGGCCAUA
154
UAUGGCCUCAGUCAGGCCC
H, Rt
[1201-1219]

(18/19)

SERPINH1_35
88
AGACACAUGGGUGCUAUUA
155
UAAUAGCACCCAUGUGUCU
H, Rt, Rh, M
[1534-1552]

(18/19)

SERPINH1_36
89
CCAUGACCUGCAGAAACAA
156
UUGUUUCUGCAGGUCAUGG
H, Rt, Rh, M
[1171-1189]

(18/19)

SERPINH1_37
90
AGAUGCAGAAGAAGGCUGA
157
UCAGCCUUCUUCUGCAUCU
H, Rt, Rh, M
[1116-1134]

(18/19)

SERPINH1_38
91
CAAGCUCUCCAGCCUCAUA
158
UAUGAGGCUGGAGAGCUUG
H, Rt, Rh,
[1015-1033]

M, P, D
(18/19)

SERPINH1_39
92
UGCAGAAGAAGGCUGUUGA
159
UCAACAGCCUUCUUCUGCA
H, Rt
[1119-1137]

(18/19)

SERPINH1_41
93
CAGCCUCAUCAUCCUCAUA
160
UAUGAGGAUGAUGAGGCUG
H, Rt, Rh,
[1024-1042]

M, D
(18/19)

SERPINH1_42
94
GACAGGCCUCUACAACUAA
161
UUAGUUGUAGAGGCCUGUC
H, Rt, Rh,
[946-964]

Rb, P
(18/19)

SERPINH1_43
95
GAUGCAGAAGAAGGCUGUA
162
UACAGCCUUCUUCUGCAUC
H, Rt, Rh, M
[1117-1135]

(18/19)

SERPINH1_44
96
ACCCAUGACCUGCAGAAAA
163
UUUUCUGCAGGUCAUGGGU
H, Rt, Rh, M
[1169-1187]

(18/19)

SERPINH1_45
97
ACUCCAAGAUCAACUUCCA
164
UGGAAGUUGAUCUUGGAGU
H, Rt, Rh,
[702-720]

M, D
(18/19)

SERPINH1_45a
98
ACUCCAAGAUCAACUUCCU
165
AGGAAGUUGAUCUUGGAGU
H, Rt, Rh,
[702-720]

M, D
(18/19)

SERPINH1_48
99
AGGCCUCUACAACUACUAA
166
UUAGUAGUUGUAGAGGCCU
H, Rt, Rh,
[949-967]

Rb, P, D
(18/19)

SERPINH1_49
100
CACUCCAAGAUCAACUUCA
167
UGAAGUUGAUCUUGGAGUG
H, Rt, Rh,
[701-719]

M, D
(18/19)

SERPINH1_51
101
UCCUGAGACACAUGGGUGA
168
UCACCCAUGUGUCUCAGGA
H, Rt, D, M
[1529-1547]

(18/19)

SERPINH1_52
102
GACAAGAUGCGAGACGAGA
169
UCUCGUCUCGCAUCUUGUC
H, Rt, Rh,
[1463-1481]

(18/19)

SERPINH1_53
103
GGUGACCCAUGACCUGCAA
170
UUGCAGGUCAUGGGUCACC
H, Rt, Rh, M
[1165-1183]

(18/19)

SERPINH1_59
104
CCGAGGUGAAGAAACCUGA
171
UCAGGUUUCUUCACCUCGG
H, Rt, Rh,
[285-303]

(18/19)

SERPINH1_51a
105
UCCUGAGACACAUGGGUGU
172
ACACCCAUGUGUCUCAGGA
H, Rt, D, M
[1529-1547]

(18/19)

SERPINH1_61
106
GCACUCCAAGAUCAACUUA
173
UAAGUUGAUCUUGGAGUGC
H, Rh, D
[700-718]

(18/19)

SERPINH1_62
107
GUGGUGGAGGUGACCCAUA
174
UAUGGGUCACCUCCACCAC
H, Rt, Rh,
[1157-1175]

M, Rb
(18/19)

SERPINH1_64
108
GCCGAGGUGAAGAAACCUA
175
UAGGUUUCUUCACCUCGGC
H, Rt, Rh,
[284-302]

(18/19)

SERPINH1_65
109
GCUCUCCAGCCUCAUCAUA
176
UAUGAUGAGGCUGGAGAGC
H, Rt, D,
[1018-1036]

M, P, Rh
(18/19)

SERPINH1_66
110
GAUGCACCGGACAGGCCUA
177
UAGGCCUGUCCGGUGCAUC
H, Rt, Rh,
[937-955]

M, Rb, P
(18/19)

SERPINH1_68
111
CUCUCCAGCCUCAUCAUCA
178
UGAUGAUGAGGCUGGAGAG
H, Rt, D,
[1019-1037]

M, P, Rh
(18/19)

SERPINH1_69
112
GCAGACCACCGACGGCAAA
179
UUUGCCGUCGGUGGUCUGC
H, Rt, D
[763-781]

(18/19)

SERPINH1_70
113
AGUCCAUCAACGAGUGGGA
180
UCCCACUCGUUGAUGGACU
H, Rt, Rh, M
[741-759]

(18/19)

SERPINH1_71
114
ACCGUGGCUUCAUGGUGAA
181
UUCACCAUGAAGCCACGGU
H, Rt, Rh, M
[891-909]

(18/19)

SERPINH1_74
115
GAAGGCUGUUGCCAUCUCA
182
UGAGAUGGCAACAGCCUUC
H, Rt,
[1126-1144]

(18/19)

SERPINH1_75
116
GAAGAUGCAGAAGAAGGCA
183
UGCCUUCUUCUGCAUCUUC
H, Rt, Rh,
[1114-1132]

Rb
(18/19)

SERPINH1_77
117
UGAUGAUGCACCGGACAGA
184
UCUGUCCGGUGCAUCAUCA
H, Rh,
[933-951]

(18/19)

SERPINH1_78
118
CCCUUUGACCAGGACAUCA
185
UGAUGUCCUGGUCAAAGGG
H, Rt, Rh,
[1322-1340]

(18/19)

SERPINH1_80
119
CAGUCCAUCAACGAGUGGA
186
UCCACUCGUUGAUGGACUG
H, Rt, Rh, M
[740-758]

(18/19)

SERPINH1_82
120
CAACCGUGGCUUCAUGGUA
187
UACCAUGAAGCCACGGUUG
H, Rt, Rh, M
[889-907]

(18/19)

SERPINH1_83
121
CGACAAGCGCAGCGCGCUA
188
UAGCGCGCUGCGCUUGUCG
H
[721-739]

(18/19)

SERPINH1_84
122
GCAGUCCAUCAACGAGUGA
189
UCACUCGUUGAUGGACUGC
H, Rt, Rh, M
[739-757]

(18/19)

SERPINH1_86
123
ACAGGCCUCUACAACUACA
190
UGUAGUUGUAGAGGCCUGU
H, Rt, Rh,
[947-965]

Rb, P, D
(18/19)

SERPINH1_87
124
AAGAUGCAGAAGAAGGCUA
191
UAGCCUUCUUCUGCAUCUU
H, Rt, Rh, M
[1115-1133]

(18/19)

SERPINH1_89
125
CAGCGCGCUGCAGUCCAUA
192
UAUGGACUGCAGCGCGCUG
H, Rt, Rh,
[730-748]

(18/19)

SERPINH1_90
126
GCGCAGCGCGCUGCAGUCA
193
UGACUGCAGCGCGCUGCGC
H, Rt, Rh,
[727-745]

(18/19)

Select siRNAs

SEQ ID
SEQ ID
Activity
Activity
Activity
IC50

siRNA
SEN
AS
0.1 nM
0.5 nM
5 nM
(nM)
Length

SERPINH1_2
60
127
65
48
7
.008
19

SERPINH1_6
63
130
164
39
5
.019
19

SERPINH1_11
68
135
119
54
6
.05
19

SERPINH1_13
69
136
91
24
4

19

SERPINH1_45
97
164
156
38
8
.07
19

SERPINH1_45a
98
165

19

SERPINH1_51
101
168
68
39
5
.05
19

SERPINH1_52
102
169
149
37
9
0.06
19

SERPINH1_86
123
190
121
61

0.27
19

SEQ
SEQ

ID
ID
Activity
Activity
Activity
Activity
Activity
Activity
Activity

siRNA
SEN
AS
0.026 nM
0.077 nM
0.23 nM
0.69 nM
2.1 nM
6.25 nM
25 nM

SERPINH1_45
97
164
102
81
55
41
28
22
16

SERPINH1_45a
98
165
107
98
84
69
36
24
16

TABLE A-19

SEQ

SEQ

ID

ID

Ident

NO

NO

Human gi

Name
SEN
Sense (5′ > 3′)
AS
Antisense (5′ > 3′)
Species
L
32454740

SERPINH1_1
194
GGACAGGCCUCUACAACUA
219
UAGUUGUAGAGGCCUGUCC
H, Rt, Rh,
19
[945-963]

Rb, P

(19/19)

SERPINH1_4
195
GAGACACAUGGGUGCUAUU
220
AAUAGCACCCAUGUGUCUC
H, Rt, Rh,
19
[1533-1551]

M, D

(19/19)

SERPINH1_12
196
ACAAGAUGCGAGACGAGUU
221
AACUCGUCUCGCAUCUUGU
H, Rt, Rh,
19
[1464-1482]

(19/19)

SERPINH1_18
197
CCUUUGACCAGGACAUCUA
222
UAGAUGUCCUGGUCAAAGG
H, Rt, Rh,
19
[1323-1341]

(19/19)

SERPINH1_29
198
GACCCAUGACCUGCAGAAA
223
UUUCUGCAGGUCAUGGGUC
H, Rt, Rh, M
19
[1168-1186]

(19/19)

SERPINH1_30
199
CGGACAGGCCUCUACAACU
224
AGUUGUAGAGGCCUGUCCG
H, Rt, Rh,
19
[944-962]

Rb, P

(19/19)

SERPINH1_40
200
ACCGGACAGGCCUCUACAA
225
UUGUAGAGGCCUGUCCGGU
H, Rt, Rh,
19
[942-960]

Rb, P,

(19/19)

SERPINH1_46
201
GCAGCGCGCUGCAGUCCAU
226
AUGGACUGCAGCGCGCUGC
H, Rt, Rh,
19
[729-747]

(19/19)

SERPINH1_47
202
GCGCGCUGCAGUCCAUCAA
227
UUGAUGGACUGCAGCGCGC
H, Rt, Rh,
19
[732-750]

(19/19)

SERPINH1_50
203
CUGAGACACAUGGGUGCUA
228
UAGCACCCAUGUGUCUCAG
H, Rt, Rh,
19
[1531-1549]

M, D

(19/19)

SERPINH1_54
204
AGAAGAAGGCUGUUGCCAU
229
AUGGCAACAGCCUUCUUCU
H, Rt
19
[1122-1140]

(19/19)

SERPINH1_55
205
AGCUCUCCAGCCUCAUCAU
230
AUGAUGAGGCUGGAGAGCU
H, Rt, D,
19
[1017-1035]

M, P, Rh

(19/19)

SERPINH1_56
206
CUGCAGUCCAUCAACGAGU
231
ACUCGUUGAUGGACUGCAG
H, Rt, Rh, M
19
[737-755]

(19/19)

SERPINH1_57
207
CGCUGCAGUCCAUCAACGA
232
UCGUUGAUGGACUGCAGCG
H, Rt, Rh,
19
[735-753]

(19/19)

SERPINH1_58
208
GACAAGAUGCGAGACGAGU
233
ACUCGUCUCGCAUCUUGUC
H, Rt, Rh,
19
[1463-1481]

(19/19)

SERPINH1_63
209
GGGCCUGACUGAGGCCAUU
234
AAUGGCCUCAGUCAGGCCC
H, Rt
19
[1201-1219]

(19/19)

SERPINH1_67
210
GAUGCAGAAGAAGGCUGUU
235
AACAGCCUUCUUCUGCAUC
H, Rt, Rh, M
19
[1117-1135]

(19/19)

SERPINH1_72
211
CACCGGACAGGCCUCUACA
236
UGUAGAGGCCUGUCCGGUG
H, Rt, Rh,
19
[941-959]

Rb, P

(19/19)

SERPINH1_73
212
AGAUGCAGAAGAAGGCUGU
237
ACAGCCUUCUUCUGCAUCU
H, Rt, Rh M
19
[1116-1134]

(19/19)

SERPINH1_76
713
AGCGCGCUGCAGUCCAUCA
238
UGAUGGACUGCAGCGCGCU
H, Rt, Rh
19
[731-749]

(19/19)

SERPINH1_79
214
GGAAGAUGCAGAAGAAGGC
239
GCCUUCUUCUGCAUCUUCC
H, Rt, Rh,
19
[1113-1131]

Rb

(19/19)

SERPINH1_81
215
GAAGAAGGCUGUUGCCAUC
240
GAUGGCAACAGCCUUCUUC
H, Rt
19
[1123-1141]

(19/19)

SERPINH1_85
216
UGCAGUCCAUCAACGAGUG
241
CACUCGUUGAUGGACUGCA
H, Rt, Rh, M
19
[738-756]

(19/19)

SERPINH1_88
217
CCUGAGACACAUGGGUGCU
242
AGCACCCAUGUGUCUCAGG
H, Rt, D, M
19
[1530-1548]

(19/19)

SERPINH1_91
218
CGCAGCGCGCUGCAGUCCA
243
UGGACUGCAGCGCGCUGCG
H, Rt, Rh,
19
[728-746]

(19/19)

Select siRNAs

SEQ ID
SEQ ID
Activity
Activity
Activity

siRNA
NO SEN
NO AS
0.1 nM
0.5 nM
5 nM
IC50 (nM)
Length

SERPINH1_4
195
220
60
35
5
.006
19

SERPINH1_12
196
221
54
42
8
.065
19

SERPINH1_18
197
222
139
43
9

19

SERPINH1_30
199
224
146
49
9
0.093
19

SERPINH1_58
208
233
na
na
8

19

SERPINH1_88
217
242
105
43
9

19

TABLE B

Additional Active 19-mer SERPINH1 siRNAs

No
SEQ ID SEN
Sense siRNA
SEQ ID AS
AntiSense siRNA
Other
human-

1
244
GGCAGACUCUGGUCAAGAA
460
UUCUUGACCAGAGUCUGCC
Rh
[2009-2027]

2
245
CAGUGAGGCGGAUUGAGAA
461
UUCUCAAUCCGCCUCACUG

[1967-1985]

3
246
AGCCUUUGUUGCUAUCAAU
462
AUUGAUAGCAACAAAGGCU
Rh
[2117-2135]

4
247
CCAUGUUCUUCAAGCCACA
463
UGUGGCUUGAAGAACAUGG
Rh, Rb, D
[837-855]

5
248
CCCUCUUCUGACACUAAAA
464
UUUUAGUGUCAGAAGAGGG

[1850-1868]

6
249
CCUCAAUCAGUAUUCAUAU
465
AUAUGAAUACUGAUUGAGG

[1774-1792]

7
250
GAGACACAUGGGUGCUAUU
466
AAUAGCACCCAUGUGUCUC
Rh, D, Rt, M
[1533-1551]

8
251
GUGACAAGAUGCGAGACGA
467
UCGUCUCGCAUCUUGUCAC
Rh
[1461-1479]

9
252
GCCACACUGGGAUGAGAAA
468
UUUCUCAUCCCAGUGUGGC
Rh, Rb, M
[850-868]

10
253
AGAUGCGAGACGAGUUAUA
469
UAUAACUCGUCUCGCAUCU
Rh
[1467-1485]

11
254
ACGACGACGAGAAGGAAAA
470
UUUUCCUUCUCGUCGUCGU

[966-984]

12
255
GCCUCUACAACUACUACGA
471
UCGUAGUAGUUGUAGAGGC
Rb, D
[951-969]

13
256
AGAUCAACUUCCGCGACAA
472
UUGUCGCGGAAGUUGAUCU
D
[708-726]

14
257
ACUACUACGACGACGAGAA
473
UUCUCGUCGUCGUAGUAGU
Rb
[960-978]

15
258
AGCCCUCUUCUGACACUAA
474
UUAGUGUCAGAAGAGGGCU

[1848-1866]

16
259
ACAAGAUGCGAGACGAGUU
475
AACUCGUCUCGCAUCUUGU
Rh, Rt
[1464-1482]

17
260
AGCCACACUGGGAUGAGAA
476
UUCUCAUCCCAGUGUGGCU
Rh, Rb, M
[849-867]

18
261
AGGACCAGGCAGUGGAGAA
477
UUCUCCACUGCCUGGUCCU
Rh
[408-426]

19
262
CAGGCAAGAAGGACCUGUA
478
UACAGGUCCUUCUUGCCUG
Rh, D
[1251-1269]

20
263
ACCUGUGAGACCAAAUUGA
479
UCAAUUUGGUCUCACAGGU
Rh
[1813-1831]

21
264
CUUUGUUGCUAUCAAUCCA
480
UGGAUUGAUAGCAACAAAG
Rh
[2120-2138]

22
265
GUGAGACCAAAUUGAGCUA
481
UAGCUCAAUUuGGUCUCAC
Rh
[1817-1835]

23
266
CCCUGAAAGUCCCAGAUCA
482
UGAUCUGGGACUUUCAGGG

[1749-1767]

24
267
CCUUUGACCAGGACAUCUA
483
UAGAUGUCCUGGUCAAAGG
Rh, Rt
[1323-1341]

25
268
GACCAGGCAGUGGAGAACA
484
UGUUCUCCACUGCCUGGUC
Rh
[410-428]

26
269
CGCGCAACGUGACCUGGAA
485
UUCCAGGUCACGUUGCGCG
M
[597-615]

27
270
AUGAGAAAUUCCACCACAA
486
UUGUGGUGGAAUUUCUCAU
Rh
[861-879]

28
271
GAAGAAACCUGCAGCCGCA
487
UGCGGCUGCAGGUUUCUUC

[292-310]

29
272
CUCUCGAGCGCCUUGAAAA
488
UUUUCAAGGCGCUCGAGAG

[1059-1077]

30
273
GGAACAUGAGCCUUUGUUG
489
CAACAAAGGCUCAUGUUCC
Rh
[2109-2127]

31
274
CUCACCUGUGAGACCAAAU
490
AUUUGGUCUCACAGGUGAG
Rh
[1810-1828]

32
275
CUACGACGACGAGAAGGAA
491
UUCCUUCUCGUCGUCGUAG
Rb
[964-982]

33
276
ACCACAAGAUGGUGGACAA
492
UUGUCCACCAUCUUGUGGU
Rh, Rb, M, P
[873-891]

34
277
CUGGCACUGCGGAGAAGUU
493
AACUUCUCCGCAGUGCCAG

[318-336]

35
278
GGUCCUAUACCGUGGGUGU
494
ACACCCACGGUAUAGGACC
Rh
[912-930]

36
279
CCAACCUCUCCCAACUAUA
495
UAUAGUUGGGAGAGGUUGG
Rh
[1896-1914]

37
280
GAGAAGGAAAAGCUGCAAA
496
UUUGCAGCUUUUCCUUCUC
Rh
[974-992]

38
281
GCCUCUCGAGCGCCUUGAA
497
UUCAAGGCGCUCGAGAGGC

[1057-1075]

39
282
AGGCCAUUGACAAGAACAA
498
UUGUUCUUGUCAAUGGCCU
Rh, D
[1212-1230]

40
283
GACCCAUGACCUGCAGAAA
499
UUUCUGCAGGUCAUGGGUC
Rh, Rt, M
[1168-1186]

41
284
CUCCUGGCACUGCGGAGAA
500
UUCUCCGCAGUGCCAGGAG

[315-333]

42
285
CGGACAGGCCUCUACAACU
501
AGUUGUAGAGGCCUGUCCG
Rh, Rb, Rt, P
[944-962]

43
286
GAUGAGAAAUUCCACCACA
502
UGUGGUGGAAUUUCUCAUC
Rh
[860-878]

44
287
CACGCAUGUCAGGCAAGAA
503
UUCUUGCCUGACAUGCGUG
Rh, D
[1242-1260]

45
288
ACCUCUCCCAACUAUAAAA
504
UUUUAUAGUUGGGAGAGGU
Rh
[1899-1917]

46
289
ACCAGGCAGUGGAGAACAU
505
AUGUUCUCCACUGCCUGGU
Rh
[411-429]

47
290
GGGAACAUGAGCCUUUGUU
506
AACAAAGGCUCAUGUUCCC
Rh
[2108-2126]

48
291
AGAAUUCACUCCACUUGGA
507
UCCAAGUGGAGUGAAUUCU
Rh
[1653-1671]

49
292
GGGCAGACUCUGGUCAAGA
508
UCUUGACCAGAGUCUGCCC
Rh
[2008-2026]

50
293
AGAAGGAAAAGCUGCAAAU
509
AUUUGCAGCUUUUCCUUCU
Rh
[975-993]

51
294
GGCAGUGGAGAACAUCCUG
510
CAGGAUGUUCUCCACUGCC
Rh
[415-433]

52
295
GGGAUGAGAAAUUCCACCA
511
UGGUGGAAUUUCUCAUCCC
Rh
[858-876]

53
296
CCAAGCUGUUCUACGCCGA
512
UCGGCGUAGAACAGCUUGG
Rh
[1365-1383]

54
297
ACCGGACAGGCCUCUACAA
513
UUGUAGAGGCCUGUCCGGU
Rh, Rb, Rt, P
[942-960]

55
298
CUGCCUCAAUCAGUAUUCA
514
UGAAUACUGAUUGAGGCAG

[1771-1789]

56
299
CAGCCCUCUUCUGACACUA
515
UAGUGUCAGAAGAGGGCUG

[1847-1865]

57
300
CCAGCCUCAUCAUCCUCAU
516
AUGAGGAUGAUGAGGCUGG
Rh, D, Rt, M
[1023-1041]

58
301
AGGGUGACAAGAUGCGAGA
517
UCUCGCAUCUUGUCACCCU
Rh, D
[1458-1476]

59
302
GGACCAGGCAGUGGAGAAC
518
GUUCUCCACUGCCUGGUCC
Rh
[409-427]

60
303
GCAGCGCGCUGCAGUCCAU
519
AUGGACUGCAGCGCGCUGC
Rh, Rt
[729-747]

61
304
GCGCGCUGCAGUCCAUCAA
520
UUGAUGGACUGCAGCGCGC
Rh, Rt
[732-750]

62
305
CCAGAUACCAUGAUGCUGA
521
UCAGCAUCAUGGUAUCUGG
Rh
[1680-1698]

63
306
CUAGUGCGGGACACCCAAA
522
UUUGGGUGUCCCGCACUAG

[1400-1418]

64
307
AGGCAGUGGAGAACAUCCU
523
AGGAUGUUCUCCACUGCCU
Rh
[414-432]

65
308
CUGAGACACAUGGGUGCUA
524
UAGCACCCAUGUGUCUCAG
Rh, D, Rt, M
[1531-1549]

66
309
GAUUGAGAAGGAGCUCCCA
525
UGGGAGCUCCUUCUCAAUC

[1977-1995]

67
310
CGCAGACCACCGACGGCAA
526
UUGCCGUCGOUGGUCUGCG
D, Rt
[762-780]

68
311
CCACACUGGGAUGAGAAAU
527
AUUUCUCAUCCCAGUGUGG
Rh
[851-869]

69
312
GCUCAGUGAGCUUCGCUGA
528
UCAGCGAAGCUCACUGAGC

[642-660]

70
313
CGCCUUUGAGUUGGACACA
529
UGUGUCCAACUCAAAGGCG
Rh
[1294-1312]

71
314
GGGUCAGCCAGCCCUCUUC
530
GAAGAGGGCUGGCUGACCC
Rh
[1839-1857]

72
315
GGGCUUCUGGGCAGACUCU
531
AGAGUCUGCCCAGAAGCCC
Rh
[2000-2018]

73
316
GGUACCUUCUCACCUGUGA
532
UCACAGGUGAGAAGGUACC
Rh
[1802-1820]

74
317
GCCUGCCUCAAUCAGUAUU
533
AAUACUGAUUGAGGCAGGC

[1769-1787]

75
318
UCUACAACUACUACGACGA
534
UCGUCGUAGUAGUUGUAGA
Rb
[954-972]

76
319
GGGAAGAUGCAGAAGAAGG
535
CCUUCUUCUGCAUCUUCCC
Rh, Rb, Rt
[1112-1130]

77
320
CGAGAAGGAAAAGCUGCAA
536
UUGCAGCUUUUCCUUCUCG
Rh
[973-991]

78
321
AGAAGAAGGCUGUUGCCAU
537
AUGGCAACAGCCUUCUUCU
Rt
[1122-1140]

79
322
CACAAGCUCUCCAGCCUCA
538
UGAGGCUGGAGAGCUUGUG
Rh, D, M, P
[1013-1031]

80
323
GGGUGACAAGAUGCGAGAC
539
GUCUCGCAUCUUGUCACCC
Rh, D
[1459-1477]

81
324
UGUUGGAGCGUGGAAAAAA
540
UUUUUUCCACGCUCCAACA

[2190-2208]

82
325
CUUUGAGUUGGACACAGAU
541
AUCUGUGUCCAACUCAAAG
Rh
[1297-1315]

83
326
AGCUCUCCAGCCUCAUCAU
542
AUGAUGAGGCUGGAGAGCU
Rh, D, Rt, M,
[1017-1035]

84
327
AGCUGUUCUACGCCGACCA
543
UGGUCGGCGUAGAACAGCU
Rh
[1368-1386]

85
328
CUGCAGUCCAUCAACGAGU
544
ACUCGUUGAUGGACUGCAG
Rh, Rt, M
[737-755]

86
329
UACGACGACGAGAAGGAAA
545
UUUCCUUCUCGUCGUCGUA

[965-983]

