HK1235821B - Modulation of hsp47 expression - Google Patents

Description

Regulation of HSP47 expression

This application is a divisional application of the PCT application with PCT application number PCT/US2010/059578 and invention name “Regulation of HSP47 Expression” and the Chinese national phase application with application number 201080060946.7 after entering the Chinese national phase.

Related patent applications

No. 61/372,072, filed August 9, 2010, U.S. Provisional Application Serial No. 61/307,412, filed February 23, 2010, and U.S. Provisional Application Serial No. 61/285,149, filed December 9, 2009, each entitled "Modulation of HSP47 Expression" and each of which is incorporated herein by reference in its entirety for all purposes.

Sequence Listing

This application contains a sequence listing entitled 220-PCT1_ST25_07-Dec-10.txt, an ASCII copy of which was created on December 7, 2010, and is 533 kb in size, which is hereby incorporated by reference in its entirety.

Field of the Invention

Provided herein are compositions and methods for modulating hsp47 expression.

Background of the Invention

Sato, Y. et al. disclosed the use of vitamin A-coupled liposomes to deliver small interfering RNA (siRNA) against gp46 (rat homolog of human heat shock protein 47) to a rat animal model of cirrhosis. Sato, Y. et al., Nature Biotechnology, Vol. 26(4), pp. 431-442 (2008).

Chen, J-J. et al. disclosed transfection of human keloid samples with HSP47-shRNA (small hairpin RNA) to examine the proliferation of keloid fibroblasts. Chen, J-J. et al., British Journal of Dermatology, Vol. 156, pp. 1188-1195 (2007).

PCT Patent Application No. WO 2006/068232 discloses a stellate cell-specific drug carrier comprising a retinoid derivative and/or a vitamin A analog.

SUMMARY OF THE INVENTION

Provided herein are compositions, methods, and kits for regulating target gene expression. In various aspects and embodiments, provided herein are compositions, methods, and kits for regulating the expression of heat shock protein 47 (hsp47), also known as SERPINH1. The compositions, methods, and kits may involve the use of nucleic acid molecules (e.g., short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), or short hairpin RNA (shRNA)) that bind to a nucleotide sequence encoding hsp47 (e.g., an mRNA sequence), such as an mRNA coding sequence of human hsp47 exemplified by SEQ ID NO: 1. In certain preferred embodiments, the compositions, methods, and kits disclosed herein inhibit the expression of hsp47. For example, siNA molecules (e.g., RISC-length dsNA molecules or Dicer-length dsNA molecules) are provided to reduce or inhibit hsp47 expression. Also provided are compositions, methods and kits for treating and/or preventing hsp47-associated diseases, conditions or disorders, such as liver fibrosis, cirrhosis, pulmonary fibrosis including pulmonary fibrosis (including ILF), renal fibrosis due to any condition (e.g., CKD including ESRD), peritoneal fibrosis, chronic liver damage, fibrillation, fibrotic diseases of other organs, abnormal scarring (keloids) associated with all possible types of accidental and iatrogenic (surgical) skin injuries; scleroderma; myocardial fibrosis, failed glaucoma filtration surgery; and intestinal adhesions.

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

In certain embodiments, the sequence of the antisense strand complementary to the sequence of human hsp47 encoding mRNA includes a sequence complementary to nucleotides 600-800, 801-899, 900-1000, or 1001-1300 of SEQ ID NO: 1, or nucleotides 650-730, or 900-975 of SEQ ID NO: 1. In some embodiments, the antisense strand includes a sequence complementary to a sequence 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 nucleotides 947-972 of SEQ ID NO: 1, or nucleotides 948-966 of SEQ ID NO: 1, or nucleotides 945-969 of SEQ ID NO: 1, or nucleotides 945-963 of SEQ ID NO: 1, or a portion thereof, encoding human hsp47.

In certain embodiments, the antisense strand of a nucleic acid molecule (e.g., a siNA molecule) 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:47 or a portion thereof. 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 and SEQ ID NO:58 or a portion thereof. In certain embodiments, the sense strand of a nucleic acid molecule (e.g., a siNA molecule) 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: 46 or a portion thereof. 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 and SEQ ID NO:57 or a portion thereof.

In certain preferred embodiments, the antisense strand of the nucleic acid molecules (e.g., siNA molecules) disclosed herein comprises a sequence corresponding to any one of the antisense sequences shown in Table A-19. In certain preferred embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in Table A-19. In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58, and SERPINH1_88. In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown 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 of the nucleic acid molecules (e.g., siNA molecules) disclosed herein comprise the sequence pair set forth in SERPINH1_4 (SEQ ID NOs: 195 and 220). In some embodiments, the antisense and sense strands of the nucleic acid molecules (e.g., siNA molecules) disclosed herein comprise the sequence pair set forth in SERPINH1_12 (SEQ ID NOs: 196 and 221). In some embodiments, the antisense and sense strands of the nucleic acid molecules (e.g., siNA molecules) disclosed herein comprise the sequence pair set forth in SERPINH1_30 (SEQ ID NOs: 199 and 224). In some embodiments, the antisense and sense strands of the nucleic acid molecules (e.g., siNA molecules) disclosed herein comprise the sequence pair 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) disclosed herein comprises a sequence corresponding to any of the antisense sequences set forth in any of Tables B or C.

In certain preferred embodiments, the antisense strand of a nucleic acid molecule (e.g., a siNA molecule) disclosed herein comprises a sequence corresponding to any one of the antisense sequences shown in Table A- 18. In certain preferred embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in Table A-18. In some embodiments, the nucleic acid molecules disclosed herein (e.g., siNA molecules) include the antisense and sense strands of a sequence pair selected from 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 strand and the sense strand are selected from the sequence pairs shown in SERPINH1_2 (SEQ ID NO: 60 and 127), SERPINH1_6 (SEQ ID NO: 63 and 130), SERPINH1_45a (SEQ ID NOS: 98 and 165) and SERPINH1_51 (SEQ ID NO: 101 and 168).

In some embodiments, the nucleic acid molecules disclosed herein (e.g., siNA molecules) include an antisense strand and a sense strand selected from the sequence pairs shown in SERPINH1_2 (SEQ ID NOs: 60 and 127). In some embodiments, the antisense strand and the sense strand include the sequence pairs shown in SERPINH1_6 (SEQ ID NOs: 63 and 130). In some embodiments, the nucleic acid molecules disclosed herein (e.g., siNA molecules) include the antisense strand and the sense strand of the sequence pairs shown in SERPINH1_11 (SEQ ID NOs: 68 and 135). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_13 (SEQ ID NOs: 69 and 136). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_45 (SEQ ID NOs: 97 and 164). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_45a (SEQ ID NOs: 98 and 165). In some embodiments, the antisense strand and the sense strand are the sequence pair shown in SERPINH1_51 (SEQ ID NOs: 101 and 168).

In certain embodiments, the antisense strand of a nucleic acid molecule (e.g., a siNA molecule) disclosed herein comprises a sequence corresponding to any of the antisense sequences set forth in any of Tables D or E.

In various embodiments of the nucleic acid molecules (e.g., siNA molecules) disclosed herein, the antisense strand can be 15-49 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 17-30 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 the nucleic acid molecules (eg, siNA molecules) disclosed herein, the antisense strand can be 19 nucleotides in length. Similarly, the sense strand of the nucleic acid molecules disclosed herein (e.g., siNA molecules) can be 15-49 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 the nucleic acid molecules disclosed herein (e.g., siNA molecules), the sense strand can be 19 nucleotides in length. In some embodiments of the nucleic acid molecules disclosed herein (e.g., siNA molecules), the antisense strand and the sense strand can be 19 nucleotides in length. The duplex region of the nucleic acid molecules disclosed herein (e.g., siNA molecules) can 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); or 15-35 nucleotides in length; or 15-30 nucleotides in length; or 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 the nucleic acid molecules (eg, siNA molecules) disclosed herein, the duplex region can be 19 nucleotides in length.

In certain embodiments, the sense and antisense strands of the nucleic acid molecules (e.g., siNA nucleic acid molecules) provided herein are separate polynucleotide chains. In some embodiments, the separate antisense and sense strands form a double-stranded structure by hydrogen bonding, such as Watson-Crick base pairing. In some embodiments, the sense and antisense strands are two separate chains covalently linked to each other. In other embodiments, the sense and antisense strands are part of a single polynucleotide chain having sense and antisense regions; in some preferred embodiments, the polynucleotide chain 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 about the overhang and has blunt ends at both ends. In other embodiments, the nucleic acid molecule (e.g., siNA molecule) is a dsNA molecule that is symmetrical about the overhang and has overhangs at both ends of the dsNA molecule; preferably, the molecule has an overhang of 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides; preferably, the molecule has an overhang of 2 nucleotides. In some embodiments, the overhang is a 5' overhang; in alternative embodiments, the overhang is a 3' overhang. In certain embodiments, the overhang nucleotides are modified with modifications disclosed herein. In some embodiments, the overhang nucleotides are 2'-deoxynucleotides.

In certain embodiments, a nucleic acid molecule (e.g., a siNA molecule) is a dsNA molecule that is asymmetric with respect to overhangs and has a blunt end at one end of the molecule and an overhang at 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, the asymmetric dsNA molecule has a 3'-overhang (e.g., a 2-nucleotide 3'-overhang) on one side of the duplex appearing on the sense strand and a blunt end on the other side of the molecule. In some preferred embodiments, the asymmetric dsNA molecule has a 5'-overhang (e.g., a 2-nucleotide 5'-overhang) on one side of the duplex appearing on the sense strand and a blunt end on the other side of the molecule. In other preferred embodiments, the asymmetric dsNA molecule has a 3'-overhang (e.g., a 2-nucleotide 3'-overhang) on one side of the duplex appearing on the antisense strand and a blunt end on the other side of the molecule. In some preferred embodiments, the asymmetric dsNA molecule has a 5'-overhang (e.g., a 2 nucleotide 5'-overhang) on one side of the duplex appearing on the antisense strand and a blunt end on the other side of the molecule. In certain preferred embodiments, the overhang is a 2'-deoxynucleotide.

In some embodiments, the nucleic acid molecule (e.g., siNA molecule) has a hairpin structure (having a sense strand and an antisense strand on one polynucleotide), wherein one end has a loop structure and the other end has a blunt end. In some embodiments, the nucleic acid molecule has a hairpin structure, wherein one end has a loop structure and the other end has an overhang (e.g., 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 in Table 1.

The nucleic acid molecules (e.g., siNA molecules) disclosed herein can include, for example, one or more modifications or modified nucleotides as described herein. For example, the nucleic acid molecules (e.g., siNA molecules) provided herein can include modified nucleotides having modified sugars; modified nucleotides having modified nucleobases; or modified nucleotides having modified phosphate groups. Similarly, the nucleic acid molecules (e.g., siNA molecules) provided herein can include modified phosphodiester backbones and/or can include modified terminal phosphate groups.

The provided nucleic acid molecules (e.g., siNA molecules) can have one or more nucleotides comprising a modified sugar moiety, such as described herein. In some preferred embodiments, the modified sugar moiety is selected from 2'-O-methyl, 2'-methoxyethoxy, 2'-deoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-(CH ₂ ) ₂ -O-2'-bridge, 2'-locked nucleic acid, and 2'-O-(N-methylcarbamate).

The provided nucleic acid molecules (e.g., siNA molecules) can have one or more modified nucleobases, such as those described herein, which can preferably be one selected from 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 adenine and guanine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and acyclic nucleotides.

The provided nucleic acid molecules (e.g., siNA molecules) can have one or more modifications to the phosphodiester backbone, such as those described herein. In some preferred embodiments, the phosphodiester bond is modified by replacing the phosphodiester bond with a phosphorothioate bond, a 3'-(or -5') deoxy-3'-(or -5') thio-phosphorothioate bond, a phosphorodithioate bond, a phosphoroselenoate bond, a 3'-(or -5') deoxyphosphinate bond, a borophosphate bond, a 3'-(or -5') deoxy-3'-(or 5'-) aminophosphoramidate bond, a hydrogenphosphonate bond, a borophosphate bond, a phosphoramidate bond, an alkyl or arylphosphonate bond, and a phosphotriester bond or a phosphorus bond.

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

In some embodiments of the provided nucleic acid molecules (e.g., siNA molecules) having modifications, the sense strand comprises a pattern of alternating modified and unmodified nucleotides, and/or the antisense strand comprises a pattern of alternating modified and unmodified nucleotides; in some preferred forms of these embodiments, the modification is a 2'-O-methyl (2'methoxy or 2'OMe) sugar moiety. The pattern of alternating modified and unmodified nucleotides can begin with a modified nucleotide at the 5' or 3' end of one of the strands; for example, the pattern of alternating modified and unmodified nucleotides can begin with a modified nucleotide at the 5' or 3' end of the sense strand and/or the pattern of alternating modified and unmodified nucleotides can begin with a modified nucleotide at the 5' or 3' end of the antisense strand. When the antisense and sense strands comprise a pattern of alternating modified and unmodified nucleotides, the pattern of modified nucleotides can be configured such that the modified nucleotides on the sense strand are opposite the modified nucleotides on the antisense strand; or there can be a phase shift in the pattern such that the modified nucleotides of the sense strand are opposite the unmodified nucleotides on the antisense strand and vice versa.

Nucleic acid molecules (e.g., siNA molecules) provided herein can include 1-3 (i.e., 1, 2, or 3) deoxynucleotides at the 3' end of the sense strand and/or antisense strand.

Nucleic acid molecules (e.g., siNA molecules) provided herein can include a phosphate group at the 5' end of the sense strand and/or the antisense strand.

In one aspect, a double-stranded nucleic acid molecule having structure (A1) is provided:

(A1)5’ (N)x–Z 3’ (antisense strand)

3’ Z’-(N’)y–z” 5’ (sense strand)

wherein N and N' are each a nucleotide which may be unmodified or modified, or an unconventional moiety;

wherein (N)x and (N')y are each oligonucleotides in which each consecutive N or N' is linked to the next N or N' by a covalent bond;

wherein Z and Z' are each independently present or absent, but if present independently comprise 1-5 consecutive nucleotides or non-nucleotide moieties, or combinations thereof, covalently linked to the 3' terminus of the chain in which they are present;

wherein z" may be present or absent, but if present is a capping moiety covalently attached to the 5' terminus of (N')y;

wherein x and y are each independently an integer between 18 and 40;

wherein the sequence of (N')y is complementary to the sequence of (N)x; and wherein (N)x comprises the antisense sequence of SEQ ID NO: 1. In some embodiments, (N)x comprises the antisense oligonucleotides present in Tables A-19. In other embodiments, (N)x is selected from the antisense oligonucleotides present in Tables B or C.

In some embodiments the covalent bond linking each consecutive N or N' is a phosphodiester bond.

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

In some embodiments of the nucleic acid molecules (eg, siNA molecules) disclosed herein, the double-stranded nucleic acid molecule is siRNA, siNA, or miRNA.

In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_4 (SEQ ID NO: 195 and 220), SERPINH1_12 (SEQ ID NO: 196 and 221), SERPINH1_30 (SEQ ID NO: 199 and 224), and SERPINH1_58 (SEQ ID NO: 208 and 233).

In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_4 (SEQ ID NO: 195 and 220). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_12 (SEQ ID NO: 196 and 221). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_30 (SEQ ID NO: 199 and 224). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_58 (SEQ ID NO: 208 and 233).

In some embodiments, the double-stranded nucleic acid molecule comprises a DNA portion at position 1 (5' end) of the antisense strand or a mismatch with the target. This structure is described herein. According to one embodiment, a modified nucleic acid molecule having the structure (A2) shown below is provided:

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

3'Z'-N ² -(N')y–z"5' (sense strand)

wherein N ² , N and N′ are each an unmodified or modified ribonucleotide or an unconventional moiety;

wherein (N)x and (N')y are each an oligonucleotide in which each consecutive N or N' is linked to an adjacent N or N' by a covalent bond;

wherein x and y are each independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)x and (N)x is complementary to a contiguous sequence in the target RNA;

wherein ^N1 is covalently bound to (N)x and is mismatched with the target RNA or is a complementary DNA portion of the target RNA;

wherein ^N1 is selected from 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 to the 5' terminus of ^N2- (N')y; and

wherein Z and Z' are each independently present or absent, but if present are independently 1-5 consecutive nucleotides, consecutive non-nucleotide moieties, or combinations thereof, covalently linked at the 3' terminus of the chain in which they are present.

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

In some embodiments, ^N1 and ^N2 form a Watson-Crick base pair. In some embodiments, ^N1 and ^N2 form 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 N ¹ -(N)x, N ¹ refers to position 1 and positions 2-19 are included in (N) _18. When y＝18 in N ² -(N′)y, N ² refers to position 19 and positions 1-18 are included in (N′) ₁₈ .

In some embodiments, ^N1 is covalently bound to (N)x and is mismatched to the target RNA. In various embodiments, ^N1 is covalently bound to (N)x and is a DNA moiety that is complementary to the target RNA.

In some embodiments, the uridine at position 1 of the antisense strand is substituted with an ^N1 selected from adenosine, deoxyadenosine, deoxyuridine (dU), or deoxythymidine. In various embodiments, ^N1 is selected from adenosine, deoxyadenosine, or deoxyuridine.

In some embodiments, the guanosine at position 1 of the antisense strand is substituted with an ^N1 selected from adenosine, deoxyadenosine, uridine, deoxyuridine, thymidine, or deoxythymidine. In various embodiments, ^N1 is selected from adenosine, deoxyadenosine, uridine, or deoxyuridine.

In some embodiments, the cytidine at position 1 of the antisense strand is substituted with an ^N1 selected from adenosine, deoxyadenosine, uridine, deoxyuridine, thymidine, or deoxythymidine. In various embodiments, ^N1 is selected from adenosine, deoxyadenosine, uridine, or deoxyuridine.

In some embodiments, the adenosine at position 1 of the antisense strand is substituted with an ^N1 selected from deoxyadenosine, deoxyuridine, thymidine, or deoxythymidine. In various embodiments, ^N1 is selected from deoxyadenosine or deoxyuridine.

In some embodiments, ^N1 and ^N2 form a base pair between uridine or deoxyuridine and adenosine or deoxyadenosine. In other embodiments, ^N1 and ^N2 form a base pair between deoxyuridine and adenosine.

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

In some embodiments, (N)x comprises an antisense oligonucleotide present in Table A-18. In some embodiments, x=y=18 and N1-(N)x comprises 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 oligonucleotide pairs shown 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 oligonucleotide pairs shown in Tables D and E. In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_2 (SEQ ID NO: 60 and 127), SERPINH1_6 (SEQ ID NO: 63 and 130), SERPINH1_11 (SEQ ID NO: 68 and 135), SERPINH1_13 (SEQ ID NO: 69 and 136), SERPINH1_45 (SEQ ID NO: 97 and 164), SERPINH1_45a (SEQ ID NO: 98 and 165), SERPINH1_51 (SEQ ID NO: 101 and 168), SERPINH1_51a (SEQ ID NO: 105 and 172), SERPINH1_52 (SEQ ID NO: 102 and 169) or SERPINH1_86 (SEQ ID NO: 123 and 190). In some preferred embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_2 (SEQ ID NO: 60 and 127), SERPINH1_6 (SEQ ID NO: 63 and 130), SERPINH1_45a (SEQ ID NO: 98 and 165), SERPINH1_51 (SEQ ID NOS: 101 and 168) and SERPINH1_51a (SEQ ID NO: 105 and 172).

In some preferred embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_2 (SEQ ID NO: 60 and 127). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_6 (SEQ ID NO: 63 and 130). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_11 (SEQ ID NO: 68 and 135). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_13 (SEQ ID NO: 69 and 136). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_45 (SEQ ID NO: 97 and 164). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_45a (SEQID NO: 98 and 165). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_51 (SEQ ID NO: 101 and 168). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_51a (SEQ ID NO: 105 and 172). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_52 (SEQ ID NO: 102 and 169). In some embodiments, the antisense strand and the sense strand are the sequence pairs shown in SERPINH1_86 (SEQ ID NO: 123 and 190). In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_2 (SEQ ID NO:60 and 127), SERPINH1_6 (SEQ ID NO:63 and 130), SERPINH1_45a (SEQ ID NO:98 and 165), SERPINH1_51 (SEQ ID NOS:101 and 168) and SERPINH1_51a (SEQ ID NO: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 riboadenosine, modified riboadenosine, deoxyriboadenosine, and modified deoxyriboadenosine. In other embodiments, N1 is selected from ribouridine, deoxyribouridine, modified ribouridine, and modified deoxyuridine.

In certain embodiments, position 1 (5' end) in the antisense strand comprises deoxyribouridine (dU) or adenosine. In some embodiments, N1 is selected from riboadenosine, modified riboadenosine, deoxyriboadenosine, modified deoxyriboadenosine and N2 is selected from ribouridine, deoxyribouridine, modified ribouridine and modified deoxyuridine. In certain embodiments, N1 is selected from riboadenosine and modified riboadenosine and N2 is selected from ribouridine and modified ribouridine.

In certain embodiments, N1 is selected from ribouridine, deoxyribouridine, modified ribouridine and modified deoxyuridine and N2 is selected from riboadenosine, modified ribouridine, deoxyriboadenosine, modified deoxyriboadenosine. In certain embodiments, N1 is selected from ribouridine and deoxyuridine and N2 is selected from riboadenosine and modified ribouridine. In certain embodiments, N1 is ribouridine and N2 is riboadenosine. In certain embodiments, N1 is deoxyuridine and N2 is riboadenosine.

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

In some embodiments of Structure (A2), N1 comprises a 2'OMe sugar-modified ribouracil or a 2'OMe sugar-modified ribouracil. In certain embodiments of Structure (A), N2 comprises a 2'OMe sugar-modified ribonucleotide.

In some embodiments, N and N' are each unmodified nucleotides. In some embodiments, at least one of N or N' comprises 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' comprises 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 comprises an antisense sequence that is fully complementary to about 17 to about 39 consecutive nucleotides in the target RNA. In other embodiments, (N)x comprises an antisense sequence that is substantially complementary to about 17 to about 39 consecutive nucleotides in the target RNA.

In some embodiments of Structure A1 and Structure A2, the compound has a blunt end, for example, wherein both Z and Z' are absent. In alternative embodiments, at least one of Z or Z' is present. Z and Z' independently comprise one or more covalently linked modified and/or unmodified nucleotides, including deoxyribonucleotides and ribonucleotides, or unconventional moieties, such as inverted abasic deoxyribose moieties or abasic ribose moieties; non-nucleotide C3, C4 or C5 moieties, amino-6 moieties, mirror nucleotides, etc. In some embodiments, Z and Z' each independently comprise a C3 moiety or an amino-C6 moiety. In some embodiments, Z' is absent and Z is present and comprises a non-nucleotide C3 moiety. In some embodiments, Z is absent and Z' is present and comprises 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 modified ribonucleotide 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 at the 2' position includes the presence of an amino, fluoro, alkoxy, or 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 alternating 2'OMe sugar-modified ribonucleotides located on one or both of the antisense and sense strands. In other embodiments, the compound includes 2'OMe sugar-modified ribonucleotides located only on the antisense strand (N)x or N1-(N)x. In certain embodiments, the middle ribonucleotide of the antisense strand; for example, the ribonucleotide at position 10 of the 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 further embodiments, the compound of Structure A1 or Structure A2 comprises modified ribonucleotides at alternating positions, wherein the sugar residue of each ribonucleotide at the 5' and 3' termini of (N)x or N1-(N)x is modified, and the sugar residue of each ribonucleotide at the 5' and 3' termini of (N')y or N2-(N')y is unmodified.

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

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

b) the N' at 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) The N' in 4, 5 or 6 consecutive positions at the 3' terminal position of (N')y comprises 2'5' nucleotides.

In some embodiments, the double-stranded molecule comprises a combination of the following modifications

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

b) The sense strand includes 2'5' nucleotides and a pseudouridine at position 9 or 10 from the 5' end.

In some embodiments, the double-stranded molecule comprises a combination of the following modifications

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

c) The sense strand comprises 4, 5 or 6 consecutive 2'5' nucleotides located at the 3' penultimate or 3' terminal position.

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

In some embodiments of Structure A1 and Structure A2, the 3' and 5' ends of the sense and antisense strands are not phosphorylated. In other embodiments, the 3' end of one or both of the sense and antisense strands is phosphorylated.

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

In some embodiments of Structure A1, (N')y includes at least one L-DNA portion. 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-DNAs at the 3' penultimate position (positions 17 and 18). In various embodiments, the unconventional portion is a nucleotide connected to an adjacent nucleotide by a 2'-5' internucleotide phosphate bond. According to various embodiments, (N')y includes 2, 3, 4, 5, or 6 consecutive ribonucleotides at the 3' terminus connected by a 2'-5' internucleotide bond. In one embodiment, the four consecutive nucleotides at the 3' terminus of (N')y are linked by three 2'-5' phosphodiester bonds, wherein one or more 2'-5' nucleotides forming the 2'-5' phosphodiester bonds further comprise a 3'-O-methyl (3'OMe) sugar modification. Preferably, the 3' terminal nucleotide of (N')y comprises a 2'OMe sugar modification. In certain embodiments, x=y=19 and (N')y comprises two or more consecutive nucleotides at positions 15, 16, 17, 18, and 19 comprising nucleotides linked to adjacent nucleotides by a 2'-5' internucleotide bond (2'-5' nucleotides). In various embodiments, the nucleotides forming the 2'-5' internucleotide bond comprise 3' deoxyribonucleotides or 3' methoxy nucleotides (3'H or 3'OMe instead of 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 2'-5' internucleotide bonds between positions 15-16, 16-17, and 17-18; or 2'-5' nucleotides at positions 15, 16, 17, 18, and 19 such that adjacent nucleotides are linked by 2'-5' internucleotide bonds between positions 15-16, 16-17, 17-18, and 18-19 and a 3' OH is available at the 3' terminal nucleotide, or 2'-5' nucleotides at positions 16, 17, and 18 such that adjacent nucleotides are linked by 2'-5' internucleotide bonds 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 positions 17 and 18, or 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 a nucleotide linked to the adjacent nucleotide by a 2'-5' internucleotide bond. In some embodiments, the antisense strand and the sense strand are selected from the sequence pair shown in SERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58 or SERPINH1_88, x=y=19 and (N')y comprises 5 consecutive nucleotides connected at the 3' terminus by 4 2'-5' linkages, particularly the linkages between nucleotide positions 15-16, 16-17, 17-18 and 18-19.

In some embodiments, the linkage includes a phosphodiester bond. In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58 or SERPINH1_88 and x=y=19, and (N') y is included in 5 consecutive nucleotides connected by 4 2'-5' linkages at the 3' end and optionally further includes Z' and z' independently selected from the reverse free base part and C3 alkyl [C3; 1,3-propylene glycol mono(dihydrogen phosphate)] cap. The C3 alkyl cap is covalently linked to the 3' or 5' terminal nucleotide. In some embodiments, the 3'C3 end cap further includes 3' phosphate. In some embodiments, the 3'C3 end cap further includes a 3' terminal hydroxyl group.

In some embodiments, the antisense strand and the sense strand are selected from the sequence pair shown in SERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58 or SERPINH1_88, x＝y＝19 and (N′)y includes L-DNA position 18; and (N′)y optionally further includes Z′ and z′ independently selected from an inverted abasic portion 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 strand and the sense strand are selected from the sequence pairs shown in SERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58 or SERPINH1_88, x=y=19 and (N)x includes a 2'OMe sugar modified ribonucleotide at position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or position 2, 4, 6, 8, 11, 13, 15, 17, 19. In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58 and SERPINH1_88, x=y=19 and (N)x includes a 2'OMe sugar modified pyrimidine. In some embodiments, all pyrimidines in (N)x include a 2'OMe sugar modification.