87
330
CCUAGUGCGGGACACCCAA
546
UUGGGUGUCCCGCACUAGG

[1399-1417]

88
331
CUUCUCACCUGUGAGACCA
547
UGGUCUCACAGGUGAGAAG
Rh
[1807-1825]

89
332
AGUUGGACACAGAUGGCAA
548
UUGCCAUCUGUGUCCAACU

[1302-1320]

90
333
CAGUGGAGAACAUCCUGGU
549
ACCAGGAUGUUCUCCACUG
Rh
[417-435]

91
334
CCAGCUAGAAUUCACUCCA
550
UGGAGUGAAUUCUAGCUGG
Rh
[1647-1665]

92
335
CGCUGCAGUCCAUCAACGA
551
UCGUUGAUGGACUGCAGCG
Rh, Rt
[735-753]

93
336
CCAAGGACCAGGCAGUGGA
552
UCCACUGCCUGGUCCUUGG
Rh
[405-423]

94
337
AGUUCUUCAAAGAUAGGGA
553
UCCCUAUCUUUGAAGAACU

[2082-2100]

95
338
CGGACCUUCCCAGCUAGAA
554
UUCUAGCUGGGAAGGUCCG
Rh
[1638-1656]

96
339
GACAAGAUGCGAGACGAGU
555
ACUCGUCUCGCAUCUUGUC
Rh, Rt
[1463-1481]

97
340
CCAAGAUCAACUUCCGCGA
556
UCGCGGAAGUUGAUCUUGG
D
[705-723]

98
341
CCCAUCACGUGGAGCCUCU
557
AGAGGCUCCACGUGAUGGG
Rh
[1044-1062]

99
342
CCAUGAUGCUGAGCCCGGA
558
UCCGGGCUCAGCAUCAUGG

[1687-1705]

100
343
AGCCUGCCUCAAUCAGUAU
559
AUACUGAUUGAGGCAGGCU

[1768-1786]

101
344
CGGCCUAAGGGUGACAAGA
560
UCUUGUCACCCUUAGGCCG
Rh
[1451-1469]

102
345
GGGCCUGACUGAGGCCAUU
561
AAUGGCCUCAGUCAGGCCC
Rt
[1201-1219]

103
346
UCACCUGUGAGACCAAAUU
562
AAUUUGGUCUCACAGGUGA
Rh
[1811-1829]

104
347
GAGGCCAUUGACAAGAACA
563
UGUUCUUGUCAAUGGCCUC
Rh, D
[1211-1229]

105
348
GCUCCUGGCACUGCGGAGA
564
UCUCCGCAGUGCCAGGAGC

[314-332]

106
349
GGCGCCUGGUCCGGCCUAA
565
UUAGGCCGGACCAGGCGCC
Rh
[1440-1458]

107
350
CCAGCCCUCUUCUGACACU
566
AGUGUCAGAAGAGGGCUGG

[1846-1864]

108
351
ACUACGACGACGAGAAGGA
567
UCCUUCUCGUCGUCGUAGU
Rb
[963-981]

109
352
CCUAUACCGUGGGUGUCAU
568
AUGACACCCACGGUAUAGG
Rh, D, P
[915-933]

110
353
GACCCAGCUCAGUGAGCUU
569
AAGCUCACUGAGCUGGGUC

[636-654]

111
354
UGGGUGUCAUGAUGAUGCA
570
UGCAUCAUCAUGACACCCA
Rh
[924-942]

112
355
CCAAGGGUGUGGUGGAGGU
571
ACCUCCACCACACCCUUGG
Rh, D
[1149-1167]

113
356
AGGUCACCAAGGACGUGGA
572
UCCACGUCCUUGGUGACCU
Rh, D
[789-807]

114
357
CCCUGGCCGCCGAGGUGAA
573
UUCACCUCGGCGGCCAGGG

[276-294]

115
358
AGCACUCCAAGAUCAACUU
574
AAGUUGAUCUUGGAGUGCU
Rh, D
[699-717]

116
359
CCUGGCACUGCGGAGAAGU
575
ACUUCUCCGCAGUGCCAGG

[317-335]

117
360
GAUGCAGAAGAAGGCUGUU
576
AACAGCCUUCUUCUGCAUC
Rh, Rt, M
[1117-1135]

118
361
CCCACAAGCUCUCCAGCCU
577
AGGCUGGAGAGCUUGUGGG
Rh, D, P
[1011-1029]

119
362
CUCUUCUGACACUAAAACA
578
UGUUUUAGUGUCAGAAGAG

[1852-1870]

120
363
ACGAGAAGGAAAAGCUGCA
579
UGCAGCUUUUCCUUCUCGU
Rh
[972-990]

121
364
UGAAAAGCUGCUAACCAAA
580
UUUGGUUAGCAGCUUUUCA

[1072-1090]

122
365
UCUCACCUGUGAGACCAAA
581
UUUGGUCUCACAGGUGAGA
Rh
[1809-1827]

123
366
CAUGAUGAUGCACCGGACA
582
UGUCCGGUGCAUCAUCAUG
Rh
[931-949]

124
367
GGAUUGAGAAGGAGCUCCC
583
GGGAGCUCCUUCUCAAUCC

[1976-1994]

125
368
CCUUCAUCUUCCUAGUGCG
584
CGCACUAGGAAGAUGAAGG

[1389-1407]

126
369
GGCCUGGCCUUCAGCUUGU
585
ACAAGCUGAAGGCCAGGCC

[374-392]

127
370
GGUCAGCCAGCCCUCUUCU
586
AGAAGAGGGCUGGCUGACC
Rh
[1840-1858]

128
371
UUCUCACCUGUGAGACCAA
587
UUGGUCUCACAGGUGAGAA
Rh
[1808-1826]

129
372
CGCAGCAGCUCCUGGCACU
588
AGUGCCAGGAGCUGCUGCG

[307-325]

130
373
GCCAUGUUCUUCAAGCCAC
589
GUGGCUUGAAGAACAUGGC
Rh, Rb, D
[836-854]

131
374
AGGCAGUGCUGAGCGCCGA
590
UCGGCGCUCAGCACUGCCU

[510-528]

132
375
CACCUGUGAGACCAAAUUG
591
CAAUUUGGUCUCACAGGUG
Rh
[1812-1830]

133
376
CACCGGACAGGCCUCUACA
592
UGUAGAGGCCUGUCCGGUG
Rh, Rb, Rt, P
[941-959]

134
377
AGCUAGAAUUCACUCCACU
593
AGUGGAGUGAAUUCUAGCU
Rh
[1649-1667]

135
378
AGAUGCAGAAGAAGGCUGU
594
ACAGCCUUCUUCUGCAUCU
Rh, Rt, M
[1116-1134]

136
379
CCCUGCUAGUCAACGCCAU
595
AUGGCGUUGACUAGCAGGG
Rh
[822-840]

137
380
ACAACUACUACGACGACGA
596
UCGUCGUCGUAGUAGUUGU
Rb
[957-975]

138
381
GCUCCUGAGACACAUGGGU
597
ACCCAUGUGUCUCAGGAGC
D
[1527-1545]

139
382
UGGAGAACAUCCUGGUGUC
598
GACACCAGGAUGUUCUCCA
Rh
[420-438]

140
383
AGCGCGCUGCAGUCCAUCA
599
UGAUGGACUGCAGCGCGCU
Rh, Rt
[731-749]

141
384
CGCCUUGAAAAGCUGCUAA
600
UUAGCAGCUUUUCAAGGCG

[1067-1085]

142
385
GCCUUUGUUGCUAUCAAUC
601
GAUUGAUAGCAACAAAGGC
Rh
[2118-2136]

143
386
CUCUACAACUACUACGACG
602
CGUCGUAGUAGUUGUAGAG
Rb
[953-971]

144
387
CGCUCACUCAGCAACUCCA
603
UGGAGUUGCUGAGUGAGCG
Rh
[575-593]

145
388
GGUACCAGCCUUGGAUACU
604
AGUAUCCAAGGCUGGUACC
Rh
[1571-1589]

146
389
GCCUGACUGAGGCCAUUGA
605
UCAAUGGCCUCAGUCAGGC
Rh
[1203-1221]

147
390
UGAGCUUCGCUGAUGACUU
606
AAGUCAUCAGCGAAGCUCA
Rh
[648-666]

148
391
CCAGCCUUGGAUACUCCAU
607
AUGGAGUAUCCAAGGCUGG
Rh
[1575-1593]

149
392
AAAGGCUCCUGAGACACAU
608
AUGUGUCUCAGGAGCCUUU

[1523-1541]

150
393
UGACCCAUGACCUGCAGAA
609
UUCUGCAGGUCAUGGGUCA
Rh, Rt, M
[1167-1185]

151
394
CCUGUGAGACCAAAUUGAG
610
CUCAAUUUGGUCUCACAGG
Rh
[1814-1832]

152
395
GCGGACCUUCCCAGCUAGA
611
UCUAGCUGGGAAGGUCCGC
Rh
[1637-1655]

153
396
GGAAGAUGCAGAAGAAGGC
612
GCCUUCUUCUGCAUCUUCC
Rh, Rb, Rt
[1113-1131]

154
397
UGCCCAAGGGUGUGGUGGA
613
UCCACCACACCCUUGGGCA
Rh, D
[1146-1164]

155
398
GGAGCCUCUCGAGCGCCUU
614
AAGGCGCUCGAGAGGCUCC

[1054-1072]

156
399
GACUCUGGUCAAGAAGCAU
615
AUGCUUCUUGACCAGAGUC
Rh
[2013-2031]

157
400
CAGGCAGUGGAGAACAUCC
616
GGAUGUUCUCCACUGCCUG
Rh
[413-431]

158
401
CAAGCCUGCCUCAAUCAGU
617
ACUGAUUGAGGCAGGCUUG
Rh
[1766-1784]

159
402
CUGGAAGCUGGGCAGCCGA
618
UCGGCUGCCCAGCUUCCAG

[610-628]

160
403
GAAGAAGGCUGUUGCCAUC
619
GAUGGCAACAGCCUUCUUC
Rt
[1123-1141]

161
404
GGGCGAGCUGCUGCGCUCA
620
UGAGCGCAGCAGCUCGCCC
Rh
[562-580]

162
405
AAGCCACACUGGGAUGAGA
621
UCUCAUCCCAGUGUGGCUU
Rh, Rb, M
[848-866]

163
406
GUGUGGUGGAGGUGACCCA
622
UGGGUCACCUCCACCACAC
Rh, D
[1155-1173]

164
407
CCGCCUUUGAGUUGGACAC
623
GUGUCCAACUCAAAGGCGG
Rh
[1293-1311]

165
408
GGCCAUUGACAAGAACAAG
624
CUUGUUCUUGUCAAUGGCC
Rh, D
[1213-1231]

166
409
UGCCUCAAUCAGUAUUCAU
625
AUGAAUACUGAUUGAGGCA

[1772-1790]

167
410
CCUUCCCAGCUAGAAUUCA
626
UGAAUUCUAGCUGGGAAGG
Rh
[1642-1660]

168
411
GGGACCUGGGCCAUAGUCA
627
UGACUAUGGCCCAGGUCCC

[1721-1739]

169
412
CGAGGUGAAGAAACCUGCA
628
UGCAGGUUUCUUCACCUCG
Rh
[286-304]

170
413
GCCUUUGAGUUGGACACAG
629
CUGUGUCCAACUCAAAGGC
Rh
[1295-1313]

171
414
AGCGGACCUUCCCAGCUAG
630
CUAGCUGGGAAGGUCCGCU
Rh
[1636-1654]

172
415
CGCAUGUCAGGCAAGAAGG
631
CCUUCUUGCCUGACAUGCG
Rh, D
[1244-1262]

173
416
ACAACUGCGAGCACUCCAA
632
UUGGAGUGCUCGCAGUUGU
Rh, D
[690-708]

174
417
GAGGCGGAUUGAGAAGGAG
633
CUCCUUCUCAAUCCGCCUC

[1971-1989]

175
418
GGCCGCCGAGGUGAAGAAA
634
UUUCUUCACCUCGGCGGCC

[280-298]

176
419
CAGCUCUAUCCCAACCUCU
635
AGAGGUUGGGAUAGAGCUG

[1886-1904]

177
420
AGCUGGGCAGCCGACUGUA
636
UACAGUCGGCUGCCCAGCU

[615-633]

178
421
GCCAUUGACAAGAACAAGG
637
CCUUGUUCUUGUCAAUGGC
Rh, D
[1214-1232]

179
422
CGCCAUGUUCUUCAAGCCA
638
UGGCUUGAAGAACAUGGCG
Rh, Rb, P
[835-853]

180
423
CCGAGGUCACCAAGGACGU
639
ACGUCCUUGGUGACCUCGG
Rh, D
[786-804]

181
424
GGACCCAGCUCAGUGAGCU
640
AGCUCACUGAGCUGGGUCC

[635-653]

182
425
CCAAUGACAUUUUGUUGGA
641
UCCAACAAAAUGUCAUUGG

[2178-2196]

183
426
AGUGAGGCGGAUUGAGAAG
642
CUUCUCAAUCCGCCUCACU

[1968-1986]

184
427
UGCAGUCCAUCAACGAGUG
643
CACUCGUUGAUGGACUGCA
Rh, Rt, M
[738-756]

185
428
UGUCACGCAUGUCAGGCAA
644
UUGCCUGACAUGCGUGACA
Rh, D
[1239-1257]

186
429
CGACGACGAGAAGGAAAAG
645
CUUUUCCUUCUCGUCGUCG

[967-985]

187
430
ACAAGAACAAGGCCGACUU
646
AAGUCGGCCUUGUUCUUGU
Rh
[1221-1239]

188
431
CUUCAAGCCACACUGGGAU
647
AUCCCAGUGUGGCUUGAAG
Rh, Rb, D
[844-862]

189
432
CCUGGGCCAUAGUCAUUCU
648
AGAAUGACUAUGGCCCAGG

[1725-1743]

190
433
UUUGUUGGAGCGUGGAAAA
649
UUUUCCACGCUCCAACAAA

[2188-2206]

191
434
AGAACAUCCUGGUGUCACC
650
GGUGACACCAGGAUGUUCU

[423-441]

192
435
ACGCCACCGCCUUUGAGUU
651
AACUCAAAGGCGGUGGCGU
Rh
[1287-1305]

193
436
GUGAGGUACCAGCCUUGGA
652
UCCAAGGCUGGUACCUCAC
Rh
[1567-1585]

194
437
GCGCCUUCUGCCUCCUGGA
653
UCCAGGAGGCAGAAGGCGC

[252-270]

195
438
GCCUGGCCUUCAGCUUGUA
654
UACAAGCUGAAGGCCAGGC

[375-393]

196
439
CCCGGAAACUCCACAUCCU
655
AGGAUGUGGAGUUUCCGGG

[1700-1718]

197
440
UCUUCAAGCCACACUGGGA
656
UCCCAGUGUGGCUUGAAGA
Rh, Rb, D
[843-861]

198
441
UGUUGCUAUCAAUCCAAGA
657
UCUUGGAUUGAUAGCAACA
Rh
[2123-2141]

199
442
GAGUGGGCCGCGCAGACCA
658
UGGUCUGCGCGGCCCACUC

[752-770]

200
443
CCUGAGACACAUGGGUGCU
659
AGCACCCAUGUGUCUCAGG
D, Rt, M
[1530-1548]

201
444
AGCCGACUGUACGGACCCA
660
UGGGUCCGUACAGUCGGCU

[623-641]

202
445
GGGCCUCAGGGUGCACACA
661
UGUGUGCACCCUGAGGCCC

[1486-1504]

203
446
ACUGGGAUGAGAAAUUCCA
662
UGGAAUUUCUCAUCCCAGU
Rh
[855-873]

204
447
AGAAUGACCUGGCCGCAGU
663
ACUGCGGCCAGGUCAUUCU

[1952-1970]

205
448
CAUAUUUAUAGCCAGGUAC
664
GUACCUGGCUAUAAAUAUG
Rh
[1788-1806]

206
449
AGGUGACCCAUGACCUGCA
665
UGCAGGUCAUGGGUCACCU
Rh, Rt, M
[1164-1182]

207
450
GCGCUGCAGUCCAUCAACG
666
CGUUGAUGGACUGCAGCGC
Rh, Rt
[734-752]

208
451
GGUGACAAGAUGCGAGACG
667
CGUCUCGCAUCUUGUCACC
Rh
[1460-1478]

209
452
CUUCAAAGAUAGGGAGGGA
668
UCCCUCCCUAUCUUUGAAG

[2086-2104]

210
453
AGCUGCAAAUCGUGGAGAU
669
AUCUCCACGAUUUGCAGCU
Rh
[984-1002]

211
454
GUGGAGAACAUCCUGGUGU
670
ACACCAGGAUGUUCUCCAC
Rh
[419-437]

212
455
GAACAAGGCCGACUUGUCA
671
UGACAAGUCGGCCUUGUUC
Rh
[1225-1243]

213
456
CAUGAUGCUGAGCCCGGAA
672
UUCCGGGCUCAGCAUCAUG

[1688-1706]

214
457
GCGCCUUGAAAAGCUGCUA
673
UAGCAGCUUUUCAAGGCGC
Rh
[1066-1084]

215
458
GCAGACUCUGGUCAAGAAG
674
CUUCUUGACCAGAGUCUGC
Rh
[2010-2028]

216
459
CCAGGCAGUGGAGAACAUC
675
GAUGUUCUCCACUGCCUGG
Rh
[412-430]

TABLE C

Cross-Species 19-mer SERPINH1 siRNAs

No.
SEQ ID SEN
Sense siRNA
SEQ ID AS
AntiSense siRNA
Other Species
human-

1
676
CACUACAACUGCGAGCACU
973
AGUGCUCGCAGUUGUAGUG
Rh, D
[686-704]

2
677
AACCGUGGCUUCAUGGUGA
974
UCACCAUGAAGCCACGGUU
Rh, Rt, M
[890-908]

3
678
GGCAAGAAGGACCUGUACC
975
GGUACAGGUCCUUCUUGCC
Rh, D, M
[1253-

4
679
GGUGGACAACCGUGGCUUC
976
GAAGCCACGGUUGUCCACC
Rh, M
[883-901]

5
680
AGGCCAUGGCCAAGGACCA
977
UGGUCCUUGGCCAUGGCCU
Rh, D
[396-414]

6
681
CGCAGCGCGCUGCAGUCCA
978
UGGACUGCAGCGCGCUGCG
Rh, Rt
[728-746]

7
682
AGCAGCAAGCAGCACUACA
979
UGUAGUGCUGCUUGCUGCU
Rh, D
[674-692]

8
683
GGCCUCUACAACUACUACG
980
CGUAGUAGUUGUAGAGGCC
Rb, D
[950-968]

9
684
GAAGAUGCAGAAGAAGGCU
981
AGCCUUCUUCUGCAUCUUC
Rh, Rb, Rt
[1114-

10
685
GGCUCCUGAGACACAUGGG
982
CCCAUGUGUCUCAGGAGCC
D
[1526-

11
686
AGCAAGCAGCACUACAACU
983
AGUUGUAGUGCUGCUUGCU
Rh, D
[677-695]

12
687
GGAGGUGACCCAUGACCUG
984
CAGGUCAUGGGUCACCUCC
Rh, Rt, M
[1162-

13
688
CCCUUUGACCAGGACAUCU
985
AGAUGUCCUGGUCAAAGGG
Rh, Rt
[1322-

14
689
CUCCUGAGACACAUGGGUG
986
CACCCAUGUGUCUCAGGAG
D
[1528-

15
690
AAGGCUCCUGAGACACAUG
987
CAUGUGUCUCAGGAGCCUU
D
[1524-

16
691
CGCGCUGCAGUCCAUCAAC
988
GUUGAUGGACUGCAGCGCG
Rh, Rt
[733-751]

17
692
AGGGUGUGGUGGAGGUGAC
989
GUCACCUCCACCACACCCU
Rh, D
[1152-

18
693
AGCACUACAACUGCGAGCA
990
UGCUCGCAGUUGUAGUGCU
Rh, D
[684-702]

19
694
GGCUCCCUGCUAUUCAUUG
991
CAAUGAAUAGCAGGGAGCC
D
[1421-

20
695
GCGCGCAACGUGACCUGGA
992
UCCAGGUCACGUUGCGCGC
M
[596-614]

21
696
GCUGCAGUCCAUCAACGAG
993
CUCGUUGAUGGACUGCAGC
Rh, Rt
[736-754]

22
697
ACCAAAGAGCAGCUGAAGA
994
UCUUCAGCUGCUCUUUGGU
Rh, Rb, P
[1085-

23
698
CCAAGGACGUGGAGCGCAC
995
GUGCGCUCCACGUCCUUGG
Rh, D
[795-813]

24
699
UGUUCUUCAAGCCACACUG
996
CAGUGUGGCUUGAAGAACA
Rh, Rb, D
[840-858]

25
700
GCCCAAGGGUGUGGUGGAG
997
CUCCACCACACCCUUGGGC
Rh, D
[1147-

26
701
ACAGGCCUCUACAACUACU
998
AGUAGUUGUAGAGGCCUGU
Rh, Rb, D, Rt, P
[947-965]

27
702
UGCGCAGCAGCAAGCAGCA
999
UGCUGCUUGCUGCUGCGCA
Rh, D
[669-687]

28
703
GGUGGAGGUGACCCAUGAC
1000
GUCAUGGGUCACCUCCACC
Rh, Rt, M
[1159-

29
704
CUUUGACCAGGACAUCUAC
1001
GUAGAUGUCCUGGUCAAAG
Rh, Rt
[1324-

30
705
AAGGGUGUGGUGGAGGUGA
1002
UCACCUCCACCACACCCUU
Rh, D
[1151-

31
706
UCCUAUACCGUGGGUGUCA
1003
UGACACCCACGGUAUAGGA
Rh, D, P
[914-932]

32
707
GCGCAGACCACCGACGGCA
1004
UGCCGUCGGUGGUCUGCGC
D
[761-779]

33
708
CGCAGCAGCAAGCAGCACU
1005
AGUGCUGCUUGCUGCUGCG
Rh, D
[671-689]

34
709
GCCUCAUCAUCCUCAUGCC
1006
GGCAUGAGGAUGAUGAGGC
Rh, D, Rt, M
[1026-

35
710
UCUCCAGCCUCAUCAUCCU
1007
AGGAUGAUGAGGCUGGAGA
Rh, D, Rt, M
[1020-

36
711
CCAUUGACAAGAACAAGGC
1008
GCCUUGUUCUUGUCAAUGG
Rh, D
[1215-

37
712
AGCAGCACUACAACUGCGA
1009
UCGCAGUUGUAGUGCUGCU
Rh, D
[681-699]

38
713
UGCACCGGACAGGCCUCUA
1010
UAGAGGCCUGUCCGGUGCA
Rh, Rb, Rt, P
[939-957]

39
714
ACUCCAAGAUCAACUUCCG
1011
CGGAAGUUGAUCUUGGAGU
Rh, D, Rt, M
[702-720]

40
715
UGGACAACCGUGGCUUCAU
1012
AUGAAGCCACGGUUGUCCA
Rh, M
[885-903]

41
716
GAGCAGCUGAAGAUCUGGA
1013
UCCAGAUCUUCAGCUGCUC
Rh, D
[1091-

42
717
CAGAAGAAGGCUGUUGCCA
1014
UGGCAACAGCCUUCUUCUG
Rt
[1121-

43
718
AGGCAAGAAGGACCUGUAC
1015
GUACAGGUCCUUCUUGCCU
Rh, D
[1252-

44
719
CCUCUACAACUACUACGAC
1016
GUCGUAGUAGUUGUAGAGG
Rb, D
[952-970]

45
720
AGCAGCUGAAGAUCUGGAU
1017
AUCCAGAUCUUCAGCUGCU
Rh, D
[1092-

46
721
AACUACUACGACGACGAGA
1018
UCUCGUCGUCGUAGUAGUU
Rb
[959-977]

47
722
GGCAAGCUGCCCGAGGUCA
1019
UGACCUCGGGCAGCUUGCC
Rh, D
[776-794]

48
723
CCGGACAGGCCUCUACAAC
1020
GUUGUAGAGGCCUGUCCGG
Rh, Rb, Rt, P
[943-961]

49
724
GCUCCCUGCUAUUCAUUGG
1021
CCAAUGAAUAGCAGGGAGC
D
[1422-

50
725
AACUGCGAGCACUCCAAGA
1022
UCUUGGAGUGCUCGCAGUU
Rh, D
[692-710]

51
726
GACACAUGGGUGCUAUUGG
1023
CCAAUAGCACCCAUGUGUC
Rh, Rt, M
[1535-

52
727
GCACCGGACAGGCCUCUAC
1024
GUAGAGGCCUGUCCGGUGC
Rh, Rb, Rt, P
[940-958]

53
728
AGCGCAGCGCGCUGCAGUC
1025
GACUGCAGCGCGCUGCGCU
Rh, Rt
[726-744]

54
729
GGACGUGGAGCGCACGGAC
1026
GUCCGUGCGCUCCACGUCC
Rh, D
[799-817]

55
730
CAGCCUCAUCAUCCUCAUG
1027
CAUGAGGAUGAUGAGGCUG
Rh, D, Rt, M
[1024-

56
731
AAGAUCAACUUCCGCGACA
1028
UGUCGCGGAAGUUGAUCUU
D
[707-725]

57
732
GCGCAACGUGACCUGGAAG
1029
CUUCCAGGUCACGUUGCGC
M
[598-616]

58
733
ACUGCGAGCACUCCAAGAU
1030
AUCUUGGAGUGCUCGCAGU
Rh, D
[693-711]

59
734
GUGGACAACCGUGGCUUCA
1031
UGAAGCCACGGUUGUCCAC
Rh, M
[884-902]

60
735
CCACAAGCUCUCCAGCCUC
1032
GAGGCUGGAGAGCUUGUGG
Rh, D, P
[1012-

61
736
CAAGAUGGUGGACAACCGU
1033
ACGGUUGUCCACCAUCUUG
Rh, Rb, M, P
[877-895]

62
737
CGAGCACUCCAAGAUCAAC
1034
GUUGAUCUUGGAGUGCUCG
Rh, D
[697-715]

63
738
AGCUGCCCGAGGUCACCAA
1035
UUGGUGACCUCGGGCAGCU
Rh, D
[780-798]

64
739
GGACAUCUACGGGCGCGAG
1036
CUCGCGCCCGUAGAUGUCC
D
[1333-

65
740
AGGACAUCUACGGGCGCGA
1037
UCGCGCCCGUAGAUGUCCU
D
[1332-

66
741
UGUCAGGCAAGAAGGACCU
1038
AGGUCCUUCUUGCCUGACA
Rh, D
[1248-

67
742
GGGUGUGGUGGAGGUGACC
1039
GGUCACCUCCACCACACCC
Rh, D
[1153-

68
743
CAAGCUCUCCAGCCUCAUC
1040
GAUGAGGCUGGAGAGCUUG
Rh, D, M, P
[1015-

69
744
GUGACCCAUGACCUGCAGA
1041
UCUGCAGGUCAUGGGUCAC
Rh, Rt, M
[1166-

70
745
GUUCUUCAAGCCACACUGG
1042
CCAGUGUGGCUUGAAGAAC
Rh, Rb, D
[841-859]

71
746
ACAUCUACGGGCGCGAGGA
1043
UCCUCGCGCCCGUAGAUGU
D, M
[1335-

72
747
UGGAGGUGACCCAUGACCU
1044
AGGUCAUGGGUCACCUCCA
Rh, Rt, M
[1161-

73
748
UGCAGAAGAAGGCUGUUGC
1045
GCAACAGCCUUCUUCUGCA
Rt
[1119-

74
749
UGUACCAGGCCAUGGCCAA
1046
UUGGCCAUGGCCUGGUACA
Rh, D
[390-408]

75
750
UGUGGUGGAGGUGACCCAU
1047
AUGGGUCACCUCCACCACA
Rh, D
[1156-

76
751
AGAAGGACCUGUACCUGGC
1048
GCCAGGUACAGGUCCUUCU
Rh, D
[1257-

77
752
AGCAGCUGCGCGACGAGGA
1049
UCCUCGUCGCGCAGCUGCU
Rh, D
[528-546]

78
753
ACGCCAUGUUCUUCAAGCC
1050
GGCUUGAAGAACAUGGCGU
Rh, Rb, P
[834-852]

79
754
ACAAGAUGGUGGACAACCG
1051
CGGUUGUCCACCAUCUUGU
Rh, Rb, M, P
[876-894]

80
755
CUGCGAGCACUCCAAGAUC
1052
GAUCUUGGAGUGCUCGCAG
Rh, D
[694-712]

81
756
GUCACGCAUGUCAGGCAAG
1053
CUUGCCUGACAUGCGUGAC
Rh, D
[1240-

82
757
ACGCAUGUCAGGCAAGAAG
1054
CUUCUUGCCUGACAUGCGU
Rh, D
[1243-

83
758
UGCUAUUCAUUGGGCGCCU
1055
AGGCGCCCAAUGAAUAGCA
D
[1428-

84
759
UGCGCGACGAGGAGGUGCA
1056
UGCACCUCCUCGUCGCGCA
Rh, D
[534-552]

85
760
GCAGCUGAAGAUCUGGAUG
1057
CAUCCAGAUCUUCAGCUGC
Rh, D
[1093-

86
761
CCAUGACCUGCAGAAACAC
1058
GUGUUUCUGCAGGUCAUGG
Rh, Rt, M
[1171-

87
762
AAGCUCUCCAGCCUCAUCA
1059
UGAUGAGGCUGGAGAGCUU
Rh, D, Rt, M, P
[1016-

88
763
CAGCAAGCAGCACUACAAC
1060
GUUGUAGUGCUGCUUGCUG
Rh, D
[676-694]

89
764
AUGUUCUUCAAGCCACACU
1061
AGUGUGGCUUGAAGAACAU
Rh, Rb, D
[839-857]

90
765
UCCUGAGACACAUGGGUGC
1062
GCACCCAUGUGUCUCAGGA
D, Rt, M
[1529-

91
766
CACUCCAAGAUCAACUUCC
1063
GGAAGUUGAUCUUGGAGUG
Rh, D, Rt, M
[701-719]

92
767
AAGGGUGACAAGAUGCGAG
1064
CUCGCAUCUUGUCACCCUU
Rh, D
[1457-

93
768
GACAGGCCUCUACAACUAC
1065
GUAGUUGUAGAGGCCUGUC
Rh, Rb, Rt, P
[946-964]

94
769
ACCCAUGACCUGCAGAAAC
1066
GUUUCUGCAGGUCAUGGGU
Rh, Rt, M
[1169-

95
770
CACCACAAGAUGGUGGACA
1067
UGUCCACCAUCUUGUGGUG
Rh, Rb, M, P
[872-890]

96
771
GCAGAAGAAGGCUGUUGCC
1068
GGCAACAGCCUUCUUCUGC
Rt
[1120-

97
772
GUGGUGGAGGUGACCCAUG
1069
CAUGGGUCACCUCCACCAC
Rh, Rb, Rt, M
[1157-

98
773
AGGCCUCUACAACUACUAC
1070
GUAGUAGUUGUAGAGGCCU
Rh, Rb, D, Rt, P
[949-967]

99
774
GGUGACCCAUGACCUGCAG
1071
CUGCAGGUCAUGGGUCACC
Rh, Rt, M
[1165-

100
775
GCCGAGGUGAAGAAACCUG
1072
CAGGUUUCUUCACCUCGGC
Rh, Rt
[284-302]

101
776
CAACUACUACGACGACGAG
1073
CUCGUCGUCGUAGUAGUUG
Rb
[958-976]

102
777
CAAGAAGGACCUGUACCUG
1074
CAGGUACAGGUCCUUCUUG
Rh, D, M
[1255-

103
778
UGUUCCACGCCACCGCCUU
1075
AAGGCGGUGGCGUGGAACA
D
[1281-

104
779
CCCUGCUAUUCAUUGGGCG
1076
CGCCCAAUGAAUAGCAGGG
D
[1425-

105
780
CCGUGGCUUCAUGGUGACU
1077
AGUCACCAUGAAGCCACGG
Rh, Rt, M
[892-910]

106
781
CUACAACUACUACGACGAC
1078
GUCGUCGUAGUAGUUGUAG
Rb
[955-973]

107
782
GCAGCACUACAACUGCGAG
1079
CUCGCAGUUGUAGUGCUGC
Rh, D
[682-700]

108
783
UGGUGGACAACCGUGGCUU
1080
AAGCCACGGUUGUCCACCA
Rh, M
[882-900]

109
784
AGACCACCGACGGCAAGCU
1081
AGCUUGCCGUCGGUGGUCU
D, Rt
[765-783]

110
785
AGAAACACCUGGCUGGGCU
1082
AGCCCAGCCAGGUGUUUCU
D
[1182-

111
786
ACCAAGGACGUGGAGCGCA
1083
UGCGCUCCACGUCCUUGGU
Rh, D
[794-812]

112
787
CCGAGGUGAAGAAACCUGC
1084
GCAGGUUUCUUCACCUCGG
Rh, Rt
[285-303]

113
788
ACUACAACUGCGAGCACUC
1085
GAGUGCUCGCAGUUGUAGU
Rh, D
[687-705]

114
789
ACAAGCUCUCCAGCCUCAU
1086
AUGAGGCUGGAGAGCUUGU
Rh, D, M, P
[1014-

115
790
AGGACGUGGAGCGCACGGA
1087
UCCGUGCGCUCCACGUCCU
Rh, D
[798-816]

116
791
GCUAUUCAUUGGGCGCCUG
1088
CAGGCGCCCAAUGAAUAGC
D
[1429-

117
792
AACUUCCGCGACAAGCGCA
1089
UGCGCUUGUCGCGGAAGUU
D
[713-731]

118
793
GCUCUCCAGCCUCAUCAUC
1090
GAUGAUGAGGCUGGAGAGC
Rh, D, Rt, M, P
[1018-

119
794
AGAAGGCUGUUGCCAUCUC
1091
GAGAUGGCAACAGCCUUCU
Rt
[1125-

120
795
GGUCACCAAGGACGUGGAG
1092
CUCCACGUCCUUGGUGACC
Rh, D
[790-808]

121
796
AGCUGCGCGACGAGGAGGU
1093
ACCUCCUCGUCGCGCAGCU
Rh, D
[531-549]

122
797
CCCGAGGUCACCAAGGACG
1094
CGUCCUUGGUGACCUCGGG
Rh, D
[785-803]

123
798
AUGUCAGGCAAGAAGGACC
1095
GGUCCUUCUUGCCUGACAU
Rh, D
[1247-

124
799
CGAGGUCACCAAGGACGUG
1096
CACGUCCUUGGUGACCUCG
Rh, D
[787-805]

125
800
GAUGCACCGGACAGGCCUC
1097
GAGGCCUGUCCGGUGCAUC
Rh, Rb, Rt, M, P
[937-955]

126
801
GCACUACAACUGCGAGCAC
1098
GUGCUCGCAGUUGUAGUGC
Rh, D
[685-703]

127
802
CCACAAGAUGGUGGACAAC
1099
GUUGUCCACCAUCUUGUGG
Rh, Rb, M, P
[874-892]

128
803
CAAGGGUGUGGUGGAGGUG
1100
CACCUCCACCACACCCUUG
Rh, D
[1150-

129
804
AGCUGAAGAUCUGGAUGGG
1101
CCCAUCCAGAUCUUCAGCU
Rh, D
[1095-

130
805
ACCAGGCCAUGGCCAAGGA
1102
UCCUUGGCCAUGGCCUGGU
Rh, D
[393-411]

131
806
CAUGUUCUUCAAGCCACAC
1103
GUGUGGCUUGAAGAACAUG
Rh, Rb, D
[838-856]

132
807
CAAGAUCAACUUCCGCGAC
1104
GUCGCGGAAGUUGAUCUUG
D
[706-724]

133
808
UCCAGCCUCAUCAUCCUCA
1105
UGAGGAUGAUGAGGCUGGA
Rh, D, Rt, M
[1022-

134
809
GCCCGAGGUCACCAAGGAC
1106
GUCCUUGGUGACCUCGGGC
Rh, D
[784-802]

135
810
UCAAGCCACACUGGGAUGA
1107
UCAUCCCAGUGUGGCUUGA
Rh, Rb
[846-864]

136
811
AGUCCAUCAACGAGUGGGC
1108
GCCCACUCGUUGAUGGACU
Rh, Rt, M
[741-759]

137
812
GACUUCGUGCGCAGCAGCA
1109
UGCUGCUGCGCACGAAGUC
Rh, D, M
[662-680]

138
813
CUCUCCAGCCUCAUCAUCC
1110
GGAUGAUGAGGCUGGAGAG
Rh, D, Rt, M, P
[1019-

139
814
GCAGACCACCGACGGCAAG
1111
CUUGCCGUCGGUGGUCUGC
D, Rt
[763-781]

140
815
AUGCAGAAGAAGGCUGUUG
1112
CAACAGCCUUCUUCUGCAU
Rt
[1118-

141
816
CAACCGUGGCUUCAUGGUG
1113
CACCAUGAAGCCACGGUUG
Rh, Rt, M
[889-907]

142
817
UACUACGACGACGAGAAGG
1114
CCUUCUCGUCGUCGUAGUA
Rb
[962-980]

143
818
GAAGGCUGUUGCCAUCUCC
1115
GGAGAUGGCAACAGCCUUC
Rt
[1126-

144
819
UCACCAAGGACGUGGAGCG
1116
CGCUCCACGUCCUUGGUGA
Rh, D
[792-810]

145
820
CAGCUGAAGAUCUGGAUGG
1117
CCAUCCAGAUCUUCAGCUG
Rh, D
[1094-

146
821
UGGGCCUGACUGAGGCCAU
1118
AUGGCCUCAGUCAGGCCCA
Rt
[1200-

147
822
ACCGUGGCUUCAUGGUGAC
1119
GUCACCAUGAAGCCACGGU
Rh, Rt, M
[891-909]

148
823
CAGUCCAUCAACGAGUGGG
1120
CCCACUCGUUGAUGGACUG
Rh, Rt, M
[740-758]

149
824
CCGACGGCAAGCUGCCCGA
1121
UCGGGCAGCUUGCCGUCGG
D
[771-789]

150
825
ACAAGCGCAGCGCGCUGCA
1122
UGCAGCGCGCUGCGCUUGU
Rh, Rt
[723-741]

151
826
GAAACACCUGGCUGGGCUG
1123
CAGCCCAGCCAGGUGUUUC
D
[1183-

152
827
AGGCUCCUGAGACACAUGG
1124
CCAUGUGUCUCAGGAGCCU
D
[1525-

153
828
CAAGGACGUGGAGCGCACG
1125
CGUGCGCUCCACGUCCUUG
Rh, D
[796-814]

154
829
GCAGUCCAUCAACGAGUGG
1126
CCACUCGUUGAUGGACUGC
Rh, Rt, M
[739-757]

155
830
AGAUGGUGGACAACCGUGG
1127
CCACGGUUGUCCACCAUCU
Rh, M
[879-897]

156
831
AAGCGCAGCGCGCUGCAGU
1128
ACUGCAGCGCGCUGCGCUU
Rh, Rt
[725-743]

157
832
CAUGUCAGGCAAGAAGGAC
1129
GUCCUUCUUGCCUGACAUG
Rh, D
[1246-

158
833
CAAGCCACACUGGGAUGAG
1130
CUCAUCCCAGUGUGGCUUG
Rh, Rb
[847-865]

159
834
AAGAUGCAGAAGAAGGCUG
1131
CAGCCUUCUUCUGCAUCUU
Rh, Rt, M
[1115-

160
835
GGCCAUGGCCAAGGACCAG
1132
CUGGUCCUUGGCCAUGGCC
Rh, D
[397-415]

161
836
GUGCGCAGCAGCAAGCAGC
1133
GCUGCUUGCUGCUGCGCAC
Rh, D
[668-686]

162
837
CAACUGCGAGCACUCCAAG
1134
CUUGGAGUGCUCGCAGUUG
Rh, D
[691-709]

163
838
UACAACUGCGAGCACUCCA
1135
UGGAGUGCUCGCAGUUGUA
Rh, D
[689-707]

164
839
CAUUGACAAGAACAAGGCC
1136
GGCCUUGUUCUUGUCAAUG
Rh, D
[1216-

165
840
CAAGCAGCACUACAACUGC
1137
GCAGUUGUAGUGCUGCUUG
Rh, D
[679-697]

166
841
GUGUUCCACGCCACCGCCU
1138
AGGCGGUGGCGUGGAACAC
D
[1280-

167
842
CCUGCUAUUCAUUGGGCGC
1139
GCGCCCAAUGAAUAGCAGG
D
[1426-

168
843
GCCCACAAGCUCUCCAGCC
1140
GGCUGGAGAGCUUGUGGGC
Rh, D, P
[1010-

169
844
CAGCAGCAAGCAGCACUAC
1141
GUAGUGCUGCUUGCUGCUG
Rh, D
[673-691]

170
845
UGAUGCACCGGACAGGCCU
1142
AGGCCUGUCCGGUGCAUCA
Rh, Rb, Rt, M, P
[936-954]

171
846
UCAACUUCCGCGACAAGCG
1143
CGCUUGUCGCGGAAGUUGA
D
[711-729]

172
847
UCAGGCAAGAAGGACCUGU
1144
ACAGGUCCUUCUUGCCUGA
Rh, D
[1250-

173
848
ACUUCGUGCGCAGCAGCAA
1145
UUGCUGCUGCGCACGAAGU
Rh, D, M
[663-681]

174
849
ACAACCGUGGCUUCAUGGU
1146
ACCAUGAAGCCACGGUUGU
Rh, Rt, M
[888-906]

175
850
AAGGCUGUUGCCAUCUCCU
1147
AGGAGAUGGCAACAGCCUU
D, Rt
[1127-

176
851
GCAGCUGCGCGAGGAGGAG
1148
CUCCUCGUCGCGCAGCUGC
Rh, D
[529-547]

177
852
UAUUCAUUGGGCGCCUGGU
1149
ACCAGGCGCCCAAUGAAUA
D
[1431-

178
853
UCCACCACAAGAUGGUGGA
1150
UCCACCAUCUUGUGGUGGA
Rh, Rb, D, P
[870-888]

179
854
CCCUGGCCCACAAGCUCUC
1151
GAGAGCUUGUGGGCCAGGG
Rh, D, P
[1005-

180
855
ACCAGGACAUCUACGGGCG
1152
CGCCCGUAGAUGUCCUGGU
D, Rt
[1329-

181
856
GAUGAUGCACCGGACAGGC
1153
GCCUGUCCGGUGCAUCAUC
Rh, Rb, Rt, M
[934-952]

182
857
CAACGCCAUGUUCUUCAAG
1154
CUUGAAGAACAUGGCGUUG
Rh, Rb, P
[832-850]

183
858
ACGGCAAGCUGCCCGAGGU
1155
ACCUCGGGCAGCUUGCCGU
Rh, D
[774-792]

184
859
CAGCGCGCUGCAGUCCAUC
1156
GAUGGACUGCAGCGCGCUG
Rh, Rt
[730-748]

185
860
CCCAAGGGUGUGGUGGAGG
1157
CCUCCACCACACCCUUGGG
Rh, D
[1148-

186
861
CAUGGCCAAGGACCAGGCA
1158
UGCCUGGUCCUUGGCCAUG
Rh, D
[400-418]

187
862
CUCCAGCCUCAUCAUCCUC
1159
GAGGAUGAUGAGGCUGGAG
Rh, D, Rt, M
[1021-

188
863
UCUACGGGCGCGAGGAGCU
1160
AGCUCCUCGCGCCCGUAGA
D, M
[1338-

189
864
GGCCCACAAGCUCUCCAGC
1161
GCUGGAGAGCUUGUGGGCC
Rh, D, P
[1009-

190
865
GUCAGGCAAGAAGGACCUG
1162
CAGGUCCUUCUUGCCUGAC
Rh, D
[1249-

191
866
CAUCUACGGGCGCGAGGAG
1163
CUCCUCGCGCCCGUAGAUG
D, M
[1336-

192
867
CGUGCGCAGCAGCAAGCAG
1164
CUGCUUGCUGCUGCGCACG
Rh, D, M
[667-685]

193
868
AGCCUCAUCAUCCUCAUGC
1165
GCAUGAGGAUGAUGAGGCU
Rh, D, Rt, M
[1025-

194
869
UUCAAGCCACACUGGGAUG
1166
CAUCCCAGUGUGGCUUGAA
Rh, Rb
[845-863]

195
870
AAGAAGGCUGUUGCCAUCU
1167
AGAUGGCAACAGCCUUCUU
Rt
[1124-

196
871
GGUGUGGUGGAGGUGACCC
1168
GGGUCACCUCCACCACACC
Rh, D
[1154-

197
872
GAGGUGACCCAUGACCUGC
1169
GCAGGUCAUGGGUCACCUC
Rh, Rt, M
[1163-

198
873
GUGGAGGUGACCCAUGACC
1170
GGUCAUGGGUCACCUCCAC
Rh, Rt, M
[1160-

199
874
CACAAGAUGGUGGACAACC
1171
GGUUGUCCACCAUCUUGUG
Rh, Rb, M, P
[875-893]

200
875
CUGGCCCACAAGCUCUCCA
1172
UGGAGAGCUUGUGGGCCAG
Rh, D, P
[1007-

201
876
GAUGACUUCGUGCGCAGCA
1173
UGCUGCGCACGAAGUCAUC
Rh, Rt, M
[659-677]

202
877
ACUUCCGCGACAAGCGCAG
1174
CUGCGCUUGUCGCGGAAGU
D
[714-732]

203
878
AACGCCAUGUUCUUCAAGC
1175
GCUUGAAGAACAUGGCGUU
Rh, Rb, P
[833-851]

204
879
GGACCUGUACCUGGCCAGC
1176
GCUGGCCAGGUACAGGUCC
Rh, D
[1261-

205
880
GCGACGAGGAGGUGCACGC
1177
GCGUGCACCUCCUCGUCGC
D
[537-555]

206
881
GCAAGCUGCCCGAGGUCAC
1178
GUGACCUCGGGCAGCUUGC
Rh, D
[777-795]

207
882
AUUCAUUGGGCGCCUGGUC
1179
GACCAGGCGCCCAAUGAAU
D
[1432-

208
883
GAGGUCACCAAGGACGUGG
1180
CCACGUCCUUGGUGACCUC
Rh, D
[788-806]

209
884
AAGAAGGACCUGUACCUGG
1181
CCAGGUACAGGUCCUUCUU
Rh, D
[1256-

210
885
GACAACCGUGGCUUCAUGG
1182
CCAUGAAGCCACGGUUGUC
Rh, Rt, M
[887-905]

211
886
CUGGGCCUGACUGAGGCCA
1183
UGGCCUCAGUCAGGCCCAG
Rt
[1199-

212
887
CUCCAAGAUCAACUUCCGC
1184
GCGGAAGUUGAUCUUGGAG
Rh, D, Rt, M
[703-721]

213
888
CAACUUCCGCGACAAGCGC
1185
GCGCUUGUCGCGGAAGUUG
D
[712-730]

214
889
CUCCCUGCUAUUCAUUGGG
1186
CCCAAUGAAUAGCAGGGAG
D
[1423-

215
890
AAGCAGCACUACAACUGCG
1187
CGCAGUUGUAGUGCUGCUU
Rh, D
[680-698]

216
891
GCGCAGCAGCAAGCAGCAC
1188
GUGCUGCUUGCUGCUGCGC
Rh, D
[670-688]

217
892
CAGGCCAUGGCCAAGGACC
1189
GGUCCUUGGCCAUGGCCUG
Rh, D
[395-413]

218
893
GUACCAGGCCAUGGCCAAG
1190
CUUGGCCAUGGCCUGGUAC
Rh, D
[391-409]

219
894
CUUCGUGCGCAGCAGCAAG
1191
CUUGCUGCUGCGCACGAAG
Rh, D, M
[664-682]

220
895
CAGCACUACAACUGCGAGC
1192
GCUCGCAGUUGUAGUGCUG
Rh, D
[683-701]

221
896
UACAACUACUACGACGACG
1193
CGUCGUCGUAGUAGUUGUA
Rb
[956-974]

222
897
GAUGGUGGACAACCGUGGC
1194
GCCACGGUUGUCCACCAUC
Rh, M
[880-898]

223
898
CUACAACUGCGAGCACUCC
1195
GGAGUGCUCGCAGUUGUAG
Rh, D
[688-706]

224
899
AAGGACCUGUACCUGGCCA
1196
UGGCCAGGUACAGGUCCUU
Rh, D
[1259-

225
900
GCUGCCCGAGGUCACCAAG
1197
CUUGGUGACCUCGGGCAGC
Rh, D
[781-799]

226
901
GACAUCUACGGGCGCGAGG
1198
CCUCGCGCCCGUAGAUGUC
D, M
[1334-

227
902
CCACCACAAGAUGGUGGAC
1199
GUCCACCAUCUUGUGGUGG
Rh, Rb, D, P
[871-889]

228
903
GCGCGACGAGGAGGUGCAC
1200
GUGCACCUCCUCGUCGCGC
Rh, D
[535-553]

229
904
CUAUUCAUUGGGCGCCUGG
1201
CCAGGCGCCCAAUGAAUAG
D
[1430-

230
905
CCAGGACAUCUACGGGCGC
1202
GCGCCCGUAGAUGUCCUGG
D, Rt
[1330-

231
906
AAGAUGGUGGACAACCGUG
1203
CACGGUUGUCCACCAUCUU
Rh, M
[878-896]

232
907
CAGGACAUCUACGGGCGCG
1204
CGCGCCCGUAGAUGUCCUG
D
[1331-

233
908
UCCAAGAUCAACUUCCGCG
1205
CGCGGAAGUUGAUCUUGGA
D
[704-722]

234
909
GUCACCAAGGACGUGGAGC
1206
GCUCCACGUCCUUGGUGAC
Rh, D
[791-809]

235
910
CUGCCCGAGGUCACCAAGG
1207
CCUUGGUGACCUCGGGCAG
Rh, D
[782-800]

236
911
GACCAGGACAUCUACGGGC
1208
GCCCGUAGAUGUCCUGGUC
D, Rt
[1328-

237
912
CCAUGGCCAAGGACCAGGC
1209
GCCUGGUCCUUGGCCAUGG
Rh, D
[399-417]

238
913
CACCAAGGACGUGGAGCGC
1210
GCGCUCCACGUCCUUGGUG
Rh, D
[793-811]

239
914
GACAAGCGCAGCGCGCUGC
1211
GCAGCGCGCUGCGCUUGUC
Rh, Rt
[722-740]

240
915
CAAGCGCAGCGCGCUGCAG
1212
CUGCAGCGCGCUGCGCUUG
Rh, Rt
[724-742]

241
916
CAGACCACCGACGGCAAGC
1213
GCUUGCCGUCGGUGGUCUG
D, Rt
[764-782]

242
917
GACCACCGACGGCAAGCUG
1214
CAGCUUGCCGUCGGUGGUC
D, Rt
[766-784]

243
918
AGGACCUGUACCUGGCCAG
1215
CUGGCCAGGUACAGGUCCU
Rh, D
[1260-

244
919
CUGCUAUUCAUUGGGCGCC
1216
GGCGCCCAAUGAAUAGCAG
D
[1427-

245
920
UCAUUGGGCGCCUGGUCCG
1217
CGGACCAGGCGCCCAAUGA
Rh, D
[1434-

246
921
GCUGCGCGACGAGGAGGUG
1218
CACCUCCUCGUCGCGCAGC
Rh, D
[532-550]