In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN_51a, SERPINH1_52 or SERPINH1_86, x=y=18 and N2 is a riboadenosine moiety.

In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN_51a, SERPINH1_52, or SERPINH1_86 and x=y=18, and N2-(N')y includes 5 consecutive nucleotides connected by 4 2'-5' linkages at the 3' terminus, particularly the linkages between nucleotide positions 15-16, 16-17, 17-18, and 18-19. In some embodiments, the linkages include phosphodiester bonds.

In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN_51a, SERPINH1_52 or SERPINH1_86 and x=y=18, and N2-(N')y includes 5 consecutive nucleotides connected by 4 2'-5' bonds at the 3' terminus and optionally further includes Z' and z' independently selected from an inverted abasic portion and a C3 alkyl [C3; 1,3-propylene glycol mono(dihydrogen phosphate)] cap.

In some embodiments, the antisense strand and the sense strand are selected from the sequence pair shown in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN_51a, SERPINH1_52 or SERPINH1_86 and x=y=18, and ^N2- (N')y includes 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, ^N2- (N')y comprises a 3' terminal phosphate. In some embodiments, ^N2- (N')y comprises a 3' terminal hydroxyl group.

In some embodiments, the antisense strand and the sense strand are selected from the sequence pair shown in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN_51a, SERPINH1_52, or SERPINH1_86 and x=y=18, and ^N1- (N)x includes a 2'OMe sugar modified ribonucleotide at position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or position 1, 3, 5, 9, 11, 13, 15, 17, 19, or position 3, 5, 9, 11, 13, 15, 17, or position 2, 4, 6, 8, 11, 13, 15, 17, 19. In some embodiments, the antisense strand and the sense strand are selected from the sequence pair shown in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN_51a, SERPINH1_52, or SERPINH1_86, x=y=18, and ^N1- (N)x includes 2'OMe sugar-modified ribonucleotides at positions 11, 13, 15, 17, and 19 (from the 5' end). In some embodiments, the antisense strand and the sense strand are selected from the sequence pair shown in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN_51a, SERPINH1_52, or SERPINH1_86, x=y=18, and ^N1- (N)x includes a 2'OMe sugar modified ribonucleotide at position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or positions 3, 5, 7, 9, 11, 13, 15, 17, 19. In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN_51a, SERPINH1_52 or SERPINH1_86, x=y=18 and N1-(N)x includes 2'OMe sugar modified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17, 19.

In some embodiments, the antisense strand and the sense strand are selected from the sequence pairs shown in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN_51a, SERPINH1_52 or SERPINH1_86, x=y=18 and N1-(N)x includes 2'OMe sugar-modified pyrimidines. In some embodiments, all pyrimidines in (N)x include 2'OMe sugar modifications. In some embodiments, the antisense strand further includes L-DNA or 2'-5' nucleotides (5'>3') at positions 5, 6 or 7. In other embodiments, the antisense strand further includes ribonucleotides (5'>3') that generate 2'5' internucleotide linkages between ribonucleotides at positions 5-6 or 6-7.

In other embodiments, N1-(N)x further comprises Z, wherein Z comprises 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 portion. 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 positions 1-15 and 18 and two consecutive L-DNAs at the 3' penultimate position (positions 16 and 17). In various embodiments, the unconventional portion is a nucleotide connected to an adjacent nucleotide by a 2'-5' internucleotide phosphate bond. According to various embodiments, (N')y includes 2, 3, 4, 5, or 6 consecutive ribonucleotides at the 3' terminus connected by a 2'-5' internucleotide bond. In one embodiment, the four consecutive nucleotides at the 3' terminus of (N')y are linked by three 2'-5' phosphodiester bonds, wherein one or more 2'-5' nucleotides forming the 2'-5' phosphodiester bonds further comprise a 3'-O-methyl (3'OMe) sugar modification. Preferably, the 3' terminal nucleotide of (N')y comprises a 2'OMe sugar modification. In certain embodiments, x=y=18 and two or more consecutive nucleotides at positions 14, 15, 16, 17 and 18 in (N')y comprise nucleotides linked to adjacent nucleotides by a 2'-5' internucleotide bond. In various embodiments, the nucleotides forming the 2'-5' internucleotide bond comprise a 3' deoxyribonucleotide or a 3' methoxy nucleotide. In some embodiments, x=y=18 and (N')y comprises nucleotides linked to adjacent nucleotides 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 a nucleotide linked to an 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 a nucleotide linked to an adjacent nucleotide by a 2'-5' internucleotide bond.

In some embodiments, the antisense strand and the sense strand are selected from the oligonucleotide pairs shown 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 comprises the antisense strand set forth in SEQ ID NO: 127 and the 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

Each "│" represents base pairing between 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 combinations thereof, covalently linked to the 3' terminus of the chain in which it is present; and

wherein z" may or may not be present, but if present is a capping moiety covalently attached to the 5' terminus of N2-(N')y.

In some embodiments, a double-stranded nucleic acid molecule is provided wherein the antisense strand (SEQ ID NO: 127) comprises one or more 2'OMe sugar-modified pyrimidines and/or purines, a 2'-5' ribonucleotide at 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) comprises 4 or 5 consecutive 2'5' nucleotides at the 3' terminus or penultimate position, a nucleotide or non-nucleotide moiety covalently linked at the 3' terminus, and a cap moiety covalently linked at the 5' terminus. In other embodiments, the sense strand (SEQ ID NO: 60) comprises one or more 2'OMe pyrimidines, a nucleotide or non-nucleotide moiety covalently linked at the 3' terminus, and a cap moiety covalently linked at the 5' terminus.

In some embodiments, a double-stranded nucleic acid molecule is provided wherein the antisense strand (SEQ ID NO: 127) comprises 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 linked to the 3' terminus; and the sense strand (SEQ ID NO: 60) is selected from the group consisting of:

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 linked at the 5' terminus; or

b) a 2'-5' ribonucleotide at positions 15, 16, 17, 18 and 19, a 3' terminal phosphate; and an inverted abasic deoxyribonucleotide moiety covalently linked 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 C3-OH 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 linked at the 3' terminus; and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' terminus.

Provided herein are double-stranded nucleic acid molecules, wherein the antisense strand (SEQ ID NO: 127) comprises 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 linked to the 3' terminus; and the sense strand (SEQ ID NO: 60) comprises 2'-5' ribonucleotides at positions 15, 16, 17, 18, and 19: a C3 3' terminal overhang; and an inverted abasic deoxyribonucleotide moiety covalently linked to the 5' terminus.

Provided herein are double-stranded nucleic acid molecules, wherein the antisense strand (SEQ ID NO: 127) comprises 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) comprises 2'-5' ribonucleotides at positions 15, 16, 17, 18, and 19: a 3' terminal phosphate; and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' terminus.

Provided herein are double-stranded nucleic acid molecules, wherein the antisense strand (SEQ ID NO: 127) comprises 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 linked to the 3' terminus; and the sense strand (SEQ ID NO: 60) comprises 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 linked at the 3' terminus; and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' terminus.

Provided herein are double-stranded nucleic acid molecules, wherein the antisense strand (SEQ ID NO: 127) comprises 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 linked to the 3' terminus; and the sense strand (SEQ ID NO: 60) comprises 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 linked at the 3' terminus; and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' terminus.

Provided herein are double-stranded nucleic acid molecules, wherein the antisense strand (SEQ ID NO: 127) comprises 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 linked to the 3' terminus; and the sense strand (SEQ ID NO: 60) comprises 2'-5' ribonucleotides at positions (5'>3') 15, 16, 17, 18, and 19: a C3-Pi moiety covalently linked to the 3' terminus; and an inverted abasic deoxyribonucleotide moiety covalently linked to the 5' terminus.

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

In some embodiments, provided herein are double-stranded nucleic acid molecules wherein the sense strand (SEQ ID NO: 60) comprises 2'-5' ribonucleotides at positions 15, 16, 17, 18, and 19: a 3' terminal phosphate and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' end, and the antisense strand (SEQ ID NO: 127) comprises an antisense strand selected from one of the following:

a) a ribonucleotide modified with a 2'OMe sugar at position (5'>3') 1, 3, 5, 9, 11, 15, 17, 19 and a C3Pi-C3OH moiety covalently linked 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 linked to the 3’ terminus.

In some embodiments, provided herein are double-stranded nucleic acid molecules comprising an antisense strand as set forth in SEQ ID NO: 130 and a sense strand as set forth in SEQ ID NO: 63; identified herein as SERPINH1_6. In some embodiments, the duplex comprises the structure:

Each "│" represents base pairing between 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 combinations thereof, covalently linked to the 3' terminus of the chain in which it is present; and

wherein z" may or may not be present, but if present is a capping moiety covalently attached to the 5' terminus of N2-(N')y.

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

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

a) a 2'OMe sugar modified ribonucleotide at position (5'>3') 1, 3, 5, 9, 11, 15, 17, 19; a 2'-5' ribonucleotide at position 7; and a C3Pi-C3OH moiety covalently linked to the 3' terminus; or

b) 2'OMe sugar-modified ribonucleotides at 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 linked to the 3' terminus; or

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

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

In some embodiments, a double-stranded nucleic acid molecule is provided, wherein the sense strand (SEQ ID NO:63) includes 2’OMe sugar-modified ribonucleotides at positions (5’>3’) 2, 14, and 18; a C3-OH moiety covalently linked at the 3’ terminus; and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5’ terminus; and the antisense strand (SEQ ID NO:130) includes 2’OMe sugar-modified ribonucleotides at 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 linked to the 3’ terminus.

In some embodiments, a duplex oligonucleotide molecule is provided wherein the sense strand (SEQ ID NO:63) comprises 2’OMe sugar-modified ribonucleotides at positions (5’>3’) 14 and 18 and optionally at 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) comprises 2’OMe sugar-modified ribonucleotides at 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.

In some embodiments, a duplex oligonucleotide molecule is provided wherein the sense strand (SEQ ID NO: 63) comprises 2'OMe sugar-modified ribonucleotides at 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) is selected from the group consisting of:

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

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

Provided herein are double-stranded nucleic acid molecules, wherein the sense strand (SEQ ID NO: 63) comprises 2'OMe sugar-modified ribonucleotides at positions (5'>3') 14 and 18; a C3-OH moiety covalently linked at the 3' terminus; and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' terminus; and the antisense strand (SEQ ID NO: 130) comprises 2'OMe sugar-modified ribonucleotides at 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 linked to the 3' terminus.

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

In some embodiments, the duplex comprises the antisense strand set forth in SEQ ID NO: 165 and the sense strand set forth in SEQ ID NO: 98; identified herein as SERPINH1_45a. In some embodiments, the duplex comprises the structure:

Each "│" represents base pairing between 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 combinations thereof, covalently linked to the 3' terminus of the chain in which it is present; and

wherein z" may or may not be present, but if present is a capping moiety covalently attached to the 5' terminus of N2-(N')y.

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

In some embodiments, the sense strand (SEQ ID NO: 98) comprises 2'-5' ribonucleotides at positions (5'>3'): C3Pi or C3-OH 3'-terminal non-nucleotide moiety and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' terminus; and the antisense strand (SEQ ID NO: 165) comprises an antisense strand selected from one of the following:

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

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

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

d) 2’OMe sugar-modified ribonucleotides at positions (5’>3’) 1, 3, 5, 9, 11, 13, 15, 17, and 19 and C3Pi-C3Pi or C3Pi-C3OH 3’ terminal overhangs.

Provided herein are double-stranded nucleic acid molecules, wherein the sense strand (SEQ ID NO: 98) comprises 2'-5' ribonucleotides at positions (5'>3') 15, 16, 17, 18 and 19: a C3-OH 3' terminal portion and an inverted abasic deoxyribonucleotide portion covalently linked at the 5' terminus; and the antisense strand (SEQ ID NO: 165) comprises 2'OMe sugar-modified ribonucleotides at positions (5'>3') 2, 4, 6, 8, 11, 13, 15, 17 and 19; a 2'-5' ribonucleotide at position 7 and a C3Pi-C3OH 3' terminal overhang.

In some embodiments, the double-stranded nucleic acid molecule comprises the antisense strand set forth in SEQ ID NO: 168 and the sense strand set forth in SEQ ID NO: 101; identified herein as SERPINH1_51. In some embodiments, the duplex comprises the structure:

Each "│" represents base pairing between 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 combinations thereof, covalently linked to the 3' terminus of the chain in which it is present; and

wherein z" may or may not be present, but if present is a capping moiety covalently attached to the 5' terminus of N2-(N')y.

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

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

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

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

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

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

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

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

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

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

In some embodiments, the double-stranded nucleic acid molecule comprises the antisense strand set forth in SEQ ID NO: 168 and the sense strand set forth in SEQ ID NO: 101; identified herein as SERPINH1_51a. In some embodiments, the duplex comprises the structure

Each "│" represents base pairing between 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 combinations thereof, covalently linked to the 3' terminus of the chain in which it is present; and

wherein z" may or may not be present, but if present is a capping moiety covalently attached to the 5' terminus of N2-(N')y.

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

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

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

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

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

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

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

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

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

Provided herein are double-stranded nucleic acid molecules, wherein the sense strand (SEQ ID NO: 105) comprises 2'OMe sugar-modified ribonucleotides at positions (5'>3') 4, 11, 13, and 17, optionally a 2'-5' ribonucleotide at position 9, a C3-OH non-nucleotide moiety covalently linked at the 3' terminus, and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' terminus; and the antisense strand (SEQ ID NO: 172) comprises 2'OMe sugar-modified ribonucleotides at positions (5'>3') 3, 8, 12, 13, and 15, a 2'5' ribonucleotide at position 6; and a C3Pi-C3OH moiety covalently linked at the 3' terminus.

In some embodiments, the antisense strand and the sense strand are selected from the oligonucleotide pairs shown 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 comprises the antisense strand set forth in SEQ ID NO: 220 and the 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:

Each "│" represents base pairing between 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 combinations thereof, covalently linked to the 3' terminus of the chain in which it is present; and

wherein z" may or may not be present, but if present is a capping moiety covalently attached to the 5' terminus of N2-(N')y.

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

a) a 2'-5' ribonucleotide at positions 15, 16, 17, 18 and 19, a C3OH moiety covalently linked to the 3' terminus; and an inverted abasic deoxyribonucleotide moiety covalently linked to the 5' terminus; or

b) a 2'-5' ribonucleotide at positions 15, 16, 17, 18 and 19, a 3' terminal phosphate; and an inverted abasic deoxyribonucleotide moiety covalently linked 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 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 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 C3Pi moiety covalently linked at the 3' terminus; and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' terminus.

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

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

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

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

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

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

In some embodiments, provided herein are double-stranded nucleic acid molecules wherein the sense strand (SEQ ID NO: 195) comprises 2'-5' ribonucleotides at positions 15, 16, 17, 18, and 19: a 3' terminal phosphate and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' end, and the antisense strand (SEQ ID NO: 220) comprises an antisense strand selected from one of the following:

a) a 2'OMe sugar modified ribonucleotide at position (5'>3') 3, 5, 7, 9, 11, 13, 15, 17, 19 and a C3Pi-C3OH moiety covalently linked 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 linked to the 3’ terminus.

In some embodiments, provided herein are double-stranded nucleic acid molecules comprising an antisense strand as set forth in SEQ ID NO: 130 and a sense strand as set forth in SEQ ID NO: 63; identified herein as SERPINH1_12. In some embodiments, the duplex comprises the structure:

Each "│" represents base pairing between 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 combinations thereof, covalently linked to the 3' terminus of the chain in which it is present; and

wherein z" may or may not be present, but if present is a capping moiety covalently attached to the 5' terminus of N2-(N')y.

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

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

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

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

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

In some embodiments, a duplex oligonucleotide molecule is provided wherein the sense strand (SEQ ID NO: 196) comprises 2'OMe sugar-modified ribonucleotides at 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) comprises an antisense strand selected from the group consisting of:

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

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

Provided herein are double-stranded nucleic acid molecules, wherein the sense strand (SEQ ID NO: 196) comprises 2'OMe sugar-modified ribonucleotides at positions (5'>3') 14 and 18; a C3-OH moiety covalently linked at the 3' terminus; and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' terminus; and the antisense strand (SEQ ID NO: 221) comprises 2'OMe sugar-modified ribonucleotides at 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 linked to the 3' terminus.

Provided herein are double-stranded nucleic acid molecules, wherein the sense strand (SEQ ID NO: 196) comprises 2'OMe sugar-modified ribonucleotides at positions (5'>3') 14 and 18; a C3-OH moiety covalently linked at the 3' terminus; and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5' terminus; and the antisense strand (SEQ ID NO: 221) comprises 2'OMe sugar-modified ribonucleotides at 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 linked to the 3' terminus.

In further embodiments of Structures A1 and A2, (N')y comprises 1-8 modified ribonucleotides, wherein the modified ribonucleotides are DNA nucleotides. In certain embodiments, (N')y comprises 1, 2, 3, 4, 5, 6, 7 or up to 8 DNA moieties.

In some embodiments, Z or Z' is present and independently comprises two non-nucleotide moieties.

In further embodiments, both Z and Z' are present and each independently comprises two non-nucleotide moieties.

In some embodiments, each of Z and Z' comprises an abasic moiety, such as a deoxyribose abasic moiety (referred to herein as a "dAb") or a ribose abasic moiety (referred to herein as a "rAb"). In some embodiments, each of Z and/or Z' comprises two covalently linked abasic moieties and is, for example, a dAb-dAb or rAb-rAb or dAb-rAb or rAb-dAb, wherein each moiety is preferably linked to the adjacent moiety via a phosphate-based bond. In some embodiments, the phosphate-based bond comprises a phosphorothioate bond, a phosphonoacetate bond, or a phosphodiester bond. In some embodiments, the phosphate-based bond comprises a phosphodiester bond.

In some embodiments, each of Z and/or Z' comprises an alkyl moiety, optionally a propane [(CH2)3] moiety or a derivative thereof, including propanol (C3-OH) and a phosphorylated derivative of propylene glycol ("C3-3'Pi"). In some embodiments, each of Z and/or Z' comprises two alkyl moieties covalently linked to the 3' end of the antisense strand or the sense strand via a phosphodiester bond or a phosphorothioate bond and covalently linked to each other via a phosphodiester bond or a phosphorothioate bond, and in some examples is C3Pi-C3Pi or C3Pi-C3OH. The 3' end of the antisense strand and/or the 3' end of the sense strand is covalently linked to the C3 moiety via a phosphate-based bond, and the C3 moiety is covalently linked to the C3-OH moiety via a phosphate-based bond. In some embodiments, the phosphate-based bond comprises a phosphorothioate bond, a phosphonoacetate bond, or a phosphodiester bond. In preferred embodiments, the phosphate-based bond comprises a phosphodiester bond.

In various embodiments of Structure A1 or Structure A2, Z and Z' are present. In other embodiments, Z or Z' is present. In some embodiments, each of Z and/or Z' independently comprises 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), propylene glycol and a phosphodiester derivative of propylene glycol ("C3Pi"). In preferred embodiments, each of Z and/or Z' comprises two hydrocarbon moieties and in some instances is C3Pi-C3OH or C3Pi-C3Pi. Each C3 is covalently conjugated to an adjacent C3 by a covalent bond, preferably by a phosphate-based bond. In some embodiments, the phosphate-based bond is a phosphorothioate bond, a phosphonoacetate bond or a phosphodiester bond.

In certain embodiments, x=y=19 and Z comprises at least one C3 alkyl overhang. In some embodiments, the C3-C3 overhang is covalently linked to the 3' terminus of (N)x or (N')y via a covalent bond, preferably a phosphodiester bond. In some embodiments, the bond between the first C3 and the second C3 is a phosphodiester bond. 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 a hydroxyl group). 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 comprising a terminal hydroxyl group, a terminal amino group or a 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, propyl phosphate, propyl thiophosphate or a combination thereof. The C3 alkyl moiety is covalently attached to the 3' end of (N')y and/or the 3' end of (N)x via a phosphodiester bond. In some embodiments, the alkyl moiety comprises propanol, propyl phosphate or propyl thiophosphate. In some embodiments, each of Z and Z' is independently selected from propanol, propyl phosphate, propyl thiophosphate, a combination thereof or a plurality thereof, especially 2 or 3 covalently attached propanol, propyl phosphate, propyl thiophosphate or a combination thereof. In some embodiments, each of Z and Z' is independently selected from propyl phosphate, propyl thiophosphate, propyl phosphate-propanol; propyl phosphate-propyl thiophosphate; propyl phosphate-propyl phosphate; (propyl phosphate) ₃ , (propyl phosphate) ₂ -propanol, (propyl phosphate) ₂ -propyl thiophosphate. Z or Z' may include any propane or propanol conjugated moiety.

The structure of an exemplary 3' terminal non-nucleotide is as follows:

In some embodiments, each of Z and Z' is independently selected from propanol, propyl phosphate, propyl thiophosphate, a combination thereof, or a plurality thereof.

In some embodiments, each of Z and Z' is independently selected from propyl phosphate, propyl thiophosphate, propyl phosphate-propanol; propyl phosphate-propyl thiophosphate; propyl phosphate-propyl phosphate; (propyl phosphate) ₃ , (propyl phosphate) ₂ -propanol, (propyl phosphate) ₂ -propyl thiophosphate. Z or Z' may include any propane or propanol conjugated moiety.

In other embodiments, each of Z and/or Z' comprises 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 (deoxyribose or ribose) and a hydrocarbon moiety. In these embodiments, each of Z and/or Z' comprises a C3-rAb or a C3-dAb, wherein each moiety is covalently bound to an adjacent moiety via a phosphate-based bond, preferably a phosphodiester bond, a phosphorothioate bond, or a phosphonoacetate bond.

In certain embodiments, the nucleic acid molecules disclosed herein include a sense oligonucleotide sequence selected from any one of oligonucleotide numbers 2-67 or 68-92 shown in Tables A-18 and A-19 below, respectively.

In certain embodiments, provided compounds 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, (e.g., Compound_1, Compound_5, and Compound_6 as described herein) a 19-mer double-stranded nucleic acid molecule is provided, wherein the antisense strand is SEQ ID NO: 127 and the sense strand is SEQ ID NO: 60. In certain embodiments, a 19-mer double-stranded nucleic acid molecule is provided, wherein the antisense strand is SEQ ID NO: 127 and comprises a 2'OMe sugar-modified ribonucleotide, a 2'-5' ribonucleotide at at least one of positions 1, 5, 6, or 7, and a 3'-terminal non-nucleotide moiety covalently linked to the 3' terminus; and the sense strand is SEQ ID NO: 60 and comprises at least one 2'5' ribonucleotide or a 2'OMe sugar-modified ribonucleotide, a non-nucleotide moiety covalently linked to the 3' terminus, and a cap moiety covalently linked to the 5' terminus. In some embodiments, a 19-mer double-stranded nucleic acid molecule is provided, 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 at position 7, and a 3' terminal C3OH non-nucleotide moiety covalently linked at the 3' terminus; and the sense strand is SEQ ID NO: 60 and includes 5 consecutive 2'5' ribonucleotides at 3' terminal positions 15, 16, 17, 18 and 19 (5'>3'), a C3Pi non-nucleotide moiety covalently linked at the 3' terminus, and an inverted abasic moiety covalently linked at the 5' terminus.

In one embodiment, compound _1 is provided which 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 at position 7, and a C3Pi-C3OH non-nucleotide portion covalently linked at the 3' end; and the sense strand is SEQ ID NO: 60 and includes 5 consecutive 2'5' ribonucleotides at 3' terminal positions 15, 16, 17, 18 and 19 (5'>3'), a C3Pi non-nucleotide portion covalently linked at the 3' end, and an inverted abasic portion covalently linked at the 5' end; and further includes a 2'OMe sugar-modified ribonucleotide at position 1 of the antisense strand.

In one embodiment, compound _6 is provided which 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 at position 7, and a C3Pi-C3OH non-nucleotide portion covalently linked at the 3' end; and the sense strand is SEQ ID NO: 60 and includes 5 consecutive 2'5' ribonucleotides at 3' terminal positions 15, 16, 17, 18 and 19 (5'>3'), a C3Pi non-nucleotide portion covalently linked at the 3' end, and an inverted abasic portion covalently linked at the 5' end; and further includes a 2'OMe sugar-modified ribonucleotide at position 1 of the antisense strand.

In one embodiment, compound _5 is provided which 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 1, 3, 5, 9, 11, 13, 15, 17 and 19 (5'>3'), a 2'-5' ribonucleotide at position 7, and a C3Pi-C3OH non-nucleotide portion covalently linked at the 3' terminus; and the sense strand is SEQ ID NO: 60 and includes 2'OMe sugar-modified ribonucleotides at 3' terminal positions (5'>3') 7, 13, 16 and 18, a 2'5' ribonucleotide at position 9, a C3OH non-nucleotide portion covalently linked at the 3' terminus, and an inverted abasic portion covalently linked at the 5' terminus.

In some embodiments (e.g., compound_2 and compound_7 as described herein), a 19-mer double-stranded nucleic acid molecule is provided, wherein the sense strand is SEQ ID NO: 63 and the antisense strand is SEQ ID NO: 130. In some embodiments, a 19-mer double-stranded nucleic acid molecule is provided, wherein the sense strand is SEQ ID NO: 63 and comprises a 2'OMe sugar-modified pyrimidine ribonucleotide, a non-nucleotide moiety covalently linked at the 3' end, and a cap moiety covalently linked at the 5' end; and the antisense strand is SEQ ID NO: 130 and comprises a 2'OMe sugar-modified ribonucleotide, a 2'-5' ribonucleotide at position 7, and a non-nucleotide moiety covalently linked at the 3' end. In some embodiments, a 19-mer double-stranded nucleic acid molecule is provided, wherein the sense strand is SEQ ID NO: 63 and includes a 2'OMe sugar-modified ribonucleotide, a non-nucleotide moiety covalently linked at the 3' end, and a cap moiety covalently linked at the 5' end; and the antisense strand is SEQ ID NO: 130 and includes a 2'OMe sugar-modified ribonucleotide, a 2'-5' ribonucleotide at at least one position 5, 6, or 7, and a non-nucleotide moiety covalently linked at the 3' end.

In one embodiment, compound _2 is provided which 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 at positions (5'>3') 2, 14 and 18, a C3OH portion covalently linked at the 3' end, and an inverted abasic deoxyribonucleotide portion covalently linked at the 5' end; and the antisense strand is SEQ ID NO: 130 and includes 2'OMe sugar-modified ribonucleotides at positions (5'>3') 1, 3, 5, 9, 12, 13 and 17, a 2'-5' ribonucleotide at at least one of positions 5, 6 or 7, and a C3Pi-C3OH non-nucleotide portion covalently linked at the 3' end.

In one embodiment, compound _7 is provided which 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 at positions (5’>3’) 2, 14 and 18, a C3OH portion covalently linked at the 3’ end and an inverted abasic deoxyribonucleotide portion covalently linked at the 5’ end; and the antisense strand is SEQ ID NO: 130 and includes 2’OMe sugar-modified ribonucleotides at positions (5’>3’) 1, 3, 5, 9, 11, 13 and 17, a 2’-5’ ribonucleotide at position 7 and a C3Pi-C3OH non-nucleotide portion covalently linked at the 3’ end.