247
922
CGGCAAGCUGCCCGAGGUC
1219
GACCUCGGGCAGCUUGCCG
Rh, D
[775-793]

248
923
CCUCAUCAUCCUCAUGCCC
1220
GGGCAUGAGGAUGAUGAGG
Rh, D, Rt, M
[1027-

249
924
CCAGGCCAUGGCCAAGGAC
1221
GUCCUUGGCCAUGGCCUGG
Rh, D
[394-412]

250
925
GCCAUGGCCAAGGACCAGG
1222
CCUGGUCCUUGGCCAUGGC
Rh, D
[398-416]

251
926
CCACCGACGGCAAGCUGCC
1223
GGCAGCUUGCCGUCGGUGG
D, Rt
[768-786]

252
927
AUGGUGGACAACCGUGGCU
1224
AGCCACGGUUGUCCACCAU
Rh, M
[881-899]

253
928
CUUCCGCGACAAGCGCAGC
1225
GCUGCGCUUGUCGCGGAAG
D
[715-733]

254
929
CGCGACGAGGAGGUGCACG
1226
CGUGCACCUCCUCGUCGCG
Rh, D
[536-554]

255
930
UGGCCCACAAGCUCUCCAG
1227
CUGGAGAGCUUGUGGGCCA
Rh, D, P
[1008-

256
931
GAGCAGCUGCGCGACGAGG
1228
CCUCGUCGCGCAGCUGCUC
Rh, D
[527-545]

257
932
UGACCAGGACAUCUACGGG
1229
CCCGUAGAUGUCCUGGUCA
Rt
[1327-

258
933
ACCACCGACGGCAAGCUGC
1230
GCAGCUUGCCGUCGGUGGU
D, Rt
[767-785]

259
934
GAAGGACCUGUACCUGGCC
1231
GGCCAGGUACAGGUCCUUC
Rh, D
[1258-

260
935
CAUUGGGCGCCUGGUCCGG
1232
CCGGACCAGGCGCCCAAUG
Rh, D
[1435-

261
936
AUGCACCGGACAGGCCUCU
1233
AGAGGCCUGUCCGGUGCAU
Rh, Rb, Rt, P
[938-956]

262
937
AUCAACUUCCGCGACAAGC
1234
GCUUGUCGCGGAAGUUGAU
D
[710-728]

263
938
CAGCUGCGCGACGAGGAGG
1235
CCUCCUCGUCGCGCAGCUG
Rh, D
[530-548]

264
939
CAGAAACACCUGGCUGGGC
1236
GCCCAGCCAGGUGUUUCUG
D
[1181-

265
940
CUACGGGCGCGAGGAGCUG
1237
CAGCUCCUCGCGCCCGUAG
D, M
[1339-

266
941
CGACGAGGAGGUGCACGCC
1238
GGCGUGCACCUCCUCGUCG
D
[538-556]

267
942
UUUGACCAGGACAUCUACG
1239
CGUAGAUGUCCUGGUCAAA
Rt
[1325-

268
943
GUCCAUCAACGAGUGGGCC
1240
GGCCCACUCGUUGAUGGAC
Rh, Rt, M
[742-760]

269
944
AUGACUUCGUGCGCAGCAG
1241
CUGCUGCGCACGAAGUCAU
Rh, Rt, M
[660-678]

270
945
UCCCUGCUAUUCAUUGGGC
1242
GCCCAAUGAAUAGCAGGGA
D
[1424-

271
946
CUGCGCGACGAGGAGGUGC
1243
GCACCUCCUCGUCGCGCAG
Rh, D
[533-551]

272
947
CAAGCUGCCCGAGGUCACC
1244
GGUGACCUCGGGCAGCUUG
Rh, D
[778-796]

273
948
AAGCUGCCCGAGGUCACCA
1245
UGGUGACCUCGGGCAGCUU
Rh, D
[779-797]

274
949
UUCUUCAAGCCACACUGGG
1246
CCCAGUGUGGCUUGAAGAA
Rh, Rb, D
[842-860]

275
950
ACACCUGGCUGGGCUGGGC
1247
GCCCAGCCCAGCCAGGUGU
D
[1186-

276
951
UCCAUCAACGAGUGGGCCG
1248
CGGCCCACUCGUUGAUGGA
Rt, M
[743-761]

277
952
AUCUACGGGCGCGAGGAGC
1249
GCUCCUCGCGCCCGUAGAU
D, M
[1337-

278
953
UCGUGCGCAGCAGCAAGCA
1250
UGCUUGCUGCUGCGCACGA
Rh, D, M
[666-684]

279
954
CGACGGCAAGCUGCCCGAG
1251
CUCGGGCAGCUUGCCGUCG
D
[772-790]

280
955
UUCAUUGGGCGCCUGGUCC
1252
GGACCAGGCGCCCAAUGAA
Rh, D
[1433-

281
956
UUGACCAGGACAUCUACGG
1253
CCGUAGAUGUCCUGGUCAA
Rt
[1326-

282
957
CCUGGCCCACAAGCUCUCC
1254
GGAGAGCUUGUGGGCCAGG
Rh, D, P
[1006-

283
958
UGACUUCGUGCGCAGCAGC
1255
GCUGCUGCGCACGAAGUCA
Rh, Rt, M
[661-679]

284
959
AUGAUGCACCGGACAGGCC
1256
GGCCUGUCCGGUGCAUCAU
Rh, Rb, Rt, M, P
[935-953]

285
960
CACCGACGGCAAGCUGCCC
1257
GGGCAGCUUGCCGUCGGUG
D, Rt
[769-787]

286
961
GACGGCAAGCUGCCCGAGG
1258
CCUCGGGCAGCUUGCCGUC
Rh, D
[773-791]

287
962
UACCAGGCCAUGGCCAAGG
1259
CCUUGGCCAUGGCCUGGUA
Rh, D
[392-410]

288
963
UCCGCGACAAGCGCAGCGC
1260
GCGCUGCGCUUGUCGCGGA
D
[717-735]

289
964
UUCCGCGACAAGCGCAGCG
1261
CGCUGCGCUUGUCGCGGAA
D
[716-734]

290
965
AAGGACGUGGAGCGCACGG
1262
CCGUGCGCUCCACGUCCUU
Rh, D
[797-815]

291
966
UUCCACCACAAGAUGGUGG
1263
CCACCAUCUUGUGGUGGAA
Rh, Rb, D, P
[869-887]

292
967
UACGGGCGCGAGGAGCUGC
1264
GCAGCUCCUCGCGCCCGUA
D, M
[1340-

293
968
AAACACCUGGCUGGGCUGG
1265
CCAGCCCAGCCAGGUGUUU
D
[1184-

294
969
AACACCUGGCUGGGCUGGG
1266
CCCAGCCCAGCCAGGUGUU
D
[1185-

295
970
AUUGGGCGCCUGGUCCGGC
1267
GCCGGACCAGGCGCCCAAU
Rh, D
[1436-

296
971
ACCGACGGCAAGCUGCCCG
1268
CGGGCAGCUUGCCGUCGGU
D
[770-788]

297
972
UUCGUGCGCAGCAGCAAGC
1269
GCUUGCUGCUGCGCACGAA
Rh, D, M
[665-683]

TABLE D

SERPINH1 Active 18 + 1-mer siRNAs

No
SEQ ID
Sense siRNA
SEQ ID
AntiSense siRNA
Other Sp
human-

1
1270
AGCCUUUGUUGCUAUCAAA
1849
UUUGAUAGCAACAAAGGCU
Rh
[2117-2135]

2
1271
GCCUAAGGGUGACAAGAUA
1850
UAUCUUGUCACCCUUAGGC
Rh
[1453-1471]

3
1272
GGCCUAAGGGUGACAAGAA
1851
UUCUUGUCACCCUUAGGCC
Rh
[1452-1470]

4
1273
CCUCAAUCAGUAUUCAUAA
1852
UUAUGAAUACUGAUUGAG

[1774-1792]

5
1274
GGCGGAUUGAGAAGGAGCA
1853
UGCUCCUUCUCAAUCCGCC

[1973-1991]

6
1275
GGCAGUGGAGAACAUCCUA
1854
UAGGAUGUUCUCCACUGCC
Rh
[415-433]

7
1276
GGGUCAGCCAGCCCUCUUA
1855
UAAGAGGGCUGGCUGACCC
Rh
[1839-1857]

8
1277
GGGUGACAAGAUGCGAGAA
1856
UUCUCGCAUCUUGUCACCC
Rh, D
[1459-1477]

9
1278
GGACCAGGCAGUGGAGAAA
1857
UUUCUCCACUGCCUGGUCC
Rh
[409-427]

10
1279
GAGACACAUGGGUGCUAUA
1858
UAUAGCACCCAUGUGUCUC
Rh, D, Rt,
[1533-1551]

11
1280
GUUGGAGCGUGGAAAAAAA
1859
UUUUUUUCCACGCUCCAAC

[2191-2208]

12
1281
GGAACAUGAGCCUUUGUUA
1860
UAACAAAGGCUCAUGUUCC
Rh
[2109-2127]

13
1282
GCCAUGUUCUUCAAGCCAA
1861
UUGGCUUGAAGAACAUGGC
Rh, Rb, D
[836-854]

14
1283
GGAUUGAGAAGGAGCUCCA
1862
UGGAGCUCCUUCUCAAUCC

[1976-1994]

15
1284
GGGAUGAACUUUUUGUUUA
1863
UAAACAAAAAGUUCAUCCC
Rh
[2048-2066]

16
1285
GCCGCAGUGAGGCGGAUUA
1864
UAAUCCGCCUCACUGCGGC

[1963-1981]

17
1286
GGACCUUCCCAGCUAGAAA
1865
UUUCUAGCUGGGAAGGUCC
Rh
[1639-1657]

18
1287
GACCUUCCCAGCUAGAAUA
1866
UAUUCUAGCUGGGAAGGUC
Rh
[1640-1658]

19
1288
CCUGUGAGACCAAAUUGAA
1867
UUCAAUUUGGUCUCACAGG
Rh
[1814-1832]

20
1289
UGGAGAACAUCCUGGUGUA
1868
UACACCAGGAUGUUCUCCA
Rh
[420-438]

21
1290
GCCUUUGUUGCUAUCAAUA
1869
UAUUGAUAGCAACAAAGGC
Rh
[2118-2136]

22
1291
CCGCCUUUGAGUUGGACAA
1870
UUGUCCAACUCAAAGGCGG
Rh
[1293-1311]

23
1292
CAGGCAGUGGAGAACAUCA
1871
UGAUGUUCUCCACUGCCUG
Rh
[413-431]

24
1293
CACCUGUGAGACCAAAUUA
1872
UAAUUUGGUCUCACAGGUG
Rh
[1812-1830]

25
1294
GGGAAGAUGCAGAAGAAGA
1873
UCUUCUUCUGCAUCUUCCC
Rh, Rb, Rt
[1112-1130]

26
1295
GGCCAUUGACAAGAACAAA
1874
UUUGUUCUUGUCAAUGGCC
Rh, D
[1213-1231]

27
1296
GCCUUUGAGUUGGACACAA
1875
UUGUGUCCAACUCAAAGGC
Rh
[1295-1313]

28
1297
AGCGGACCUUCCCAGCUAA
1876
UUAGCUGGGAAGGUCCGCU
Rh
[1636-1654]

29
1298
GAAGAAGGCUGUUGCCAUA
1877
UAUGGCAACAGCCUUCUUC
Rt
[1123-1141]

30
1299
ACAAGAUGCGAGACGAGUA
1878
UACUCGUCUCGCAUCUUGU
Rh, Rt
[1464-1482]

31
1300
GAGGCGGAUUGAGAAGGAA
1879
UUCCUUCUCAAUCCGCCUC

[1971-1989]

32
1301
GGACAACCGUGGCUUCAUA
1880
UAUGAAGCCACGGUUGUCC
Rh, M
[886-904]

33
1302
CAUAUUUAUAGCCAGGUAA
1881
UUACCUGGCUAUAAAUAUG
Rh
[1788-1806]

34
1303
CGACGACGAGAAGGAAAAA
1882
UUUUUCCUUCUCGUCGUCG

[967-985]

35
1304
CUCACCUGUGAGACCAAAA
1883
UUUUGGUCUCACAGGUGAG
Rh
[1810-1828]

36
1305
GCGGCUCCCUGCUAUUCAA
1884
UUGAAUAGCAGGGAGCCGC

[1419-1437]

37
1306
AGAACAUCCUGGUGUCACA
1885
UGUGACACCAGGAUGUUCU

[423-441]

38
1307
CACACUGGGAUGAGAAAUA
1886
UAUUUCUCAUCCCAGUGUG
Rh
[852-870]

39
1308
GCUAGAAUUCACUCCACUA
1887
UAGUGGAGUGAAUUCUAGC
Rh
[1650-1668]

40
1309
CCUUCAUCUUCCUAGUGCA
1888
UGCACUAGGAAGAUGAAGG

[1389-1407]

41
1310
UGCUAUCAAUCCAAGAACA
1889
UGUUCUUGGAUUGAUAGCA
Rh
[2126-2144]

42
1311
GGAAGAUGCAGAAGAAGGA
1890
UCCUUCUUCUGCAUCUUCC
Rh, Rb, Rt
[1113-1131]

43
1312
CAUGAGCCUUUGUUGCUAA
1891
UUAGCAACAAAGGCUCAUG
Rh
[2113-2131]

44
1313
GCGGAUUGAGAAGGAGCUA
1892
UAGCUCCUUCUCAAUCCGC

[1974-1992]

45
1314
UGCAGUCCAUCAACGAGUA
1893
UACUCGUUGAUGGACUGCA
Rh, Rt, M
[738-756]

46
1315
GCACUGCGGAGAAGUUGAA
1894
UUCAACUUCUCCGCAGUGC

[321-339]

47
1316
CCAGGCAGUGGAGAACAUA
1895
UAUGUUCUCCACUGCCUGG
Rh
[412-430]

48
1317
GGCAAGAAGGACCUGUACA
1896
UGUACAGGUCCUUCUUGCC
Rh, D, M
[1253-1271]

49
1318
CUCUACAACUACUACGACA
1897
UGUCGUAGUAGUUGUAGA
Rb
[953-971]

50
1319
CUUCCCAGCUAGAAUUCAA
1898
UUGAAUUCUAGCUGGGAAG
Rh
[1643-1661]

51
1320
AGGCGGAUUGAGAAGGAGA
1899
UCUCCUUCUCAAUCCGCCU

[1972-1990]

52
1321
GGUCCUAUACCGUGGGUGA
1900
UCACCCACGGUAUAGGACC
Rh
[912-930]

53
1322
GCAAGAAGGACCUGUACCA
1901
UGGUACAGGUCCUUCUUGC
Rh, D, M
[1254-1272]

54
1323
CCGUGGGUGUCAUGAUGAA
1902
UUCAUCAUGACACCCACGG
Rh
[921-939]

55
1324
GAUGCGAGACGAGUUAUAA
1903
UUAUAACUCGUCUCGCAUC
Rh
[1468-1486]

56
1325
GGCAGUGCUGAGCGCCGAA
1904
UUCGGCGCUCAGCACUGCC

[511-529]

57
1326
CAGCUAGAAUUCACUCCAA
1905
UUGGAGUGAAUUCUAGCUG
Rh
[1648-1666]

58
1327
GAGCUUCGCUGAUGACUUA
1906
UAAGUCAUCAGCGAAGCUC
Rh
[649-667]

59
1328
CUUUGAGUUGGACACAGAA
1907
UUCUGUGUCCAACUCAAAG
Rh
[1297-1315]

60
1329
GGUGGACAACCGUGGCUUA
1908
UAAGCCACGGUUGUCCACC
Rh, M
[883-901]

61
1330
GCCUCAUCAUCCUCAUGCA
1909
UGCAUGAGGAUGAUGAGGC
Rh, D, Rt,
[1026-1044]

62
1331
ACCAGGCAGUGGAGAACAA
1910
UUGUUCUCCACUGCCUGGU
Rh
[411-429]

63
1332
CCUGCCUCAAUCAGUAUUA
1911
UAAUACUGAUUGAGGCAGG

[1770-1788]

64
1333
GAUCAAGCCUGCCUCAAUA
1912
UAUUGAGGCAGGCUUGAUC
Rh
[1763-1781]

65
1334
CAGACUCUGGUCAAGAAGA
1913
UCUUCUUGACCAGAGUCUG
Rh
[2011-2029]

66
1335
CGCGCUGCAGUCCAUCAAA
1914
UUUGAUGGACUGCAGCGCG
Rh, Rt
[733-751]

67
1336
CUGGCACUGCGGAGAAGUA
1915
UACUUCUCCGCAGUGCCAG

[318-336]

68
1337
CCAGCUCUAUCCCAACCUA
1916
UAGGUUGGGAUAGAGCUG

[1885-1903]

69
1338
AGGGUGUGGUGGAGGUGAA
1917
UUCACCUCCACCACACCCU
Rh, D
[1152-1170]

70
1339
AGUGAGGCGGAUUGAGAAA
1918
UUUCUCAAUCCGCCUCACU

[1968-1986]

71
1340
CGGACAGGCCUCUACAACA
1919
UGUUGUAGAGGCCUGUCCG
Rh, Rb, Rt,
[944-962]

72
1341
CGACGAGAAGGAAAAGCUA
1920
UAGCUUUUCCUUCUCGUCG
Rh
[970-988]

73
1342
AGGCCAAGGCAGUGCUGAA
1921
UUCAGCACUGCCUUGGCCU
Rh
[504-522]

74
1343
GCCUCAGGGUGCACACAGA
1922
UCUGUGUGCACCCUGAGGC

[1488-1506]

75
1344
GGAUGAGAAAUUCCACCAA
1923
UUGGUGGAAUUUCUCAUCC
Rh
[859-877]

76
1345
AGAAGGAAAAGCUGCAAAA
1924
UUUUGCAGCUUUUCCUUCU
Rh
[975-993]

77
1346
AGCUCUAUCCCAACCUCUA
1925
UAGAGGUUGGGAUAGAGC
Rh
[1887-1905]

78
1347
UGACAAGAUGCGAGACGAA
1926
UUCGUCUCGCAUCUUGUCA
Rh
[1462-1480]

79
1348
AGAAGGAGCUCCCAGGAGA
1927
UCUCCUGGGAGCUCCUUCU

[1982-2000]

80
1349
CCUUCUCACCUGUGAGACA
1928
UGUCUCACAGGUGAGAAGG
Rh
[1806-1824]

81
1350
GGCUUCUGGGCAGACUCUA
1929
UAGAGUCUGCCCAGAAGCC
Rh
[2001-2019]

82
1351
CCAGCCUCAUCAUCCUCAA
1930
UUGAGGAUGAUGAGGCUG
Rh, D, Rt,
[1023-1041]

83
1352
CCAAAGGCUCCUGAGACAA
1931
UUGUCUCAGGAGCCUUUGG

[1521-1539]

84
1353
GGACCUGGGCCAUAGUCAA
1932
UUGACUAUGGCCCAGGUCC

[1722-1740]

85
1354
GGGUGUCAUGAUGAUGCAA
1933
UUGCAUCAUCAUGACACCC
Rh
[925-943]

86
1355
GUACCAGCCUUGGAUACUA
1934
UAGUAUCCAAGGCUGGUAC
Rh
[1572-1590]

87
1356
GGCUGUUGCCAUCUCCUUA
1935
UAAGGAGAUGGCAACAGCC

[1129-1147]

88
1357
CGCAGUGAGGCGGAUUGAA
1936
UUCAAUCCGCCUCACUGCG

[1965-1983]

89
1358
CCAAGGACGUGGAGCGCAA
1937
UUGCGCUCCACGUCCUUGG
Rh, D
[795-813]

90
1359
GGCUCCUGAGACACAUGGA
1938
UCCAUGUGUCUCAGGAGCC
D
[1526-1544]

91
1360
GCUGCAGUCCAUCAACGAA
1939
UUCGUUGAUGGACUGCAGC
Rh, Rt
[736-754]

92
1361
CCAGGUACCUUCUCACCUA
1940
UAGGUGAGAAGGUACCUGG
Rh
[1799-1817]

93
1362
GCAGCGCGCUGCAGUCCAA
1941
UUGGACUGCAGCGCGCUGC
Rh, Rt
[729-747]

94
1363
GAGACCAAAUUGAGCUAGA
1942
UCUAGCUCAAUUUGGUCUC
Rh
[1819-1837]

95
1364
GCCGCCGAGGUGAAGAAAA
1943
UUUUCUUCACCUCGGCGGC

[281-299]

96
1365
GCAGACUCUGGUCAAGAAA
1944
UUUCUUGACCAGAGUCUGC
Rh
[2010-2028]

97
1366
CUAGAAUUCACUCCACUUA
1945
UAAGUGGAGUGAAUUCUA
Rh
[1651-1669]

98
1367
GCAGUGGAGAACAUCCUGA
1946
UCAGGAUGUUCUCCACUGC
Rh
[416-434]

99
1368
CGCAUGUCAGGCAAGAAGA
1947
UCUUCUUGCCUGACAUGCG
Rh, D
[1244-1262]

10
1369
CGGAUUGAGAAGGAGCUCA
1948
UGAGCUCCUUCUCAAUCCG

[1975-1993]

10
1370
AGGUGAGGUACCAGCCUUA
1949
UAAGGCUGGUACCUCACCU
Rh
[1565-1583]

10
1371
CCACACUGGGAUGAGAAAA
1950
UUUUCUCAUCCCAGUGUGG
Rh
[851-869]

10
1372
GCCAUUGACAAGAACAAGA
1951
UCUUGUUCUUGUCAAUGGC
Rh, D
[1214-1232]

10
1373
GCGCUGCAGUCCAUCAACA
1952
UGUUGAUGGACUGCAGCGC
Rh, Rt
[734-752]

10
1374
CUCCCAACUAUAAAACUAA
1953
UUAGUUUAUAGUUGGGA
Rh
[1903-1921]

10
1375
GGUGACAAGAUGCGAGACA
1954
UGUCUCGCAUCUUGUCACC
Rh
[1460-1478]

10
1376
GGCCGACUUGUCACGCAUA
1955
UAUGCGUGACAAGUCGGCC
Rh
[1231-1249]

10
1377
CCUAAGGGUGACAAGAUGA
1956
UCAUCUUGUCACCCUUAGG
Rh
[1454-1472]

10
1378
UGAGACACAUGGGUGCUAA
1957
UUAGCACCCAUGUGUCUCA
Rh, D, Rt,
[1532-1550]

11
1379
GGGUGGAAAAACAGACCGA
1958
UCGGUCUGUUUUUCCACCC

[1601-1619]

11
1380
GGUGGAGGUGACCCAUGAA
1959
UUCAUGGGUCACCUCCACC
Rh, Rt, M
[1159-1177]

11
1381
CUUUGACCAGGACAUCUAA
1960
UUAGAUGUCCUGGUCAAAG
Rh, Rt
[1324-1342]

11
1382
GAACAUGAGCCUUUGUUGA
1961
UCAACAAAGGCUCAUGUUC
Rh
[2110-2128]

11
1383
AGCCUUGGAUACUCCAUGA
1962
UCAUGGAGUAUCCAAGGCU
Rh
[1577-1595]

11
1384
GGAGGUGACCCAUGACCUA
1963
UAGGUCAUGGGUCACCUCC
Rh, Rt, M
[1162-1180]

11
1385
AGAUCAAGCCUGCCUCAAA
1964
UUUGAGGCAGGCUUGAUCU
Rh
[1762-1780]

11
1386
GCCCAAGGGUGUGGUGGAA
1965
UUCCACCACACCCUUGGGC
Rh, D
[1147-1165]

11
1387
AGAACAAGGCCGACUUGUA
1966
UACAAGUCGGCCUUGUUCU
Rh
[1224-1242]

11
1388
GUGGCUUCAUGGUGACUCA
1967
UGAGUCACCAUGAAGCCAC
Rh
[894-912]

12
1389
CUCCUGAGACACAUGGGUA
1968
UACCCAUGUGUCUCAGGAG
D
[1528-1546]

12
1390
CAGCCUUGGAUACUCCAUA
1969
UAUGGAGUAUCCAAGGCUG
Rh
[1576-1594]

12
1391
AAGGCUCCUGAGACACAUA
1970
UAUGUGUCUCAGGAGCCUU
D
[1524-1542]

12
1392
AGAAGAAGGCUGUUGCCAA
1971
UUGGCAACAGCCUUCUUCU
Rt
[1122-1140]

12
1393
CUACUACGACGACGAGAAA
1972
UUUCUCGUCGUCGUAGUAG
Rb
[961-979]

12
1394
CCUUUGUUGCUAUCAAUCA
1973
UGAUUGAUAGCAACAAAGG
Rh
[2119-2137]

12
1395
AGGCAGUGGAGAACAUCCA
1974
UGGAUGUUCUCCACUGCCU
Rh
[414-432]

12
1396
CCAUCACGUGGAGCCUCUA
1975
UAGAGGCUCCACGUGAUGG
Rh
[1045-1063]

12
1397
AGCUCUCCAGCCUCAUCAA
1976
UUGAUGAGGCUGGAGAGCU
Rh, D, Rt,
[1017-1035]

12
1398
GGCUCCCUGCUAUUCAUUA
1977
UAAUGAAUAGCAGGGAGCC
D
[1421-1439]

13
1399
GGGAACAUGAGCCUUUGUA
1978
UACAAAGGCUCAUGUUCCC
Rh
[2108-2126]

13
1400
GGGCCAUAGUCAUUCUGCA
1979
UGCAGAAUGACUAUGGCCC

[1728-1746]