In some embodiments (e.g., compound_3 as described herein), a 19-mer double-stranded nucleic acid molecule is provided, wherein the sense strand is SEQ ID NO: 98 and the antisense strand is SEQ ID NO: 165. In some embodiments, a 19-mer double-stranded nucleic acid molecule is provided, wherein the sense strand is SEQ ID NO: 98 and comprises a 2'-5' ribonucleotide at the 3' terminal position: a non-nucleotide moiety covalently linked at the 3' terminus, and a cap moiety covalently linked at the 5' terminus; and the antisense strand is SEQ ID NO: 165 and comprises a 2'OMe sugar-modified ribonucleotide, a 2'-5' ribonucleotide at at least one of positions 5, 6, or 7, and a non-nucleotide moiety covalently linked at the 3' terminus. In one embodiment, compound _3 is provided which is a 19-mer double-stranded nucleic acid molecule, wherein the sense strand is SEQ ID NO: 98 and includes 2’-5’ ribonucleotides at positions (5’>3’) 15, 16, 17, 18 and 19: a C3-OH 3’ moiety covalently linked at the 3’ end and an inverted abasic deoxyribonucleotide moiety covalently linked at the 5’ end; and the antisense strand is SEQ ID NO: 165 and includes 2’OMe sugar-modified ribonucleotides at positions (5’>3’), 2’-5’ ribonucleotide at position 7 and C3Pi-C3OH covalently linked at the 3’ end.

In some embodiments (e.g., compound 4, compound 8, and compound 9 as described herein), a 19-mer double-stranded nucleic acid molecule is provided, wherein the sense strand is SEQ ID NO: 101 and the antisense strand is SEQ ID NO: 168. In some embodiments, a 19-mer double-stranded nucleic acid molecule is provided, wherein the sense strand is SEQ ID NO: 101 and comprises a 2'OMe sugar-modified pyrimidine ribonucleotide, an optional 2'-5' ribonucleotide at one of positions 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 comprises a 2'OMe sugar-modified ribonucleotide, a 2'5' ribonucleotide at at least one of positions 5, 6, or 7, and a non-nucleotide moiety covalently attached at the 3' terminus.

In one embodiment, compound _4 is provided which 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 at positions (5’>3’) 4, 11, 13 and 17, a 2’-5’ ribonucleotide at position 9, a C3OH non-nucleotide portion covalently linked at the 3’ end, and an inverted abasic deoxyribonucleotide portion covalently linked at the 5’ end; and the antisense strand is SEQ ID NO: 168 and includes 2’OMe sugar-modified ribonucleotides at positions (5’>3’) 1, 4, 8, 11 and 15, a 2’5’ ribonucleotide at position 6; and a 3’C3Pi-C3OH overhang covalently linked at the 3’ end.

In one embodiment, compound _8 is provided which 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 at positions (5’>3’) 4, 11, 13 and 17, a C3OH non-nucleotide portion covalently linked at the 3’ end and an inverted abasic deoxyribonucleotide portion covalently linked at the 5’ end; and the antisense strand is SEQ ID NO: 168 and includes 2’OMe sugar-modified ribonucleotides at positions (5’>3’) 1, 4, 8, 13 and 15, a 2’5’ ribonucleotide at position 6 and a 3’C3Pi-C3OH overhang covalently linked at the 3’ end.

In one embodiment, compound _9 is provided which 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 at positions (5’>3’) 2, 4, 11, 13 and 17, a C3OH non-nucleotide portion covalently linked at the 3’ end, and an inverted abasic deoxyribonucleotide portion covalently linked at the 5’ end; and the antisense strand is SEQ ID NO: 168 and includes 2’OMe sugar-modified ribonucleotides at positions (5’>3’) 1, 4, 8, 11 and 15, a 2’5’ ribonucleotide at position 6, and a 3’C3Pi-C3OH non-nucleotide portion covalently linked at the 3’ end.

In another aspect, methods are provided for reducing hsp47 expression in cells by introducing into the cells a nucleic acid molecule as provided herein in an amount sufficient to reduce hsp47 expression. In one embodiment, the cells are hepatic stellate cells. In another embodiment, the cells are stellate cells in kidney or lung tissue. In certain embodiments, the methods are performed in vitro, and in other embodiments, the methods are performed in vivo.

In yet another aspect, a method for treating an individual with an hsp47-related disease is provided. The method comprises administering to the individual, for example, a nucleic acid molecule as provided herein, in an amount sufficient to reduce hsp47 expression. In certain embodiments, the hsp47-related disease is a disease selected from the group consisting of liver fibrosis, cirrhosis, pulmonary fibrosis including pulmonary fibrosis (including ILF), any condition causing renal fibrosis (e.g., CKD including ESRD), peritoneal fibrosis, chronic liver damage, fibrillation, fibrotic diseases of other organs, abnormal scarring (keloids) associated with all possible types of accidental and iatrogenic (surgical) skin injuries; scleroderma; myocardial fibrosis, failure of glaucoma filtration surgery; and intestinal adhesions. In some embodiments, the compound can be used to treat organ-specific indications, for example, indications including those shown in Table 2 below:

Table 2

In some embodiments, preferred indications include cirrhosis due to hepatitis C following liver transplantation; cirrhosis due to nonalcoholic steatohepatitis (NASH); idiopathic pulmonary fibrosis; radiation pneumonitis leading to pulmonary fibrosis; diabetic nephropathy; peritoneal sclerosis associated with continuous ambulatory peritoneal dialysis (CAPD) and ocular cicatricial pemphigoid.

Indications of fibrotic liver include alcoholic cirrhosis, hepatitis B cirrhosis, hepatitis C cirrhosis, hepatitis C (Hep C) cirrhosis after orthotopic liver transplantation (OLTX), NASH/NAFLD, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia, alpha-1 antitrypsin deficiency syndrome (A1AD), copper storage diseases (Wilson's disease), fructosemia, galactosemia, glycogen storage diseases (especially types III, IV, VI, IX, and X), iron overload syndromes (hemochromatosis), dyslipidemias (e.g., Gaucher's disease), peroxidase disorders (e.g., Zellweger syndrome), tyrosinemia, congenital hepatic fibrosis, bacterial infections (e.g., brucellosis), parasitic diseases (e.g., echinococcosis), Budd-Chiari syndrome syndrome)(hepatic veno-occlusive disease).

Pulmonary indications include idiopathic pulmonary fibrosis, silicosis, pneumoconiosis, bronchopulmonary dysplasia after neonatal respiratory distress syndrome, bleomycin/chemotherapy-induced lung injury, bronchiolitis obliterans (BOS) after lung transplantation, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma.

Cardiac indications include cardiomyopathy, atherosclerosis (such as Buerger's disease), myocardial endocardial fibrosis, atrial fibrillation, and scar formation after myocardial infarction (MI).

Other chest indications include radiation-induced capsular tissue reaction around textured breast implants and oral submucosal fibrosis.

Renal indications include autosomal dominant polycystic kidney disease (ADPKD), diabetic nephropathy (diabetic glomerulosclerosis), FSGS (collapse and other histologic variants), IgA nephropathy (Buerg's disease), lupus nephritis, Wegner's disease, scleroderma, Goodpasture's syndrome, tubulointerstitial fibrosis: drug-induced (prophylactic) penicillin, cephalosporin, analgesic nephropathy, membranous proliferative glomerulonephritis (MPGN), Henoch-Schonlein purpura, congenital kidney diseases: medullary cystic disease, nail-patella syndrome, and Alport syndrome.

Bone marrow indications include lymphangioleiomyomatosis (LAM), chronic graft-versus-host disease, polycythemia vera, essential thrombocythemia, and myelofibrosis.

Ocular indications include retinopathy of prematurity (RoP), ocular cicatricial pruritic eczema, lacrimal gland fibrosis, retinal annexation, corneal opacity, herpetic keratitis, pterygium, glaucoma, age-related macular degeneration (AMD/ARMD), and diabetic retinopathy (DM) associated with retinal fibrosis.

Gynecologic indications include endometriosis, which occurs with hormonal therapy to prevent STD fibrosis/post-salpingitis scarring.

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

Injury-related fibrotic diseases include burns (including chemical-induced) scarring and contraction of the skin and soft tissues, radiation-induced scarring of the skin and organs after radiation therapy for cancer treatment, and keloids (skin).

Indications for surgery include peritoneal dialysis catheters, corneal transplants, cochlear transplants, other transplants, peritoneal fibrosis after breast silicone transplants, chronic sinusitis; adhesion of dialysis grafts, and pseudointimal hyperplasia.

Other indications include chronic pancreatitis.

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

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

In some embodiments, methods of achieving regression of fibrosis are provided, comprising administering to a subject in need thereof an effective amount of a nucleic acid molecule disclosed herein, thereby achieving regression of fibrosis.

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

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

In some embodiments, a method for treating a subject suffering from pulmonary fibrosis is provided, the method comprising administering to the subject an effective amount of a nucleic acid molecule disclosed herein, thereby treating pulmonary fibrosis. In some embodiments, the subject suffers from interstitial pulmonary fibrosis (ILF). In some embodiments, the subject suffers from radiation pneumonitis causing pulmonary fibrosis. In some embodiments, the subject suffers from drug-induced pulmonary fibrosis.

In some embodiments, the nucleic acid molecules disclosed herein are used to prepare a medicament for treating pulmonary fibrosis. In some embodiments, the pulmonary fibrosis is ILF. In some embodiments, the pulmonary fibrosis is drug- or radiation-induced pulmonary fibrosis.

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

In a related aspect, a composition or kit comprising a nucleic acid molecule (e.g., siNA molecule) packaged for use with a patient is provided. The packaging may be labeled or include a label or package insert that indicates the contents of the package and provides certain information about how the nucleic acid molecule (e.g., siNA molecule) should or can be used by the patient, for example, the label may include dosing information and/or instructions for use. In certain embodiments, the content of the label will have a notice in a form prescribed by a government agency (e.g., the U.S. Food and Drug Administration). In certain embodiments, the label may indicate that the nucleic acid molecule (e.g., siNA molecule) is suitable for treating a patient suffering from an hsp47-related disease; for example, the label may indicate that the nucleic acid molecule (e.g., siNA molecule) is suitable for treating a fibroid; or, for example, the label may indicate that the nucleic acid molecule (e.g., siNA molecule) is suitable for treating a disease selected from the group consisting of fibrosis, liver fibrosis, cirrhosis, pulmonary fibrosis, renal fibrosis, peritoneal fibrosis, chronic liver injury, and fibrillation.

As used herein, the terms "heat shock protein 47," "hsp47," or "HSP47" are used interchangeably and refer to any heat shock protein 47, peptide, or polypeptide having any hsp4 protein activity. Heat shock protein 47 is a serine protease inhibitor (serpin), also known as, for example, serpin peptidase inhibitor, clade H, member 1 (SERPINH1), SERPINH2, collagen binding protein 1 (CBP1), CBP2, gp46; arsenic transforming activating protein 3 (AsTP3); HSP47; proliferation inducing gene 14 (PIG14); PPROM; rheumatoid arthritis antigen A-47 (RA-A47); collagen binding protein-1; and collagen binding protein-2. In certain preferred embodiments, "hsp47" refers to human hsp47. Heat shock protein 47 (or more specifically, human hsp47) may have an amino acid sequence that is identical or substantially identical to SEQ ID NO. 2 ( FIG. 7 ).

As used herein, the term "nucleotide sequence encoding hsp47" refers to a nucleotide sequence encoding an hsp47 protein or a portion thereof. The term "nucleotide sequence encoding hsp47" is also intended to include hsp47 coding sequences, such as hsp47 isoforms, mutant hsp47 genes, splice mutants of hsp47 genes, and hsp47 gene polymorphisms. Nucleic acid sequences encoding hsp47 include mRNA sequences encoding hsp47, also referred to as "hsp47 mRNA." An exemplary sequence of human hsp47 mRNA is SEQ ID NO: 1.

As used herein, the terms "nucleic acid molecule" or "nucleic acid" are used interchangeably and refer to an oligonucleotide, a nucleotide, or a polynucleotide. Variations of "nucleic acid" are described in more detail herein. Nucleic acid molecules encompass modified nucleic acid molecules and unmodified nucleic acid molecules as described herein. Nucleic acid molecules can 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 its analog or modified sugar), a nucleotide base (or its analog or modified base) and a phosphate group (or its analog or modified phosphate group). Nucleotides encompass modified nucleotides or unmodified nucleotides as described herein. As used herein, nucleotides may include deoxyribonucleotides (e.g., unmodified deoxyribonucleotides), ribonucleotides (e.g., unmodified ribonucleotides), modified nucleotide analogs, particularly including locked nucleic acids and unlocked nucleic acids, peptide nucleic acids, L-nucleotides (also known as mirror nucleotides), ethylene bridged nucleic acids (ENA), arabinoside, PACE, nucleotides with 6-carbon sugars, and nucleotide analogs (including abasic nucleotides) that are often considered non-nucleotides. In some embodiments, nucleotides may be modified in the sugar, nucleotide base, and/or phosphate group by any modification known in the art, such as the modifications described herein. As used herein, "polynucleotide" or "oligonucleotide" refers to a chain of linked nucleotides; likewise polynucleotides and oligonucleotides may have modifications in the nucleotide sugars, nucleotide bases, and phosphate backbones as are well known in the art and/or disclosed herein.

As used herein, the terms "short interfering nucleic acid," "siNA," or "short interfering nucleic acid molecule" refer to any nucleic acid molecule capable of modulating gene expression or viral replication. Preferably, siNA inhibits or downregulates gene expression or viral replication. siNA includes, but is not limited to, nucleic acid molecules capable of mediating sequence-specific RNAi, such as short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotides, short interfering nucleic acids, short interfering modified oligonucleotides, 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" have the meanings described in more detail elsewhere herein.

As used herein, the term "complementary" refers to a nucleic acid that can form hydrogen bonds with another nucleic acid sequence by conventional Watson-Crick or other unconventional types. With regard to nucleic acid molecules disclosed herein, the binding free energy of a nucleic acid molecule to its complementary sequence is sufficient to allow the relevant functions of the nucleic acid to proceed, such as RNAi activity. The determination of nucleic acid molecule binding free energy is well known in the art (see, for example, 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). 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 of a total of 10 nucleotides in a first oligonucleotide base pair with a second nucleic acid sequence having 10 nucleotides, representing 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). "Fully complementary" means that all contiguous residues of a nucleic acid molecule will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, the nucleic acid molecules disclosed herein include 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 that is complementary (partially or completely) to the antisense region of a siNA molecule. The sense strand of a siNA molecule can include a nucleic acid sequence that has homology to a target nucleic acid sequence. As used herein, "sense strand" refers to a nucleic acid molecule that includes the sense region and may also include additional nucleotides.

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

As used herein, the term "RNA" refers to a molecule comprising at least one ribonucleotide residue.

As used herein, the term "duplex region" refers to a region in which two complementary or substantially complementary oligonucleotides form base pairs by Watson-Crick base pairing or any other method of allowing a duplex between complementary or substantially complementary oligonucleotide chains. For example, an oligonucleotide chain with 21 nucleotide units can base pair with another oligonucleotide chain of 21 nucleotide units, but only 19 bases are complementary or substantially complementary on each chain, so that a "duplex region" consists of 19 base pairs. The remaining base pairs can exist, for example, as 5' and 3' overhangs. Further, in the duplex region, 100% complementarity is not required; substantial complementarity can be allowed in the duplex region. Substantial complementarity refers to the complementarity between chains that enables them to anneal under biological conditions. The technology of empirically determining whether two chains can anneal under biological conditions is well known in the art. Alternatively, two chains can be synthesized under biological conditions and added together to determine whether they anneal to each other.

As used herein, the term "non-pairing nucleotide analog" refers to a nucleotide analog that includes a non-base pairing portion, including but not limited to: 6-aminoadenosine (Nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me ribo T, 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, the non-base pairing nucleotide analog is a deoxyribonucleotide.

As used herein, the term "terminal functional group" includes, but is not limited to, halogen, alcohol, amine, carboxyl, ester, amide, aldehyde, ketone, and ether groups.

As used herein, the term "abasic nucleotide" or "abasic nucleotide analog" may also be generally referred to herein and in the art as a pseudonucleotide or unconventional moiety. Although nucleotides are monomeric units of nucleic acids, they are generally composed of ribose or deoxyribose, a phosphate and a base (adenine, guanine, thymine or cytosine in DNA; adenine, guanine, uracil or cytosine in RNA). Abasic or pseudonucleotides lack a base and are therefore not strictly speaking nucleotides as the term is commonly used in the art. Abasic deoxyribose moieties include (for example) abasic deoxyribose-3'-phosphate; 1,2-dideoxy-D-ribofuranosyl-3-phosphate; 1,4-anhydro-2-deoxy-D-ribitol-3-phosphate. Inverted abasic deoxyribose moieties include inverted abasic deoxyribose; 3',5' inverted deoxy abasic 5'-phosphate.

As used herein, the term "capping moiety" includes moieties that can be covalently linked to the 5' terminus of (N')y and includes abasic ribose moieties, abasic deoxyribose moieties, modified abasic ribose and abasic deoxyribose moieties including 2'O alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and modifications thereof; C6-imino-Pi; mirror nucleotides including L-DNA and L-RNA; 5'OMe nucleotides; and nucleotide analogs including 4',5'-methylene nucleotides; 1-(β-D-erythrofuranyl) nucleotides; 4'-thio nucleotides, carbonyl nucleotides, thiocyanate ... Cyclic nucleotides; 5'-amino-alkyl phosphates; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotides; α-nucleotides; threo-pentofuranosyl nucleotides; acyclic 3',4'-openonucleotides; 3,4-dihydroxybutyl nucleotides; 3,5-dihydroxypentyl nucleotides; 5'-5'-inverted abasic moieties; 1,4-butanediol phosphate; 5'-amino; and bridged or non-bridged methylphosphonate and 5'-thiol moieties.

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

As used herein, the term "unconventional moiety" refers to a non-nucleotide moiety, including abasic moieties, inverted abasic moieties, hydrocarbon (alkyl) moieties, and derivatives thereof, and further includes deoxyribonucleotides, modified deoxyribonucleotides, mirror nucleotides (L-DNA or L-RNA), non-base pairing nucleotide analogs, and nucleotides linked to adjacent nucleotides by 2'-5' internucleotide phosphate bonds; bridged nucleic acids including LNA and ethylene bridged nucleic acids, linkage-modified (e.g., PACE) and base-modified nucleotides, as well as additional moieties specifically disclosed herein as unconventional moieties.

As used herein, the terms "inhibit," "downregulate," or "reduce" with respect to gene expression refer to a decrease in gene expression, or the level of an RNA molecule or equivalent RNA molecule (e.g., mRNA) encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, below the activity observed in the absence of an inhibitory factor (e.g., a nucleic acid molecule having structural features described herein, e.g., siNA); for example, expression can be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less than the expression observed in the absence of the inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a bar graph showing the effect of GFP siNA on various reporter cell lines. Cell lines were created by lentiviral induction of human HSP47 cDNA-GFP or rat GP46 cDNA-GFP constructs into HEK293, human fibrosarcoma cell line HT1080, human HSC line hTERT or NRK cell line. Negative control siNA or anti-GFP siNA was introduced into the cells and GFP fluorescence was measured. The results showed that anti-GFP siNA knocked down fluorescence to varying degrees in different cell lines. 293_HSP47-GFP and 293_GP46-GFP cell lines were selected for siHsp47 screening because they are easy to transfect and sensitive to fluorescence knockdown.

Figure 2 is a series of bar graphs showing the cytotoxicity and knockdown efficiency of each siHsp47 in the 293_HSP47-GFP and 293_GP46-GFP cell lines. The results show that siHsp47-C, siHsp47-2, and siHsp47-2d effectively knock down human HSP47 and rat GP46 (human hsp47 homolog) without being generally cytotoxic. siGp46A, which is anti-GP46, does not knock down human HSP47. In addition, the newly designed siHsp47 is superior to siGp46A in knocking down rat GP46.

Figure 3 is a bar graph showing the knockdown effect of each siHsp47 on hsp47 mRNA measured by qPCR using the human HSC cell line hTERT. The Y axis represents the remaining mRNA level of hsp47. HSP47-C was the most effective of all hsp47 siNAs tested.

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

FIG5 shows that the fibrotic area of the liver is reduced in animals treated with siHSP47.

FIG6 shows an exemplary nucleic acid sequence of human hsp47 mRNA cDNA (SEQ ID NO: 1; based on the cDNA disclosed in GenBank Accession No.: NM_001235).

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

FIG8 is a protein-coding nucleic acid sequence of human hsp47 cDNA (SEQ ID NO: 59), which corresponds to nucleotides 230-1486 of SEQ ID NO: 1.

Figures 9A-9I show the 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.

Figures 10A-10I show the on-target and off-target activities of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, and Compound 9, respectively. AS_CM shows the activity of the antisense strand of the compound against a plasmid containing a perfect match insert; AS_CM shows the activity of the antisense strand of the compound against a plasmid containing a seed sequence insert; S_CM shows the activity of the sense strand of the compound against a plasmid containing a perfect match insert. All assays were performed in human cells, except for the data shown in Figure 10F, which were 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 RNA (siRNA) (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 generally referred to as post-transcriptional gene silencing (PTG) or RNA silencing. It is believed that the process of post-transcriptional gene silencing is an evolutionarily conservative cellular defense mechanism for preventing the expression of exogenous genes (Fire et al., 1999, Trends Genet., 15, 358). This protection against exogenous gene expression may be generated in response to double-stranded RNA (dsRNA) derived from viral infection or random integration of transposon elements into the host genome, evolving by specifically destroying cellular responses to homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers RNAi responses through mechanisms that have not yet been fully characterized. This mechanism appears to be distinct from other known mechanisms involving double-stranded RNA-specific ribonucleases, such as the interferon response resulting from dsRNA-mediated activation of the protein kinase PKR and 2',5'-oligoadenylate synthetase, which results in nonspecific cleavage of the mRNA by ribonuclease L (see, e.g., U.S. Patent 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 dsRNA in cells stimulates the activity of an RNase III enzyme called 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 processing dsRNA into short dsRNA fragments called short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise a duplex of about 19 base pairs (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 sequential RNAs (stRNAs) from precursor RNAs of conserved structures implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi reaction is also characteristic of an endonuclease complex, often referred to as an RNA-induced silencing complex (RISC), which mediates the cleavage of single-stranded RNAs having a sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA occurs 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, first observed RNAi in Caenorhabditis elegans. Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283, and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe dsRNA-mediated RNAi 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 introducing duplexes of synthetic 21-nucleotide RNA into cultured mammalian cells, including human embryonic kidney and HeLa cells. Recent studies on Drosophila embryo lysates (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164) have revealed certain requirements for siRNA length, structure, chemical composition, and sequence necessary to mediate efficient RNAi activity.

Nucleic acid molecules (e.g., having the structural features disclosed herein) can inhibit or downregulate 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).

The siNA nucleic acid molecule can be assembled from two separate polynucleotide strands, wherein one strand is a sense strand and the other is an antisense strand, wherein the antisense strand and the sense strand are self-complementary (i.e., each strand includes a nucleotide sequence that is complementary to a nucleotide sequence on the other strand); for example, wherein the antisense strand and the sense strand form a duplex or double-stranded structure having any length and structure as described herein for the nucleic acid molecules as provided, for example, wherein the double-stranded region (duplex region) is about 15 to about 49 base pairs (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, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 64, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 5, 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 comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule (i.e., hsp47 mRNA) or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 17 to about 49 or more nucleotides of a nucleic acid molecule herein are complementary to the target nucleic acid or a portion thereof).

In certain aspects and embodiments, the nucleic acid molecules (eg, siNA molecules) provided herein can be "RISC length" molecules or can be Dicer substrates as described in more detail below.

The siNA nucleic acid molecule may comprise separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linking molecules as known in the art, or alternatively non-covalently linked by ionic interactions, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and/or stacking interactions. The nucleic acid molecule may comprise a nucleotide sequence that is complementary to the nucleotide sequence of the target gene. The nucleic acid molecule may interact with the nucleotide sequence of the target gene in a manner that inhibits expression of the target gene.

Alternatively, the siNA nucleic acid molecule can be assembled from a single polynucleotide, wherein the self-complementary sense and antisense regions of the nucleic acid molecule are connected by a nucleic acid-based or non-nucleic acid-based linker, i.e., the antisense strand and the sense strand are a portion of a single polynucleotide having an antisense region and a sense region that fold to form a duplex region (e.g., forming a "hairpin" structure well known in the art). Such siNA nucleic acid molecules can be polynucleotides having a duplex, an asymmetric duplex, a hairpin, or an asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to the nucleotide sequence of a separate target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence (e.g., the sequence of hsp47 mRNA). Such a siNA nucleic acid molecule can be a circular single-stranded polynucleotide having two or more loops and a backbone comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide is processed in vivo or in vitro to generate an active nucleic acid molecule capable of mediating RNAi.

The following nomenclature is commonly used in the art to describe the length and overhangs of siNA molecules and will be used throughout the specification and examples. The name assigned to the duplex indicates the length of the oligomer and the presence or absence of overhangs. For example, a "21+2" duplex contains two nucleic acid chains each 21 nucleotides in length, also referred to as a 21-mer siRNA duplex or 21-mer nucleic acid, and has a 2-nucleotide 3'-overhang. The "21-2" design refers to a 21-mer nucleic acid duplex with a 2-nucleotide 5'-overhang. The 21-0 design is a 21-mer nucleic acid duplex with no overhangs (blunt ends). "21+2UU" is a 21-mer duplex with a 2-nucleotide 3'-overhang and both terminal nucleotides at the 3' end are U residues (which may result in mismatches with the target sequence). The above nomenclature can be applied to siNA molecules with various chain lengths, duplexes, and overhangs (e.g., 19-0, 21+2, 27+2, etc.). In an alternative but similar nomenclature, "25/27" is an asymmetric duplex having a 25-base sense strand and a 27-base antisense strand with a 2-nucleotide 3'-overhang. "27/25" is an asymmetric duplex having a 27-base sense strand and a 25-base antisense strand.

Chemical modification

In certain aspects and embodiments, the nucleic acid molecules (e.g., siNA molecules) provided herein include one or more modifications (or chemical modifications). In certain embodiments, these modifications include any changes in a nucleic acid molecule or polynucleotide that make the molecule different from a standard ribonucleotide or RNA molecule (i.e., a ribonucleotide or RNA molecule that includes a standard adenosine, cytosine, uracil, or guanosine moiety); standard ribonucleotides may be referred to as "unmodified" ribonucleotides or unmodified ribonucleic acids. Conventional DNA bases and polynucleotides having 2'-deoxysugars represented by adenosine, cytosine, thymine, or guanosine moieties may be referred to as "unmodified deoxyribonucleotides" or "unmodified deoxyribonucleic acids"; therefore, unless clearly indicated to the contrary, as used herein, the terms "unmodified nucleotides" or "unmodified nucleic acids" refer to "unmodified ribonucleotides" or "unmodified ribonucleic acids." Such modifications may be present in the nucleotide sugar, nucleotide base, nucleotide phosphate group, and/or phosphate backbone of the polynucleotide.

In certain embodiments, the modifications disclosed herein can be used to enhance the RNAi activity of the molecule and/or enhance the stability of the molecule in vivo, particularly in serum, and/or enhance the bioavailability of the molecule. Non-limiting examples of modifications include, but are not limited to, internucleotide or internucleoside linkages; deoxynucleotides and dideoxyribonucleotides at any position and on any chain of the nucleic acid molecule; nucleic acids (e.g., ribonucleic acids) having a modification at the 2'-position preferably selected from amino, fluoro, methoxy, alkoxy, and alkyl; 2'-deoxyribonucleotides, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluororibonucleotides, "universal base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, biotin groups, and terminal glyceryl and/or inverted deoxy abasic residue incorporation, sterically hindered molecules, such as fluorescent molecules, and the like. Other nucleotide modifications can 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 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC), and 2',3'-didehydro-2',3'-dideoxythymidine (d4T) monophosphate nucleotides. More details about the various modifications are described in more detail below.

Modified nucleotides include nucleotides having a Northern conformation (e.g., a Northern pseudorotational loop, see, e.g., Saenger, Principles of Nucleic Acid Structure, Springer-Verlag, ed., 1984). Non-limiting examples of nucleotides having a northern conformation 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 LNAs, 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, WO 98/39352, and WO 2004/083430. In one embodiment, the LNA is incorporated at the 5' end of the sense strand.

Chemical modifications also include unlocked nucleic acids or UNAs that are non-nucleotide, acyclic analogs in which the C2'-C3' bond is absent (although UNAs are not true nucleotides, UNAs are clearly included within the scope of "modified" nucleotides or modified nucleic acids contemplated herein). In certain embodiments, nucleic acid molecules with overhangs can be modified to have a UNA at the overhang position (i.e., a 2 nucleotide overhang). In other embodiments, a UNA is included at the 3'- or 5'-end. The UNA can be located anywhere along the nucleic acid chain, i.e., position 7. Nucleic acid molecules may contain one or more UNAs. Exemplary UNAs are disclosed in Nucleic Acids Symposium Series No. 52, p. 133-134 (2008). In certain embodiments, nucleic acid molecules (e.g., siNA molecules) described herein include one or more UNAs; or one UNA. In some embodiments, nucleic acid molecules (e.g., siNA molecules) with 3'-overhangs as described herein 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 (e.g., one UNA) in the antisense strand; for example, at position 6 or position 7 of the antisense strand. Chemical modifications also include, for example, non-pairing nucleotide analogs as described herein. Chemical modifications further include unconventional moieties disclosed herein.

Chemical modifications also include terminal modifications at the 5' and/or 3' portion of the oligonucleotide and are also referred to as capping moieties. Such terminal modifications are selected from nucleotides, modified nucleotides, lipids, peptides, and sugars.

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- esters 6:1457-1460). International Patent Publication No. WO 2006/047842 discloses oligonucleotides comprising six-membered ring nucleotide analogs, including altritol nucleoside monomers.

Chemical modifications also include "mirror" nucleotides that have reversed handedness compared to normal natural nucleotides; i.e., mirror nucleotides that can be "L-nucleotide" analogs of naturally occurring D-nucleotides (see U.S. Patent No. 6,602,858). Mirror nucleotides can further include at least one sugar or base modification and/or backbone modification, such as those described herein, such as a phosphorothioate or phosphonate moiety. U.S. Patent No. 6,602,858 discloses nucleic acid catalysts that include at least one L-nucleotide substitution. Mirror nucleotides include, for example, L-DNA (L-deoxyriboadenosine-3'-phosphate (mirror image dA); L-deoxyribocytidine-3'-phosphate (mirror image dC); L-deoxyriboguanosine-3'-phosphate (mirror image dG); L-deoxyribothymidine-3'-phosphate (mirror image dT)) and L-RNA (L-riboadenosine-3'-phosphate (mirror image rA); L-ribocytidine-3'-phosphate (mirror image rC); L-riboguanosine-3'-phosphate (mirror image rG); L-ribouracil-3'-phosphate (mirror image dU).

In some embodiments, the modified ribonucleotides include modified deoxyribonucleotides, such as 5'OMeDNA (5-methyl-deoxyriboguanosine-3'-phosphate), which can be used as the nucleotide at 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 the nucleic acid molecules disclosed herein, for example, in the sense strand, the antisense strand, or both strands. In certain embodiments, the antisense strand may include modifications and the sense strand may include only unmodified RNA.

Nucleobases

The nucleobases of the nucleic acids disclosed herein may include unmodified ribonucleotides (purines and pyrimidines) such as adenine, guanine, cytosine, and uracil. Nucleobases in one or both chains may 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, sulfhydryl, thioalkyl, hydroxyl, and other 8-substituted uracils. The bases include 5-(1-methyl)-1,2-difluoro-2-nitro-1,2-difluoro-3-nitro-1,2-difluoro-4-nitro-2-nitro-1,2-difluoro-5-nitro-2-nitro-1,2-difluoro-6-nitro-1,2-difluoro-7-nitro-1,2-difluoro-8-nitro-2,2-difluoro-9-nitro-1,2-difluoro-1,2-difluoro-1,2-difluoro-2 ...

Sugar part

The sugar moiety in the nucleic acids disclosed herein may include a 2'-hydroxy-pentofuranosyl sugar moiety without any modification. Alternatively, the sugar moiety may be modified, such as a 2'-deoxy-pentofuranosyl sugar moiety, D-ribose, hexose, a 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=C H2), 2'-propargyl, 2'-propyl, ethynyl, vinyl, propenyl, CF, cyano, imidazole, carboxylate, thioate, C1-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, in particular as described in European Patent EP 0 586 520 B1 or EP 0 618 925 B1.

Alkyl groups include 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, straight-chain or branched alkyl groups have 6 or fewer carbon atoms in their backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and more preferably 4 or fewer. Similarly, preferred cycloalkyl groups may have 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in their ring structure. The term C1-C6 includes alkyl groups containing 1-6 carbon atoms. The alkyl group may be a substituted alkyl group, for example, an alkyl moiety having a substituent replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and urea), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonic acid, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Alkoxy groups include substituted and unsubstituted alkyl, alkenyl and alkynyl groups covalently attached to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropoxy, propoxy, butoxy and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy group can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonic acid, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Examples of halogen-substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, and the like.

In some embodiments, the pentofuranosyl ring can be substituted with an acyclic derivative lacking the C2′–C3′ bond of the pentofuranosyl ring. For example, an acyclic nucleotide can substitute a 2-hydroxyethoxymethyl group for the 2′-deoxyribofuranosyl sugar typically present in dNMPs.

Halogen includes fluorine, bromine, chlorine and iodine.

Main Chain

The nucleoside subunits of nucleic acid disclosed herein can be interconnected by phosphodiester bonds.Phosphodiester bonds can be optionally replaced with other linkages.For example, phosphorothioate, phosphorothioate-D-ribose entity, triester, thioester, 2'-5' bridged backbone (also may be referred to as 5'-2' or 2'5' nucleotides or 2'5' ribonucleotides), PACE, 3'-(or-5') deoxy-3'-(or-5') thio-phosphorothioate, phosphorodithioate, selenophosphate, 3'-(or-5') deoxyphosphinate, borophosphate, 3'-(or-5') deoxy-3'-(or 5'-) aminophosphoramidate, hydrogen phosphonate, Phosphonate, boronate, phosphoramidate, alkyl or arylphosphonate and phosphotriester modifications, such as alkylphosphotriester, phosphotriester phosphorus linkage, 5'-ethoxyphosphodiester, p-alkoxyphosphotriester, methylphosphonate and non-phosphorus-containing linkages, such as carbonate linkages, carbamate linkages, silyl linkages, sulfide linkages, sulfonate linkages, sulfonamide linkages, formaldehyde linkages, thioformaldehyde linkages, oxime linkages, methyleneimino linkages, methylenemethylimino linkages, methylenehydrazono linkages, methylenedimethylhydrazono linkages and methyleneoxymethylimino linkages.

The nucleic acid molecules disclosed herein may include a peptide nucleic acid (PNA) backbone. The PNA backbone comprises repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Various bases, such as purines, pyrimidines, natural and synthetic bases, are linked to the backbone via methylene carbonyl bonds.

terminal phosphate

Modifications can be made at the terminal phosphate group. Non-limiting examples of different stabilization chemistries that can be used, for example, to stabilize the 3' end of a nucleic acid sequence include (1) [3'-3']-inverted deoxyribose; (2) deoxyribonucleotides; (3) [5'-3']-3'-deoxyribonucleotides; (4) [5'-3']-ribonucleotides; (5) [5'-3']-3'-O-methyl ribonucleotides; (6) 3'-glyceryl; (7) [3'-5']-3'-deoxyribonucleotides; (8) [3'-3']-deoxyribonucleotides; (9) [5'-2']-deoxyribonucleotides; and (10) [5'-3']-dideoxyribonucleotides. Additionally, unmodified backbone chemistries can be combined with one or more of the different backbone modifications described herein.

Exemplary chemically modified terminal phosphate groups include the groups shown below:

conjugate

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

Linker

The nucleic acid molecules provided herein (e.g., siNA molecules) may include nucleotides, non-nucleotides, or mixed nucleotide/non-nucleotide linkers connecting the sense region of the nucleic acid and the antisense region of the nucleic acid. The nucleotide linker may be a linker of ≥2 nucleotides in length, such as a linker of 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. The nucleotide linker may be a nucleic acid aptamer. As used herein, "aptamer" or "nucleic acid aptamer" refers to a nucleic acid molecule that specifically binds to a target molecule, wherein the nucleic acid molecule includes a sequence that is recognized by the target molecule in its natural environment. Alternatively, an aptamer may be a nucleic acid molecule that binds to a target molecule (e.g., hsp47 mRNA), wherein the target molecule is not naturally bound to the nucleic acid. For example, an aptamer can be used to bind to a binding domain of a protein, thereby preventing the interaction of a natural ligand with the protein. This is a non-limiting example and those skilled in the art will recognize that other embodiments can be readily produced 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.

Non-nucleotide linkers may include abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons, or other polymeric compounds (eg, polyethylene glycol having 2-100 ethylene glycol units). Specific examples include 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 Linkers described in 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' end, 3' end and overhang

The nucleic acid molecules disclosed herein (e.g., siNA molecules) can have blunt ends on both sides, overhangs on both sides, or a combination of blunt ends and overhangs. Overhangs can occur on the 5'- or 3'-end of the sense or antisense strand.

The 5'- and/or 3'-ends of a double-stranded nucleic acid molecule (e.g., siNA) can have blunt ends or overhangs. The 5'-end of the sense strand or the antisense strand can have a blunt end and the 3'-end can have an overhang. In other embodiments, the 3'-end of the sense strand or the antisense strand can have a blunt end and the 5'-end can have an overhang. In yet other embodiments, both the 5'- and 3'-ends have blunt ends or both the 5'- and 3'-ends have overhangs.

The 5'- and/or 3'-ends of one or both strands of the nucleic acid may include free hydroxyl groups. The 5'- and/or 3'-ends of any nucleic acid molecule strand may be modified to include chemical modifications. Such modifications stabilize nucleic acid molecules, for example, at the 3'-end stability may have enhanced stability due to the presence of the nucleic acid molecule modification. Examples of terminal modifications (e.g., terminal caps) include, but are not limited to, abasic, deoxyabasic, inverted (deoxy)abasic, glyceryl, dinucleotide, acyclic nucleotide, amino, fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioester, C1-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, among others, as described in European Patents EP 586,520 and EP 618,925 and others disclosed herein.

Nucleic acid molecules include nucleic acid molecules having blunt ends, i.e., ends that do not include any protruding nucleotides. Nucleic acid molecules can include one or more blunt ends. The number of base pairs in a blunt-ended nucleic acid molecule is equal to the number of nucleotides present in each strand of the nucleic acid molecule. For example, when the 5'-end of the antisense strand and the 3'-end of the sense strand do not have any protruding nucleotides, the nucleic acid molecule can include one blunt end. For example, when the 3'-end of the antisense strand and the 5'-end of the sense strand do not have any protruding nucleotides, the nucleic acid molecule can include one blunt end. For example, when the 3'-end of the antisense strand and the 5'-end of the sense strand and the 5'-end of the antisense strand and the 3'-end of the sense strand do not have any protruding nucleotides, the nucleic acid molecule can include two blunt ends. 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 in mediating 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 (e.g., 1-8 overhanging 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 the 3' end or both. The overhang may be present on either or both of the sense and antisense strands of the nucleic acid molecule. The length of the overhang may be as short as 1 nucleotide and as long as 1-8 or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides); in some preferred embodiments, the overhang is 2, 3, 4, 5, 6, 7, or 8 nucleotides; for example, the overhang may be 2 nucleotides. The nucleotides forming the overhang may include deoxyribonucleotides, ribonucleotides, natural and non-natural nucleobases, or any nucleotide modified in a sugar, base, or phosphate group, such as disclosed herein. A double-stranded nucleic acid molecule may have 5'- and 3'-overhangs. The overhangs on the 5'- and 3'-ends may be of different lengths. The overhang may include at least one nucleic acid modification that may be a deoxyribonucleotide. The one or more deoxyribonucleotides may be at the 5'-terminus. The 3'-terminus of each corresponding strand of the nucleic acid molecule may have no overhang, more preferably no deoxyribonucleotide overhang. The one or more deoxyribonucleotides may be at the 3'-terminus. The 5'-terminus of each corresponding strand of the dsRNA may have no overhang, more preferably no deoxyribonucleotide overhang. The overhang at the 5' or 3'-terminus of the strand may be 1-8 (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 can include duplex nucleic acid molecules having overhangs 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, such as about 21-nucleotide duplexes having about 19 base pairs and 3'-terminal mononucleotide, dinucleotide or trinucleotide overhangs. Nucleic acid molecules herein can include duplex nucleic acid molecules having blunt ends, wherein both ends are blunt ends, or alternatively, wherein one end is blunt end. Nucleic acid molecules disclosed herein can include one or more blunt ends, i.e., wherein the blunt ends do not have any overhanging nucleotides. In one embodiment, the number of base pairs of a blunt-ended nucleic acid molecule is equal to the number of nucleotides present in each strand of the nucleic acid molecule. For example, when 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 can include one blunt end. For example, when the 3'-end of the antisense strand and the 5'-end of the sense strand do not have any overhanging nucleotides, the nucleic acid molecule may include one blunt end. For example, when the 3'-end of the antisense strand and the 5'-end of the sense strand do not have any overhanging nucleotides, and 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 two blunt ends. In certain preferred embodiments, the nucleic acid compound has blunt ends. Other nucleotides present in the blunt-ended siNA molecule may include, for example, mismatches, bulges, loops, or wobble base pairs to modulate the activity of the nucleic acid molecule in mediating RNA interference.

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

Amount, location, and pattern of modification

The nucleic acid molecules disclosed herein (e.g., siNA molecules) can include modified nucleotides as a percentage of the total number of nucleotides present in the nucleic acid molecule. Thus, a nucleic acid molecule can include from 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 percentage modification can be based on the total number of nucleotides present in the single-stranded nucleic acid molecule. Similarly, if the nucleic acid molecule is double-stranded, the percentage modification can be based on the total number of nucleotides present in the sense strand, the antisense strand, or both.

Nucleic acid molecules disclosed herein can include unmodified RNA as a percentage of the total nucleotides in the nucleic acid molecule. Thus, a nucleic acid molecule can include from 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 the total nucleotides present in the nucleic acid molecule).

A nucleic acid molecule (e.g., a siNA molecule) can include a sense strand comprising about 1 to about 5, particularly 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 sense strand; and wherein The antisense strand includes about 1 to about 5 or more, particularly about 1, 2, 3, 4, or 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 antisense strand. The sense and/or antisense nucleic acid strand of the nucleic acid molecule can include about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides, 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 (e.g., about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages, and/or a terminal cap molecule present at the 3'-end, the 5'-end, or both, in the same or different strands.

The nucleic acid molecule may comprise from about 1 to about 5 or more (particularly about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages in each strand of the nucleic acid molecule.

Nucleic acid molecules can include, for example, 2'-5' internucleotide linkages at the 3'-end, the 5'-end, or both of one or both nucleic acid sequence strands. In addition, 2'-5' internucleotide linkages can be present at various other positions in one or both nucleic acid sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the internucleotide linkages for each pyrimidine nucleotide in one or both strands of the siNA molecule can include 2'-5' internucleotide linkages, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the internucleotide linkages for each purine nucleotide in one or both strands of the siNA molecule can include 2'-5' internucleotide linkages.

A chemically-modified short interfering nucleic acid (siNA) molecule can 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 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 a plurality of purine nucleotides are 2'-deoxy purine nucleotides).

A chemically-modified short interfering nucleic acid (siNA) molecule can 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 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 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 a recombinant in vitro system may include a sense region and an antisense 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 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-2'-fluoro pyrimidine nucleotides). -deoxy purine nucleotides or a plurality of purine nucleotides are 2'-deoxy purine nucleotides), 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 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 a plurality of purine nucleotides are 2'-O-methyl purine nucleotides). The sense and antisense regions may have terminal cap modifications, such as any modifications that are optionally present at the 3'-end, 5'-end, or both of the sense and/or antisense sequences. The sense and antisense regions may optionally further include a 3'-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxy nucleotides. The overhanging nucleotides may further comprise one or more (e.g., about 1, 2, 3, 4, or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages. Alternatively, the purine nucleotides in the sense region may be 2'-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl purine nucleotides or 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 a plurality of purine nucleotides are 2'-O-methyl purine nucleotides). Alternatively, one or more purine nucleotides in the sense region may be purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or 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 a plurality of purine nucleotides are 2'-O-methyl purine nucleotides). Alternatively, one or more purine nucleotides in the sense region and/or present in the antisense region are selected from 2'-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from 2'-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides, or a plurality of purine nucleotides are selected from 2'-deoxynucleotides, 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 (e.g., one modified nucleotide) in the antisense strand, e.g., at position 6 or position 7 of the antisense strand.

Modification patterns and alternating modifications

The nucleic acid molecules (e.g., siNA molecules) provided herein can have modified and unmodified nucleic acid patterns. The modification pattern of nucleotides in a stretch of adjacent nucleotides can be modifications contained in a single nucleotide or a group of nucleotides covalently linked to each other by standard phosphodiester bonds or at least in part by phosphorothioate bonds. Thus, a "pattern" as contemplated herein does not necessarily, although it may, involve repeating units. Examples of modification patterns that can be used in conjunction with the nucleic acid molecules (e.g., siNA molecules) provided herein include modification patterns disclosed in Giese, U.S. Patent No. 7,452,987. For example, the nucleic acid molecules (e.g., siNA molecules) provided herein include nucleic acid molecules having, for example, a modification pattern similar to or identical to the pattern illustrated in Figure 2 of Giese, U.S. Patent No. 7,452,987.

The modified nucleotide or group of modified nucleotides may be at the 5'-end or 3'-end of the sense strand or antisense strand, with flanking nucleotides or groups of nucleotides arranged on either side of the modified nucleotide or group, wherein the flanking nucleotides or groups are unmodified or do not have the same modification as the aforementioned nucleotide or group of nucleotides. However, the flanking nucleotides or groups of nucleotides may have different modifications. Separately, a sequence of modified nucleotides or groups of modified nucleotides and a sequence of unmodified or differently modified nucleotides or groups of unmodified or differently modified nucleotides may be repeated one or more times.

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

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

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

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

In some patterns, the second (penultimate) nucleotide from the end of the strand is an unmodified nucleotide, or the beginning of an unmodified nucleotide group. Preferably, this unmodified nucleotide or unmodified group of nucleotides is located at the 5' end of either or both the sense and antisense strands, and even more preferably at the end of the sense strand. An unmodified nucleotide or unmodified group of nucleotides 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, 2'-O-methyl modified nucleotides and unmodified nucleotides, preferably nucleotides that are not 2'-O-methyl modified, are incorporated into the two strands in an alternating manner, resulting in a pattern of alternating 2'-O-methyl modified nucleotides and unmodified, or at least no 2'-O-methyl modified, nucleotides. In certain embodiments, the same sequence of 2'-O-methyl modified and unmodified nucleotides is present on the second strand; in other embodiments, the alternating 2'-O-methyl modified nucleotides are present only in the sense strand and not in the antisense strand; and in still other embodiments, the alternating 2'-O-methyl modified nucleotides are present only in the antisense strand and not in the sense strand. In certain embodiments, there is a phase shift between the two strands, such that 2'-O-methyl modified nucleotides on the first strand base pair with unmodified nucleotides on the second strand and vice versa. This particular arrangement, i.e., base pairing of 2'-O-methyl modified and unmodified nucleotides on both strands, is particularly preferred in certain embodiments. In certain embodiments, the alternating 2'-O-methyl modified nucleotide pattern is present throughout the entire nucleic acid molecule; or throughout the entire duplex region. In other embodiments, the alternating 2'-O-methyl modified nucleotide pattern is present only in a portion of the nucleic acid; or throughout the entire duplex region.

In the "phase shift" mode, it may be preferred that the 5' end of the antisense strand begins with a 2'-O-methyl modified nucleotide, so that the second nucleotide is unmodified, and thus the third, fifth, seventh, etc. nucleotides are again 2'-O-methyl modified nucleotides, while the second, fourth, sixth, eighth, etc. nucleotides are unmodified nucleotides.

Exemplary modification positions and patterns

While exemplary patterns are provided in more detail below, all possible pattern arrangements of nucleic acid molecules disclosed herein and known in the art are encompassed (e.g., features include, but are not limited to, sense strand length, antisense strand length, duplex region length, overhang length, whether one or both ends of the double-stranded nucleic acid molecule are blunt-ended or have overhangs, the position of modified nucleic acids, the number of modified nucleic acids, the type of modification, whether the double-overhanging nucleic acid molecule has the same or different number of nucleotides on each side of the overhang, whether one or more types of modifications are used for the nucleic acid molecule, and the number of adjacent modified/unmodified nucleotides). With respect to the detailed examples provided below, although the duplex region is shown to be 19 nucleotides, the nucleic acid molecules provided herein can have duplex regions of 1-49 nucleotides in length, as each strand of the duplex region can independently be 17-49 nucleotides in length. Exemplary patterns are provided herein.

The nucleic acid molecule may have blunt ends (when n is 0) at both ends, including a single or group of adjacent modified nucleic acids. The modified nucleic acids may be located anywhere along the sense or antisense strand. The nucleic acid molecule 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 adjacent modified nucleotides. The modified nucleic acid may comprise 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 the nucleic acid strand. The modified nucleic acids of the examples immediately below may be in the sense strand only, in the antisense strand only, or in both the sense and antisense strands.

A general nucleic acid pattern is shown below, where X = sense strand nucleotides in the duplex region; _Xa = 5'-overhanging nucleotides in the sense strand; _Xb = 3'-overhanging nucleotides in the sense strand; Y = antisense strand nucleotides in the duplex region; _Ya = 3'-overhanging nucleotides in the sense strand; _Yb = 5'-overhanging nucleotides in the sense strand; and M = modified nucleotides in the duplex region. Each a and b is independently 0-8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, or 8). Each X, Y, a, and b is independently modified or unmodified. The sense and antisense strands are each independently 17-49 nucleotides in length. The examples provided below have a duplex region of 19 nucleotides; however, the nucleic acid molecules disclosed herein can have any number of duplex regions between 17 and 49 nucleotides and wherein each strand is independently 17-49 nucleotides in length.

5' X _a XXXXXXXXXXXXXXXXXXXX _b

3' Y _b YYYYYYYYYYYYYYYYYYY _a

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

The nucleic acid molecule may have blunt ends at both ends with alternating modified nucleic acids.The modified nucleic acids may be located anywhere along the sense or antisense strand.

Nucleic acid molecules having a 5'-blunt end and a 3'-end overhang terminate in a single modified nucleic acid.

Nucleic acid molecules having a 5'-terminal overhang and a 3'-blunt end terminate in a single modified nucleic acid.

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

5’ XXXXXXXXXXXXXXXXXXXM

3’ MYYYYYYYYYYYYYYYYYY

A nucleic acid molecule having overhangs at both ends, and some of the overhang nucleotides are modified nucleotides. In the schema immediately below, M is the number n of modified nucleic acids, x is the number n of unmodified overhang nucleotides in the sense strand, y is the number n of unmodified overhang nucleotides in the antisense strand, wherein each n is independently an integer from 0 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, and 8), and wherein each overhang is at most 20 nucleotides; preferably at most 8 nucleotides (modified and/or unmodified).

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

Overhang at the 5' end of the sense region

Overhang at the 3' end of the antisense region

Modified nucleotides in the sense region

Exemplary nucleic acid molecules are provided below, along with equivalent general structures consistent with the notation used above.

siHSP47-C siRNAs for human and rat hsp47 have a duplex region of 19 nucleotides (i.e., 19-mer) and overhangs of 2 modified nucleotides (i.e., deoxynucleotides) at the 3' ends of both the sense and antisense strands.

siHSP47-Cd siRNA for human and rat hsp47, which has a 25-mer duplex region, a 2 nucleotide overhang at the 3'-end of the antisense strand, and 2 modified nucleotides at the 5'-terminus and penultimate position of the sense strand.

siHSP47-1 siRNA to human and rat hsp47 cDNA 719-737, which has a 19-mer duplex region and overhangs of 2 modified nucleotides (i.e., deoxynucleotides) at the 3'-ends of both the sense and antisense strands.

siHSP47-1d siRNA of human hsp47 cDNA 719-743, which has a 25-mer with a blunt end at the 3'-end of the sense strand, a 2-nucleotide overhang at the 3'-end of the antisense strand, and 2 modified nucleotides at the 5'-end and the penultimate position of the sense strand.

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

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

siHSP47-2d rat siRNA to rat Gp46 cDNA 466-490, which has a 25-mer duplex region with a blunt end at the 3'-end of the sense strand, a 2 nucleotide overhang at the 3'-end of the antisense strand, and 2 modified nucleotides at the 5'-terminus and penultimate position of the sense strand.

siHSP47-3 siRNA of human hsp47 cDNA 980-998, having a 19-mer duplex region and overhangs of two modified nucleotides (i.e., deoxynucleotides) at the 3'-ends of the sense and antisense strands.

siHSP47-3d siRNA of human hsp47 cDNA 980-1004, which has a 25-mer duplex region with a blunt end at the 3'-end of the sense strand, a 2 nucleotide overhang at the 3'-end of the antisense strand, and 2 modified nucleotides at the 5'-end and the penultimate position of the sense strand.

siHSP47-4 siRNA of human hsp47 cDNA 735-753, having a 19-mer duplex region and overhangs of two modified nucleotides (i.e., deoxynucleotides) at the 3'-ends of the sense and antisense strands.

siHSP47-4d siRNA against human hsp47 cDNA 735-759, which has a 25-mer duplex region with a blunt end at the 3'-end of the sense strand, a 2 nucleotide overhang at the 3'-end of the antisense strand, and 2 modified nucleotides at the 5'-end and penultimate position of the sense strand.

siHSP47-5 siRNA of human hsp47 cDNA 621-639, having a 19-mer duplex region and overhangs of two modified nucleotides (i.e., deoxynucleotides) at the 3'-ends of the sense and antisense strands.

siHSP47-6 siRNA of human hsp47 cDNA 446-464, having a 19-mer duplex region and overhangs of two modified nucleotides (i.e., deoxynucleotides) at the 3'-ends of the sense and antisense strands.

siHSP47-7 siRNA of human hsp47 cDNA 692-710, having a 19-mer duplex region and overhangs of two modified nucleotides (i.e., deoxynucleotides) at the 3'-ends of the sense and antisense strands.