13
1401
CCAAAGAGCAGCUGAAGAA
1980
UUCUUCAGCUGCUCUUUGG
Rh, Rb, P
[1086-1104]

13
1402
GACGAGAAGGAAAAGCUGA
1981
UCAGCUUUUCCUUCUCGUC
Rh
[971-989]

13
1403
GGGCUUCUGGGCAGACUCA
1982
UGAGUCUGCCCAGAAGCCC
Rh
[2000-2018]

13
1404
CAAGGACCAGGCAGUGGAA
1983
UUCCACUGCCUGGUCCUUG
Rh
[406-424]

13
1405
CUGUGAGACCAAAUUGAGA
1984
UCUCAAUUUGGUCUCACAG
Rh
[1815-1833]

13
1406
GACUGAGGCCAUUGACAAA
1985
UUUGUCAAUGGCCUCAGUC
Rh
[1207-1225]

13
1407
GACUUGUCACGCAUGUCAA
1986
UUGACAUGCGUGACAAGUC
Rh
[1235-1253]

13
1408
GAGGUGAGGUACCAGCCUA
1987
UAGGCUGGUACCUCACCUC

[1564-1582]

14
1409
CAGAUACCAUGAUGCUGAA
1988
UUCAGCAUCAUGGUAUCUG
Rh
[1681-1699]

14
1410
AGGCAAGAAGGACCUGUAA
1989
UUACAGGUCCUUCUUGCCU
Rh, D
[1252-1270]

14
1411
CUGGGAUGAGAAAUUCCAA
1990
UUGGAAUUUCUCAUCCCAG
Rh
[856-874]

14
1412
AGGUACCAGCCUUGGAUAA
1991
UUAUCCAAGGCUGGUACCU
Rh
[1570-1588]

14
1413
CAGCCAGCCCUCUUCUGAA
1992
UUCAGAAGAGGGCUGGCUG

[1843-1861]

14
1414
GUGUCAUGAUGAUGCACCA
1993
UGGUGCAUCAUCAUGACAC
Rh
[927-945]

14
1415
CCUCUACAACUACUACGAA
1994
UUCGUAGUAGUUGUAGAG
Rb, D
[952-970]

14
1416
CCGCCGAGGUGAAGAAACA
1995
UGUUUCUUCACCUCGGCGG
Rh
[282-300]

14
1417
GCUAUCAAUCCAAGAACUA
1996
UAGUUCUUGGAUUGAUAGC
Rh
[2127-2145]

14
1418
AGCCUGCCUCAAUCAGUAA
1997
UUACUGAUUGAGGCAGGCU

[1768-1786]

15
1419
GGUCCGGCCUAAGGGUGAA
1998
UUCACCCUUAGGCCGGACC
Rh
[1447-1465]

15
1420
GAAGGAAAAGCUGCAAAUA
1999
UAUUUGCAGCUUUUCCUUC
Rh
[976-994]

15
1421
GGCCUCUACAACUACUACA
2000
UGUAGUAGUUGUAGAGGCC
Rb, D
[950-968]

15
1422
UGUUCUUCAAGCCACACUA
2001
UAGUGUGGCUUGAAGAACA
Rh, Rb, D
[840-858]

15
1423
GGCCAAGGCAGUGCUGAGA
2002
UCUCAGCACUGCCUUGGCC
Rh
[505-523]

15
1424
AGAAAUUCCACCACAAGAA
2003
UUCUUGUGGUGGAAUUUCU
Rh
[864-882]

15
1425
CUGCAGUCCAUCAACGAGA
2004
UCUCGUUGAUGGACUGCAG
Rh, Rt, M
[737-755]

15
1426
CCAGCGUGUUCCACGCCAA
2005
UUGGCGUGGAACACGCUGG

[1275-1293]

15
1427
GCUCCCUCCUGCUUCUCAA
2006
UUGAGAAGCAGGAGGGAGC

[234-252]

15
1428
CCGGACAGGCCUCUACAAA
2007
UUUGUAGAGGCCUGUCCGG
Rh, Rb, Rt,
[943-961]

16
1429
CCCAUCACGUGGAGCCUCA
2008
UGAGGCUCCACGUGAUGGG
Rh
[1044-1062]

16
1430
CCGGCCUAAGGGUGACAAA
2009
UUUGUCACCCUUAGGCCGG
Rh
[1450-1468]

16
1431
CCUAUACCGUGGGUGUCAA
2010
UUGACACCCACGGUAUAGG
Rh, D, P
[915-933]

16
1432
CAGUGGAGAACAUCCUGGA
2011
UCCAGGAUGUUCUCCACUG
Rh
[417-435]

16
1433
CACUGGGAUGAGAAAUUCA
2012
UGAAUUUCUCAUCCCAGUG
Rh
[854-872]

16
1434
AUCCAAAGGCUCCUGAGAA
2013
UUCUCAGGAGCCUUUGGAU

[1519-1537]

16
1435
UGAGAAAUUCCACCACAAA
2014
UUUGUGGUGGAAUUUCUCA
Rh
[862-880]

16
1436
GGUGGAAAAACAGACCGGA
2015
UCCGGUCUGUUUUUCCACC

[1602-1620]

16
1437
GCUGGGCAGCCGACUGUAA
2016
UUACAGUCGGCUGCCCAGC

[616-634]

16
1438
CCAUAGUCAUUCUGCCUGA
2017
UCAGGCAGAAUGACUAUGG

[1731-1749]

17
1439
GCACCGGACAGGCCUCUAA
2018
UUAGAGGCCUGUCCGGUGC
Rh, Rb, Rt,
[940-958]

17
1440
GUUGGACACAGAUGGCAAA
2019
UUUGCCAUCUGUGUCCAAC

[1303-1321]

17
1441
GCCUGCCUCAAUCAGUAUA
2020
UAUACUGAUUGAGGCAGGC

[1769-1787]

17
1442
GAUCAACUUCCGCGACAAA
2021
UUUGUCGCGGAAGUUGAUC
D
[709-727]

17
1443
GGCCGCAGUGAGGCGGAUA
2022
UAUCCGCCUCACUGCGGCC

[1962-1980]

17
1444
CUGCGGAGAAGUUGAGCCA
2023
UGGCUCAACUUCUCCGCAG

[324-342]

17
1445
GCAUCCAAAGGCUCCUGAA
2024
UUCAGGAGCCUUUGGAUGC

[1517-1535]

17
1446
GCUUCUGGGCAGACUCUGA
2025
UCAGAGUCUGCCCAGAAGC
Rh
[2002-2020]

17
1447
CCAGCCCUCUUCUGACACA
2026
UGUGUCAGAAGAGGGCUGG

[1846-1864]

17
1448
GCUCUAUCCCAACCUCUCA
2027
UGAGAGGUUGGGAUAGAG
Rh
[1888-1906]

18
1449
GGACGUGGAGCGCACGGAA
2028
UUCCGUGCGCUCCACGUCC
Rh, D
[799-817]

18
1450
CCAAGGCAGUGCUGAGCGA
2029
UCGCUCAGCACUGCCUUGG
Rh
[507-525]

18
1451
GCAGAAGAAGGCUGUUGCA
2030
UGCAACAGCCUUCUUCUGC
Rt
[1120-1138]

18
1452
GACAUUUUGUUGGAGCGUA
2031
UACGCUCCAACAAAAUGUC

[2183-2201]

18
1453
CGAGCACUCCAAGAUCAAA
2032
UUUGAUCUUGGAGUGCUCG
Rh, D
[697-715]

18
1454
UCAUGAUGAUGCACCGGAA
2033
UUCCGGUGCAUCAUCAUGA
Rh
[930-948]

18
1455
CCUGCUUCUCAGCGCCUUA
2034
UAAGGCGCUGAGAAGCAGG

[241-259]

18
1456
CCCAACCUCUCCCAACUAA
2035
UUAGUUGGGAGAGGUUGG
Rh
[1895-1913]

18
1457
UGGGCAGACUCUGGUCAAA
2036
UUUGACCAGAGUCUGCCCA
Rh
[2007-2025]

18
1458
CUCUGGUCAAGAAGCAUCA
2037
UGAUGCUUCUUGACCAGAG
Rh
[2015-2033]

19
1459
GAGCCUCUCGAGCGCCUUA
2038
UAAGGCGCUCGAGAGGCUC

[1055-1073]

19
1460
AGAAGGCUGUUGCCAUCUA
2039
UAGAUGGCAACAGCCUUCU
Rt
[1125-1143]

19
1461
CCCUGCUAGUCAACGCCAA
2040
UUGGCGUUGACUAGCAGGG
Rh
[822-840]

19
1462
GCCUUCAGCUUGUACCAGA
2041
UCUGGUACAAGCUGAAGGC

[380-398]

19
1463
GCUGCUAACCAAAGAGCAA
2042
UUGCUCUUUGGUUAGCAGC

[1078-1096]

19
1464
CCCACAAGCUCUCCAGCCA
2043
UGGCUGGAGAGCUUGUGGG
Rh, D, P
[1011-1029]

19
1465
GCUCCCUGCUAUUCAUUGA
2044
UCAAUGAAUAGCAGGGAGC
D
[1422-1440]

19
1466
GUUCUUCAAAGAUAGGGAA
2045
UUCCCUAUCUUUGAAGAAC

[2083-2101]

19
1467
GUCAGCCAGCCCUCUUCUA
2046
UAGAAGAGGGCUGGCUGAC
Rh
[1841-1859]

19
1468
GCGGGACACCCAAAGCGGA
2047
UCCGCUUUGGGUGUCCCGC

[1405-1423]

20
1469
AGCGCAGCGCGCUGCAGUA
2048
UACUGCAGCGCGCUGCGCU
Rh, Rt
[726-744]

20
1470
CCGGAAACUCCACAUCCUA
2049
UAGGAUGUGGAGUUUCCGG

[1701-1719]

20
1471
CCAUUGACAAGAACAAGGA
2050
UCCUUGUUCUUGUCAAUGG
Rh, D
[1215-1233]

20
1472
GGACAUCUACGGGCGCGAA
2051
UUCGCGCCCGUAGAUGUCC
D
[1333-1351]

20
1473
GACACAUGGGUGCUAUUGA
2052
UCAAUAGCACCCAUGUGUC
Rh, Rt, M
[1535-1553]

20
1474
CCUGGCACUGCGGAGAAGA
2053
UCUUCUCCGCAGUGCCAGG

[317-335]

20
1475
GGGCCUGACUGAGGCCAUA
2054
UAUGGCCUCAGUCAGGCCC
Rt
[1201-1219]

20
1476
ACACUGGGAUGAGAAAUUA
2055
UAAUUUCUCAUCCCAGUGU
Rh
[853-871]

20
1477
GGUCAGCCAGCCCUCUUCA
2056
UGAAGAGGGCUGGCUGACC
Rh
[1840-1858]

20
1478
GUGAGGCGGAUUGAGAAGA
2057
UCUUCUCAAUCCGCCUCAC

[1969-1987]

21
1479
UCACCUGUGAGACCAAAUA
2058
UAUUUGGUCUCACAGGUGA
Rh
[1811-1829]

21
1480
AGCUGCAAAUCGUGGAGAA
2059
UUCUCCACGAUUUGCAGCU
Rh
[984-1002]

21
1481
GGUGCACACAGGAUGGCAA
2060
UUGCCAUCCUGUGUGCACC
Rh
[1495-1513]

21
1482
GGGUGUGGUGGAGGUGACA
2061
UGUCACCUCCACCACACCC
Rh, D
[1153-1171]

21
1483
CCAGCCUUGGAUACUCCAA
2062
UUGGAGUAUCCAAGGCUGG
Rh
[1575-1593]

21
1484
CCACAAGCUCUCCAGCCUA
2063
UAGGCUGGAGAGCUUGUGG
Rh, D, P
[1012-1030]

21
1485
AAAGGCUCCUGAGACACAA
2064
UUGUGUCUCAGGAGCCUUU

[1523-1541]

21
1486
AGGAAAAGCUGCAAAUCGA
2065
UCGAUUUGCAGCUUUUCCU
Rh
[978-996]

21
1487
CGCAGCAGCUCCUGGCACA
2066
UGUGCCAGGAGCUGCUGCG

[307-325]

21
1488
GGUGUCAUGAUGAUGCACA
2067
UGUGCAUCAUCAUGACACC
Rh
[926-944]

22
1489
CCUCUUCUGACACUAAAAA
2068
UUUUUAGUGUCAGAAGAG

[1851-1869]

22
1490
AGCUAGAAUUCACUCCACA
2069
UGUGGAGUGAAUUCUAGCU
Rh
[1649-1667]

22
1491
CGCUGGGCGGCAAGGCGAA
2070
UUCGCCUUGCCGCCCAGCG

[474-492]

22
1492
GGCCUGGCCUUCAGCUUGA
2071
UCAAGCUGAAGGCCAGGCC

[374-392]

22
1493
AGACACAUGGGUGCUAUUA
2072
UAAUAGCACCCAUGUGUCU
Rh, Rt, M
[1534-1552]

22
1494
CGUGGGUGUCAUGAUGAUA
2073
UAUCAUCAUGACACCCACG
Rh
[922-940]

22
1495
GUGGGUGUCAUGAUGAUGA
2074
UCAUCAUCAUGACACCCAC
Rh
[923-941]

22
1496
GAGAAGGAGCUCCCAGGAA
2075
UUCCUGGGAGCUCCUUCUC

[1981-1999]

22
1497
GACUCUGGUCAAGAAGCAA
2076
UUGCUUCUUGACCAGAGUC
Rh
[2013-2031]

22
1498
CACUAAAACACCUCAGCUA
2077
UAGCUGAGGUGUUUUAGU

[1861-1879]

23
1499
GGAGGCAUCCAAAGGCUCA
2078
UGAGCCUUUGGAUGCCUCC

[1513-1531]

23
1500
GACCCAGCUCAGUGAGCUA
2079
UAGCUCACUGAGCUGGGUC

[636-654]

23
1501
CCAUGACCUGCAGAAACAA
2080
UUGUUUCUGCAGGUCAUGG
Rh, Rt, M
[1171-1189]

23
1502
AGAUGCAGAAGAAGGCUGA
2081
UCAGCCUUCUUCUGCAUCU
Rh, Rt, M
[1116-1134]

23
1503
CAGCAAGCAGCACUACAAA
2082
UUUGUAGUGCUGCUUGCUG
Rh, D
[676-694]

23
1504
CAAGCUCUCCAGCCUCAUA
2083
UAUGAGGCUGGAGAGCUUG
Rh, D, M, P
[1015-1033]

23
1505
UGCAGAAGAAGGCUGUUGA
2084
UCAACAGCCUUCUUCUGCA
Rt
[1119-1137]

23
1506
GGCGCGAGGAGCUGCGCAA
2085
UUGCGCAGCUCCUCGCGCC
Rh, D, M
[1344-1362]

23
1507
GGUACCAGCCUUGGAUACA
2086
UGUAUCCAAGGCUGGUACC
Rh
[1571-1589]

23
1508
GCAGCCGACUGUACGGACA
2087
UGUCCGUACAGUCGGCUGC

[621-639]

24
1509
CAGCCUCAUCAUCCUCAUA
2088
UAUGAGGAUGAUGAGGCU
Rh, D, Rt,
[1024-1042]

24
1510
GCCACCGCCUUUGAGUUGA
2089
UCAACUCAAAGGCGGUGGC
Rh
[1289-1307]

24
1511
AGAAGGACCUGUACCUGGA
2090
UCCAGGUACAGGUCCUUCU
Rh, D
[1257-1275]

24
1512
GGUGAAGAAACCUGCAGCA
2091
UGCUGCAGGUUUCUUCACC
Rh
[289-307]

24
1513
GUACCUUCUCACCUGUGAA
2092
UUCACAGGUGAGAAGGUAC
Rh
[1803-1821]

24
1514
GGCCAAGGACCAGGCAGUA
2093
UACUGCCUGGUCCUUGGCC
Rh
[403-421]

24
1515
GGCGGCAAGGCGACCACGA
2094
UCGUGGUCGCCUUGCCGCC

[479-497]

24
1516
AGCACUCCAAGAUCAACUA
2095
UAGUUGAUCUUGGAGUGCU
Rh, D
[699-717]

24
1517
AUAUUUAUAGCCAGGUACA
2096
UGUACCUGGCUAUAAAUAU
Rh
[1789-1807]

24
1518
GGCAGCCGACUGUACGGAA
2097
UUCCGUACAGUCGGCUGCC

[620-638]

25
1519
GUCACGCAUGUCAGGCAAA
2098
UUUGCCUGACAUGCGUGAC
Rh, D
[1240-1258]

25
1520
GACAGGCCUCUACAACUAA
2099
UUAGUUGUAGAGGCCUGUC
Rh, Rb, Rt,
[946-964]

25
1521
GAUGCAGAAGAAGGCUGUA
2100
UACAGCCUUCUUCUGCAUC
Rh, Rt, M
[1117-1135]

25
1522
ACCCAUGACCUGCAGAAAA
2101
UUUUCUGCAGGUCAUGGGU
Rh, Rt, M
[1169-1187]

25
1523
GGCUUCAUGGUGACUCGGA
2102
UCCGAGUCACCAUGAAGCC
Rh
[896-914]

25
1524
UGCCUCAAUCAGUAUUCAA
2103
UUGAAUACUGAUUGAGGCA

[1772-1790]

25
1525
GUUCUUCAAGCCACACUGA
2104
UCAGUGUGGCUUGAAGAAC
Rh, Rb, D
[841-859]

25
1526
ACUCCAAGAUCAACUUCCA
2105
UGGAAGUUGAUCUUGGAG
Rh, D, Rt,
[702-720]

25
1527
GCUGUUCUACGCCGACCAA
2106
UUGGUCGGCGUAGAACAGC
Rh
[1369-1387]

25
1528
UAGUCAACGCCAUGUUCUA
2107
UAGAACAUGGCGUUGACUA
Rh
[828-846]

26
1529
CCGUGUGCCUGAGCGGACA
2108
UGUCCGCUCAGGCACACGG
Rh
[1625-1643]

26
1530
AGGCCUCUACAACUACUAA
2109
UUAGUAGUUGUAGAGGCCU
Rh, Rb, D,
[949-967]

26
1531
GCUUCAUGGUGACUCGGUA
2110
UACCGAGUCACCAUGAAGC
Rh
[897-915]

26
1532
GGUCAAGAAGCAUCGUGUA
2111
UACACGAUGCUUCUUGACC
Rh
[2019-2037]

26
1533
CUGCGAGCACUCCAAGAUA
2112
UAUCUUGGAGUGCUCGCAG
Rh, D
[694-712]

26
1534
GUCCUAUACCGUGGGUGUA
2113
UACACCCACGGUAUAGGAC
Rh
[913-931]

26
1535
GGCCUGACUGAGGCCAUUA
2114
UAAUGGCCUCAGUCAGGCC
Rh
[1202-1220]

26
1536
CACUCCAAGAUCAACUUCA
2115
UGAAGUUGAUCUUGGAGU
Rh, D, Rt,
[701-719]

26
1537
GCGUCGCAGGCCAAGGCAA
2116
UUGCCUUGGCCUGCGACGC

[497-515]

26
1538
AAGGGUGACAAGAUGCGAA
2117
UUCGCAUCUUGUCACCCUU
Rh, D
[1457-1475]

27
1539
CAAGCUGUUCUACGCCGAA
2118
UUCGGCGUAGAACAGCUUG
Rh
[1366-1384]

27
1540
CCUGCUAGUCAACGCCAUA
2119
UAUGGCGUUGACUAGCAGG
Rh
[823-841]

27
1541
CCAAGGGUGUGGUGGAGGA
2120
UCCUCCACCACACCCUUGG
Rh, D
[1149-1167]

27
1542
CACACAGGAUGGCAGGAGA
2121
UCUCCUGCCAUCCUGUGUG
Rh
[1499-1517]

27
1543
UCCUGAGACACAUGGGUGA
2122
UCACCCAUGUGUCUCAGGA
D, Rt, M
[1529-1547]

27
1544
CUACAACUACUACGACGAA
2123
UUCGUCGUAGUAGUUGUAG
Rb
[955-973]

27
1545
GACAAGAUGCGAGACGAGA
2124
UCUCGUCUCGCAUCUUGUC
Rh, Rt
[1463-1481]

27
1546
CCUGGAAGCUGGGCAGCCA
2125
UGGCUGCCCAGCUUCCAGG

[609-627]

27
1547
CUUCAAGCCACACUGGGAA
2126
UUCCCAGUGUGGCUUGAAG
Rh, Rb, D
[844-862]

27
1548
GCGAGACGAGUUAUAGGGA
2127
UCCCUAUAACUCGUCUCGC
Rh
[1471-1489]

28
1549
GAAGCUGGGCAGCCGACUA
2128
UAGUCGGCUGCCCAGCUUC

[613-631]

28
1550
GUGCCUGAGCGGACCUUCA
7129
UGAAGGUCCGCUCAGGCAC
Rh
[1629-1647]

28
1551
GGUGACCCAUGACCUGCAA
2130
UUGCAGGUCAUGGGUCACC
Rh, Rt, M
[1165-1183]

28
1552
AUGAGCCUUUGUUGCUAUA
2131
UAUAGCAACAAAGGCUCAU
Rh
[2114-2132]

28
1553
CAACUACUACGACGACGAA
2132
UUCGUCGUCGUAGUAGUUG
Rb
[958-976]

28
1554
GCUGCGCUCACUCAGCAAA
2133
UUUGCUGAGUGAGCGCAGC
Rh
[571-589]

28
1555
GAGAACAUCCUGGUGUCAA
2134
UUGACACCAGGAUGUUCUC

[422-440]

28
1556
CCCAAGCUGUUCUACGCCA
2135
UGGCGUAGAACAGCUUGGG
Rh
[1364-1382]

28
1557
CAGCUCUAUCCCAACCUCA
2136
UGAGGUUGGGAUAGAGCU

[1886-1904]

28
1558
UGAGCUUCGCUGAUGACUA
2137
UAGUCAUCAGCGAAGCUCA
Rh
[648-666]

29
1559
CCCAAGGCGGCCACGCUUA
2138
UAAGCGUGGCCGCCUUGGG
Rh
[341-359]

29
1560
CUAUACCGUGGGUGUCAUA
2139
UAUGACACCCACGGUAUAG
Rh
[916-934]

29
1561
CAUUGACAAGAACAAGGCA
2140
UGCCUUGUUCUUGUCAAUG
Rh, D
[1216-1234]

29
1562
GGACCCAGCUCAGUGAGCA
2141
UGCUCACUGAGCUGGGUCC

[635-653]

29
1563
GACGACGAGAAGGAAAAGA
2142
UCUUUUCCUUCUCGUCGUC
Rh
[968-986]

29
1564
GCGGCAAGGCGACCACGGA
2143
UCCGUGGUCGCCUUGCCGC

[480-498]

29
1565
GGGACACCCAAAGCGGCUA
2144
UAGCCGCUUUGGGUGUCCC

[1407-1425]

29
1566
GGGAGGUGAGGUACCAGCA
2145
UGCUGGUACCUCACCUCCC

[1562-1580]

29
1567
GCAGCACUACAACUGCGAA
2146
UUCGCAGUUGUAGUGCUGC
Rh, D
[682-700]

29
1568
GCGCAACGUGACCUGGAAA
2147
UUUCCAGGUCACGUUGCGC
M
[598-616]

30
1569
GGGCUGGGCCUGACUGAGA
2148
UCUCAGUCAGGCCCAGCCC

[1196-1214]

30
1570
CCUGAGCGGACCUUCCCAA
2149
UUGGGAAGGUCCGCUCAGG
Rh
[1632-1650]

30
1571
GCAGCUGAAGAUCUGGAUA
2150
UAUCCAGAUCUUCAGCUGC
Rh, D
[1093-1111]

30
1572
AGUGGAGAACAUCCUGGUA
2151
UACCAGGAUGUUCUCCACU
Rh
[418-436]

30
1573
GCAAGCAGCACUACAACUA
2152
UAGUUGUAGUGCUGCUUGC
Rh, D
[678-696]

30
1574
AGCUCAGUGAGCUUCGCUA
2153
UAGCGAAGCUCACUGAGCU

[641-659]

30
1575
CCGACUUGUCACGCAUGUA
2154
UACAUGCGUGACAAGUCGG
Rh
[1233-1251]

30
1576
CCGAGGUCACCAAGGACGA
2155
UCGUCCUUGGUGACCUCGG
Rh, D
[786-804]

30
1577
GGAGCCUCUCGAGCGCCUA
2156
UAGGCGCUCGAGAGGCUCC

[1054-1072]

30
1578
GGCCGCGCAGACCACCGAA
2157
UUCGGUGGUCUGCGCGGCC

[757-775]

31
1579
GGAAACUCCACAUCCUGUA
2158
UACAGGAUGUGGAGUUUCC
Rh
[1703-1721]

31
1580
CAAAGCGGCUCCCUGCUAA
2159
UUAGCAGGGAGCCGCUUUG

[1415-1433]

31
1581
GCUCCUGAGACACAUGGGA
2160
UCCCAUGUGUCUCAGGAGC
D
[1527-1545]

31
1582
CCUGGGCCAUAGUCAUUCA
2161
UGAAUGACUAUGGCCCAGG

[1725-1743]

31
1583
CGUGGAGCCUCUCGAGCGA
2162
UCGCUCGAGAGGCUCCACG

[1051-1069]

31
1584
CCUCCUGCUUCUCAGCGCA
2163
UGCGCUGAGAAGCAGGAGG

[238-256]

31
1585
AGUCCCAGAUCAAGCCUGA
2164
UCAGGCUUGAUCUGGGACU
Rh
[1756-1774]

31
1586
UACCGUGGGUGUCAUGAUA
2165
UAUCAUGACACCCACGGUA
Rh
[919-937]