Gaps and gaps in nucleic acid chains

The nucleic acid molecules (e.g., siNA molecules) provided herein may have a strand with a gap or a gap, preferably a sense strand. Thus, the nucleic acid molecule may have three or more strands, such as the partially duplex RNA (mdRNA) disclosed in International Patent Application No. PCT/US07/081836. The nucleic acid molecule having a strand with a gap or a gap may be about 1-49 nucleotides, or may have a RISC length (e.g., about 15-25 nucleotides) or, for example, a Dicer substrate length disclosed herein (e.g., about 25-30 nucleotides).

A nucleic acid molecule having three or more strands, e.g., an 'A' (antisense) strand, an 'S1' (second) strand, and an 'S2' (third) strand, wherein the 'S1' and 'S2' strands are complementary to and form base pairs with non-overlapping regions of the 'A' strand (e.g., an mdRNA may have the form A:S1S2). The S1, S2, or multiple strands together form a sense strand that is substantially similar to the 'A' antisense strand. The double-stranded region formed by annealing the 'S1' and 'A' strands is distinct from and does not overlap with the double-stranded region formed by annealing the 'S2' and 'A' strands. The nucleic acid molecule (e.g., a siNA molecule) may be a "gapped" molecule, meaning that the "gap" ranges from 0 nucleotides to 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 created by 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 different from any one or more unpaired nucleotides at the 3’ end of one or more of ‘A’, ‘S1’, or ‘S2’. The A:S1 duplex may be separated from the A:B2 duplex by a zero nucleotide gap (i.e., a gap in which only the phosphodiester bond between two nucleotides in the polynucleotide molecule is broken or missing) between the A:S1 duplex and the A:S2 duplex, which may also be referred to as a gapped dsRNA (ndsRNA). For example, A:S1S2 can include a dsRNA having at least two double-stranded regions that combine to 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 with blunt ends, or A:S1S2 can include a dsRNA having at least two double-stranded regions separated by a gap of up to 10 nucleotides, wherein at least one double-stranded region comprises about 5 base pairs to 13 base pairs.

Dicer substrates

In certain embodiments, the nucleic acid molecules (e.g., siNA molecules) provided herein can be precursor "Dicer substrate" molecules (e.g., double-stranded nucleic acids) that are processed in vivo to produce active nucleic acid molecules, such as described in Rossi, U.S. Patent Application No. 20050244858. Under certain conditions and circumstances, it has been found that these relatively long dsRNA siNA species, such as dsRNA siNA species of about 25 to about 30 nucleotides, can produce unexpectedly effective results in terms of potency and duration of action. Without wishing to be bound by any particular theory, it is believed that the longer dsRNA species serve as substrates for the enzyme Dicer in the cytoplasm of cells. In addition to cleaving double-stranded nucleic acids into shorter fragments, Dicer can promote the incorporation of single-stranded cleavage products from the cleaved dsRNA into the RNA-induced silencing complex (RISC complex) responsible for destroying cytoplasmic RNA from the target gene.

Dicer substrates may have certain properties that enhance their processing by Dicer. Dicer substrates are of sufficient length to allow them to be processed by Dicer to produce active nucleic acid molecules and may further include one or more of the following properties: (i) the dsRNA is asymmetric, such as having a 3' overhang on the first strand (the antisense strand) and (ii) the dsRNA has a modified 3' end on the second strand (the sense strand) to direct the orientation of Dicer binding and processing of the dsRNA into active siRNA. In certain embodiments, the longest strand in a Dicer substrate may be 24-30 nucleotides.

Dicer substrates can be symmetrical or asymmetrical. A Dicer substrate can have a sense strand comprising 22-28 nucleotides and an antisense strand comprising 24-30 nucleotides; thus, in some embodiments, the resulting Dicer substrate can have an overhang at the 3' end of the antisense strand. A Dicer substrate can have a sense strand that is 25 nucleotides in length and an antisense strand that is 27 nucleotides in length and has a 2-base 3'-overhang. The overhang can be 1-3 nucleotides, for example, 2 nucleotides. The sense strand can also have a 5' phosphate.

Asymmetric Dicer substrates may further contain two deoxynucleotides instead of two ribonucleotides at the 3'-end of the sense strand. Some exemplary Dicer substrate 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 the Dicer substrate can be 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 to about 29, or about 27 to about 28 nucleotides in length. In certain preferred embodiments, the Dicer substrate contains a sense strand and an antisense strand that are at least about 25 nucleotides in length and no longer than about 30 nucleotides, about 26 to about 29 nucleotides in length, or 27 nucleotides in length. The sense strand and antisense strand can be the same length (blunt ends), different lengths (with overhangs), or a combination. The sense strand and antisense strand may be present on the same polynucleotide or on different polynucleotides. The Dicer substrate can have a duplex region of about 19, 20, 21, 22, 23, 24, 25, or 27 nucleotides.

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

Dicer substrates can have any modification to the nucleotide bases, sugars, or phosphate backbones known in the art and/or described herein for other nucleic acid molecules (e.g., siNA molecules). In certain embodiments, the Dicer substrate can have a sense strand modified by a suitable modifier located at the 3' end of the sense strand for Dicer processing, i.e., the dsRNA is designed to direct the orientation of Dicer binding and processing. Suitable modifiers include nucleotides, such as deoxyribonucleotides, dideoxyribonucleotides, acyclic nucleotides, and the like, and sterically hindering molecules, such as fluorescent molecules. Acyclic nucleotides replace the 2'-deoxyribofuranosyl sugar typically present in dNMPs with a 2-hydroxyethoxymethyl group. Other nucleotide modifiers that can be used for 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'-dideoxyribothymidine (d4T) and 3'-azido-3'-deoxyribothymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T) monophosphate nucleotides. In one embodiment, deoxynucleotides are used as modifiers. When nucleotide modifiers are used, they can replace ribonucleotides (e.g., replacing the ribonucleotides on the 3' end of the sense strand with 1-3 nucleotide modifiers or 2 nucleotide modifiers) such that the length of the Dicer substrate remains unchanged. When steric hindrance molecules are utilized, they can be linked to the ribonucleotides on the 3' end of the antisense strand. Thus, in certain embodiments, the length of the chain does not change with the incorporation of the modifying factor. In certain embodiments, two DNA bases in the dsRNA are substituted to direct the orientation of the antisense strand Dicer processing. In another embodiment, two ribonucleotides forming the blunt ends of the duplex on the 3' end of the sense strand and the 5' end of the antisense strand are substituted with two terminal DNA bases, and the 2-nucleotide RNA overhang is located at the 3' end of the antisense strand. This is an asymmetric combination of the DNA on the blunt end and the RNA base on the overhang.

In certain embodiments, the Dicer substrate includes modifications such that the modifications do not hinder the nucleic acid molecule from serving as a substrate for Dicer. In one embodiment, one or more modifications are made to enhance Dicer processing of the Dicer substrate. One or more modifications may be made to cause more effective RNAi production. One or more modifications may be made to support a stronger RNAi effect. One or more modifications may be made to allow each Dicer substrate to be delivered to a cell to produce a stronger effect. Modifications may be incorporated into the 3'-terminal region, the 5'-terminal region, the 3'-terminal region, and the 5'-terminal region or various positions in the sequence. Any number and combination of modifications may be incorporated into the Dicer substrate, as long as the modifications do not hinder the nucleic acid molecule from serving as a substrate for Dicer. When multiple modifications are present, the modifications may be the same or different. Modifications to bases, sugar moieties, phosphate backbones, and combinations thereof are encompassed. Any 5'-end may be phosphorylated.

Examples of modifications to the phosphate backbone of Dicer substrates include phosphonates, including methylphosphonates, phosphorothioates, and phosphotriesters, such as alkylphosphotriesters. Examples of modifications to the sugar moiety of Dicer substrates include 2'-alkylpyrimidines (e.g., 2'-O-methyl), 2'-fluoro, amino, and deoxy modifications (see, e.g., Amarzguioui et al., 2003). Examples of modifications to the bases of Dicer substrates include abasic sugars, 2'-O-alkyl-modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil. Locked nucleic acids, or LNAs, may also be incorporated.

The sense strand may be modified by suitable modifiers located at the 3' end of the sense strand to facilitate Dicer processing, i.e., the dsRNA is designed to direct the orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclic nucleotides, and sterically hindering molecules such as fluorescent molecules. Acyclic nucleotides replace the 2'-deoxyribofuranosyl sugar typically present in dNMPs with a 2-hydroxyethoxymethyl group. Other nucleotide modification factors include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxyribothymidine (AZT), 2', 3'-dideoxyinosine (ddI), 2', 3'-dideoxy-3'-thiacytidine (3TC), 2', 3'-didehydro-2', 3'-dideoxythymidine (d4T) and 3'-azido-3'-deoxythymidine (AZT), 2', 3'-dideoxy-3'-thiacytidine (3TC) and 2', 3'-didehydro-2', 3'-dideoxythymidine (d4T) monophosphate nucleotides. In one embodiment, deoxynucleotides are used as modification factors. When nucleotide modification factors are utilized, 1-3 nucleotide modification factors or 2 nucleotide modification factors are used to replace the ribonucleotides on the 3' end of the sense strand. When steric hindrance molecules are utilized, they can be linked to the ribonucleotides on the 3' end of the antisense strand. Therefore, the length of the chain does not change with the incorporation of the modifying factor. In another embodiment, the present invention contemplates replacing two DNA bases in the Dicer substrate to direct the orientation of Dicer processing of the antisense strand. In another embodiment of the present invention, the two ribonucleotides at the 3' end of the sense strand that form the blunt end of the duplex at the 3' end of the sense strand and the 5' end of the antisense strand are replaced with two terminal DNA bases, and the 2-nucleotide RNA overhang is located at the 3' end of the antisense strand. This is an asymmetric combination of DNA on the blunt end and RNA bases on the overhang.

The antisense strand may be modified by suitable modifiers located at the 3' end of the antisense strand to facilitate Dicer processing, i.e., the dsRNA is designed to direct the orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclic nucleotides, and sterically hindering molecules such as fluorescent molecules. Acyclic nucleotides replace the 2'-deoxyribofuranosyl sugar typically present in dNMPs with a 2-hydroxyethoxymethyl group. Other nucleotide modification factors 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 3'-azido-3'-deoxythymidine (AZT), 2', 3'-dideoxy-3'-thiacytidine (3TC) and 2', 3'-didehydro-2', 3'-dideoxythymidine (d4T) monophosphate nucleotides. In one embodiment, deoxynucleotides are used as modification factors. When nucleotide modification factors are utilized, 1-3 nucleotide modification factors or 2 nucleotide modification factors are used to replace the ribonucleotides on the 3' end of the antisense strand. When steric hindrance molecules are utilized, they can be connected to the ribonucleotides on the 3' end of the antisense strand. Therefore, the length of the chain does not change with the incorporation of the modifying factor. In another embodiment, the present invention contemplates replacing two DNA bases in the Dicer substrate to direct the orientation of Dicer processing. In another invention, the two terminal DNA bases at the 3' end of the antisense strand replace the two ribonucleotides that form the blunt ends of the duplex at the 5' end of the sense strand and the 3' end of the antisense strand, and the 2-nucleotide RNA overhang is located at the 3' end of the sense strand. This is an asymmetric combination with DNA on the blunt end and RNA bases on the overhang.

The Dicer substrate with a sense strand and an antisense strand can be connected by a tertiary structure. The tertiary structure will not block the Dicer activity on the Dicer substrate and will not interfere with the directional destruction of the RNA transcribed by the target gene. The tertiary structure can be a chemical linking group. Suitable chemical linking groups are known and available in the art. Alternatively, the tertiary structure can be an oligonucleotide that connects the two oligonucleotides of the dsRNA in a manner such that a hairpin structure is generated once the two oligonucleotides constituting the Dicer substrate are annealed. The hairpin structure preferably does not block the Dicer activity on the Dicer substrate and will not interfere with the directional destruction of the RNA transcribed by the target gene.

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

A Dicer substrate can have certain properties that enhance its processing by Dicer. A Dicer substrate can be of sufficient length to allow it to be processed by Dicer to produce an active nucleic acid molecule (e.g., siRNA) and can have one or more of the following properties: (i) the Dicer substrate is asymmetric, e.g., has a 3' overhang on the first strand (the antisense strand) and (ii) the Dicer substrate has a modified 3' end on the second strand (the sense strand) to direct the orientation of Dicer binding and processing the Dicer substrate into an active siRNA. The Dicer substrate can be asymmetric such that the sense strand comprises 22-28 nucleotides and the antisense strand comprises 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, e.g., 2 nucleotides. The sense strand can also have a 5' phosphate.

The Dicer substrate may have an overhang at the 3' end of the antisense strand and the sense strand may be 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 those found in the cytoplasm of a cell. A region of one of the strands of the Dicer substrate (particularly the antisense strand) may have a sequence length of at least 19 nucleotides, wherein these nucleotides are in a 21-nucleotide region adjacent to the 3' end of the antisense strand and are fully complementary to the nucleotide sequence of the RNA produced by the target gene. The Dicer substrate may also have one or more of the following additional properties: (a) the antisense strand is right-shifted from the corresponding 21-mer (i.e., the antisense strand includes nucleotides that are 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 a single mismatch and (c) the 5' end of the sense strand may include a base modification, such as a locked nucleic acid.

The antisense strand of the Dicer substrate nucleic acid molecule can be modified to include 1-9 nucleotides at the 5' end to produce a length of 22-28 nucleotides. When the antisense strand has a length of 21 nucleotides, 1-7 ribonucleotides or 2-5 ribonucleotides and/or 4 ribonucleotides can be added to the 3' end. The added ribonucleotides can have any sequence. Although the added ribonucleotides may be complementary to the target gene sequence, it is not necessary for the target sequence to be completely complementary to the antisense strand. That is, the resulting antisense strand is fully complementary to the target sequence. The sense strand can then have 24-30 nucleotides. The sense strand may be fully complementary to the antisense strand to anneal to the antisense strand under biological conditions. In one embodiment, the antisense strand can be synthesized to contain a modified 3' end to guide Dicer processing. The sense strand can have a 3' overhang. The antisense strand can be synthesized to contain a modified 3' end so that Dicer binds and processes and the sense strand has a 3' overhang.

Heat shock protein 47

Heat shock protein 47 (HSP47) is a collagen-specific chaperone that resides in the endoplasmic reticulum. HSP47 interacts with procollagen during folding, assembly, and transport 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 be upregulated in various tissue fibrosis, 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) (Koide et al. J Biol Chem 1999; 274: 34523-26). An exemplary nucleic acid sequence of the target human hsp47 cDNA is disclosed based on GenBank Accession No.: NM_001235 and the corresponding mRNA sequence (e.g., as set forth in SEQ ID NO: 1). One of ordinary skill in the art will understand that the designated sequence may change over time and therefore incorporate any desired changes into the nucleic acid molecules herein.

HSP47's specific association with different types of collagen makes it a potential target for fibrosis treatment. Inhibiting hsp47 expression can prevent extracellular collagen I secretion. Sato et al. (Nat Biotechnol 2008;26:431–442) explored this possibility by using siRNA to inhibit hsp47 expression and prevent the progression of liver fibrosis in rats. Similarly, Chen et al. (Br JDermatol 2007;156:1188-1195) and Wang et al. (Plast. Reconstr Surg 2003;111:1980-7) investigated the inhibition of hsp47 expression using RNA interference.

Methods and compositions for inhibiting hsp47

Provided are compositions and methods for inhibiting hsp47 expression by using small nucleic acid molecules that can mediate or mediate RNA interference with hsp47 gene expression, such as short interfering nucleic acid (siNA), interfering RNA (RNAi), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) and short hairpin RNA (shRNA) molecules. The compositions and methods disclosed herein are also useful for treating various fibrosis, such as liver fibrosis, pulmonary fibrosis and renal fibrosis.

The nucleic acid molecules and/or methods of the invention are used to downregulate expression of a gene encoding, for example, the RNA designated by GenBank accession NM_001235.

The compositions, methods, and kits provided herein can include one or more nucleic acid molecules (e.g., siNA) and methods that independently or in combination regulate (e.g., downregulate) the expression of hsp47 proteins and/or genes encoding hsp47 proteins, proteins, and/or genes encoding hsp47 that are associated with the maintenance and/or development of hsp47-related diseases, conditions, or disorders (e.g., genes encoding sequences including those designated by GenBank Accession No. NM_001235), or members of the hsp47 gene family in which the genes or gene family sequences share sequence homology. Various aspects and embodiments are described with respect to the exemplary gene hsp47. However, various aspects and embodiments are also directed to other related hsp47 genes (e.g., homologous genes and transcript variants) and polymorphisms (e.g., single nucleotide polymorphisms) associated with certain hsp47 genes. Thus, various aspects and embodiments are also directed to other genes involved in hsp47-mediated signal transduction pathways or gene expression involved in the maintenance or development of, for example, a disease, trait, or condition described herein. Target sites for these additional genes can be identified using the methods described herein for the hsp47 gene. Thus, regulation of other genes and the effects of such other gene regulation can be performed, determined, and measured as described herein.

In one embodiment, the compositions and methods provided herein include a double-stranded short interfering nucleic acid (siNA) molecule that downregulates expression of an hsp47 gene (eg, human hsp47 exemplified by SEQ ID NO: 1), wherein the nucleic acid molecule comprises about 15 to about 49 base pairs.

In one embodiment, the disclosed nucleic acids can be used to inhibit the expression of the hsp47 gene or the hsp47 gene family in which the gene or gene family sequence has a consensus sequence homology. Such homologous sequences can be identified, for example, using sequence alignment as known in the art. Nucleic acid molecules can be designed to target such homologous sequences, for example, using perfect complementary sequences or by incorporating non-canonical base pairs (e.g., mismatches and/or wobble base pairs) that can provide additional target sequences. In the case of identifying mismatches, non-canonical base pairs (e.g., 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 (e.g., UU and CC base pairs) are used to generate nucleic acid molecules that can target the sequence of the hsp47 target that distinguishes the consensus sequence homology. Therefore, one advantage of using siNA disclosed herein is that a single nucleic acid can be designed to include a nucleic acid sequence that is complementary to a nucleotide sequence that is conserved between homologous genes. In this method, a single nucleic acid can be used to inhibit the expression of more than one gene without using more than one nucleic acid molecule to target different genes.

Nucleic acid molecules can be used to target conserved sequences corresponding to gene families (e.g., hsp47 family genes). Similarly, nucleic acid molecules targeting multiple hsp47 targets can provide therapeutic effects. In addition, nucleic acid can be used to characterize pathways of gene function in various applications. For example, in gene function analysis, mRNA function analysis, or translation analysis, nucleic acid molecules can be used to inhibit the activity of target genes in determining pathways for uncharacterized gene function. Nucleic acid molecules can be used to determine potential target gene pathways for pharmaceutical research and development involved in various diseases and conditions. Nucleic acid molecules can be used to understand pathways of gene expression involved in, for example, fibrosis (e.g., liver, kidney, or pulmonary fibrosis) and/or inflammation and proliferative traits, diseases, disorders, and/or conditions.

In one embodiment, the compositions and methods provided herein include nucleic acid molecules that have RNAi activity against hsp47 RNA, wherein the nucleic acid molecule comprises a sequence complementary to any RNA having an hsp47 coding sequence, such as those having a sequence as shown in Table 1. In another embodiment, the nucleic acid molecule may have RNAi activity against hsp47 RNA, wherein the nucleic acid molecule comprises a sequence complementary to an RNA having a variant hsp47 coding sequence, such as other mutant hsp47 genes not shown in Table 1 but known in the art to be associated with the maintenance and/or progression of fibrosis. The chemical modifications shown in Table 1 or described herein can be applied to any nucleic acid construct disclosed herein. In another embodiment, the nucleic acid molecules disclosed herein comprise a nucleotide sequence that can interact with a nucleotide sequence of an hsp47 gene, thereby mediating silencing of hsp47 gene expression, for example, wherein the nucleic acid molecule mediates regulation of hsp47 gene expression by modulating the chromatin structure or methylation pattern of the hsp47 gene and preventing cellular processes that transcribe the hsp47 gene.

The nucleic acid molecules disclosed herein can have RNAi activity against hsp47 RNA, wherein the nucleic acid molecules include sequences complementary to any RNA having an hsp47 coding sequence (e.g., a sequence having GenBank Accession No. NM_001235). The nucleic acid molecules can have RNAi activity against hsp47 RNA, wherein the nucleic acid molecules include sequences complementary to RNA having a variant hsp47 coding sequence, such as other mutant hsp47 genes known in the art to be associated with the maintenance and/or development of fibrosis. The nucleic acid molecules disclosed herein include nucleotide sequences that can interact with the nucleotide sequence of the hsp47 gene, thereby mediating the silencing of hsp47 gene expression, for example, wherein the nucleic acid molecules mediate the regulation of hsp47 gene expression by regulating the chromatin structure or methylation pattern of the hsp47 gene and preventing cellular processes that transcribe the hsp47 gene.

Treatment

HSP47's specific association with different types of collagen makes it a target for fibrosis treatment. Inhibiting hsp47 expression can prevent extracellular collagen I secretion. Sato et al. (Nat Biotechnol 2008;26:431–442) explored this possibility by using siRNA to inhibit hsp47 expression and prevent the progression of liver 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 of hsp47 expression using RNA interference.

In one embodiment, nucleic acid molecules can be used to downregulate or inhibit the expression of hsp47 and/or hsp47 protein and/or hsp47 haplotype polymorphisms 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 at risk of developing a trait, condition, or disease described herein. These subjects are amenable to treatment, for example, with the nucleic acid molecules disclosed herein and any other compositions for treating diseases associated with hsp47 and/or hsp47 gene expression. Thus, analysis of hsp47 and/or hsp47 protein or RNA levels can be used to determine the type and course of treatment when treating a subject. Monitoring of hsp47 and/or hsp47 protein or RNA levels can be used to predict treatment outcomes and determine the effects 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 inhibiting hsp47 expression by using small nucleic acid molecules provided herein that can mediate or mediate RNA interference with hsp47 gene expression, such as short interfering nucleic acid (siNA), interfering RNA (RNAi), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) and short hairpin RNA (shRNA) molecules. The compositions and methods disclosed herein are also useful for treating various fibrosis, such as liver fibrosis, pulmonary fibrosis and renal fibrosis.

The nucleic acid molecules disclosed herein can be used alone or in combination or with other drugs to prevent or treat hsp47-associated diseases, traits, conditions or disorders, such as liver fibrosis, cirrhosis, pulmonary fibrosis, renal fibrosis, peritoneal fibrosis, chronic liver injury and fibrillation.

The nucleic acid molecules disclosed herein are capable of inhibiting the expression of hsp47 in a sequence-specific manner.The nucleic acid molecules can include a sense strand and an antisense strand comprising contiguous nucleotides that are at least partially complementary to hsp47 mRNA.

In some embodiments, dsRNA specific for hsp47 can be used along with other dsRNAs specific for other molecular chaperones that help newly synthesized proteins fold (e.g., 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).

The nucleic acid molecules disclosed herein can be used to perform RNA interference to treat fibrosis. Exemplary fibrosis includes liver fibrosis, peritoneal fibrosis, pulmonary fibrosis, and renal fibrosis. The nucleic acid molecules disclosed herein can inhibit the expression of hsp47 in a sequence-specific manner.

Treatment of fibrosis can be monitored by measuring 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 measuring the level of hsp47 mRNA or the level of HSP47 protein in the affected tissue cells. Treatment can also be monitored by non-invasive scanning of the affected organ or tissue, such as by computer-assisted tomography or magnetic resonance elastography.

Methods of treating or preventing an hsp47-associated disease or condition in a subject or organism can comprise contacting the subject or organism with a nucleic acid molecule provided herein under conditions suitable for modulating hsp47 gene expression in the subject or organism.

A method of treating or preventing fibrosis in a subject or organism can comprise contacting the subject or organism with a nucleic acid molecule under conditions suitable for modulating expression of an hsp47 gene in the subject or organism.

A method of treating or preventing one or more fibrosis selected from liver fibrosis, kidney fibrosis, and lung fibrosis in a subject or organism may comprise contacting the subject or organism with a nucleic acid molecule under conditions suitable for modulating expression of an hsp47 gene in the subject or organism.

fibrotic diseases

Fibrotic diseases are generally characterized by excessive deposition of fibrous material in the extracellular matrix, which leads to abnormal changes in tissue architecture and interference with normal organ function.

All tissues damaged by trauma initiate a wound healing program response. When the normal self-limiting process of the wound healing response is disturbed, fibrosis (a class of conditions characterized by excessive scarring) occurs and causes excessive production and deposition of collagen. As a result, normal organ tissue is replaced by scar tissue, which ultimately leads to organ failure.

Fibrosis can develop in various organs for a variety of reasons. Liver cirrhosis, pulmonary fibrosis, sarcoidosis, keloids, and renal fibrosis are all chronic conditions associated with progressive fibrosis, leading to a continuous loss of normal tissue function.

Acute fibrosis (usually with sudden, severe onset and short duration) occurs as a normal response to various forms of trauma, including accidental injury (especially to the spine and central nervous system), infection, surgery, ischemic disease (e.g., scarring of the heart after a heart attack), burns, environmental pollution, alcohol and other types of toxins, acute respiratory distress syndrome, radiation therapy, and chemotherapy.

Fibrosis, fibrosis-related pathologies, or pathologies associated with abnormal cross-linking of cellular proteins can be treated by the siRNA disclosed herein. Fibrotic diseases or diseases in which fibrosis is evident (fibrosis-related pathologies) include acute and chronic forms of organ fibrosis, including all pathogenic variants of the following: pulmonary fibrosis (including interstitial lung disease and fibrosing lung disease), liver fibrosis, cardiac fibrosis (including myocardial fibrosis), renal fibrosis (including chronic renal failure), skin fibrosis (including scleroderma, keloids, and hypertrophic scars); myelofibrosis (myelofibrosis); all types of ocular scarring, including proliferative vitreoretinopathy (PVR) and scarring caused by surgery to treat cataracts or glaucoma; inflammatory bowel disease of variable etiology, macular degeneration, Graves' ophthalmopathy, drug-induced ergotism, keloids, scleroderma, psoriasis, Li-Fraumeni syndrome (Li-Fraumeni syndrome); syndrome), sporadic glioblastoma, myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative syndrome, gynecological cancer, Kaposi's sarcoma, leprosy (Hansen's disease), and collagenous colitis.

In various embodiments, the compounds (nucleic acid molecules) disclosed herein can be used to treat, for example, fibrotic diseases disclosed herein, as well as many other diseases and conditions disclosed herein in addition to fibrotic diseases. Other conditions to be treated include fibrotic diseases of other organs - renal fibrosis for any reason (CKD including ESRD); pulmonary fibrosis (including ILF); myelofibrosis, abnormal scarring (keloids) associated with all possible types of accidental and iatrogenic (surgical) skin injuries; scleroderma; myocardial fibrosis, failed glaucoma filtration surgery; intestinal adhesions.