31
1587
GCCAGCCCUCUUCUGACAA
2166
UUGUCAGAAGAGGGCUGGC

[1845-1863]

31
1588
CCGAGGUGAAGAAACCUGA
2167
UCAGGUUUCUUCACCUCGG
Rh, Rt
[285-303]

32
1589
UCCUGGCACUGCGGAGAAA
2168
UUUCUCCGCAGUGCCAGGA

[316-334]

32
1590
CCCGGAAACUCCACAUCCA
2169
UGGAUGUGGAGUUUCCGGG

[1700-1718]

32
1591
ACUCUGGUCAAGAAGCAUA
2170
UAUGCUUCUUGACCAGAGU
Rh
[2014-2032]

32
1592
CCCAGAUACCAUGAUGCUA
2171
UAGCAUCAUGGUAUCUGGG
Rh
[1679-1697]

32
1593
CCUGAGACACAUGGGUGCA
2172
UGCACCCAUGUGUCUCAGG
D, Rt, M
[1530-1548]

32
1594
GCACUACAACUGCGAGCAA
2173
UUGCUCGCAGUUGUAGUGC
Rh, D
[685-703]

32
1595
CCACAAGAUGGUGGACAAA
2174
UUUGUCCACCAUCUUGUGG
Rh, Rb, M,
[874-892]

32
1596
GGACACAGAUGGCAACCCA
2175
UGGGUUGCCAUCUGUGUCC

[1306-1324]

32
1597
GAAAAGCUGCUAACCAAAA
2176
UUUUGGUUAGCAGCUUUUC

[1073-1091]

32
1598
ACUACAACUGCGAGCACUA
2177
UAGUGCUCGCAGUUGUAGU
Rh, D
[687-705]

33
1599
GCACUCCAAGAUCAACUUA
2178
UAAGUUGAUCUUGGAGUGC
Rh, D
[700-718]

33
1600
GCCUUGAAAAGCUGCUAAA
2179
UUUAGCAGCUUUUCAAGGC

[1068-1086]

33
1601
GUGACUCGGUCCUAUACCA
2180
UGGUAUAGGACCGAGUCAC
Rh
[905-923]

33
1602
GUGGUGGAGGUGACCCAUA
2181
UAUGGGUCACCUCCACCAC
Rh, Rb, Rt,
[1157-1175]

33
1603
AUGCGAGACGAGUUAUAGA
2182
UCUAUAACUCGUCUCGCAU
Rh
[1469-1487]

33
1604
ACCUUCCCAGCUAGAAUUA
2183
UAAUUCUAGCUGGGAAGGU
Rh
[1641-1659]

33
1605
CCCAGCUAGAAUUCACUCA
2184
UGAGUGAAUUCUAGCUGGG
Rh
[1646-1664]

33
1606
GGUCACCAAGGACGUGGAA
2185
UUCCACGUCCUUGGUGACC
Rh, D
[790-808]

33
1607
GGCCUCAGGGUGCACACAA
2186
UUGUGUGCACCCUGAGGCC

[1487-1505]

33
1608
UGAGGUACCAGCCUUGGAA
2187
UUCCAAGGCUGGUACCUCA
Rh
[1568-1586]

34
1609
CAUGGUGACUCGGUCCUAA
2188
UUAGGACCGAGUCACCAUG
Rh
[901-919]

34
1610
GGUGAGGUACCAGCCUUGA
2189
UCAAGGCUGGUACCUCACC
Rh
[1566-1584]

34
1611
GCCGAGGUGAAGAAACCUA
2190
UAGGUUUCUUCACCUCGGC
Rh, Rt
[284-302]

34
1612
GUACGGACCCAGCUCAGUA
2191
UACUGAGCUGGGUCCGUAC

[631-649]

34
1613
CAAGAAGGACCUGUACCUA
2192
UAGGUACAGGUCCUUCUUG
Rh, D, M
[1255-1273]

34
1614
GAGCACUCCAAGAUCAACA
2193
UGUUGAUCUUGGAGUGCUC
Rh, D
[698-716]

34
1615
CAUGUUCUUCAAGCCACAA
2194
UUGUGGCUUGAAGAACAUG
Rh, Rb, D
[838-856]

34
1616
CCCUCCUGCUUCUCAGCGA
2195
UCGCUGAGAAGCAGGAGGG

[237-255]

34
1617
AUGUCAGGCAAGAAGGACA
2196
UGUCCUUCUUGCCUGACAU
Rh, D
[1247-1265]

34
1618
CAAGAUCAACUUCCGCGAA
2197
UUCGCGGAAGUUGAUCUUG
D
[706-724]

35
1619
GCGUGUUCCACGCCACCGA
2198
UCGGUGGCGUGGAACACGC

[1278-1296]

35
1620
CGGACCCAGCUCAGUGAGA
2199
UCUCACUGAGCUGGGUCCG

[634-652]

35
1621
CCUUCAGCUUGUACCAGGA
2200
UCCUGGUACAAGCUGAAGG

[381-399]

35
1622
GCUCUCCAGCCUCAUCAUA
2201
UAUGAUGAGGCUGGAGAGC
Rh, D, Rt,
[1018-1036]

35
1623
CCCUGGCCCACAAGCUCUA
2202
UAGAGCUUGUGGGCCAGGG
Rh, D, P
[1005-1023]

35
1624
GCCCGAGGUCACCAAGGAA
2203
UUCCUUGGUGACCUCGGGC
Rh, D
[784-802]

35
1625
GUGGAGAACAUCCUGGUGA
2204
UCACCAGGAUGUUCUCCAC
Rh
[419-437]

35
1626
GCUCACUCAGCAACUCCAA
2205
UUGGAGUUGCUGAGUGAGC
Rh
[576-594]

35
1627
ACGCCAUGUUCUUCAAGCA
2206
UGCUUGAAGAACAUGGCGU
Rh, Rb, P
[834-852]

35
1628
ACACAUGGGUGCUAUUGGA
2907
UCCAAUAGCACCCAUGUGU
Rh
[1536-1554]

36
1629
CCAGCUCAGUGAGCUUCGA
2208
UCGAAGCUCACUGAGCUGG

[639-657]

36
1630
CCCAGCUCAGUGAGCUUCA
2209
UGAAGCUCACUGAGCUGGG

[638-656]

36
1631
GGGCGGCAAGGCGACCACA
2210
UGUGGUCGCCUUGCCGCCC

[478-496]

36
1632
CAGGGUGCACACAGGAUGA
2211
UCAUCCUGUGUGCACCCUG

[1492-1510]

36
1633
AGGUGAAGAAACCUGCAGA
2212
UCUGCAGGUUUCUUCACCU
Rh
[288-306]

36
1634
CCUCUCCCAACUAUAAAAA
2213
UUUUUAUAGUUGGGAGAG
Rh
[1900-1918]

36
1635
GACUGUACGGACCCAGCUA
2214
UAGCUGGGUCCGUACAGUC

[627-645]

36
1636
GAAGGAGCUCCCAGGAGGA
2215
UCCUCCUGGGAGCUCCUUC

[1983-2001]

36
1637
ACGCAUGUCAGGCAAGAAA
2216
UUUCUUGCCUGACAUGCGU
Rh, D
[1243-1261]

36
1638
GACUCGGUCCUAUACCGUA
2217
UACGGUAUAGGACCGAGUC
Rh
[907-925]

37
1639
CACUACAACUGCGAGCACA
2218
UGUGCUCGCAGUUGUAGUG
Rh, D
[686-704]

37
1640
AGCUCCUGGCACUGCGGAA
2219
UUCCGCAGUGCCAGGAGCU

[313-331]

37
1641
CUAAGGGUGACAAGAUGCA
2220
UGCAUCUUGUCACCCUUAG
Rh
[1455-1473]

37
1642
UGUGAGACCAAAUUGAGCA
2221
UGCUCAAUUUGGUCUCACA
Rh
[1816-1834]

37
1643
GCCGACUUGUCACGCAUGA
2222
UCAUGCGUGACAAGUCGGC
Rh
[1232-1250]

37
1644
CAGGAUGGCAGGAGGCAUA
2223
UAUGCCUCCUGCCAUCCUG

[1503-1521]

37
1645
ACAAGAACAAGGCCGACUA
2224
UAGUCGGCCUUGUUCUUGU
Rh
[1221-1239]

37
1646
UGCGCUCCCUCCUGCUUCA
2225
UGAAGCAGGAGGGAGCGCA

[231-249]

37
1647
GGCGAGCUGCUGCGCUCAA
2226
UUGAGCGCAGCAGCUCGCC
Rh
[563-581]

37
1648
GAUGCACCGGACAGGCCUA
2227
UAGGCCUGUCCGGUGCAUC
Rh, Rb, Rt,
[937-955]

38
1649
CGUGUCGCUGGGCGGCAAA
2228
UUUGCCGCCCAGCGACACG

[469-487]

38
1650
AUCCCAACCUCUCCCAACA
2229
UGUUGGGAGAGGUUGGGA
Rh
[1893-1911]

38
1651
UGUUCUACGCCGACCACCA
2230
UGGUGGUCGGCGUAGAACA
Rh
[1371-1389]

38
1652
CGGCCUGGCCUUCAGCUUA
2231
UAAGCUGAAGGCCAGGCCG

[373-391]

38
1653
GUCGCAGGCCAAGGCAGUA
2232
UACUGCCUUGGCCUGCGAC

[499-517]

38
1654
AGUCAUUCUGCCUGCCCUA
2233
UAGGGCAGGCAGAAUGACU

[1735-1753]

38
1655
CCCAGAAUGACCUGGCCGA
2234
UCGGCCAGGUCAUUCUGGG

[1949-1967]

38
1656
ACAAGAUGGUGGACAACCA
2235
UGGUUGUCCACCAUCUUGU
Rh, Rb, M,
[876-894]

38
1657
GCUAGUCAACGCCAUGUUA
2236
UAACAUGGCGUUGACUAGC
Rh
[826-844]

38
1658
ACGCCACCGCCUUUGAGUA
2237
UACUCAAAGGCGGUGGCGU
Rh
[1287-1305]

39
1659
GCCGCGCAGACCACCGACA
2238
UGUCGGUGGUCUGCGCGGC

[758-776]

39
1660
GCUAUUCAUUGGGCGCCUA
2239
UAGGCGCCCAAUGAAUAGC
D
[1429-1447]

39
1661
CUCAGUGAGCUUCGCUGAA
2240
UUCAGCGAAGCUCACUGAG

[643-661]

39
1662
GGAGGUGAGGUACCAGCCA
2241
UGGCUGGUACCUCACCUCC

[1563-1581]

39
1663
GCCAAGGCAGUGCUGAGCA
2242
UGCUCAGCACUGCCUUGGC
Rh
[506-524]

39
1664
CUCUCCAGCCUCAUCAUCA
2243
UGAUGAUGAGGCUGGAGA
Rh, D, Rt,
[1019-1037]

39
1665
GAAUGACCUGGCCGCAGUA
2244
UACUGCGGCCAGGUCAUUC

[1953-1971]

39
1666
UGGUGACUCGGUCCUAUAA
2245
UUAUAGGACCGAGUCACCA
Rh
[903-921]

39
1667
CAGGUACCUUCUCACCUGA
2246
UCAGGUGAGAAGGUACCUG
Rh
[1800-1818]

39
1668
GUUCCACGCCACCGCCUUA
2247
UAAGGCGGUGGCGUGGAAC
D
[1282-1300]

40
1669
CCGACUGUACGGACCCAGA
2248
UCUGGGUCCGUACAGUCGG

[625-643]

40
1670
GCAGACCACCGACGGCAAA
2249
UUUGCCGUCGGUGGUCUGC
D, Rt
[763-781]

40
1671
AAGAUGCGAGACGAGUUAA
2250
UUAACUCGUCUCGCAUCUU
Rh
[1466-1484]

40
1672
CAAAGAGCAGCUGAAGAUA
2251
UAUCUUCAGCUGCUCUUUG
Rh
[1087-1105]

40
1673
ACGACGAGAAGGAAAAGCA
2252
UGCUUUUCCUUCUCGUCGU
Rh
[969-987]

40
1674
CACUCCACUUGGACAUGGA
2253
UCCAUGUCCAAGUGGAGUG
Rh
[1659-1677]

40
1675
AGUCCAUCAACGAGUGGGA
2254
UCCCACUCGUUGAUGGACU
Rh, Rt, M
[741-759]

40
1676
GCGCCGGCCUGGCCUUCAA
2255
UUGAAGGCCAGGCCGGCGC
Rh
[369-387]

40
1677
GGAAAAGCUGCAAAUCGUA
2256
UACGAUUUGCAGCUUUUCC
Rh
[979-997]

40
1678
ACAUUUUGUUGGAGCGUGA
2257
UCACGCUCCAACAAAAUGU

[2184-2202]

41
1679
ACCGUGGCUUCAUGGUGAA
2258
UUCACCAUGAAGCCACGGU
Rh, Rt, M
[891-909]

41
1680
CCCUUCAUCUUCCUAGUGA
2259
UCACUAGGAAGAUGAAGGG

[1388-1406]

41
1681
GAAAUUCCACCACAAGAUA
2260
UAUCUUGUGGUGGAAUUUC
Rh
[865-883]

41
1682
CUAUAAAACUAGGUGCUGA
2261
UCAGCACCUAGUUUUAUAG
Rh
[1910-1928]

41
1683
GGAGGUGCACGCCGGCCUA
2262
UAGGCCGGCGUGCACCUCC

[544-562]

41
1684
GCAGGCCAAGGCAGUGCUA
2263
UAGCACUGCCUUGGCCUGC

[502-520]

41
1685
UGAGACCAAAUUGAGCUAA
2264
UUAGCUCAAUUUGGUCUCA
Rh
[1818-1836]

41
1686
GCCAUAGUCAUUCUGCCUA
2265
UAGGCAGAAUGACUAUGGC

[1730-1748]

41
1687
AGCUGAAGAUCUGGAUGGA
2266
UCCAUCCAGAUCUUCAGCU
Rh, D
[1095-1113]

41
1688
CCAUCUCCUUGCCCAAGGA
2267
UCCUUGGGCAAGGAGAUGG
Rh
[1137-1155]

42
1689
CCCAGAUCAAGCCUGCCUA
2268
UAGGCAGGCUUGAUCUGGG
Rh
[1759-1777]

42
1690
GCUGUUGCCAUCUCCUUGA
2269
UCAAGGAGAUGGCAACAGC

[1130-1148]

42
1691
CGAGGUCACCAAGGACGUA
2270
UACGUCCUUGGUGACCUCG
Rh, D
[787-805]

42
1692
CAACUAUAAAACUAGGUGA
2271
UCACCUAGUUUUAUAGUUG
Rh
[1907-1925]

42
1693
GAAGGCUGUUGCCAUCUCA
2272
UGAGAUGGCAACAGCCUUC
Rt
[1126-1144]

42
1694
UGCGGAGAAGUUGAGCCCA
2273
UGGGCUCAACUUCUCCGCA

[325-343]

42
1695
CUCCUUGCCCAAGGGUGUA
2274
UACACCCUUGGGCAAGGAG
Rh
[1141-1159]

42
1696
GCCCUGAAAGUCCCAGAUA
2275
UAUCUGGGACUUUCAGGGC

[1748-1766]

42
1697
CAAGGGUGUGGUGGAGGUA
2276
UACCUCCACCACACCCUUG
Rh, D
[1150-1168]

42
1698
AAGAGCAGCUGAAGAUCUA
2277
UAGAUCUUCAGCUGCUCUU
Rh
[1089-1107]

43
1699
GAAGAUGCAGAAGAAGGCA
2278
UGCCUUCUUCUGCAUCUUC
Rh, Rb, Rt
[1114-1132]

43
1700
CGGAAACUCCACAUCCUGA
2279
UCAGGAUGUGGAGUUUCCG

[1702-1720]

43
1701
AGUCAACGCCAUGUUCUUA
2280
UAAGAACAUGGCGUUGACU
Rh
[829-847]

43
1702
CGAGCGCCUUGAAAAGCUA
2281
UAGCUUUUCAAGGCGCUCG

[1063-1081]

43
1703
AUACCGUGGGUGUCAUGAA
2282
UUCAUGACACCCACGGUAU
Rh
[918-936]

43
1704
GACCUGGGCCAUAGUCAUA
2283
UAUGACUAUGGCCCAGGUC

[1723-1741]

43
1705
CAUGUCAGGCAAGAAGGAA
2284
UUCCUUCUUGCCUGACAUG
Rh, D
[1246-1264]

43
1706
UGCGAGACGAGUUAUAGGA
2285
UCCUAUAACUCGUCUCGCA
Rh
[1470-1488]

43
1707
CGCAACGUGACCUGGAAGA
2286
UCUUCCAGGUCACGUUGCG

[599-617]

43
1708
AGCAAGCAGCACUACAACA
2287
UGUUGUAGUGCUGCUUGCU
Rh, D
[677-695]

44
1709
GCUGCUGCGCUCACUCAGA
2288
UCUGAGUGAGCGCAGCAGC
Rh
[568-586]

44
1710
UGAUGAUGCACCGGACAGA
2289
UCUGUCCGGUGCAUCAUCA
Rh
[933-951]

44
1711
UUGUUGCUAUCAAUCCAAA
2290
UUUGGAUUGAUAGCAACAA
Rh
[2122-2140]

44
1712
CCUUGAAAAGCUGCUAACA
2291
UGUUAGCAGCUUUUCAAGG

[1069-1087]

44
1713
CCCUUUGACCAGGACAUCA
2292
UGAUGUCCUGGUCAAAGGG
Rh, Rt
[1322-1340]

44
1714
GAGGUGAAGAAACCUGCAA
2293
UUGCAGGUUUCUUCACCUC
Rh
[287-305]

44
1715
CCCAAGGGUGUGGUGGAGA
2294
UCUCCACCACACCCUUGGG
Rh, D
[1148-1166]

44
1716
CCCUGCUAUUCAUUGGGCA
2295
UGCCCAAUGAAUAGCAGGG
D
[1425-1443]

44
1717
CUGAAAGUCCCAGAUCAAA
2296
UUUGAUCUGGGACUUUCAG

[1751-1769]

44
1718
GCUGCAAAUCGUGGAGAUA
2297
UAUCUCCACGAUUUGCAGC
Rh
[985-1003]

45
1719
CAAGCCUGCCUCAAUCAGA
2298
UCUGAUUGAGGCAGGCUUG
Rh
[1766-1784]

45
1720
CGAGCAGCUGCGCGACGAA
2299
UUCGUCGCGCAGCUGCUCG

[526-544]

45
1721
AGGCCGACUUGUCACGCAA
2300
UUGCGUGACAAGUCGGCCU
Rh
[1230-1248]

45
1722
GCAGCAGCUCCUGGCACUA
2301
UAGUGCCAGGAGCUGCUGC

[308-326]

45
1723
GGCCAUAGUCAUUCUGCCA
2302
UGGCAGAAUGACUAUGGCC

[1729-1747]

45
1724
CCCGUGUGCCUGAGCGGAA
2303
UUCCGCUCAGGCACACGGG
Rh
[1624-1642]

45
1725
CAGCUGAAGAUCUGGAUGA
2304
UCAUCCAGAUCUUCAGCUG
Rh, D
[1094-1112]

45
1726
CAAGCCACACUGGGAUGAA
2305
UUCAUCCCAGUGUGGCUUG
Rh, Rb
[847-865]

45
1727
GAAUUCACUCCACUUGGAA
2306
UUCCAAGUGGAGUGAAUUC
Rh
[1654-1672]

45
1728
CGGCGCCCUGCUAGUCAAA
2307
UUUGACUAGCAGGGCGCCG
Rh
[817-835]

46
1729
UGGAAGCUGGGCAGCCGAA
2308
UUCGGCUGCCCAGCUUCCA

[611-629]

46
1730
GGCAAGGCGACCACGGCGA
2309
UCGCCGUGGUCGCCUUGCC
Rh
[482-500]

46
1731
CACUGCGGAGAAGUUGAGA
2310
UCUCAACUUCUCCGCAGUG

[322-340]

46
1732
GGCAGGAGGCAUCCAAAGA
2311
UCUUUGGAUGCCUCCUGCC

[1509-1527]

46
1733
GGUGACUCGGUCCUAUACA
2312
UGUAUAGGACCGAGUCACC
Rh
[904-922]

46
1734
UUUAUAGCCAGGUACCUUA
2313
UAAGGUACCUGGCUAUAAA
Rh
[1792-1810]

46
1735
GGCCAUGGCCAAGGACCAA
2314
UUGGUCCUUGGCCAUGGCC
Rh, D
[397-415]

46
1736
CAAAGAUAGGGAGGGAAGA
2315
UCUUCCCUCCCUAUCUUUG

[2089-2107]

46
1737
UCUUCUGACACUAAAACAA
2316
UUGUUUUAGUGUCAGAAG

[1853-1871]

46
1738
CUUCUGACACUAAAACACA
2317
UGUGUUUUAGUGUCAGAA

[1854-1872]

47
1739
UCACGUGGAGCCUCUCGAA
2318
UUCGAGAGGCUCCACGUGA

[1048-1066]

47
1740
CAGUCCAUCAACGAGUGGA
2319
UCCACUCGUUGAUGGACUG
Rh, Rt, M
[740-758]

47
1741
AGACCAAAUUGAGCUAGGA
2320
UCCUAGCUCAAUUUGGUCU

[1820-1838]

47
1742
GGGUUCCCGUGUGCCUGAA
2321
UUCAGGCACACGGGAACCC
Rh
[1619-1637]

47
1743
UUGCUAUCAAUCCAAGAAA
2322
UUUCUUGGAUUGAUAGCAA
Rh
[2125-2143]

47
1744
CAACCGUGGCUUCAUGGUA
2323
UACCAUGAAGCCACGGUUG
Rh, Rt, M
[889-907]

47
1745
CUGUACGGACCCAGCUCAA
2324
UUGAGCUGGGUCCGUACAG

[629-647]

47
1746
CAGCAGCAAGCAGCACUAA
2325
UUAGUGCUGCUUGCUGCUG
Rh, D
[673-691]

47
1747
CCUGCAGCCGCAGCAGCUA
2326
UAGCUGCUGCGGCUGCAGG

[299-317]

47
1748
GACACUAAAACACCUCAGA
2327
UCUGAGGUGUUUUAGUGUC

[1859-1877]

48
1749
CAACUGCGAGCACUCCAAA
2328
UUUGGAGUGCUCGCAGUUG
Rh, D
[691-709]

48
1750
ACUGCGGAGAAGUUGAGCA
2329
UGCUCAACUUCUCCGCAGU

[323-341]

48
1751
GCGCCCUGCUAGUCAACGA
2330
UCGUUGACUAGCAGGGCGC
Rh
[819-837]

48
1752
GGAAGCUGGGCAGCCGACA
2331
UGUCGGCUGCCCAGCUUCC

[612-630]

48
1753
AGGCUCCUGAGACACAUGA
2332
UCAUGUGUCUCAGGAGCCU
D
[1525-1543]

48
1754
CGACAAGCGCAGCGCGCUA
2333
UAGCGCGCUGCGCUUGUCG

[721-739]

48
1755
UCAGUGAGCUUCGCUGAUA
2334
UAUCAGCGAAGCUCACUGA

[644-662]

48
1756
UUGAGAAGGAGCUCCCAGA
2335
UCUGGGAGCUCCUUCUCAA

[1979-1997]

48
1757
ACUGCGAGCACUCCAAGAA
2336
UUCUUGGAGUGCUCGCAGU
Rh, D
[693-711]

48
1758
CAUCCUGGUGUCACCCGUA
2337
UACGGGUGACACCAGGAUG

[427-445]

49
1759
GUGCGCAGCAGCAAGCAGA
2338
UCUGCUUGCUGCUGCGCAC
Rh, D
[668-686]

49
1760
CACGCCACCGCCUUUGAGA
2339
UCUCAAAGGCGGUGGCGUG
Rh
[1286-1304]

49
1761
UCUCGAGCGCCUUGAAAAA
2340
UUUUUCAAGGCGCUCGAGA

[1060-1078]

49
1762
GCUUCGCUGAUGACUUCGA
2341
UCGAAGUCAUCAGCGAAGC
Rh
[651-669]

49
1763
UCUCCUUGCCCAAGGGUGA
2342
UCACCCUUGGGCAAGGAGA
Rh
[1140-1158]

49
1764
GCAGUCCAUCAACGAGUGA
2343
UCACUCGUUGAUGGACUGC
Rh, Rt, M
[739-757]

49
1765
AGAUGGUGGACAACCGUGA
2344
UCACGGUUGUCCACCAUCU
Rh, M
[879-897]

49
1766
CGGCUCCCUGCUAUUCAUA
2345
UAUGAAUAGCAGGGAGCCG

[1420-1438]

49
1767
AUACCAUGAUGCUGAGCCA
2346
UGGCUCAGCAUCAUGGUAU

[1684-1702]

49
1768
AGCCAGGUACCUUCUCACA
2347
UGUGAGAAGGUACCUGGCU
Rh
[1797-1815]

50
1769
GAGCCCGGAAACUCCACAA
2348
UUGUGGAGUUUCCGGGCUC

[1697-1715]

50
1770
GCAGCUCCUGGCACUGCGA
2349
UCGCAGUGCCAGGAGCUGC

[311-329]

50
1771
CCCGAGGUCACCAAGGACA
2350
UGUCCUUGGUGACCUCGGG
Rh, D
[785-803]

50
1772
CCUGACUGAGGCCAUUGAA
2351
UUCAAUGGCCUCAGUCAGG
Rh
[1204-1222]

50
1773
UGCUGAGCCCGGAAACUCA
2352
UGAGUUUCCGGGCUCAGCA

[1693-1711]

50
1774
GCCAUCUCCUUGCCCAAGA
2353
UCUUGGGCAAGGAGAUGGC
Rh
[1136-1154]