Ocular surgery and fibrotic complications

Scar tissue contracture caused by eye surgery may often occur. Glaucoma surgery to establish new drainage channels often fails due to scarring and contraction of tissue, which may block the established drainage system, necessitating additional surgical intervention. Current anti-scarring regimens (mitomycin C or 5FU) are limited by complications such as blindness, see, for example, Cordeiro MF et al., Human anti-transforming growth factor-beta2 antibody: a new glaucoma anti-scarring agent Invest Ophthalmol Vis Sci. 1999 Sep; 40(10): 2225-34. Scar tissue formed after corneal trauma or corneal surgery (such as laser or surgical treatment of myopia or refractive error) may also contract, where the contraction of the tissue may lead to inaccurate results. Scar tissue may form in the vitreous humor or on/in the retina, which may eventually cause blindness in some diabetic patients, and may form after detachment surgery, a condition known as proliferative vitreoretinopathy (PVR). PVR is the most common complication after retinal detachment and is associated with retinal holes or breaks. PVR refers to the growth of cell membranes in the vitreous cavity and in the front and back of the retina containing retinal pigment epithelial (RPE) cells. These membranes, which are essentially scar tissue, exert traction on the retina and may cause recurrence of retinal detachment even after initially successful retinal detachment surgery.

Scar tissue may form in the eye socket or on the eye and eyelid muscles after strabismus, orbital or eyelid surgery, or thyroid eye disease, and conjunctival scarring may occur, as may occur after glaucoma surgery or in scarring diseases, inflammatory diseases (e.g., pemphigoid), infectious diseases (e.g., trachoma). Further eye diseases associated with the contraction of tissues including collagen are lens capsule opacification and contracture after cataract extraction. The important role of MMPs in eye diseases has been recognized, including wound healing, dry eye, sterile corneal ulcers, recurrent epithelial erosions, corneal neovascularization, pterygium, conjunctivochalasis, glaucoma, PVR, and ocular fibrosis.

Liver fibrosis

Liver fibrosis (LF) is an irreversible consequence of liver damage from a variety of causes. In Western societies, the major etiologies are alcoholic liver disease (30-50%), viral hepatitis (30%), biliary tract disease (5-10%), primary hemochromatosis (5%), as well as drug-related cirrhosis and cryptogenic cirrhosis of unknown etiology (10-15%). Wilson's disease, _α1 -antitrypsin deficiency, and other rare diseases also present with fibrosis as a symptom. Advanced cirrhosis and fibrosis often require liver transplantation and are among the top ten causes of death in Western societies.

Renal fibrosis and related conditions

Chronic renal failure (CRF)

Chronic renal failure is the progressive loss of the kidneys' ability to excrete waste, concentrate urine, and conserve electrolytes. CRF is a slowly progressive disease. It is most often caused by any disease that leads to a gradual loss of kidney function, and fibrosis is the primary pathology that causes CRF.

diabetic nephropathy

Diabetic nephropathy, characterized by glomerulosclerosis and tubulointerstitial fibrosis, is the single most common cause of advanced renal disease in modern society, and diabetic patients constitute the largest group on dialysis. This treatment is expensive and far from ideal. Transplantation offers better outcomes, but there is 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 associated with an increased risk of cardiovascular disease and chronic renal failure (CRF).

The National Kidney Foundation (NKF) Kidney Disease Outcomes Quality Initiative (K/DOQI) defines chronic kidney disease as kidney damage or a decrease in glomerular filtration rate (GFR) for 3 or more months. Other markers of CKD are also known and used for diagnosis. Typically, destruction of renal masses with irreversible sclerosis and nephron damage leads to a progressive decrease in GFR. Recently, K/DOQI has published a classification of CKD stages as follows:

Stage 1: Renal injury with normal or elevated GFR (>90 mL/min/1.73 m2)

Stage 2: Slightly decreased GFR (60-89 mL/min/1.73 m2)

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

Stage 4: Severely decreased GFR (15-29 mL/min/1.73 m2)

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

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

Pathophysiology of CKD

There are approximately one million nephrons in each kidney, each of which contributes to the total GFR. Regardless of the cause of renal injury, as the nephrons are gradually destroyed, the kidneys are able to maintain the GFR through ultrafiltration and compensatory hypertrophy of the remaining healthy nephrons. This nephron adaptability allows for continued normal clearance of plasma solutes, so that substances such as urine and creatinine begin to show a significant increase in plasma levels only after the total GFR drops to 50%, when the renal reserve is depleted. Plasma creatinine values nearly double, and GFR decreases by 50%. Therefore, a doubling of plasma creatinine from a baseline value of 0.6 mg/dL to 1.2 mg/dL in a patient actually represents a 50% loss of functional nephron material.

Although favorable for the reasons mentioned, it is believed that residual nephron hyperfiltration and hypertrophy represent the main causes of progressive renal dysfunction. It is believed that this occurs due to elevated glomerular capillary pressure, which damages the capillaries and initially leads to focal segmental glomerulosclerosis and ultimately to global glomerulosclerosis. This hypothesis has been based on studies in 5/6 nephrectomized rats, which exhibited lesions consistent with those observed in humans with CKD.

The two most common causes of chronic kidney disease are diabetes and hypertension. Other contributing factors include acute trauma due to nephrotoxins (including contrast agents) or decreased perfusion; proteinuria; increased renal ammonia production with interstitial damage; hyperlipidemia; hyperphosphatemia with calcium phosphate deposition; decreased nitrous oxide levels; and smoking.

The incidence and prevalence of chronic kidney disease (CKD) are increasing in the United States, resulting in adverse consequences and high costs to the healthcare system. Kidney disease is the ninth leading cause of death in the United States. This high mortality rate prompted the US Surgeon General's mandate for America's citizenry, Healthy People 2010, to include a chapter focusing on CKD. The purpose of this chapter is to identify goals and provide solutions to reduce the incidence, morbidity, mortality, and health costs of chronic kidney disease in the United States.

Internationally, the incidence of end-stage renal disease (ESRD) has also been steadily increasing since 1989. The United States has the highest ESRD incidence, followed by Japan. The prevalence per million people is highest in Japan, followed by the United States.

The mortality rate associated with hemodialysis is alarming, and the life expectancy of patients who are instructed to undergo hemodialysis is significantly shortened. At each age, the mortality rate of ESRD patients undergoing dialysis significantly increases when compared with non-dialysis patients and individuals without nephropathy. At the age of 60, healthy people can expect to live more than 20 years, while the life expectancy of 60-year-old patients who begin 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 lung scarring caused by various inhaled agents, including mineral particles, organic dust, and oxidizing gases, or by unknown causes (idiopathic pulmonary fibrosis). Millions of people worldwide suffer from this disease, and there is no effective treatment. This lack of effective treatment is primarily due to the inadequate understanding of the disease's molecular mechanisms for designing appropriate therapeutic targets (Lasky JA, Brody AR (2000), "Interstitial fibrosis and growth factors", Environ Health Perspect.; 108 Suppl 4:751-62).

Cardiac fibrosis

Heart failure is unique among the major cardiovascular diseases in that its prevalence has increased while other conditions have decreased significantly. Some of this increase in heart failure can be attributed to the aging of the population in the United States and Europe. The ability to rescue patients with myocardial injury is also a major factor, as these patients may develop left ventricular dysfunction due to adverse cardiac remodeling.

Normal myocardium is composed of a variety of cells, cardiomyocytes and non-cardiomyocytes (which include endothelial and vascular smooth muscle cells and fibroblasts).

Structural remodeling of the ventricular wall is a key determinant of the clinical outcome of heart disease. This remodeling involves the production and destruction of extracellular matrix proteins, cell proliferation and migration, and apoptosis and necrotic cell death. Cardiac fibroblasts are crucially involved in these processes, producing growth factors and cytokines as autocrine and paracrine factors, as well as extracellular matrix proteins and proteases. Recent studies have shown that the interaction between cardiac fibroblasts and cardiomyocytes is essential for the progression of cardiac remodeling, and the net effect of cardiac remodeling is the deterioration of 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 that may occur in fibrotic diseases is tissue contraction, such as scar contraction. Contraction of tissues comprising extracellular matrix components, particularly collagen, may occur in connection with many different pathological conditions and surgical or plastic surgery procedures. For example, contraction of scars may cause physiological problems, thereby leading to the need for medical treatment, or may lead to purely cosmetic problems. Collagen is the main component of scars and other contractile tissues and is also an important structural component to be considered. However, scars and other contractile tissues also include other structural components that may also contribute to tissue contraction, particularly other extracellular matrix components, such as elastin.

Contraction of tissue, including collagen (and possibly other extracellular matrix components), often occurs during burn healing. Burns can be chemical, thermal, or radiation-induced and can occur in the eye, on the surface of the skin, or on the skin and underlying tissue. Burns can also occur on internal tissues, for example, as a result of radiation therapy. Contraction of burn tissue is often a problem and can lead to physical and/or cosmetic problems, such as loss of mobility and/or disfigurement.

Skin grafts may be applied for a variety of reasons and often undergo shrinkage after application. Shrinkage can cause physical and cosmetic problems, as does tissue healing after burns. This is a particularly serious problem in cases of severe burns, for example, where many skin grafts are required.

Shrinkage is also a problem in the production of artificial skin. To produce true artificial skin, the epidermis, composed of epithelial cells (keratinocytes), and the dermis, composed of collagen, need to be grown by fibroblasts. Having both cell types is important because they use growth factors for signaling and stimulate each other. When grown by fibroblasts, the collagen component of artificial skin often shrinks to less than 1/10 of its original area.

Scar contraction is common and is caused by the shrinkage of scar fibrous tissue. In some cases, scars may become malignant scars, a type of scar in which contraction causes severe deformity. The hourglass contraction caused by the contraction of scar tissue formed when a gastric ulcer heals may effectively separate the patient's stomach into two separate cavities. Due to scar tissue contraction, obstruction of channels and ducts and scar stenosis may occur. Vascular contraction may be due to primary obstruction or surgical trauma, for example, after surgery or angioplasty. Stenosis of other hollow viscera (e.g., ureters) may also occur. Whether caused by accidental injury or surgery, problems may arise when any form of scarring occurs. Skin and tendon conditions involving the contraction of tissues including collagen include post-traumatic conditions caused by surgery or accidents (e.g., tendon injuries in the hands or feet), post-transplant conditions, and pathological conditions such as scleroderma, Dupuytren's contracture, and epidermolysis bullosa. Scarring and contraction of ocular tissue may occur in a variety of conditions, such as retinal detachment and the sequelae of diabetic eye disease (described above). Shrinkage of the eye socket, found in the skull that holds the eyeball and associated structures (including the extraocular muscles and eyelids), can occur if there is traumatic or inflammatory damage. The tissues within the eye socket shrink, causing a variety of problems, including double vision and an unsightly appearance.

For more 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 WF and Lyle PA. (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 BrennerDA. (2001), "Hepatic stellates as a target for the treatment of liverfibrosis", Semin Liver Dis.21(3):437-51; Gross TJ and Hunninghake GW, (2001) "Idiopathic pulmonary fibrosis", N Engl J Med.345(7):517-25; Frohlich ED. (2001) "Fibrosis and ischemia: the real risks in hypertensive heart disease", Am JHypertens; 14(6Pt 2):194S-199S; Friedman SL. (2003), "Liver fibrosis-from bench to bedside", J Hepatol.38 Supplement 1:S38-53; Albanis E et al., (2003), “Treatment of hepatic fibrosis: almost there", Curr Gastroenterol Rep. 5(1): 48-56; (Weber KT. (2000), "Fibrosis and hypertensive heart disease", Curr Opin Cardiol. 15(4): 264-72).

Delivery of nucleic acid molecules and pharmaceutical preparations

The nucleic acid molecules can be made suitable for use alone or in combination with other therapies for the prevention or treatment of fibrotic (e.g., liver, kidney, peritoneal and lung) diseases, traits, conditions and/or disorders, and/or any other traits, diseases, disorders or conditions associated with or responsive to hsp47 levels in cells or tissues. The nucleic acid molecules can include a delivery vehicle (including liposomes), a carrier and a diluent, and salts thereof, for administration to a subject, and/or can be present in a pharmaceutically acceptable formulation.

The nucleic acid molecules disclosed herein can be delivered or administered directly with a carrier or diluent without any delivery vehicle that acts to assist, promote or facilitate entry into cells, including viral vectors, viral particles, lipofectin formulations, liposomal agents or precipitation agents, etc.

Nucleic acid molecules can be delivered or administered to a subject by directly applying the nucleic acid molecule together with a carrier or diluent or any delivery vehicle that acts to assist, promote or facilitate entry into cells, including viral sequences, viral particles, liposome preparations, liposome reagents or precipitation agents, etc. Polypeptides facilitate the introduction of nucleic acids into the intended subject, such as those described in U.S. Application Publication No. 20070155658 (e.g., melamine derivatives (e.g., 2,4,6-triguanidinotriazine and 2,4,6-triamidocarnoylmelamine), polyarginine polypeptides, and polypeptides comprising alternating glutamine and asparagine residues).

Akhtar et al., Trends Cell Bio., 2:139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, Akhtar ed. (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. Patent 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 describe methods for delivering nucleic acid molecules. These methods can be used to deliver nearly all nucleic acid molecules. Nucleic acid molecules can be administered to cells by various methods known in the art, including but not limited 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), polylactic-co-glycolic acid (PLGA) and PLCA microspheres (see, e.g., U.S. Patent No. 6,447,796 and U.S. Application Publication No. 2002130430), biodegradable nanocapsules and bioadhesive microspheres, or by protein carriers (O'Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, the nucleic acid/vehicle combination is delivered locally by direct injection or using an infusion pump. Whether injected subcutaneously, intramuscularly, or directly intradermally, the nucleic acid molecules of the invention can be administered using standard needle and syringe methods or by needle-free techniques 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 invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate, or treat (relieve symptoms to a certain degree, preferably all symptoms) a disease state in a subject.

Nucleic acid molecules can be complexed with cationic lipids, encapsulated in liposomes, or otherwise delivered to target cells or tissues. Nucleic acids or nucleic acid complexes can be topically administered to relevant tissues in vitro or in vivo via direct dermal application, transdermal application, or injection, with or without incorporation into a biopolymer. Nucleic acid molecules of the present invention can include the sequences shown in Table 1. Examples of such nucleic acid molecules consist essentially of the sequences provided in Table 1.

Delivery systems include surface-modified liposomes containing polyethylene glycol lipids (PEG-modified or long-circulating liposomes or stealth liposomes). These formulations provide a method for increasing drug accumulation in target tissues. Such drug carriers resist opsonization and elimination by the mononuclear phagocyte system (MPS or RES), thereby conferring longer blood circulation time and enhancing tissue exposure of 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 can be formulated or complexed with polyethyleneimine (e.g., linear or branched PEI) and/or polyethyleneimine derivatives, including, for example, polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives, grafted PEI, 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, US Patent No. 6,586,524 and US Patent Application Publication No. 20030077829.

Nucleic acid molecules can be complexed with membrane disruptive agents, such as those described in U.S. Patent Application Publication No. 20010007666. Membrane disruptive agents and nucleic acid molecules can also be complexed with cationic lipids or helper lipid molecules, such as those described in U.S. Patent No. 6,235,310.

Nucleic acid molecules can be administered by pulmonary delivery, for example, by inhalation of an aerosol or spray dry formulation administered via an inhalation device or nebulizer, thereby providing rapid local uptake of the nucleic acid molecule to the relevant lung tissue. Solid particulate compositions containing respirable dry particles of micronized nucleic acid compositions can be prepared by grinding dry or lyophilizing the nucleic acid composition and then passing the micronized composition through, for example, a 400 mesh sieve to break up or separate out large agglomerates. The solid particulate composition comprising the nucleic acid composition of the present invention may optionally contain a dispersant to facilitate the formation of an aerosol and other therapeutic compounds. A suitable dispersant is lactose, which can be mixed with the nucleic acid compound in any suitable ratio (e.g., a 1:1 ratio by weight).

Aerosols of liquid particles can include the nucleic acid molecules disclosed herein and can be generated by any suitable means, such as using a nebulizer (see, for example, U.S. Patent No. 4,501,729). Nebulizers are commercially available devices that convert solutions or suspensions of the active ingredient into an aerosol mist by accelerating a compressed gas (usually air or oxygen) through a narrow venturi orifice or by ultrasonic agitation. Suitable formulations for nebulizers include the active ingredient in a liquid carrier in an amount up to 40% w/w of the formulation, preferably less than 20% w/w. The carrier is typically water or a dilute aqueous alcohol solution, preferably made isotonic with body fluids by adding, for example, sodium chloride or other suitable salts. If the formulation is not prepared sterile, optional additives include preservatives, such as methyl hydroxybenzoates, antioxidants, flavorings, volatile oils, buffers, and emulsifiers and other formulation surfactants. Similarly, any solid particle aerosol generator can be used to generate an aerosol of solid particles comprising the active composition and a surfactant. Aerosol generators used to administer solid particulate therapeutic agents to a subject generate respirable particles as described above and produce a large volume of aerosol containing a predetermined dose of the 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 divided powders that can be delivered with the aid of an insufflator. In an insufflator, the powder, for example, in a therapeutically effective dosage as described herein, is contained in a capsule or cartridge, typically made of gelatin or plastic, pierced or opened in situ, and delivered by drawing air through the device during inhalation or with the aid of a manually operated pump. Powders employed in insufflators consist solely of the active ingredient or of a powder mixture comprising the active ingredient, a suitable powder diluent (e.g., lactose), and optionally a surfactant. Formulations typically contain 0.1-100% w/w of the active ingredient. A second illustrative type of aerosol generator includes a metered-dose inhaler. A metered-dose inhaler is a pressurized aerosol dispenser that typically contains a suspension or solution formulation of the active ingredient in a liquefied propellant. During use, these devices discharge the formulation through a valve suitable for delivering a metered volume to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation may additionally contain one or more cosolvents (e.g., ethanol), emulsifiers and other formulation surfactants (e.g., oleic acid or sorbitan trioleate), antioxidants and suitable flavorings. For example, other methods of pulmonary delivery are described in U.S. Patent Application No. 20040037780 and U.S. Patent Nos. 6,592,904; 6,582,728; 6,565,885. PCT Patent Publication No. WO2008/132723 generally relates to aerosol delivery of oligonucleotides, particularly siRNA, to the respiratory system.

Nucleic acid molecules can be administered to the central nervous system (CNS) or the peripheral nervous system (PNS). Experiments have demonstrated efficient in vivo uptake of nucleic acids by neurons. See, for example, 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. Pharmacol., 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 can thus undergo delivery to and be taken up by cells in the CNS and/or PNS.

Delivery of nucleic acid molecules to the CNS is provided by a variety of different approaches: conventional CNS delivery methods that can be used include, but are not limited to, intrathecal and intraventricular administration, implanted catheters and pumps, direct injection or infusion at the site of injury or damage, injection into the cerebral arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other methods may include the use of various transport and carrier systems, for example, through the use of conjugates and biodegradable polymers. Furthermore, gene therapy methods, such as those described in Kaplitt et al., U.S. Patent No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.

Delivery systems include, for example, aqueous and non-aqueous gels, creams, composite emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon bases, and powders, and may contain excipients (e.g., solubilizers), penetration enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols, and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. Examples of liposomes useful in the present invention include the following: (1) CellFectin, a 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmitoyl-spermamine and dioleoylphosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, a 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-trimethyl-methylammonium sulfate) (Boehringer Manheim); and (4) Lipofectamine, a 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA, the neutral lipid DOPE (GIBCO BRL) and a di-alkylated amino acid (DiLA2).

Delivery systems include patches, tablets, suppositories, pessaries, gels, creams, and may contain excipients such as cosolvents 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 hydroxypropyl methylcellulose and hyaluronic acid)).

Nucleic acid molecules can be formulated or complexed with polyethyleneimine (e.g., linear or branched PEI) and/or polyethyleneimine derivatives, including, for example, grafted PEI, 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 Pharm Sci, 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. Patent No. 6,586,524.

Nucleic acid molecules can include bioconjugates, such as those 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; and U.S. Pat. No. 5,138,045.

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

Nucleic acid molecules that interact with target RNA molecules and down-regulate genes encoding target RNA molecules (e.g., target RNA molecules indicated by gene bank accession numbers herein) can be expressed by transcription units inserted into DNA or RNA vectors. Recombinant vectors can be DNA plasmids or viral vectors. Viral vectors that can express nucleic acid molecules based on, but not limited to, adeno-associated viruses, retroviruses, adenoviruses, or alphavirus constructs. Recombinant vectors capable of expressing nucleic acid molecules can be delivered as described herein and retained in target cells. Alternatively, viral vectors can be used for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered if necessary. Once expressed, nucleic acid molecules can bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of nucleic acid molecule expression vectors can be systemic, for example, by intravenous or intramuscular administration, by administration to cells explanted from a subject, and then reintroduced into the subject's body, or by any other means that allow introduction into the desired target cells.

An expression vector may include a nucleic acid sequence encoding at least one nucleic acid molecule disclosed herein in a manner that allows the expression of the nucleic acid molecule. For example, a vector may contain sequences encoding both strands of a nucleic acid molecule, including a duplex. A vector may also contain sequences 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, online preview version doi: 10.1038/nm725. Expression vectors may also be included in mammalian (e.g., human) cells.

The expression vector can include the nucleic acid sequence of two or more identical or different nucleic acid molecules of coding.The expression vector can include the sequence of the nucleic acid molecule complementary to the nucleic acid molecule referred to by GenBank Accession No. NM_001235, such as those shown in Table 1.

The expression vector can encode one or both strands of a nucleic acid duplex or can self-hybridize to form a single, self-complementary strand of a nucleic acid duplex. The nucleic acid sequence encoding the nucleic acid molecule can be operably linked in a manner that allows expression of the nucleic acid molecule (see, e.g., 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, online preview version doi: 10.1038/nm725).

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

Transcription of a nucleic acid sequence can be driven by 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 level 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 can also be used, as long as the prokaryotic RNA polymerase is expressed in the appropriate cell (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 researchers have demonstrated that nucleic acid molecules expressed from this promoter 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. USA, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4). Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units, such as those derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA), and adenovirus VARNA, are used to produce 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. Transcriptase 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 limited to plasmid DNA vectors, viral DNA vectors (e.g., adenovirus or adeno-associated virus vectors), or viral RNA vectors (e.g., retrovirus or alphavirus vectors) (see Couture and Stinchcomb, 1996 supra).

Nucleic acid molecules can be expressed in 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). One skilled in the art will recognize that any nucleic acid can be expressed in eukaryotic cells from an appropriate DNA/RNA vector. The activity of such a nucleic acid can be enhanced by releasing the nucleic acid from the primary transcript with an 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).

The viral construct packaged in viral particles will achieve efficient introduction of the expression construct into cells and transcription of the dsRNA construct encoded by the expression construct.

Methods for oral introduction include directly mixing the RNA with the organism's food and engineering species used as food to express the RNA, which is then fed to the affected organism. Physical methods can be used to introduce solutions of nucleic acid molecules into cells. Physical methods for introducing nucleic acids include injecting solutions containing nucleic acid molecules, bombarding with particles coated with nucleic acid molecules, immersing cells or organisms in RNA solutions, or electroporating cell membranes in the presence of nucleic acid molecules.

Other methods known in the art for introducing nucleic acids into cells can be used, such as lipid-mediated vector transport, chemical-mediated transport, such as calcium phosphate, etc. Thus, nucleic acid molecules can be introduced along with components that achieve one or more of the following activities: enhancing RNA uptake by the cell, promoting annealing of duplex strands, stabilizing annealed strands, or enhancing repression of the target gene.

Suitable preparations can be used to introduce nucleic acid molecules or vector constructs into cells. A preferred preparation is a lipid formulation, such as Lipofectamine ^™ 2000 (Invitrogen, CA, USA), vitamin A coupled lipids (Sato et al., Nat Biotechnol 2008; 26: 431–442, PCT Patent Publication No. WO 2006/068232). It is also possible (for example) to administer lipid formulations to animals by intravenous, intramuscular or intraperitoneal injection, or orally or by inhalation or other methods as known in the art. When the preparation is suitable for administration to animals (such as mammals), especially in the human body, it is also directly pharmaceutically acceptable. Pharmaceutically acceptable preparations for administering oligonucleotides are known and can be used. In some cases, dsRNA can preferably be formulated in a buffer or saline solution and the prepared dsRNA can be directly injected into the cell, as in the research using oocytes. Direct injection of dsRNA duplexes can also be performed. For suitable methods of introducing dsRNA, see U.S. Published Patent Application Nos. 2004/0203145, 20070265220, incorporated herein by reference.

Polymeric nanocapsules or microcapsules facilitate encapsulation or binding of dsRNA for transport and release into cells. Polymeric nanocapsules or microcapsules comprise a polymeric material and a monomeric substance, particularly polybutylcyanoacrylate. A summary of materials and manufacturing methods has been published (see Kreuter, 1991). The polymeric material formed from monomeric and/or oligomeric precursors in the polymerization/nanoparticle formation step is known in the art, and the molecular weight and molecular weight distribution of the polymeric material can be appropriately selected by those skilled in the art of nanoparticle production according to common techniques.

Nucleic acid molecules can be formulated as microemulsions. A microemulsion is a system of water, oil, and amphiphilic molecules that is a single optically isotropic and thermodynamically stable liquid solution. Microemulsions are typically prepared by first dispersing the oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component (usually a medium-chain alcohol) to form a transparent system.

Surfactants that can be used to prepare microemulsions alone or in combination with co-surfactants include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 9, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglyceryl monolaurate (ML310), tetraglyceryl monooleate (MO310), hexaglyceryl monooleate (PO310), hexaglyceryl pentaoleate (PO500), decaglyceryl monocaprate (MCA750), decaglyceryl monooleate (MO750), decaglyceryl sesquioleate (SO750), decaglyceryl decaoleate (DA0750). Co-surfactants, typically short-chain alcohols (e.g., ethanol, 1-propanol, and 1-butanol), act to enhance interfacial fluidity by penetrating into the surfactant film and thereby creating a disordered film due to the creation of void spaces between the surfactant molecules.

Water-soluble cross-linked polymer

The delivery formulation may include a water-soluble degradable cross-linked polymer comprising one or more degradable cross-linked lipid moieties, one or more PEI moieties, and/or one or more mPEG (a methyl ether derivative of PEG (methoxypoly(ethylene glycol))).

The degradable lipid moiety preferably comprises a compound having the following structural motif:

In the above formula, the ester bond is a biodegradable group, R represents a relatively hydrophobic "lipid" group, and the structural motif shown appears m times, where m ranges from about 1 to about 30. For example, in certain embodiments, R is selected from 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, E is a saturated or unsaturated alkyl or sterol having 4-30 carbon atoms, more preferably 8-24 carbon atoms, preferably a cholesteryl moiety. In preferred embodiments, R is oleic acid, lauric acid, myristic acid, palmitic heptadecanoic acid, stearic acid, arachidic acid, behenic acid, or tetracosanoic acid. In the most preferred embodiment, R is oleic acid.

N in formula (A) may have an electron pair or a bond to a hydrogen atom. When N has an electron pair, the repeating unit may have cationicity at low pH.

The degradable cross-linked lipid moiety can be reacted with polyethyleneimine (PEI) as shown in Scheme A below:

Plan A

In Scheme (A), R has the same meaning as described above. PEI may contain repeating units of formula (B) wherein 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.

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

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

The molar ratio of PEI to diacrylate is preferably in the range of about 1:2 to about 1:20. The 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. The molecular weight can be determined by size exclusion chromatography using PEG standards or by agarose gel electrophoresis.

The cationic lipopolymer is preferably degradable, more preferably biodegradable, for example degradable by a mechanism selected from hydrolysis, enzymatic cleavage, reduction, photolysis and ultrasonic degradation. Although not wishing to be bound by any particular theory, it is believed that the degradation of the cationic lipopolymer of formula (II) within the cell occurs by enzymatic cleavage and/or hydrolysis of the ester bond.

The synthesis can be carried out by reacting a degradable lipid moiety with the PEI moiety described above. Then mPEG (a methyl ether derivative of PEG (methoxy poly (ethylene glycol))) is added to form a degradable cross-linked polymer. In a preferred embodiment, the reaction is carried out at room temperature. The reaction product can be separated by any means known in the art (including chromatographic techniques). In a preferred embodiment, the reaction product can be removed by precipitation and then centrifugation.

dose

As will be apparent to those skilled in the art, useful dosages to be administered and the specific mode of administration will vary with factors such as the cell type or, for in vivo use, the age, weight, and specific animal and part thereof to be treated, the specific nucleic acid used and the method of delivery, the intended therapeutic or diagnostic use, and the form of the formulation, such as a suspension, emulsion, micelle, or liposome. Typically, the dosage is administered at lower levels and increased until the desired effect is achieved.

When lipids are used to deliver nucleic acids, the amount of lipid compound administered can vary and generally depends on the amount of nucleic acid 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 from about 5:1 to about 10:1 being more preferred.

The dosage unit of the nucleic acid molecule can be in the range of about 0.001-0.25 mg/kg body weight of the recipient per day, or in the range of about 0.01-20 mg/kg body weight per day, or in the range of about 0.01-10 mg/kg body weight per day, or in the range of about 0.10-5 mg/kg body weight per day, or in the range of about 0.1-2.5 mg/kg body weight per day.

Appropriate amounts of nucleic acid molecules can be introduced and these amounts can be determined empirically using standard methods. The effective concentration of individual nucleic acid molecule species in the cellular environment can be about 1 fmol, about 50 fmol, 100 fmol, 1 pmol, 1.5 pmol, 2.5 pmol, 5 pmol, 10 pmol, 25 pmol, 50 pmol, 100 pmol, 500 pmol, 1 nmol, 2.5 nmol, 5 nmol, 10 nmol, 25 nmol, 50 nmol, 100 nmol, 500 nmol, 1 μmol, 2.5 μmol, 5 μmol, 10 μmol, 100 μmol or more.

The dosage can be 0.01 g to 1 g per kg of body weight (e.g., 0.1 g, 0.25 g, 0.5 g, 0.75 g, 1 g, 2.5 g, 5 g, 10 g, 25 g, 50 g, 100 g, 250 g, 500 g, 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 about 0.1 mg to about 140 mg per kg of body weight per day are useful for treating the conditions indicated above (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 dose varies depending upon the host treated and the particular mode of administration. Unit dosage forms generally contain from about 1 mg to about 500 mg of the active ingredient.

It is understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, route of administration and rate of excretion, drug combination, and the severity of the specific condition being treated.

The pharmaceutical composition comprising the nucleic acid molecules disclosed herein can be administered once a day, four times a day, three times a day, twice a day, or once a day or at any interval and for any duration that is medically appropriate. However, the therapeutic agent can also be administered in dosage units containing 2, 3, 4, 5, 6 or more sub-doses administered at appropriate intervals throughout the day. In this case, the nucleic acid molecules contained in each sub-dose can be correspondingly smaller to reach a daily total dosage unit. For example, a conventional sustained-release formulation that provides a sustained, stable release of dsRNA for several days can be used to compound the dosage unit into a single dose over several days. Sustained-release formulations are well known in the art. The dosage unit can contain a corresponding plurality of daily doses. The composition can be compounded in such a way that the sum of the plurality of nucleic acid units contains sufficient dosage.

Pharmaceutical compositions, reagents and containers

Also provided are compositions, kits, containers, and formulations comprising nucleic acid molecules (e.g., siNA molecules) for reducing hsp47 expression as provided herein, for use in administering or dispensing the nucleic acid molecules to a patient. The kit may include at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials, such as glass, metal, or plastic. The container may contain an amino acid sequence, a small molecule, a nucleic acid sequence, a cell population, and/or an antibody. In one embodiment, the container contains a polynucleotide for examining the mRNA expression profile of a cell and reagents for this purpose. In another embodiment, the container includes an antibody, binding fragment thereof, or specific binding protein for assessing hsp47 protein expression in a cell nucleus or tissue, or for related experimental, prognostic, diagnostic, preventive, and therapeutic purposes; instructions and/or instructions for such use may be included on or with such container, as may reagents and other compositions or tools for such purposes. The kit may further include relevant instructions and/or instructions; reagents and other compositions or tools for such purposes may also be included.

The container can optionally hold a composition effective for treating, diagnosing, prognosing or preventing 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 ingredient in the composition can be a nucleic acid molecule capable of specifically binding to hsp47 and/or modulating hsp47 function.

The kit further includes a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and/or dextrose solution. The second container may further include other substances desirable from a commercial and user perspective, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with instructions for use and/or directions.

The unit dose ampoule or multidose container in which the nucleic acid molecule is packaged prior to use can include a sealed container filled with a certain amount of polynucleotide or a solution containing a polynucleotide suitable for its pharmaceutically effective dose or multiple effective doses. The polynucleotide is packaged as a sterile preparation, and the sealed container is designed to maintain the sterility of the preparation until use.

The container containing the polynucleotide comprising a sequence encoding a cellular immune response element or a fragment thereof may include labeled packaging, and the label may carry a notice in the form prescribed by a government agency, such as the U.S. Food and Drug Administration, reflecting approval by the agency under federal law for the manufacture, use, or sale of the polynucleotide material therein for human administration.

Federal law requires that pharmaceutical compositions be approved by a federal agency for use in human therapy. In the United States, the U.S. Food and Drug Administration is responsible for enforcing the regulations governing obtaining such approval, as detailed in 21 U.S.C. §301-392. Regulations for biological materials, including products made from animal tissue, are provided under 42 U.S.C. §262. Most foreign countries require similar approvals. While regulations vary from country to country, the individual procedures are well known in the art and the compositions and methods provided herein comply accordingly.

The dosage to be administered depends largely on the condition and the size of the subject being treated, as well as the frequency of treatment and the route of administration. The initial response and clinical judgment may guide the continued treatment regimen, including dosage and frequency. Although other parenteral routes may be required in specific administrations, such as inhalation of aerosol formulations, for example, to the mucous membranes of the nose, throat, bronchial tissue, or lungs, parenteral injection into the interstitial spaces of the tissues is preferred.

Therefore, the present invention provides a pharmaceutical product, which includes: a polynucleotide comprising a sequence encoding a cellular immune response element or a fragment thereof in a pharmaceutically acceptable injectable carrier in the form of a solution, and suitable for being introduced into a tissue through the interstitium to cause the cells of the tissue to express the cellular immune response element or the fragment thereof; a container containing the solution and a notice in a form prescribed by a government agency regulating the production, use or sale of pharmaceuticals attached to the container, wherein the notice reflects the approval of the agency to produce, use or sell the polynucleotide solution for human administration.

Indications

The nucleic acid molecules disclosed herein can be used alone or in combination with other therapies to treat diseases, conditions, or disorders associated with hsp47, such as liver fibrosis, cirrhosis, pulmonary fibrosis, renal fibrosis, peritoneal fibrosis, chronic liver injury, and fibrillation, and any other disease or condition associated with or responsive to hsp47 levels in cells or tissues. Thus, the compositions, kits, and methods disclosed herein can include packaging of the nucleic acid molecules disclosed herein, including a label or package insert. The label can include instructions for use of the nucleic acid molecule, such as for use alone or in combination with other therapies to treat or prevent liver fibrosis, peritoneal fibrosis, renal fibrosis, and pulmonary fibrosis, and any other disease or condition associated with or responsive to hsp47 levels in cells or tissues. The label can include instructions for reducing hsp47 expression. "Package insert" is used to refer to information typically included in commercial packaging for a therapeutic product containing information regarding indications, use, dosage, administration, contraindications, other therapeutic products combined with the packaged product, and/or warnings regarding the use of such therapeutic product.

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

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

Example 1

Select hsp47 nucleic acid molecule sequence:

Nucleic acid molecules (e.g., siNA ≤ 25 nucleotides) against Hsp47 were designed using several computer programs, including Whitehead's siRNA (Whitehead Institute for Biomedical Research), IDT siRNA Design (Integrated DNA Technologies), BLOCK-iTRNAi Designer (Invitrogen), siDESIGN Center (Dharmacon), and IOPREDsi (Friedrich Miescher Institute for Biomedical Research (part of the Novartis Research Foundation, available at http://www.biopredsi.org/start.html). The sequences of high-scoring siRNAs from these programs were compared and selected based on algorithms and sequence homology between human and rat (see Table 1). Candidate sequences can be tested by in vitro knockdown assays.

For selecting nucleic acid molecule (e.g., 21-mer siRNA) sequences, several parameters are considered. Exemplary parameters include:

1) Thermodynamic stability (RISC prefers chains with less stable 5' ends)

2) 30-52% GC content

3) Nucleotide position priority: (C/G)NNNNNNNN(A/U)10NNNNNNNN(A/U), where N is any nucleotide

4) Lack of putative immunostimulatory motifs

5) 2-nucleotide 3' overhang

6) Location of siRNA in the transcript (preferably in the cDNA region)

7) Sequence specificity (checked using BLAST)

8) Variations in single nucleotides identified by checking the SNP database

siRNA sequences with ≤25 nucleotides were designed based on the aforementioned methods. The corresponding Dicer substrate siRNA was designed based on a smaller sequence (e.g., ≥26 nucleotides) and the target site of the siNA was extended by ≤25 nucleotides by adding 4 bases to the 3'-end of the sense strand and 6 bases to the 5'-end of the antisense strand. The resulting Dicer substrate typically has a 25-base sense strand, a 27-base antisense strand, and an asymmetric molecule with blunt ends and 3'-overhangs. The sequences of the sense and antisense strands without base modification (base sequence) and with base modification (experimental sequence) are provided in Table 1.

Example 2

In order to screen out the potential of various siNA molecules against human and rat hsp47 genes, human HSP47cDNA-green fluorescent protein (GFP) or rat GP46 cDNA-GFP constructs were introduced into 293, HT1080, human HSC line hTERT or NRK cell lines by lentivirus to establish various reporter cell lines. These cell lines were further evaluated with anti-GFP siRNA. The remaining fluorescence signal was measured and standardized to mixed siRNA (Ambion), and then standardized to cell viability. The results showed that anti-GFP siRNA knocked down fluorescence to varying degrees in different cell lines (Figure 1). 293_HSP47-GFP and 293_GP46-GFP cell lines were selected for siHsp47 screening because they are easy to transfect and sensitive to fluorescence knockdown.

siRNA transfection

Cells were transfected with 1.5 pmol of anti-GFP siNA per well of a 96-well tissue culture plate using Lipofectamine RNAiMAX (Invitrogen) in a reverse transfection format. Cells were seeded at 6,000 cells/well and mixed with the siNA complex. After a 72-h incubation period, fluorescence readings were obtained using a Synergy 2 Multi-Mode Microplate Reader (BioTek).

Cell viability assay

After 72 h of incubation, the viability of cells treated with or without siNA was measured using the 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 the inhibition efficiency of siHsp47 on hsp47 expression in reporter cell lines

The inhibitory efficacy of anti-hsp47 siNAs in the 293_HSP47-GFP and 293_GP46-GFP cell lines was assessed by evaluating changes in the fluorescent signal from the reporter GFP. The experiments were performed as described in Example 2. The fluorescent signal was normalized to the fluorescent signal from cells treated with a scrambled siRNA (Ambion) as a control. The results showed that the experimental hsp47 siNA molecules were effective in inhibiting hsp47 mRNA in both cell lines. However, siNAs against GP46 mRNA (as published in the 2008 paper by Sato et al.) were only effective in the 293_GP46-GFP cell line. The results are shown in Figures 2A-B.

The viability of 293_HSP47-GFP and 293_GP46-GFP cell lines treated with siRNA against hsp47 and gp46 was assessed using the method described in Example 2. Cell viability was normalized to cells treated with a scrambled siRNA (Ambion). The results showed that treatment with siNA molecules did not significantly affect cell viability. However, the cell viability of the 293_HSP47-GFP cell line treated with different hsp47 siNA molecules varied depending on the siNA molecule used, while the viability of the 293_GP46-GFP cell line was similar. For cells treated with siHsp47-6 and Hsp47-7, the viability of 293_HSP47-GFP cells was lower than that of the remaining cells. The results are shown in Figures 2C-D.

Example 4

pass qPCR evaluation of the inhibitory effect of siHsp47 on hsp47 mRNA

In Example 3, the knockdown efficiency of siHsp47 in reporter cell lines was assessed by changes in fluorescence signals. To verify the results at the mRNA level, siRNA targeting endogenous hsp47 was transfected into cells of the human HSC cell line hTERT using Lipofectamine RNAiMAX (Invitrogen) in the reverse transfection method described in Example 2.

hsp47 mRNA levels were assessed for knockdown efficiency of various siHsp47 siNA molecules. Briefly, 72 h after transfection, mRNA was isolated from hTERT cells using the RNeasy micro kit (Qiagen). The level of hsp47 mRNA was determined using a probe by reverse transcription combined with quantitative PCR. Briefly, cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (ABI) according to the manufacturer's instructions, and TaqMan gene expression assays (ABI, hsp47 assay IDHs01060395_g1) were performed. The level of hsp47 mRNA was normalized to the level of GAPDH mRNA according to the manufacturer's instructions (ABI). The results showed that siHsp47-C was the most effective of all hsp47 siRNAs, followed by siHsp47-2 and siHsp47-2d. The combination of siHsp47-1 and siHsp47-2 or siHsp47-1 and siHsp47-2d was more effective than siHsp47-1 alone. The results are shown in Figure 3.

Example 5

Assaying the knockdown effect of siHsp47 at the protein level

The inhibitory effect of different Hsp47 siNA molecules (siHsp47) on hsp47 mRNA expression was determined at the protein level by measuring HSP47 in hTERT cells transfected with different siHsp47. As described in Example 2, hTERT cells were transfected with different siHsp47. The transfected hTERT cells were lysed and the cell lysates were clarified by centrifugation. The proteins in the clarified cell lysates were resolved by SDS polyacrylamide gel electrophoresis. The HSP47 protein levels in the cell lysates were determined using an anti-HSP47 antibody (AssayDesigns) as the primary antibody and a goat anti-mouse IgG conjugated to HRP (Millipore) as the secondary antibody, followed by detection using a Supersignal West Pico chemiluminescent kit (Pierce). An anti-actin antibody (Abcam) was used as a protein loading control. The results showed that the levels of Hsp47 protein in cells treated with either siHsp47-C alone, siHsp47-2d, or the combination of siHsp47-1 and siHsp47-2d were significantly reduced.

Example 6

Downregulation of collagen I expression by hsp47 siRNA

To determine the effect of siHsp47 on collagen I expression levels, collagen I mRNA levels in hTERT cells treated with different siRNAs against hsp47 were measured. In brief, hTERT cells were transfected with different siHsp47s as described in Example 2. After 72 h, cells were dissolved and mRNA was isolated using the RNeasy micro kit according to the manual (Qiagen). The level of collagen I mRNA was determined using a probe in conjunction with quantitative PCR using reverse transcription. In brief, cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (ABI) according to the manual, and TaqMan gene expression assays were performed (ABI, COL1A1 assay IDHs01076780_g1). The level of collagen I mRNA was standardized to the level of GAPDH mRNA according to the manufacturer's instructions (ABI). Signals were standardized to the signal obtained from cells transfected with scrambled siNA. The results showed that collagen I mRNA levels were significantly decreased in cells treated with some candidates siHsp47-2, siHsp47-2d and their combination with siHsp47-1 and are shown in FIG4 .

Example 7

Immunofluorescence staining of hTERT cells treated with hsp47 siRNA

To visualize the expression of two fibrosis markers, collagen I and α-smooth muscle actin (SMA), in hTERT cells transfected with or without siHsp47, cells were stained with rabbit anti-collagen I antibody (Abcam) and mouse anti-α-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 α-SMA (red). Hoescht was used to visualize cell nuclei (blue). The results showed a correlation between siRNA knockdown of some target genes and collagen/SMA expression.

Example 8

In vivo testing of siHSP47 in an animal model of liver fibrosis

siRNA for the treatment of rat liver cirrhosis

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

Sense (5'->3') ggacaggccucuacaacuaTT

Antisense (5'->3') uaguuguagaggccuguccTT

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

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

siRNA treatment: siRNA treatment was initiated on day 32 with five intravenous injections. Specifically, rats were treated with siRNA on days 32, 34, 36, 38, and 40. Rats were then sacrificed on day 42 or 43. Three different siRNA doses were tested (1.5 mg siRNA/kg body weight, 2.25 mg siRNA/kg body weight, and 3.0 mg siRNA/kg body weight). Details of the experimental groups and the number of animals in each group are as follows:

1) Induction of cirrhosis by DMN injection followed by 5% glucose injection instead of siRNA (n=10)

2) VA-Lip-siHSP47C 1.5mg/kg (n=10)

3) VA-Lip-siHSP47C 2.25mg/kg (n=10)

4) VA-Lip-siHSP47C 3.0mg/Kg (n=10)

5) Sham treatment group (PBS injection instead of DMN. 5% glucose injection instead of siRNA) (n=6)

6) Untreated control (intact) (n=6)

VA-Lip refers to Vitamin A-liposome complex.

Evaluation of therapeutic effect: On day 43, 2 out of 10 animals in the "sick rat" group and 1 out of 10 animals in the "VA-Lip-siHSP47C siRNA 1.5 mg/kg" group died due to the development of cirrhosis. The remaining animals survived. After the rats were sacrificed, the liver tissue was fixed in 10% formalin. Then, the left lobule of each liver was embedded in paraffin for histological studies. Tissue slides were stained with Sirius red and hematoxylin and eosin (HE). Sirius red staining was used to visualize collagen precipitation and determine the level of cirrhosis. HE staining was used as a contrast stain for cell nuclei and cytoplasm. Each slide was observed under a microscope (BZ-8000, Keyence Corp. Japan) and the percentage of Sirius red stained area on each slide was determined by image analysis software connected to the microscope. At least 4 slides were prepared for each liver for image analysis, and all areas of each slide (liver section) were captured with a camera and analyzed. Statistical analysis was performed by Student's t-test.

Results: Figure 5 shows the area of fibrosis. The area of fibrosis in the "sick rats" was larger than that in the "sham-treated" or "untreated control" groups. Thus, DMN treatment induces collagen deposition in the liver, which is typically observed in liver fibrosis. However, treatment with siRNA targeting the HSP47 gene significantly reduced the area of fibrosis compared to the "sick rat" group (Figure 5). This result demonstrates that the siRNA disclosed herein has therapeutic effects in actual diseases.

Additional siRNA compounds were tested in an animal model of liver fibrosis and shown to reduce the area of fibrosis in the liver.

Example 9

The sequences of the HSP47/SERPINH1 active double-stranded RNA compounds were generated and shown in Tables A-18, A-19, and B-E. siRNA generation

Using a proprietary algorithm and the known sequence of the target gene, the sequences of many potential siRNAs are generated. Sequences generated using this method are complementary to the corresponding mRNA sequence.

Duplexes were generated by annealing complementary single-stranded oligonucleotides. In a laminar flow hood, a ~500 μM single-stranded oligonucleotide stock solution was prepared by diluting in WFI (water for injection, Norbrook). The actual ssRNA concentration was determined by diluting each 500 μM ssRNA at 1:200 using WFI and measuring the OD using a Nano Drop. This step was repeated 3 times and the average concentration was calculated. The stock solution was then diluted to a final concentration of 250 μM. The complementary single strands were annealed by heating to 85°C and cooling to room temperature for at least 45 minutes. Complete annealing of the duplexes was tested by testing 5 μl of the duplex on a 20% polyacrylamide gel and staining. The 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 that are prioritized based on their scores in a proprietary algorithm to best target human gene expression.

The following abbreviations are used in Tables A-18, A-19, and B-E herein: "Other species" refers to the cross-species identity of other animals: D - dog, Rt - rat, Rb - rabbit, Rh - macaque, P - pig, M - mouse; ORF: open reading frame. 19-mer and 18+1-mer refer to oligomers with 19 and 18+1 RNAs in length, respectively (U in position 1 of the antisense strand and A in position 19 of the sense strand).

In vitro testing of target genes of siRNA compounds

Low throughput screening (LTS) of siRNA oligonucleotides targeting human and rat SERPINH1 genes.

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

Approximately 2 × 10 ⁵ human PC-3 cells endogenously expressing the SERPINH1 gene were seeded in 1.5 mL of growth medium and allowed to reach 30-50% confluence after 24 hours. The cells were transfected with Lipofectamine ^™ 2000 reagent to a final concentration of 0.01-5 nM per transfected cell. The cells were incubated at 37 ± 1°C for 48 hours. The siRNA-transfected cells were harvested and RNA was isolated using the EZ-RNA kit [Biological Industries (#20-410-100)].

Reverse transcription was performed as follows: cDNA synthesis was performed and human SERPINH1 mRNA levels were determined by real-time qPCR and normalized to the level of cyclophilin A (CYNA, PPIA) mRNA for each sample. siRNA activity was determined based on the ratio of SERPINH1 mRNA levels in siRNA-treated samples to non-transfected control samples.

Select the most active sequence by other determinations.From Table A-18, select siRNA compound SERPINH1_2, SERPINH1_6, SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPINH1_51a, SERPINH1_52 and SERPINH1_86 as preferred compounds.From Table A-19, select siRNA compound SERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58 and SERPINH1_88 as preferred compounds.

Other preferred compounds include SERPINH1_50, SERPINH1_67, SERPINH1_73, and SERPINH1_74.

IC50 values of LTS-selected SERPINH1 siRNA oligonucleotides

Approximately 2 × 10 ⁵ human PC-3 cells endogenously expressing the SERPINH1 gene or 0.9 × 10 ⁵ rat REF52 cells were seeded in 1.5 mL of growth medium to a confluence of 30-50%. Cells were transfected with SERPINH1 double-stranded RNA compounds (i.e., SERPINH1_2, 4, 6, 12, 13, 18, 45, 51, 58, 88) using Lipofectamine ^™ 2000 reagent to a final transfection concentration ranging from 0.0029 to 100 nM. As a negative control, cells were treated with Lipofectamine ^™ 2000 reagent or synthetic randomized sequence non-targeting siRNA at a final concentration of 20-100 nM. Cy3-labeled siRNA transfected cells were used as a positive control for transfection efficiency.

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

The IC50 value of the RNAi activity was determined by constructing a dose-response curve using the activity results obtained with various final siRNA concentrations. The dose-response curve was constructed by plotting the relative amount of the remaining SERPINH1 mRNA relative to the logarithm of the transfection siRNA concentration. The curve was calculated by fitting the best sigmoidal curve to the measured data. The sigmoidal fitting method is also referred to as 3-point curve fitting.

Where Y is the remaining SERPINH1 mRNA response, X is the logarithm of the transfected siRNA concentration, Bot is the Y value at the bottom plateau, LogIC50 is the X value when Y is midway between the bottom and top plateaus, and HillSlope is the slope of the curve.

qPCR was used to analyze target genes in cells expressing endogenous genes to determine the percentage of gene expression inhibited by a specific siRNA. Other siRNA compounds according to Tables A-18, A-19, and B-E were tested in vitro and demonstrated to inhibit gene expression. Activity was expressed as a percentage of remaining mRNA; therefore, lower values reflect better activity.

To test the stability of 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 were collected at 0.5, 1, 3, 6, 8, 10, 16, and 24 hours. Migration patterns were determined by polyacrylamide gel electrophoresis (PAGE) at each collection time.

Table 3 shows the IC50 (or activity when IC50 is not calculated) of unmodified double-stranded nucleic acid compounds (unmodified sense and antisense strands, dTdT 3'-terminal overhang) selected from Tables A-18 and A-19 in human cell lines.

Table 3

siRNA knockdown activity

In 6-well plates, approximately 2 × 10^⁵ human PC-3 cells expressing endogenous SERPINH1 genes were seeded per well and grown for approximately 24 hours to a confluence of 30-70%. Cells were transfected with the siRNA tested at various concentrations using Lipofectamine ^™ 2000 reagent (Invitrogen). Cells were cultured in a 5% CO _₂ incubator at 37°C for 48 hours or 72 hours. 48-72 hours after transfection, cells were harvested and cellular RNA was extracted. Cy3-labeled siRNA duplexes were used as a positive control for transfection efficiency. The mock cells treated with Lipofectamine ^™ 2000 reagent were defined as "control inactive samples" (negative controls) and the cells treated with a known active siRNA (HSP47-C) at a final concentration of 5 nM were defined as "control active samples" (positive controls). Z' and control multiples {multiples = mean (negative) / mean (positive)} are methods for describing assay efficiency.

The percentage inhibition of target gene expression by each siRNA assayed was determined by qPCR analysis of the target mRNA from the cells. Reverse transcription was performed by synthesizing cDNA from the cells and determining the target gene mRNA level by real-time qPCR. The measured cell mRNA levels were standardized to the level of cyclophilin A (CYNA, PPIA) mRNA for each sample. siRNA activity was determined based on the ratio of the target gene mRNA amount in the sample treated with siRNA to the non-transfected control sample. Z' and control multiples {multiples = mean (negative) / mean (positive)} are ways to describe assay efficiency.

qPCR results must pass QC criteria, i.e., the slope of the standard curve must be within the range [-4, -3], R² > 0.99, and there must be no primer dimers. Results that fail QC will be disqualified from analysis.

Dose response curves were constructed using the activity results obtained with various final siRNA concentrations to determine IC50 values for test RNAi activity. As described above, dose response curves were constructed by plotting the relative amount of remaining SERPINH1 mRNA against the logarithm of the transfected siRNA concentration.

On-target and off-target assays for double-stranded RNA molecules

The psiCHECK system assesses on-target and off-target effects of the guide (GS) (antisense) and passenger (PS) strands by monitoring changes in target sequence expression levels. Four constructs based on siCHECK ^™ -2 (Promega) were prepared to assess the on-target activity and potential off-target activity of each test molecule's GS and PS strands. In each construct, one or three copies of the complete target or seed-target sequence of the test molecule, PS or GS, were cloned into the multiple cloning site located downstream of the translation stop codon of Renilla luciferase in the 3'-UTR.

The resulting vector is called:

1-GS-CM (guide strand, perfect match) vector, containing 1 copy of the complete target sequence (a nucleotide sequence that is completely complementary to the entire 19-base sequence of the GS of the test molecule);

2-PS-CM (passenger strand, perfect match) vector, containing 1 copy of the complete target sequence (a nucleotide sequence that is completely complementary to the entire 19-base sequence of the PS of the test molecule);

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

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

name:

Guide strand: The siRNA strand that enters the RISC complex and directs cleavage/silencing of the complementary RNA sequence

Seed sequence: nucleotides 2-8 from the 5' end of the guide strand

cm (perfect match): A DNA fragment that is completely complementary to the guide strand of the siRNA. This DNA fragment is cloned into the 3' UTR of a reporter gene and used as a target for direct RNA silencing.

sm (seed match): A 19-mer DNA fragment whose nucleotides ns 12-18 are completely complementary to ns 2-8 of the siRNA guide strand. This DNA fragment is cloned in the 3' UTR of the reporter gene and used as a target for "off-target" silencing.

X1: One copy of cm or sm cloned into the 3’UTR of the reporter gene.