50
1775
CAAGCAGCACUACAACUGA
2354
UCAGUUGUAGUGCUGCUUG
Rh, D
[679-697]

50
1776
CAAGGCAGUGCUGAGCGCA
2355
UGCGCUCAGCACUGCCUUG
Rh
[508-526]

50
1777
CAAUGACAUUUUGUUGGAA
2356
UUCCAACAAAAUGUCAUUG

[2179-2197]

50
1778
AGUGAGCUUCGCUGAUGAA
2357
UUCAUCAGCGAAGCUCACU

[646-664]

51
1779
AUGAUGAUGCACCGGACAA
2358
UUGUCCGGUGCAUCAUCAU
Rh
[932-950]

51
1780
GAAACACCUGGCUGGGCUA
2359
UAGCCCAGCCAGGUGUUUC
D
[1183-1201]

51
1781
CCUGCUAUUCAUUGGGCGA
2360
UCGCCCAAUGAAUAGCAGG
D
[1426-1444]

51
1782
CGCCACCGCCUUUGAGUUA
2361
UAACUCAAAGGCGGUGGCG
Rh
[1288-1306]

51
1783
GCUUCUCAGCGCCUUCUGA
2362
UCAGAAGGCGCUGAGAAGC

[244-262]

51
1784
UGAUGCUGAGCCCGGAAAA
2363
UUUUCCGGGCUCAGCAUCA

[1690-1708]

51
1785
UGACCUGGCCGCAGUGAGA
2364
UCUCACUGCGGCCAGGUCA

[1956-1974]

51
1786
UGCAGAAACACCUGGCUGA
2365
UCAGCCAGGUGUUUCUGCA

[1179-1197]

51
1787
GCAGUGCUGAGCGCCGAGA
2366
UCUCGGCGCUCAGCACUGC

[512-530]

51
1788
CGGCGCGCAACGUGACCUA
2367
UAGGUCACGUUGCGCGCCG

[594-612]

52
1789
AGUGCUGAGCGCCGAGCAA
2368
UUGCUCGGCGCUCAGCACU

[514-532]

52
1790
ACAGGCCUCUACAACUACA
2369
UGUAGUUGUAGAGGCCUGU
Rh, Rb, D,
[947-965]

52
1791
GCAGCUGCGCGACGAGGAA
2370
UUCCUCGUCGCGCAGCUGC
Rh, D
[529-547]

52
1792
AUUGAGAAGGAGCUCCCAA
2371
UUGGGAGCUCCUUCUCAAU

[1978-1996]

52
1793
CGCGCAGACCACCGACGGA
2372
UCCGUCGGUGGUCUGCGCG

[760-778]

52
1794
CCUGUACCUGGCCAGCGUA
2373
UACGCUGGCCAGGUACAGG
Rh
[1264-1282]

52
1795
CUGAGCGGACCUUCCCAGA
2374
UCUGGGAAGGUCCGCUCAG
Rh
[1633-1651]

52
1796
GGCCUUCAGCUUGUACCAA
2375
UUGGUACAAGCUGAAGGCC

[379-397]

52
1797
CACCCAAAGCGGCUCCCUA
2376
UAGGGAGCCGCUUUGGGUG

[1411-1429]

52
1798
GCCAAGGACCAGGCAGUGA
2377
UCACUGCCUGGUCCUUGGC
Rh
[404-422]

53
1799
CUCAGGGUGCACACAGGAA
2378
UUCCUGUGUGCACCCUGAG

[1490-1508]

53
1800
CGAGCUGCUGCGCUCACUA
2379
UAGUGAGCGCAGCAGCUCG
Rh
[565-583]

53
1801
GGCUGGGCCUGACUGAGGA
2380
UCCUCAGUCAGGCCCAGCC

[1197-1215]

53
1802
CCGCAGCAGCUCCUGGCAA
2381
UUGCCAGGAGCUGCUGCGG

[306-324]

53
1803
UGUGGGACCUGGGCCAUAA
2382
UUAUGGCCCAGGUCCCACA

[1718-1736]

53
1804
AAGAUGCAGAAGAAGGCUA
2383
UAGCCUUCUUCUGCAUCUU
Rh, Rt, M
[1115-1133]

53
1805
CCACGGCGCGCAACGUGAA
2384
UUCACGUUGCGCGCCGUGG
Rh
[591-609]

53
1806
ACCUUCUCACCUGUGAGAA
2385
UUCUCACAGGUGAGAAGGU
Rh
[1805-1823]

53
1807
UGAAGAAACCUGCAGCCGA
2386
UCGGCUGCAGGUUUCUUCA

[291-309]

53
1808
CAGCACUACAACUGCGAGA
2387
UCUCGCAGUUGUAGUGCUG
Rh, D
[683-701]

54
1809
GCGACAAGCGCAGCGCGCA
2388
UGCGCGCUGCGCUUGUCGC

[720-738]

54
1810
UAGAAUUCACUCCACUUGA
2389
UCAAGUGGAGUGAAUUCUA
Rh
[1652-1670]

54
1811
GUGGAAAAACAGACCGGGA
2390
UCCCGGUCUGUUUUUCCAC

[1603-1621]

54
1812
ACGUGGAGCCUCUCGAGCA
2391
UGCUCGAGAGGCUCCACGU

[1050-1068]

54
1813
GGCGCGCAACGUGACCUGA
2392
UCAGGUCACGUUGCGCGCC

[595-613]

54
1814
UGGACAACCGUGGCUUCAA
2393
UUGAAGCCACGGUUGUCCA
Rh, M
[885-903]

54
1815
CUAGUCAACGCCAUGUUCA
2394
UGAACAUGGCGUUGACUAG
Rh
[827-845]

54
1816
AGAAUGACCUGGCCGCAGA
2395
UCUGCGGCCAGGUCAUUCU

[1952-1970]

54
1817
AGCUGCUGCGCUCACUCAA
2396
UUGAGUGAGCGCAGCAGCU
Rh
[567-585]

54
1818
CUCUAUCCCAACCUCUCCA
2397
UGGAGAGGUUGGGAUAGA
Rh
[1889-1907]

55
1819
GCGAGCUGCUGCGCUCACA
2398
UGUGAGCGCAGCAGCUCGC
Rh
[564-582]

55
1820
CGCAGCAGCAAGCAGCACA
2399
UGUGCUGCUUGCUGCUGCG
Rh, D
[671-689]

55
1821
GGCUGGGCUGGGCCUGACA
2400
UGUCAGGCCCAGCCCAGCC

[1192-1210]

55
1822
UCUCCAGCCUCAUCAUCCA
2401
UGGAUGAUGAGGCUGGAG
Rh, D, Rt,
[1020-1038]

55
1823
CAACGCCAUGUUCUUCAAA
2402
UUUGAAGAACAUGGCGUUG
Rh, Rb, P
[832-850]

55
1824
UGGCACUGCGGAGAAGUUA
2403
UAACUUCUCCGCAGUGCCA

[319-337]

55
1825
UUUGAGUUGGACACAGAUA
2404
UAUCUGUGUCCAACUCAAA

[1298-1316]

55
1826
UGGGCGAGCUGCUGCGCUA
2405
UAGCGCAGCAGCUCGCCCA
Rh
[561-579]

55
1827
CUGCUAACCAAAGAGCAGA
2406
UCUGCUCUUUGGUUAGCAG

[1079-1097]

55
1828
AACGUGACCUGGAAGCUGA
2407
UCAGCUUCCAGGUCACGUU

[602-620]

56
1829
AUGACAUUUUGUUGGAGCA
2408
UGCUCCAACAAAAUGUCAU

[2181-2199]

56
1830
CAGGAGGCAUCCAAAGGCA
2409
UGCCUUUGGAUGCCUCCUG

[1511-1529]

56
1831
AUCUCCUUGCCCAAGGGUA
2410
UACCCUUGGGCAAGGAGAU
Rh
[1139-1157]

56
1832
UGGGAUGAGAAAUUCCACA
2411
UGUGGAAUUUCUCAUCCCA
Rh
[857-875]

56
1833
AAAGCUGCUAACCAAAGAA
2412
UUCUUUGGUUAGCAGCUUU

[1075-1093]

56
1834
AGGAGGCAUCCAAAGGCUA
2413
UAGCCUUUGGAUGCCUCCU

[1512-1530]

56
1835
CACCGCCUUUGAGUUGGAA
2414
UUCCAACUCAAAGGCGGUG
Rh
[1291-1309]

56
1836
CCAACUAUAAAACUAGGUA
2415
UACCUAGUUUUAUAGUUGG
Rh
[1906-1924]

56
1837
CAAGAAGCAUCGUGUCUGA
2416
UCAGACACGAUGCUUCUUG
Rh
[2022-2040]

56
1838
AGCAGCUGAAGAUCUGGAA
2417
UUCCAGAUCUUCAGCUGCU
Rh, D
[1092-1110]

57
1839
GCGCUCCCUCCUGCUUCUA
2418
UAGAAGCAGGAGGGAGCGC

[232-250]

57
1840
UGCUAGUCAACGCCAUGUA
2419
UACAUGGCGUUGACUAGCA
Rh
[825-843]

57
1841
CGCCGAGCAGCUGCGCGAA
2420
UUCGCGCAGCUGCUCGGCG

[523-541]

57
1842
CCGCGCAGACCACCGACGA
2421
UCGUCGGUGGUCUGCGCGG

[759-777]

57
1843
UAGCCAGGUACCUUCUCAA
2422
UUGAGAAGGUACCUGGCUA
Rh
[1796-1814]

57
1844
UGCUUCUCAGCGCCUUCUA
2423
UAGAAGGCGCUGAGAAGCA

[243-261]

57
1845
CUCCCUCCUGCUUCUCAGA
2424
UCUGAGAAGCAGGAGGGAG

[235-253]

57
1846
CGCAGGCCAAGGCAGUGCA
2425
UGCACUGCCUUGGCCUGCG

[501-519]

57
1847
GCAAGGCGACCACGGCGUA
2426
UACGCCGUGGUCGCCUUGC
Rh
[483-501]

57
1848
GCAGCCGCAGCAGCUCCUA
2427
UAGGAGCUGCUGCGGCUGC

[302-320]

TABLE E

SERPINH1 Cross-Species 18 + 1-mer siRNAs

No.
SEQ ID NO
Sense siRNA
SEQ
AntiSense siRNA
Other
human-

1
2428
UCACCAAGGACGUGGAGCA
2576
UGCUCCACGUCCUUGGU
Rh, D
[792-810]

2
2429
CAGCGCGCUGCAGUCCAUA
2577
UAUGGACUGCAGCGCGC
Rh, Rt
[730-748]

3
2430
CAUCUACGGGCGCGAGGAA
2578
UUCCUCGCGCCCGUAGA
D, M
[1336-1354]

4
2431
CUCCAGCCUCAUCAUCCUA
2579
UAGGAUGAUGAGGCUGG
Rh, D, Rt, M
[1021-1039]

5
2432
GACAUCUACGGGCGCGAGA
2580
UCUCGCGCCCGUAGAUG
D, M
[1334-1352]

6
2433
CGUGCGCAGCAGCAAGCAA
2581
UUGCUUGCUGCUGCGCA
Rh, D, M
[667-685]

7
2434
GUCACCAAGGACGUGGAGA
2582
UCUCCACGUCCUUGGUG
Rh, D
[791-809]

8
2435
CCGCGACAAGCGCAGCGCA
2583
UGCGCUGCGCUUGUCGC
D
[718-736]

9
2436
GCGCAGCGCGCUGCAGUCA
2584
UGACUGCAGCGCGCUGC
Rh, Rt
[727-745]

10
2437
GGCCCACAAGCUCUCCAGA
2585
UCUGGAGAGCUUGUGGG
Rh, D, P
[1009-1027]

11
2438
CAAGGACGUGGAGCGCACA
2586
UGUGCGCUCCACGUCCU
Rh, D
[796-814]

12
2439
AGCCUCAUCAUCCUCAUGA
2587
UCAUGAGGAUGAUGAGG
Rh, D, Rt, M
[1025-1043]

13
2440
GGUGUGGUGGAGGUGACCA
2588
UGGUCACCUCCACCACA
Rh, D
[1154-1172]

14
2441
GCAAGCUGCCCGAGGUCAA
2589
UUGACCUCGGGCAGCUU
Rh, D
[777-795]

15
2442
GUGGAGGUGACCCAUGACA
2590
UGUCAUGGGUCACCUCC
Rh, Rt, M
[1160-1178]

16
2443
CACAAGAUGGUGGACAACA
2591
UGUUGUCCACCAUCUUG
Rh, Rb, M, P
[875-893]

17
2444
GCGAGGAGCUGCGCAGCCA
2592
UGGCUGCGCAGCUCCUC
D, M
[1347-1365]

18
2445
UACUACGACGACGAGAAGA
2593
UCUUCUCGUCGUCGUAG
Rb
[962-980]

19
2446
GAGGUGACCCAUGACCUGA
2594
UCAGGUCAUGGGUCACC
Rh, Rt, M
[1163-1181]

20
2447
ACUUCCGCGACAAGCGCAA
2595
UUGCGCUUGUCGCGGAA
D
[714-732]

21
2448
GCCCACAAGCUCUCCAGCA
2596
UGCUGGAGAGCUUGUGG
Rh, D, P
[1010-1028]

22
2449
GCGCAGCAGCAAGCAGCAA
2597
UUGCUGCUUGCUGCUGC
Rh, D
[670-688]

23
2450
CGAGGAGCUGCGCAGCCCA
2598
UGGGCUGCGCAGCUCCU
D, M
[1348-1366]

24
2451
AACGCCAUGUUCUUCAAGA
2599
UCUUGAAGAACAUGGCG
Rh, Rb, P
[833-851]

25
2452
GUCAGGCAAGAAGGACCUA
2600
UAGGUCCUUCUUGCCUG
Rh, D
[1249-1267]

26
2453
GCCUGGGCGAGCUGCUGCA
2601
UGCAGCAGCUCGCCCAG
Rh, D
[558-576]

27
2454
GAUGAUGCACCGGACAGGA
2602
UCCUGUCCGGUGCAUCA
Rh, Rb, Rt, M
[934-952]

28
2455
GGACCUGUACCUGGCCAGA
2603
UCUGGCCAGGUACAGGU
Rh, D
[1261-1279]

29
2456
GCGACGAGGAGGUGCACGA
2604
UCGUGCACCUCCUCGUC
D
[537-555]

30
2457
UGUGGUGGAGGUGACCCAA
2605
UUGGGUCACCUCCACCA
Rh, D
[1156-1174]

31
2458
UUCAAGCCACACUGGGAUA
2606
UAUCCCAGUGUGGCUUG
Rh, Rb
[845-863]

32
2459
CAAGAUGGUGGACAACCGA
2607
UCGGUUGUCCACCAUCU
Rh, Rb, M, P
[877-895]

33
2460
UCAACUUCCGCGACAAGCA
2608
UGCUUGUCGCGGAAGUU
D
[711-729]

34
2461
AUUCAUUGGGCGCCUGGUA
2609
UACCAGGCGCCCAAUGA
D
[1432-1450]

35
2462
CUCCAAGAUCAACUUCCGA
2610
UCGGAAGUUGAUCUUGG
Rh, D, Rt, M
[703-721]

36
2463
CAGGCCAUGGCCAAGGACA
2611
UGUCCUUGGCCAUGGCC
Rh, D
[395-413]

37
2464
GUACCAGGCCAUGGCCAAA
2612
UUUGGCCAUGGCCUGGU
Rh, D
[391-409]

38
2465
UGUCAGGCAAGAAGGACCA
2613
UGGUCCUUCUUGCCUGA
Rh, D
[1248-1266]

39
2466
CUUCGUGCGCAGCAGCAAA
2614
UUUGCUGCUGCGCACGA
Rh, D, M
[664-682]

40
2467
CAACUUCCGCGACAAGCGA
2615
UCGCUUGUCGCGGAAGU
D
[712-730]

41
2468
CCACCACAAGAUGGUGGAA
2616
UUCCACCAUCUUGUGGU
Rh, Rb, D, P
[871-889]

42
2469
GCGCGACGAGGAGGUGCAA
2617
UUGCACCUCCUCGUCGC
Rh, D
[535-553]

43
2470
CUACAACUGCGAGCACUCA
2618
UGAGUGCUCGCAGUUGU
Rh, D
[688-706]

44
2471
UGGAGGUGACCCAUGACCA
2619
UGGUCAUGGGUCACCUC
Rh, Rt, M
[1161-1179]

45
2472
GAGGUCACCAAGGACGUGA
2620
UCACGUCCUUGGUGACC
Rh, D
[788-806]

46
2473
AAGAAGGACCUGUACCUGA
2621
UCAGGUACAGGUCCUUC
Rh, D
[1256-1274]

47
2474
GACAACCGUGGCUUCAUGA
2622
UCAUGAAGCCACGGUUG
Rh, Rt, M
[887-905]

48
2475
ACCAGGACAUCUACGGGCA
2623
UGCCCGUAGAUGUCCUG
D, Rt
[1329-1347]

49
2476
GCUGCCCGAGGUCACCAAA
2624
UUUGGUGACCUCGGGCA
Rh, D
[781-799]

50
2477
AUGCAGAAGAAGGCUGUUA
2625
UAACAGCCUUCUUCUGC
Rt
[1118-1136]

51
2478
GGCCUGGGCGAGCUGCUGA
2626
UCAGCAGCUCGCCCAGG
Rh, D
[557-575]

52
2479
GAUGGUGGACAACCGUGGA
2627
UCCACGGUUGUCCACCA
Rh, M
[880-898]

53
2480
CUCCCUGCUAUUCAUUGGA
2628
UCCAAUGAAUAGCAGGG
D
[1423-1441]

54
2481
GAAGGACCUGUACCUGGCA
2629
UGCCAGGUACAGGUCCU
Rh, D
[1258-1276]

55
2482
CCACCGACGGCAAGCUGCA
2630
UGCAGCUUGCCGUCGGU
D, Rt
[768-786]

56
2483
UGCUAUUCAUUGGGCGCCA
2631
UGGCGCCCAAUGAAUAG
D
[1428-1446]

57
2484
AUGUUCUUCAAGCCACACA
2632
UGUGUGGCUUGAAGAAC
Rh, Rb, D
[839-857]

58
2485
CCAGGACAUCUACGGGCGA
2633
UCGCCCGUAGAUGUCCU
D, Rt
[1330-1348]

59
2486
GCGCGAGGAGCUGCGCAGA
2634
UCUGCGCAGCUCCUCGC
Rh, D, M
[1345-1363]

60
2487
GAGCAGCUGCGCGACGAGA
2635
UCUCGUCGCGCAGCUGC
Rh, D
[527-545]

61
2488
CUAUUCAUUGGGCGCCUGA
2636
UCAGGCGCCCAAUGAAU
D
[1430-1448]

62
2489
ACAAGCUCUCCAGCCUCAA
2637
UUGAGGCUGGAGAGCUU
Rh, D, M, P
[1014-1032]

63
2490
GCUGAAGAUCUGGAUGGGA
2638
UCCCAUCCAGAUCUUCA
Rh, D
[1096-1114]

64
2491
GACCAGGACAUCUACGGGA
2639
UCCCGUAGAUGUCCUGG
D, Rt
[1328-1346]

65
2492
CAAGCGCAGCGCGCUGCAA
2640
UUGCAGCGCGCUGCGCU
Rh, Rt
[724-742]

66
2493
CCAUGGCCAAGGACCAGGA
2641
UCCUGGUCCUUGGCCAU
Rh, D
[399-417]

67
2494
CACCAAGGACGUGGAGCGA
2642
UCGCUCCACGUCCUUGG
Rh, D
[793-811]

68
2495
CCGUGGCUUCAUGGUGACA
2643
UGUCACCAUGAAGCCAC
Rh, Rt, M
[892-910]

69
2496
UGACCAGGACAUCUACGGA
2644
UCCGUAGAUGUCCUGGU
Rt
[1327-1345]

70
2497
AGACCACCGACGGCAAGCA
2645
UGCUUGCCGUCGGUGGU
D, Rt
[765-783]

71
2498
GACAAGCGCAGCGCGCUGA
2646
UCAGCGCGCUGCGCUUG
Rh, Rt
[722-740]

72
2499
AGAAACACCUGGCUGGGCA
2647
UGCCCAGCCAGGUGUUU
D
[1182-1200]

73
2500
AAGAUGGUGGACAACCGUA
2648
UACGGUUGUCCACCAUC
Rh, M
[878-896]

74
2501
CAGACCACCGACGGCAAGA
2649
UCUUGCCGUCGGUGGUC
D, Rt
[764-782]

75
2502
AGGACCUGUACCUGGCCAA
2650
UUGGCCAGGUACAGGUC
Rh, D
[1260-1278]

76
2503
CUGCUAUUCAUUGGGCGCA
2651
UGCGCCCAAUGAAUAGC
D
[1427-1445]

77
2504
GUCCAUCAACGAGUGGGCA
2652
UGCCCACUCGUUGAUGG
Rh, Rt, M
[742-760]

78
2505
CCAGGCCAUGGCCAAGGAA
2653
UUCCUUGGCCAUGGCCU
Rh, D
[394-412]

79
2506
AAGCAGCACUACAACUGCA
2654
UGCAGUUGUAGUGCUGC
Rh, D
[680-698]

80
2507
UGUUCCACGCCACCGCCUA
2655
UAGGCGGUGGCGUGGAA
D
[1281-1299]

81
2508
UACAACUACUACGACGACA
2656
UGUCGUCGUAGUAGUUG
Rb
[956-974]

82
2509
CCUCAUCAUCCUCAUGCCA
2657
UGGCAUGAGGAUGAUGA
Rh, D, Rt, M
[1027-1045]

83
2510
UGGUGGACAACCGUGGCUA
2658
UAGCCACGGUUGUCCAC
Rh, M
[882-900]

84
2511
GACCACCGACGGCAAGCUA
2659
UAGCUUGCCGUCGGUGG
D, Rt
[766-784]

85
2512
AGCUGCGCGACGAGGAGGA
2660
UCCUCCUCGUCGCGCAG
Rh, D
[531-549]

86
2513
CGGCAAGCUGCCCGAGGUA
2661
UACCUCGGGCAGCUUGC
Rh, D
[775-793]

87
2514
UGGCCCACAAGCUCUCCAA
2662
UUGGAGAGCUUGUGGGC
Rh, D, P
[1008-1026]

88
2515
CAGCUGCGCGACGAGGAGA
2663
UCUCCUCGUCGCGCAGC
Rh, D
[530-548]

89
2516
CUUCCGCGACAAGCGCAGA
2664
UCUGCGCUUGUCGCGGA
D
[715-733]

90
2517
UGGGCCUGACUGAGGCCAA
2665
UUGGCCUCAGUCAGGCC
Rt
[1200-1218]

91
2518
GCUGCGCGACGAGGAGGUA
2666
UACCUCCUCGUCGCGCA
Rh, D
[532-550]

92
2519
CAGGACAUCUACGGGCGCA
2667
UGCGCCCGUAGAUGUCC
D
[1331-1349]

93
2520
GCCAUGCCGAAGGACCAGA
2668
UCUGGUCCUUGGCCAUG
Rh, D
[398-416]

94
2521
UCCAAGAUCAACUUCCGCA
2669
UGCGGAAGUUGAUCUUG
D
[704-722]

95
2592
ACCACCGACGGCAAGCUGA
2670
UCAGCUUGCCGUCGGUG
D, Rt
[767-785]

96
2523
AUCUACGGGCGCGAGGAGA
2671
UCUCCUCGCGCCCGUAG
D, M
[1337-1355]

97
2524
CUGCCCGAGGUCACCAAGA
2672
UCUUGGUGACCUCGGGC
Rh, D
[782-800]

98
2525
AUCAACUUCCGCGACAAGA
2673
UCUUGUCGCGGAAGUUG
D
[710-728]

99
2526
UCAUUGGGCGCCUGGUCCA
2674
UGGACCAGGCGCCCAAU
Rh, D
[1434-1452]

100
2527
CAUUGGGCGCCUGGUCCGA
2675
UCGGACCAGGCGCCCAA
Rh, D
[1435-1453]

101
2528
GUGUUCCACGCCACCGCCA
2676
UGGCGGUGGCGUGGAAC
D
[1280-1298]

102
2529
AUGAUGCACCGGACAGGCA
2677
UGCCUGUCCGGUGCAUC
Rh, Rb, Rt, M
[935-953]

103
2530
CGACGAGGAGGUGCACGCA
2678
UGCGUGCACCUCCUCGU
D
[538-556]

104
2531
CAGAAACACCUGGCUGGGA
2679
UCCCAGCCAGGUGUUUC
D
[1181-1199]

105
2532
UGAUGCACCGGACAGGCCA
2680
UGGCCUGUCCGGUGCAU
Rh, Rb, Rt, M
[936-954]

106
2533
AAGGCUGUUGCCAUCUCCA
2681
UGGAGAUGGCAACAGCC
D, Rt
[1127-1145]

107
2534
AUGACUUCGUGCGCAGCAA
2682
UUGCUGCGCACGAAGUC
Rh, Rt, M
[660-678]

108
2535
UCAGGCAAGAAGGACCUGA
2683
UCAGGUCCUUCUUGCCU
Rh, D
[1250-1268]

109
2536
CUCAUCAUCCUCAUGCCCA
2684
UGGGCAUGAGGAUGAUG
Rh, Rt, M
[1028-1046]

110
2537
CGCGACGAGGAGGUGCACA
2685
UGUGCACCUCCUCGUCG
Rh, D
[536-554]

111
2538
ACAACCGUGGCUUCAUGGA
2686
UCCAUGAAGCCACGGUU
Rh, Rt, M
[888-906]

112
2539
UUGACCAGGACAUCCACGA
2687
UCGUAGAUGUCCUGGUC
Rt
[1326-1344]

113
2540
CAAGCUGCCCGAGGUCACA
2688
UGUGACCUCGGGCAGCU
Rh, D
[778-796]

114
2541
UCCCUGCUAUUCAUUGGGA
2689
UCCCAAUGAAUAGCAGG
D
[1424-1442]

115
2542
UAUUCAUUGGGCGCCUGGA
2690
UCCAGGCGCCCAAUGAA
D
[1431-1449]

116
2543
CUGCGCGACGAGGAGGUGA
2691
UCACCUCCUCGUCGCGC
Rh, D
[533-551]

117
2544
CUACGGGCGCGAGGAGCUA
2692
UAGCUCCUCGCGCCCGU
D, M
[1339-1357]

118
2545
CGCGAGGAGCUGCGCAGCA
2693
UGCUGCGCAGCUCCUCG
D, M
[1346-1364]

119
2546
ACACCUGGCUGGGCUGGGA
2694
UCCCAGCCCAGCCAGGU
D
[1186-1204]

120
2547
UCUACGGGCGCGAGGAGCA
2695
UGCUCCUCGCGCCCGUA
D, M
[1338-1356]

121
2548
UUCUUCAAGCCACACUGGA
2696
UCCAGUGUGGCUUGAAG
Rh, Rb, D
[842-860]

122
2549
CCUGGGCGAGCUGCUGCGA
2697
UCGCAGCAGCUCGCCCA
Rh, D
[559-577]

123
2550
AAGAAGGCUGUUGCCAUCA
2698
UGAUGGCAACAGCCUUC
Rt
[1124-1142]

124
2551
CGACGGCAAGCUGCCCGAA
2699
UUCGGGCAGCUUGCCGU
D
[772-790]

125
2552
GACGGCAAGCUGCCCGAGA
2700
UCUCGGGCAGCUUGCCG
Rh, D
[773-791]

126
2553
UUCAUUGGGCGCCUGGUCA
2701
UGACCAGGCGCCCAAUG
Rh, D
[1433-1451]

127
2554
AAGCGCAGCGCGCUGCAGA
2702
UCUGCAGCGCGCUGCGC
Rh, Rt
[725-743]

128
2555
CCUGGCCCACAAGCUCUCA
2703
UGAGAGCUUGUGGGCCA
Rh, D, P
[1006-1024]

129
2556
ACGGCAAGCUGCCCGAGGA
2704
UCCUCGGGCAGCUUGCC
Rh, D
[774-792]

130
2557
UUUGACCAGGACAUCUACA
2705
UGUAGAUGUCCUGGUCA
Rt
[1325-1343]

131
2558
UGACUUCGUGCGCAGCAGA
2706
UCUGCUGCGCACGAAGU
Rh, Rt, M
[661-679]

132
2559
AAGGACGUGGAGCGCACGA
2707
UCGUGCGCUCCACGUCC
Rh, D
[797-815]

133
2560
UCCAUCAACGAGUGGGCCA
2708
UGGCCCACUCGUUGAUG
Rt, M
[743-761]

134
2561
CACCGACGCCAACCUGCCA
2709
UGGCAGCUUGCCGUCGG
D, Rt
[769-787]

135
2562
ACGGGCGCGAGGAGCUGCA
2710
UGCAGCUCCUCGCGCCC
D, M
[1341-1359]

136
2563
UCCGCGACAAGCGCAGCGA
2711
UCGCUGCGCUUGUCGCG
D
[717-735]

137
2564
UUGGGCGCCUGGUCCGGCA
2712
UGCCGGACCAGGCGCCC
Rh, D
[1437-1455]

138
2565
AUGGUGGACAACCGUGGCA
2713
UGCCACGGUUGUCCACC
Rh, M
[881-899]

139
2566
AUUGGGCGCCUGGUCCGGA
2714
UCCGGACCAGGCGCCCA
Rh, D
[1436-1454]

140
2567
UACGGGCGCGAGGAGCUGA
2715
UCAGCUCCUCGCGCCCG
D, M
[1340-1358]

141
2568
AUGCACCGGACAGGCCUCA
2716
UGAGGCCUGUCCGGUGC
Rh, Rb, Rt, P
[938-956]

142
2569
UUCCACCACAAGAUGGUGA
2717
UCACCAUCUUGUGGUGG
Rh, Rb, D, P
[869-887]

143
2570
UUCCGCGACAAGCGCAGCA
2718
UGCUGCGCUUGUCGCGG
D
[716-734]

144
2571
UACCAGGCCAUGGCCAAGA
2719
UCUUGGCCAUGGCCUGG
Rh, D
[392-410]

145
2572
AAACACCUGGCUGGGCUGA
2720
UCAGCCCAGCCAGGUGU
D
[1184-1202]

146
2573
ACCGACGGCAAGCUGCCCA
2721
UGGGCAGCUUGCCGUCG
D
[770-788]

147
2574
AACACCUGGCUGGGCUGGA
2722
UCCAGCCCAGCCAGGUG
D
[1185-1203]

148
2575
UUCGUGCGCAGCAGCAAGA
2723
UCUUGCUGCUGCGCACG
Rh, D, M
[665-683]

EXAMPLE 10
Animal Models

Model Systems of Fibrotic Conditions

Testing the active siRNAs of the invention may be done in predictive animal models. Rat diabetic and aging models of kidney fibrosis include Zucker diabetic fatty (ZDF) rats, aged fa/fa (obese Zucker) rats, aged Sprague-Dawley (SD) rats, and Goto Kakizaki (GK) rats; GK rats are an inbred strain derived from Wistar rats, selected for spontaneous development of NIDDM (diabetes type II). Induced models of kidney fibrosis include the permanent unilateral ureteral obstruction (UUO) model which is a model of acute interstitial fibrosis occurring in healthy non-diabetic animals; renal fibrosis develops within days following the obstruction. Another induced model of kidney fibrosis is 5/6 nephrectomy.

Two models of liver fibrosis in rats are the Bile Duct Ligation (BDL) with sham operation as controls, and CCl4 poisoning, with olive oil fed animals as controls, as described in the following references: Lotersztajn S, et al Hepatic Fibrosis: Molecular Mechanisms and Drug Targets. Annu Rev Pharmacol Toxicol. 2004 Oct. 7; Uchio K, et al., Down-regulation of connective tissue growth factor and type I collagen mRNA expression by connective tissue growth factor antisense oligonucleotide during experimental liver fibrosis. Wound Repair Regen. 2004 January-February; 12 (1):60-6; Xu X Q, et al., Molecular classification of liver cirrhosis in a rat model by proteomics and bioinformatics Proteomics. 2004 October; 4 (10):3235-45.

Models for ocular scarring are well known in the art e.g. Sherwood M B et al., J. Glaucoma. 2004 October; 13 (5):407-12. A new model of glaucoma filtering surgery in the rat; Miller M H et al., Ophthalmic Surg. 1989 May; 20(5):350-7. Wound healing in an animal model of glaucoma fistulizing surgery in the Rb; vanBockxmeer F M et al., Retina. 1985 Fall-Winter; 5 (4): 239-52. Models for assessing scar tissue inhibitors; Wiedemann P et al., J Pharmacol Methods. 1984 August; 12 (1): 69-78. Proliferative vitreoretinopathy: the Rb cell injection model for screening of antiproliferative drugs.

Models of cataract are described in the following publications: The role of Src family kinases in cortical cataract formation. Zhou J, Menko A S. Invest Ophthalmol V is Sci. 2002 July; 43 (7):2293-300; Bioavailability and anticataract effects of a topical ocular drug delivery system containing disulfuram and hydroxypropyl-beta-cyclodextrin on selenite-treated rats. Wang S, et al. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15370367 Curr Eye Res. 2004 July; 29 (1):51-8; and Long-term organ culture system to study the effects of UV-A irradiation on lens transglutaminase. Weinreb O, Dovrat A.; Curr Eye Res. 2004 July; 29 (1):51-8.

The compounds of Table A-18 and Table A-19 are tested in these models of fibrotic conditions, in which it is found that they are effective in treating liver fibrosis and other fibrotic conditions.

Model Systems of Glaucoma

Testing the active siRNA of the invention for treating or preventing glaucoma is preformed in rat animal model for optic nerve crush described for example in: Maeda, K. et al., “A Novel Neuroprotectant against Retinal Ganglion Cell Damage in a Glaucoma Model and an Optic Nerve Crush Model in the rat”, Investigative Ophthalmology and visual Science (IOVS), March 2004, 45(3)851. Specifically, for optic nerve transection the orbital optic nerve (ON) of anesthetized rats is exposed through a supraorbital approach, the meninges severed and all axons in the ON transected by crushing with forceps for 10 seconds, 2 mm from the lamina cribrosa.

Nucleic acid molecules as disclosed herein are tested in this animal model and the results show that these siRNA compounds are useful in treating and/or preventing glaucoma.

Rat Optic Nerve Crush (ONC) Model: Intravitreal siRNA Delivery and Eye Drop Delivery

For optic nerve transsection the orbital optic nerve (ON) of anesthetized rats is exposed through a supraorbital approach, the meninges severed and all axons in the ON transected by crushing with forceps for 10 seconds, 2 mm from the lamina cribrosa.

The siRNA compounds are delivered alone or in combination in 5 uL volume (10 ug/uL) as eye drops. Immediately after optic nerve crush (ONC), 20 ug/10 ul test siRNA or 10 ul PBS is administered to one or both eyes of adult Wistar rats and the levels of siRNA taken up into the dissected and snap frozen whole retinae at 5 h and 1 d, and later at 2 d, 4 d, 7 d, 14 d and 21 d post injection is determined. Similar experiments are performed in order to test activity and efficacy of siRNA administered via eye drops.

Model Systems of Ischemia Reperfusion Injury Following Lung Transplantation in Rats

Lung ischemia/reperfusion injury is achieved in a rat animal model as described in Mizobuchi et al., The Journal of Heart and Lung Transplantation, Vol 23 No. 7 (2004) and in Kazuhiro Yasufuku et al., Am. J. Respir. Cell Mol Biol, Vol 25, pp 26-34 (2001).

Specifically, after inducing anesthesia with isofluorane, the trachea is cannulated with a 14-gauge Teflon catheter and the rat is mechanically ventilated with rodent ventilator using 100% oxygen, at a rate of 70 breaths per minute and 2 cm H2O of positive end-respiratory pressure. The left pulmonary artery, veins and main stem bronchus are occluded with a Castaneda clamp. During the operation, the lung is kept moist with saline and the incision is covered to minimize evaporative losses. The period of ischemia is 60 minutes long. At the end of the ischemic period the clamp is removed and the lung is allowed to ventilate and reperfuse for further 4 h, 24 h, and 5 d post induction of lung ischemia. At the end of the experiment, the lungs are gently harvested and either frozen for RNA extraction or fixed in glutaraldehyde cocktail for subsequent histological analysis.

The Bleomycin Animal Model as a Model for Idiopathic Pulmonary Fibrosis (IPF).

Testing feasibility of lung and liver delivery of vitamin A-Coatsome formulated siRNA administered by intravenous injection and intratracheal administration of siRNA-vitamin A-Coatsome complex to a healthy mice and bleomycine-treated mice

Objective: To test two administration routes for feasibility of vitamin A-Coatsome formulated siRNA delivery to normal and fibrotic mouse lungs. The main hypothesis to be tested in the current study is whether systemic administration of vitamin A-Coatsome formulated modified siRNA provides efficient uptake and cell-specific distribution in the fibrotic and normal mouse lungs. Intratracheal route of vitamin A-Coatsome formulated modified siRNA will be tested in parallel. siRNA detection and cell-specific distribution in the lungs and liver will be performed by in situ hybridization (ISH)

Background: The Bleomycin model of pulmonary fibrosis has been well developed and characterized over the last three decades (Moeller, et al. Int J Biochem Cell Biol, 40:362-382, 2008; Chua et al., Am J Respir Cell Mol Biol 33:9-13, 2005). Histological hallmarks, such as intra-alveolar buds, mural incorporation of collagen and obliteration of alveolar space are present in BLM-treated animals similar to IPF patients. Early studies demonstrated that C57/B1 mice were consistently prone to BLM-induced lung fibrosis, whereas Balb/C mice were inheritantly resistant. Depending on the route of administration, different fibrotic pattern develops. Intratracheal instillation of BLM results in bronchiocentric accentuated fibrosis, whereas intravenous or intraperitoneal administration induces subpleural scarring similar to human disease (Chua et al. ibid). A mouse model of usual interstitial pneumonia (UIP) is used. This model shows a heterogenous distribution of fibroproliferation, distributed mainly subpleurally, forming similar lesions to those observed in the lungs of patients with idiopathic pulmonary fibrsosis (IPF) (Onuma, et al., Tohoku J Exp Med 194: 147-156, 2001 and Yamaguchi and Ruoslahti, Nature 336: 244-246, 1988). UIP will be induced by intraperitoneal injection of bleomycin every other day for 7 days for a constant composition of subpleural fibroproliferation in the mouse lung (Swiderski et al. Am J Pathol 152: 821-828, 1998 and Shimizukawa et al., Am J Physiol Lung Cell Mol Physiol 284: L526-L532, 2003).

As was previously demonstrated, vitamin A-loaded liposomes containing siRNA interact with retinol-binding protein (RBP) and provide efficient delivery to the hepatic stellate cells via RBP receptor (Sato et al. Nat Biotechnol 26:431-442, 2008). This study is planned to test whether vitA-Coatsome-siRNA complex will be efficiently taken up by an RBP receptor-expressing activated myofibroblasts in the lungs of bleomycin-treated mice. In addition, local administration route (intratracheal instillation) will be tested.

General Study Design

Mice—C57 B1 male

Starting N (BLM I.P.)—40 (6 for the first pilot group, 34 for the study, taling in consideration anticipated 25% mortality)

Starting N (Total)—60

Test siRNA: SERPINHI compounds disclosed herein.

Groups:

BLM

dose,

Termination,

mg/kg

siRNA

post

BW, in
BLM

dose,
siRNA

last
N (before

0.1 ml
adm.
BLM
mg/kg
adm
siRNA
siRNA
siRNA

No
saline
route
regime
BW
route
regime
adm
administration

1
0.75
I.P.
dd 0, 2, 4, 6
4.5
I.V.
2 daily
2 h
4

2
0.75
I.P.
dd 0, 2, 4, 6
4.5
I.V.
2 daily
24 h
4

3
0.75
I.P.
dd 0, 2, 4, 6
2.25
I.T.
2 daily
2 h
4

4
0.75
I.P.
dd 0, 2, 4, 6
2.25
I.T.
2 daily
24 h
4

5

intact
n/a
4.5
I.V.
2 daily
2 h
4

6

intact
n/a
4.5
I.V
2 daily
24 h
4

7

intact
n/a
2.25
I.T
2 daily
2 h
4

8

intact
n/a
2.25
I.T.
2 daily
24 h
4

9
0.75
I.P.
dd 0, 2, 4, 6
n/a
I.V.
2 daily
2 h
3

vehicle

10
0.75
I.P.
dd 0, 2, 4, 6
n/a
I/T/vehicle
2 daily
24 h
3

11

Intact
n/a
n/a
intact
n/a
Any time
3

Bleomycin-induced pulmonary fibrosis. Pulmonary fibrosis of 12-wk-old female C57BL/6 mice will be induced by intraperitoneal instillation of bleomycin chlorate: 0.75 mg/kg body weight dissolved in 0.1 ml of saline every other day for 7 days, on days 0, 2, 4, and 6.

Model follow up and monitoring. The mice will be weighed before the BLM treatment, and twice weekly for the period of study duration.

Pilot evaluation of the establishment of fibrosis. The mice (N=30) are subjected to BLM treatment in groups, to allow for a one week time interval between the first treated group (N=5) and the rest of the animals. On day 14, two mice from the first group are sacrificed and the lungs harvested for the fast HE stain and quick histopathological evaluation of fibrosis. When lung fibrosis is confirmed, the remaining rats are sorted into the groups and treated with siRNA on Day 14 after the first BLM treatment. In case that no sufficient fibrosis develops in the lungs by day 14, the remaining mice from the first treated group are sacrificed on day 21, followed by quick histopathology evaluation of fibrosis. The rest of the animals are treated with test siRNA complex starting from day 21 after the BLM treatment.

siRNA administration. On day 14 or day 21 after the first BLM administration (TBD during the study, based on pilot evaluation of establishment of fibrosis), the animals are group sorted, according to BW. The animals from groups 1 and 2 are administered intravenously (tail vein injection) with siRNA/vitA/Coatsome complex, at an siRNA concentration of 4.5 mg/kg BW. Intact animals of the same age (Groups 5 and 6) are treated in the same manner. BLM treated animals (Group 9) will be used as vitA-coatsome vehicle control). In 24 hours, the injection is repeated to all the animals, as above.

The BLM animals from the groups 3 and 4, and intact mice from groups 7 and 8 are anesthetized with isoflutrane and subjected to intratracheal instillation of 2.25 mg/kg BW siRNA formulated in vitA-loaded liposomes. Mice from the BLM group 10 are administered with vitA/Coatsome vehicle only. The inttratracheal instillation is repeated after 24 hours.

Study termination. The animals from the groups 1, 3, 5, 7, 9 are sacrificed at 2 hours after the second siRNA complex injection or instillation. The animals from the groups 2, 4, 6, 8, 10 are sacrificed at 24 hours after the second siRNA complex injection or instillation.

Upon animals sacrifice, the mice are perfused transcardially with 10% neutral buffered formalin. The lungs are inflated with 0.8-1.0 ml of 10% NBF, and the trachea ligated. The lungs are excised and fixed for 24 h in 10% NBF. The liver is harvested from each animal and fixed in 10% NBF for 24 h.

Sectioning and evaluation. Consequent sections are prepared from the lungs and livers. First consequent section are stained with hematoxylin and eosin for assessment of lung and liver morphology, second section are stained with Sirius Red (trichrome) to identify collagen The third consequent sections are subjected to in situ hybridization (ISH) for detection of siRNA.

The compounds as described herein are tested in this animal model and the results show that these siRNA compounds are useful in treating and/or preventing ischemia reperfusion injury following lung transplantation.

TABLE 1

List of siRNA sequences

Experimental

Target

Base sequence [corresponding
sequence [corresponding

siRNA
region

nucleotides of SEQ ID NO: 1]
nucleotides of SEQ ID NO: 1]

siHSP47-C
human/rat
sense
5′ GGACAGGCCUCUACAACUAUU (SEQ ID
5′ GGACAGGCCUCUACAACUAdTdT (SEQ

hsp47

NO: 3)
ID NO: 5)

[945-963]
[945-963]

anti-
5′ UAGUUGUAGAGGCCUGUCCUU (SEQ
5′ UAGUUGUAGAGGCCUGUCCdTdT (SEQ

sense
ID NO: 4)
ID NO: 6)

[945-963]
[945-963]

siHSP47-
human/rat
sense
5′ GGACAGGCCUCUACAACUACUACGA
5′ GGACAGGCCUCUACAACUACUACdGdA

Cd
hsp47

(SEQ ID NO: 7)
(SEQ ID NO: 9)

[945-969]
[945-969]

anti-
5′
5′

sense
UCGUAGUAGUUGUAGAGGCCUGUCCUU
UCGUAGUAGUUGUAGAGGCCUGUCCUU

(SEQ ID NO: 8)
(SEQ ID NO: 10)

[945-969]
[945-969]

siHSP47-1
human/rat
sense
5′ CAGGCCUCUACAACUACUAUU (SEQ ID
5′ CAGGCCUCUACAACUACUAdTdT (SEQ

hsp47

NO: 11)
ID NO: 13)

[948-966]
[948-966]

anti-
5′ UAGUAGUUGUAGAGGCCUGUU (SEQ
5′ UAGUAGUUGUAGAGGCCUGdTdT (SEQ

sense
ID NO: 12)
ID NO: 14)

[948-966]
[948-966]

siHSP47-
human
sense
5′ CAGGCCUCUACAACUACUACGACGA
5′ CAGGCCUCUACAACUACUACGACdGdA

1d
hsp47

(SEQ ID NO: 15)
(SEQ ID NO: 17)

[948-972]
[948-972]

anti-
5′
5′

sense
UCGUCGUAGUAGUUGUAGAGGCCUGUU
UCGUCGUAGUAGUUGUAGAGGCCUGUU

(SEQ ID NO: 16)
(SEQ ID NO: 18)

[948-972]
[948-972]

siHsp47-2
human
sense
5′ GAGCACUCCAAGAUCAACUUU (SEQ ID
5′ GAGCACUCCAAGAUCAACUdTdT (SEQ

hsp47

NO: 19)
ID NO: 21)

[698-717]
[698-717]

anti-
5′ AGUUGAUCUUGGAGUGCUCUU (SEQ
5′ AGUUGAUCUUGGAGUGCUCdTdT (SEQ

sense
ID NO: 20)
ID NO: 22)

[698-716]
[698-716]

siHsp47-2d
human
sense
5′ GAGCACUCCAAGAUCAACUUCCGCG
5′ GAGCACUCCAAGAUCAACUUCCGdCdG

hsp47

(SEQ ID NO: 23)
(SEQ ID NO: 25)

[698-722]
[698-722]

anti-
5′
5′

sense
CGCGGAAGUUGAUCUUGGAGUGCUCUU
CGCGGAAGUUGAUCUUGGAGUGCUCUU

(SEQ ID NO: 24)
(SEQ ID NO: 26)

[698-722]
[698-722]

siHsp47-2d
rat Gp46
sense
5′ GAACACUCCAAGAUCAACUUCCGAG
5′ GAACACUCCAAGAUCAACUUCCGdAdG

rat

(SEQ ID NO: 27)
(SEQ ID NO: 29)

[587-611]
[587-611]

anti-
5′
5′

sense
CUCGGAAGUUGAUCUUGGAGUGUUCUU
CUCGGAAGUUGAUCUUGGAGUGUUCUU

(SEQ ID NO: 28)
(SEQ ID NO: 30)

[587-611]
[587-611]

siHsp47-3
human
sense
5′ CUGAGGCCAUUGACAAGAAUU (SEQ
5′ CUGAGGCCAUUGACAAGAAdTdT (SEQ

hsp47

ID NO: 31)
ID NO: 33)

[1209-1227]
[1209-1227]

anti-
5′ UUCUUGUCAAUGGCCUCAGUU (SEQ
5′ UUCUUGUCAAUGGCCUCAGdTdT (SEQ

sense
ID NO: 32)
ID NO: 34)

[1209-1227]
[1209-1227]

siHsp47-3d
human
sense
5′ CUGAGGCCAUUGACAAGAACAAGGC
5′

hsp47

(SEQ ID NO: 35)
CUGAGGCCAUUGACAAGAACAAGdGdC

[1209-1233]
(SEQ ID NO: 37)

[1209-1233]

anti-
5′
5′

sense
GCCUUGUUCUUGUCAAUGGCCUCAGUU
GCCUUGUUCUUGUCAAUGGCCUCAGUU

(SEQ ID NO: 36)
(SEQ ID NO: 38)

[1209-1233]
[1209-1233]

siHsp47-4
human
sense
5′ CUACGACGACGAGAAGGAAUU (SEQ
5′ CUACGACGACGAGAAGGAAdTdT (SEQ

hsp47

ID NO: 39)
ID NO: 41)

[964-982]
[964-982]

anti-
5′ UUCCUUCUCGUCGUCGUAGUU (SEQ ID
5′ UUCCUUCUCGUCGUCGUAGdTdT (SEQ

sense
NO: 40)
ID NO: 42)

[964-982]
[964-982]

siHsp47-4d
human
sense
5′ CUACGACGACGAGAAGGAAAAGCUG
5′

hsp47

(SEQ ID NO: 43)
CUACGACGACGAGAAGGAAAAGCdTdG

[964-988]
(SEQ ID NO: 45)

[964-988]

anti-
5′
5′

sense
CAGCUUUUCCUUCUCGUCGUCGUAGUU
CAGCUUUUCCUUCUCGUCGUCGUAGUU

(SEQ ID NO: 44)
(SEQ ID NO: 46)

[964-988]
[964-988]

siHsp47-5
human
sense
5′ GCCACACUGGGAUGAGAAAUU (SEQ
5′ GCCACACUGGGAUGAGAAAdTdT (SEQ

hsp47

ID NO: 47)
ID NO: 49)

[850-870]
[850-870]

anti-
5′ UUUCUCAUCCCAGUGUGGCUU (SEQ ID
5′ UUUCUCAUCCCAGUGUGGCdTdT (SEQ

sense
NO: 48)
ID NO: 50)

[850-868]
[850-868]

siHsp47-6
human
sense
5′ GCAGCAAGCAGCACUACAAUU (SEQ ID
5′ GCAGCAAGCAGCACUACAAdTdT (SEQ

hsp47

NO: 51)
ID NO: 53)

[675-693]
[675-693]

anti-
5′ UUGUAGUGCUGCUUGCUGCUU (SEQ
5′ UUGUAGUGCUGCUUGCUGCdTdT (SEQ

sense
ID NO: 52)
ID NO: 54)

[675-693]
[675-693]

siHsp47-7
human
sense
5′ CCGUGGGUGUCAUGAUGAUUU (SEQ
5′ CCGUGGGUGUCAUGAUGAUdTdT (SEQ

hsp47

ID NO: 55)
ID NO: 57)

[921-939]
[921-939]

anti-
5′ AUCAUCAUGACACCCACGGUU (SEQ ID
5′ AUCAUCAUGACACCCACGGdTdT (SEQ

sense
NO: 56)
ID NO: 58)

[921-939]
[921-939]

The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can include improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying nucleic acid molecules with improved RNAi activity.

The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising”, “having,” “including,” containing”, etc. shall be read expansively and without limitation (e.g., meaning “including, but not limited to,”). Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and docs not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

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Modulation of HSP47 expression

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