X3: Three copies of cm or sm cloned into the 3'UTR of the reporter gene separated by 4 nucleotides

Table 4: Non-limiting examples of psiCHECK cloning targets

The target sequence was cloned using XhoI and NotI compatible restriction enzyme sites. The annealing mixture was prepared in a sealed 0.5 ml Eppendorf tube, heated to 85°C in a water bath, immersed in a boiling water bath, and finally gradually cooled to room temperature.

Ligation: The double-stranded oligonucleotides generated by the annealing step were ligated to linearized (with XhoI and NotI) psiCHECK ^™ -2 and transfected into cells using standard techniques. Positive clones were identified and sequenced to verify the insert sequence. Table 5 shows the nucleotide sequences of the insert oligonucleotides.

Table 5

The above-mentioned related chains were cloned in the Renilla luciferase in the 3'UTR of the reporter mRNA, psiCHECK ^™ -2 (Promega) vector. Standard molecular biology techniques were used with XhoI and NotI as cloning sites. Each chain was chemically synthesized and annealed by heating to 100°C and cooling to room temperature. Standard molecular biology techniques were used to connect for 3 hours and transform into Escherichia coli (E. coli) DH5a cells. The presence of the resulting colony plasmid construct was screened by colony-PCR using the relevant primers. Each plasmid (vector) was purified from a positive colony and its sequence was verified.

Approximately 1.3 × 10 ⁶ human HeLa cells were seeded in a 10 cm dish. The cells were then incubated for 24 hours in a 37 ± 1°C, 5% CO 2 incubator. After one day of incubation, the growth medium was replaced with 8 mL of fresh growth medium, and each plate was transfected with one of the aforementioned plasmids using Lipofectamine ^™ 2000 reagent according to the manufacturer's instructions and incubated for 5 hours at 37 ± 1°C and 5% CO 2 . Following incubation, the cells were seeded in 96-well plates at a final concentration of 5 × 10 ³ cells/well in 80 μL of growth medium. After 16 hours, the cells were transfected with SERPINH1 siRNA using Lipofectamine ^™ 2000 reagent at concentrations ranging from 0.001 nM to 5 nM in a final volume of 100 μL. Mock cells treated with Lipofectamine ^™ 2000 reagent and the corresponding psiCHECK ^™ -2 plasmid were defined as "control inactive samples" (negative controls), while cells treated with a known active siRNA (HSP47-C) at a final concentration of 5 nM were defined as "control active samples" (positive controls). Z' and control fold {fold = mean (negative) / mean (positive)} are ways to describe assay efficiency.

Then cultivate cells at 37 ± 1 ℃ for 48h and use Dual-Assay Kit (Promega, catalog number (Cat. No.) E1960) to measure Renilla and Firefly luciferase activity in each siRNA transfection sample according to manufacturer's procedure. The activity of synthetic siRNA to the target sequence causes fusion mRNA cracking and subsequent degradation, or causes translation inhibition of the encoded protein. Therefore, measuring Renilla luciferase activity reduces the convenient way to provide monitoring siRNA effects, and Firefly luciferase standardizes Renilla luciferase expression. Renilla luciferase activity value is divided by the Firefly luciferase activity value (standardization) of each sample. Finally, Renilla luciferase activity is expressed as the percentage relative to the standardized activity in " control inactive sample " test sample.

Results for TNFα and IL-6 levels in peripheral blood mononuclear cells (PMNCs) exposed to unmodified or modified siRNA/Lipofectamine ^™ 2000. Results are presented as pg/ml values calculated based on a standard curve. "Control Lipofectamine™ 2000" refers to the level of cytokine secretion induced by the transfection reagent Lipofectamine ^™ 2000. None of the modified compounds produced levels of the cytokines TNFα or IL-6 above those produced by the control transfection reagent.

Data on the induction of interferon (IFN) response genes MX1 and IFIT1 by unmodified and modified double-stranded nucleic acid compounds. As with the human PMNC assay, the results are presented as the remaining (fold of control Lipofectamine 2000-treated cells) human IFIT1 and MX1 genes. The data show that all modified compounds induced very low levels of IFN downstream genes compared to the unmodified (_S709) compound.

The table below shows the activity of Compound 1, Compound 2, Compound 3, and Compound 4 compared to the unmodified (_S709) compound in rat cells. Results are shown as the remaining target (% of control Lipofectamine ^™ 2000-treated cells) rat SERPINH1 gene in REF52 cells. Results from two separate experiments are shown. Knockdown or target gene in rat cells is relevant to testing compounds in animal models of human disease.

Serum stability assay

The duplex thermostability of the modified compounds according to the present invention in human serum or human tissue extracts was tested as follows:

siRNA molecules were incubated at 37°C in 100% human serum (Sigma catalog number H4522) at a final concentration of 7 μM. (A 100 μM siRNA stock solution was diluted 1:14.29 in human serum or human tissue extracts from various tissue types). At various time points (e.g., 0, 30 min, 1 h, 3 h, 6 h, 8 h, 10 h, 16 h, and 24 h), 5 μl of 1.5× TBE loading buffer was added to 15 μl. Samples were immediately frozen in liquid nitrogen and stored at -20°C.

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

Exonuclease stability assay

To investigate the stabilizing effect of the 3' non-nucleotide moiety on nucleic acid molecules, sense, antisense, and annealed siRNA duplexes were incubated in cytosolic extracts prepared from different cell types.

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

Extraction buffer: 25 mM Hepes, pH 7.3, 8 mM MgCl, 150 mM NaCl, and 1 mM DTT at 37°C, added immediately before use.

Methods: 3.5 ml of test siRNA (100 mM) was mixed with 46.5 ml of HCT116 cytosolic extract containing 120 mg. The 46.5 ml was composed of 12 ml of HCT116 extract and 34.5 ml of extract buffer supplemented with DTT and protease inhibitor cocktail/100 (Calbiochem, set III-539134). The final concentration of siRNA in the incubation tube was 7 mM. Samples were incubated at 37°C, and at designated time points, 5 ml of sample was transferred to a new tube, mixed with 15 ml of 1X TBE-50% glycerol loading buffer, and flash-frozen in liquid N2. The final concentration of siRNA in the loading buffer was 1.75 mM (21 ng siRNA/ml). For analysis by native PAGE and EtBr staining, 50 ng was loaded per band. For Northern analysis, 1 ng of test siRNA was loaded per band.

Innate immune response to SERPINH1 siRNA molecules

Fresh human blood (at room temperature) was mixed with sterile 0.9% NaCl in a 1:1 ratio at room temperature and gently loaded onto Ficoll (Lymphoprep, Axis-Shield catalog number 1114547) (1:2 ratio). Samples were centrifuged in a swinging centrifuge at room temperature (22°C, 800g) for 30 minutes, washed with RPMI1640 medium, and centrifuged (RT, 250g) for 10 minutes. Cells were counted and plated in growth medium (RPMI1640 + 10% FBS + 2mM L-glutamine + 1% Pen-Strep) at a final concentration of 1.5× ¹⁰ cells/ml and incubated at 37°C for 1 hour prior to siRNA treatment.

The cells were then treated with the siRNA tested at different concentrations using Lipofectamine ^™ 2000 reagent (Invitrogen) according to the manufacturer's instructions and incubated at 37° C. in a 5% CO 2 incubator for 24 h.

As a positive control for IFN response, cells were treated with poly(I:C) at a final concentration of 0.25-5.0 μg/mL or thiazolaquinolone (CLO75) at a final concentration of 0.075-2 μg/mL. Poly(I:C) is a synthetic analog of double-stranded RNA (dsRNA) for TLR3 ligands (InvivoGen catalog number tlrl-pic), and thiazolaquinolone is a TLR 7/8 ligand (InvivoGen catalog number tlrl-c75). Cells treated with Lipofectamine ^™ 2000 reagent served as a negative (reference) control for IFN response.

After about 24 hours of cultivation, cells were collected and the supernatant was transferred to a new tube. The samples were immediately frozen in liquid nitrogen and the secretion of IL-6 and TNF-α was tested according to the manufacturer's instructions using IL-6, DuoSet ELISA kit (R&D System DY2060) and TNF-α, DuoSet ELISA kit (R&D System DY210). RNA was extracted from the cell pellet and the mRNA levels of the human genes IFIT1 (interferon-induced protein with triangular tetrapeptide repeats 1) and MX1 (myxovirus (influenza virus) resistance 1, interferon-induced protein p78) were measured by qPCR. The measured mRNA amount was standardized to the mRNA amount of the reference gene peptide prolyl isomerase A (cyclophilin A; CycloA). The induction of IFN signaling was assessed by comparing the mRNA amounts of the IFIT1 and MX1 genes from treated cells with those of non-treated cells. qPCR results must pass QC criteria, i.e., the slope of the standard curve must be within the range [-4, -3], R² > 0.99, and there must be no primer dimers. Results that fail QC will be disqualified from analysis.

Table 6 shows the siSERPINH1 compounds. Activity and stability data for some compounds are presented in Table 6. The symbols for the sense and antisense strand structures are presented in Table 7 below.

Table 6:

Table 7: Codes for modified nucleotides/unconventional moieties used in the tables herein

The siRNA oligonucleotides used to generate double-stranded RNA molecules are disclosed in Tables A-18, A-19, and B-E below.

SERPINH1 oligonucleotide sequences for preparing siRNA compounds

Table A-18:

Table A-18 Selection of siRNA

Table A-19:

Table A-19 Selection of siRNA

Table B : Additional active 19-mer SERPINH1 siRNAs

Table C : Cross-species 19-mer SERPINH1 siRNA

Table D : SERPINH1 active 18+1-mer siRNA

Table E : SERPINH1 cross-species 18+1-mer siRNA

Example 10

Animal models

Model systems for fibrotic pathologies

Active siRNAs of the present invention can be tested in predictive animal models. Rat diabetic and aging models of renal fibrosis include Zucker diabetic fatty (ZDF) rats, aged fa/fa (fatty Zucker) rats, aged Sprague-Dawley (SD) rats, and Goto Kakizaki (GK) rats; GK rats are an inbred strain derived from Wistar rats selected for the natural development of NIDDM (type II diabetes). Inducible models of renal fibrosis include the permanent unilateral ureteral obstruction (UUO) model, which is a model of acute interstitial fibrosis that occurs in healthy non-diabetic animals; renal fibrosis develops within a few days of obstruction. Another induced model of renal fibrosis is 5/6 nephrectomy.

Two models of liver fibrosis in rats were bile duct ligation (BDL) with sham surgery as a control and CCl4 intoxication with olive oil-fed animals as a control, 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 Jan-Feb;12(1):60-6; Xu XQ et al., Molecular classification of liver cirrhosis in a rat model by proteomics and bioinformatics Proteomics. 2004 Oct;4(10):3235-45.

Models of ocular scarring are well known in the art, for example, Herwood MB et al., J Glaucoma. 2004 Oct; 13(5): 407-12. A new model of glaucoma filtering surgery in the rat; Miller MH et al., Ophthalmic Surg. 1989 May; 20(5): 350-7. Wound healing in an animal model of glaucoma fistulizing surgery in the Rb; van Bockxmeer FM et al., Retina. 1985 Autumn-Winter; 5(4): 239-52. Models for assessing scar tissue inhibitors; Wiedemann P et al., J Pharmacol Methods. 1984 Aug; 12(1): 69-78. Proliferative vitreoretinopathy: the Rb cell injection model for screening of antiproliferative drugs.

Cataract models are described in the following publications: The role of Src family kinases incortical cataract formation. Zhou J, Menko AS. Invest Ophthalmol Vis Sci. 2002 Jul; 43(7): 2293-300; Bioavailability and anticataract effects of a topicalocular drug delivery system containing disulfiram and hydroxypropyl-beta-cyclodextrin on selenite-treated rats. Wang S et al., http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?utm_source=ncbi&utm_source=ncbi&utm_source=ncbi&utm_source=ncbi&utm_source=ncbi&utm_source=ncbi&utm_source=ncbi&utm_source=ncbi cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15370367 Curr Eye Res. 2004 Jul;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 were tested in these models of fibrotic conditions and were found to be effective in treating liver fibrosis and other fibrotic conditions.

Glaucoma model system

For example, active siRNAs of the present invention for treating or preventing glaucoma were tested in a rat model for optic nerve crush as described in Maeda 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 was exposed through a supraorbital approach, the meninges were cut, and all axons in the ON were transected 2 mm from the cribriform plate by crushing with forceps for 10 s.

The nucleic acid molecules disclosed herein were tested in this animal model and the results showed that these siRNA compounds are effective in treating and/or preventing glaucoma.

Rat Optic Nerve Crush (ONC) Model: Intravitreal siRNA Delivery and Eye Drop Delivery

For optic nerve transection, the orbital optic nerve (ON) of anesthetized rats was exposed through a supraorbital approach, the meninges were cut and all axons in the ON were transected 2 mm from the cribriform lamina by pinching with forceps for 10 s.

siRNA compounds were delivered as eye drops either alone or in combination (10 μg/μL) in a 5 μL volume. 20 μg/10 μl of test siRNA or 10 μl of PBS was administered to one or both eyes of adult Wistar rats immediately after optic nerve crush (ONC) and the levels of siRNA uptake in whole dissected, snap-frozen retinas were determined 5 h and 1 day after injection, and then on days 2, 4, 7, 14, and 21. Similar experiments were performed to test the activity and efficacy of siRNA administered by eye drops.

A model system for ischemia-reperfusion injury after rat lung transplantation

Lung ischemia/reperfusion injury was obtained in a rat animal model as described in Mizobuchi et al., The Journal of Heart and Lung Transplantation, Vol. 23, No. 7 (2004) and Kazuhiro Yasufuku et al., Am. J. Respir. Cell Mol Biol, Vol. 25, pp. 26-34 (2001).

Specifically, after induction of anesthesia with isoflurane, a No. 14 Teflon cannula was inserted into the trachea and the rats were mechanically ventilated using a rodent respirator with 100% oxygen at a rate of 70 breaths/min and a positive end-expiratory pressure of 2 cmH2O. The left pulmonary artery, vein, and main bronchus were occluded with a Castaneda clamp. During surgery, the lungs were kept moist with saline and the incisions were covered to minimize evaporative losses. The ischemic period lasted up to 60 min. At the end of the ischemic period, the clamps were removed and the lungs were ventilated and reperfused for 4 h, 24 h, and 5 days after induction of pulmonary ischemia. At the end of the experiment, the lungs were gently removed and frozen for RNA extraction or fixed in a glutaraldehyde mixture for subsequent histological analysis.

Bleomycin animal model as a model of idiopathic pulmonary fibrosis (IPF)

Testing the feasibility of siRNA delivery via intravenous injection of vitamin A-Coatsome formulated siRNA and intratracheal administration of siRNA-vitamin A-Coatsome complexes to the lungs and liver in healthy and bleomycin-treated mice

Objective: To test the feasibility of two routes of administration of siRNA formulated with Vitamin A-Coatsome to the lungs of normal and fibrotic mice. The main hypothesis tested in the current study is whether systemic administration of modified siRNA formulated with Vitamin A-Coatsome provides effective uptake and cell-specific distribution in the lungs of fibrotic and normal mice. In parallel, the intratracheal route of modified siRNA formulated with Vitamin A-Coatsome will be tested. siRNA detection and cell-specific distribution in the lungs and liver will be performed by in situ hybridization (ISH).

Background: Over the past three decades, the bleomycin model of pulmonary fibrosis has been well characterized (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). Similar to IPF patients, BLM-treated animals exhibit histological features such as alveolar budding, collagen incorporation into the luminal wall, and effacement of alveolar spaces. Early studies demonstrated that C57/Bl mice are consistently susceptible to BLM-induced pulmonary fibrosis, while Balb/C mice are genetically resistant. Different patterns of fibrosis develop depending on the route of administration. Intratracheal instillation of BLM induces bronchocentric, enhanced fibrosis, while intravenous or intraperitoneal administration induces subpleural scarring similar to human disease (Chua et al., supra). A mouse model of usual interstitial pneumonia (UIP) was used. This model shows that fibrosis is unevenly distributed, mainly in the subperitoneum, forming lesions similar to those observed in the lungs of patients with idiopathic pulmonary fibrosis (IPF) (Onuma et al., Tohoku J Exp Med 194:147-156, 2001 and Yamaguchi and Ruoslahti, Nature 336:244-246, 1988). UIP is induced by intraperitoneal injection of bleomycin every other day for 7 days, so that the composition of subperitoneal fibrosis in the mouse lung is constant (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 previously demonstrated, siRNA-containing microbial A-coatsome liposomes interact with retinol-binding protein (RBP) and provide efficient delivery to hepatic stellate cells via the RBP receptor (Sato et al. Nat Biotechnol 26:431–442, 2008). This study was planned to test whether vitamin A-coatsome-siRNA complexes are efficiently taken up by activated myofibroblasts expressing the RBP receptor in the lungs of bleomycin-treated mice. In addition, a local route of administration (intratracheal instillation) will be tested.

General study design

Mouse-C57 Bl male

Starting number (BLM I.P.) – 40 (6 in the first experimental group, 34 in the study, taking into account the expected 25% mortality rate)

Starting number (total) – 60

Test siRNA: SERPINHI compounds disclosed herein

Group:

Bleomycin-induced pulmonary fibrosis Pulmonary fibrosis was induced in 12-week-old female C57BL/6 mice by intraperitoneal instillation of bleomycin chlorate: 0.75 mg/kg body weight dissolved in 0.1 ml saline, intraperitoneally on days 0, 2, 4, and 6, every other day for 7 days.

Model Tracking and Monitoring. Mice were weighed before BLM treatment and twice weekly for the duration of the study.

Preliminary assessment of fibrosis establishment. Mice (N=30) were grouped for BLM treatment to allow a one-week interval between the first treatment group (N=5) and the remaining animals. On the 14th day, two mice from Group 1 were sacrificed and the lungs were removed for rapid HE staining and rapid histopathological assessment of fibrosis. When pulmonary fibrosis was confirmed, the remaining rats were grouped and treated with siRNA on the 14th day after the first BLM treatment. If there was no sufficient fibrosis development in the lungs by the 14th day, the remaining mice from the first treatment group were sacrificed on the 21st day and then a rapid histopathological assessment of fibrosis was performed. The remaining animals were treated with the test siRNA complex starting on the 21st day after BLM treatment.

siRNA administration. On the 14th or 21st day after the first BLM administration (TBD during the study, based on the preliminary assessment of fibrosis establishment), animals were grouped according to BW. Animals from Groups 1 and 2 were administered siRNA/vitamin A/Coatsome complexes intravenously (tail vein injection) at a siRNA concentration of 4.5 mg/kg BW. Animals of the same age (Groups 5 and 6) were treated in the same manner. Animals treated with BLM (Group 9) will be used as vitamin A-coatsome vehicle controls. Within 24 hours, all animals were injected repeatedly.

BLM animals from Groups 3 and 4 and naive mice from Groups 7 and 8 were anesthetized with isoflurane and intratracheally instilled with 2.25 mg/kg BW of siRNA formulated in vitamin A-loaded liposomes. Only mice from BLM Group 10 were administered the vitamin A/Coatsome vehicle. The intratracheal instillation was repeated 24 h later.

The study was terminated. Animals from Groups 1, 3, 5, 7, and 9 were sacrificed 2 h after the second injection or instillation of siRNA complexes. Animals from Groups 2, 4, 6, 8, and 10 were sacrificed 24 h after the second injection or instillation of siRNA complexes.

Upon sacrifice, mice were perfused cardiacally with 10% neutral buffered formalin. Lungs were inflated with 0.8-1.0 ml of 10% NBF, and the trachea was ligated. Lungs were excised and fixed in 10% NBF for 24 h. The liver was removed from each animal and fixed in 10% NBF for 24 h.

Sectioning and Assessment. Prepare anterograde sections from the lung and liver. Stain the first anterograde section with hematoxylin and eosin to assess lung and liver morphology, and stain the second anterograde section with Sirius Red (Trichrome) to identify collagen. Perform in situ hybridization (ISH) on the third anterograde section to detect siRNA.

The compounds described herein were tested in this animal model and the results showed that these siRNA compounds are useful in treating and/or preventing pulmonary fibrosis.

The contents of articles, patents and patent applications and 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 paper, patent, patent application or other physical and electronic file.

It will be apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Therefore, these additional embodiments are within the scope of the present invention and the claims below. The present invention teaches those skilled in the art to test various combinations and/or substitutions of the chemical modifications described herein to generate nucleic acid constructs with enhanced activity that mediate RNAi activity. Such enhanced activity may include enhanced stability, improved bioavailability, and/or enhanced activation of cellular responses that mediate RNAi. Therefore, the specific embodiments described herein are not limiting and those skilled in the art will readily appreciate that specific combinations of modifications described herein may be tested without undue experimentation to identify nucleic acid molecules with enhanced RNAi activity.

The invention illustratively described herein may be suitable for implementation in the absence of any elements or limitations not specifically disclosed herein. Therefore, unless otherwise noted herein or clearly contradicted by the context, for example, the terms "a", "an", and "the", as well as similar indicators in the context of describing the present invention (particularly in the context of the following claims), are to be interpreted as including both the singular and the plural. The terms "comprising", "having", "including", "containing", etc. should be understood broadly and without limitation (e.g., meaning "including, but not limited to"). Unless otherwise noted herein, the description of the value range herein is merely intended to serve as a shorthand method for individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if each individual value were individually cited herein. Unless otherwise noted herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "for example") provided herein is intended only to better illustrate the present invention and does not limit the scope of the invention unless otherwise required. The language in the specification should not be interpreted as indicating that any non-required element is necessary for the practice of the present invention. Additionally, the terms and expressions employed herein have been used as terms of description rather than limitation, and it is not intended that these terms and expressions be used to exclude 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 claimed invention. Thus, it is understood that while the present invention has been particularly disclosed by preferred embodiments and optional features, modifications and variations of the invention embodied therein disclosed herein will be resorted to by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

The invention is described broadly and generically herein. Each of the narrower species and subgeneric groupings disclosed herein also forms part of the invention. This includes the generic description of the invention with the proviso or negative limitation that removes any subject matter from the genus, regardless of whether or not specific reference is made herein to excised material. Other embodiments are in the following claims. In addition, where features or aspects of the invention are described in terms of Markush groupings, those skilled in the art will recognize that the invention is also thereby described in terms of individual members or subgroups of members of the Markush group.

Claims (30)

1. A double-stranded nucleic acid molecule having the structure shown below: Antisense chain: 5' N ¹ -(N)xZ 3' Significant chain: 3'Z'-N ² -(N')yz” 5' ^N2 , N, and N' are each independently an unmodified or modified ribonucleotide or an unconventional moiety; Where (N)x and (N’)y are each of the oligonucleotides in which each consecutive N or N’ is covalently linked to an adjacent N or N’; Where x = y = 18; The sequence of (N’)y is complementary to the sequence of (N)x, and the sequence of (N)x is complementary to the continuous sequence in the mRNA encoding hsp47. ^N1 is covalently bound to (N)x and mismatched with the mRNA encoding hsp47; ^N1 is a fraction selected from natural or modified uridine, deoxyribouridine, ribothymidine, deoxyribothymidine, adenosine or deoxyadenosine; Where z” may or may not exist, but if it exists, it is the capped part covalently connected at the 5’ end of N ² - (N’)y; Z and Z’ may or may not exist independently, but if they do exist, they are independently 1-5 consecutive nucleotides, consecutive non-nucleotide portions, or combinations thereof covalently linked to the 3’ end of the strand in which they exist. The non-standard portion is selected from the group consisting of: debased portions, reverse debased portions, hydrocarbon portions and their derivatives, deoxyribonucleotides, modified deoxyribonucleotides, mirror nucleotides, non-base-paired nucleotide analogs, and nucleotides linked to adjacent nucleotides by 2'-5' phosphate bonds; bridging nucleic acids, chain-modified nucleotides, and base-modified nucleotides; and The sense strand and the antisense strand are selected from the sequence pairs shown in SEQ ID NO:98 and SEQ ID NO:165 or the sequence pairs shown in SEQ ID NO:101 and SEQ ID NO:168. 2. The double-stranded nucleic acid molecule of claim 1, wherein ^N1 , ^N2 , N and N' are each unmodified ribonucleotides, and wherein z”, Z and Z' are absent. 3. The double-stranded nucleic acid molecule of claim 1, wherein Z and Z’ each exist. 4. The double-stranded nucleic acid molecule of claim 3, wherein Z and Z’ each independently comprise a nucleotide overhang or a non-nucleotide overhang. 5. The double-stranded nucleic acid molecule of claim 4, wherein Z and Z’ each independently comprise non-nucleotide overhangs. 6. The double-stranded nucleic acid molecule of claim 5, wherein the non-nucleotide overhang comprises an alkyl portion. 7. The double-stranded nucleic acid molecule of claim 6, wherein the non-nucleotide overhang is selected from the group consisting of: C3OH, C3Pi, C3Pi-C3OH, C3Pi-C3Pi and C3Pi-C3P-C3OH. 8. The double-stranded nucleic acid molecule of claim 1, wherein z” is present and includes a capped portion covalently linked to the 5’ end of the sense strand. 9. The double-stranded nucleic acid molecule of claim 8, wherein the capped portion is selected from the debased ribose portion, the reverse debased ribose portion, the reverse debased deoxyribose portion, the debased deoxyribose portion, and modifications thereof; C6-imino-Pi; mirror nucleotide; 5’OMe nucleotide; 4’,5’-methylene nucleotide; 1-β-D-erythrofuranyl nucleotide; 4’-thionucleotide, carbocyclic nucleotide; 5’-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate Esters, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-dehydrated hexitol nucleotide; α-nucleotide; threo-pentafuranosyl nucleotide; acyclic 3',4'-open ring nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide; 5'-5'-reverse debasement moiety; 1,4-butanediol phosphate; 5'-amino; and bridged or unbridged methylphosphonates and 5'-mercaptoyl moiety. 10. The double-stranded nucleic acid molecule of claim 9, wherein the capped portion comprises a reverse debasing deoxyribose portion. 11. The double-stranded nucleic acid molecule of claim 1, having the following structure: or Each "|" represents a base pairing between ribonucleotides; A, C, G, and U are each an unmodified or modified ribonucleotide, or an unconventional part; Each A, C, G, and U is covalently connected to its neighboring A, C, G, or U; Z and Z' may or may not exist independently, but if present, they are independently 1-5 consecutive nucleotides or non-nucleotide motifs or combinations thereof covalently linked to the 3' end of the strand in which they exist; and Where z” may or may not exist, but if it exists, it is the capped portion covalently connected to the 5’ end of the sense chain. 12. The double-stranded nucleic acid molecule of claim 1, wherein ^N1 , ^N2 , N and/or N' comprises modified or modified nucleotides. 13. The double-stranded nucleic acid molecule of claim 12, wherein the modified nucleotide comprises a modified sugar moiety. 14. The double-stranded nucleic acid molecule of claim 13, wherein the modified sugar moiety is independently selected from the group consisting of: 2’-O-methyl, 2’-methoxyethoxy, 2’-deoxy, 2’-fluorine, 2’-allyl, 2’-O-[2-(methylamino)-2-oxoethyl], 4’-thio, 4’-(CH2)2-O-2’-bridge, 2’-locked nucleic acid, or 2’-O-(N-methylcarbamate). 15. The double-stranded nucleic acid molecule of claim 12, wherein the modified nucleotide comprises modified nucleobases. 16. The double-stranded nucleic acid molecule of claim 15, wherein the modified nucleobase is 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-halogenated uracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil, 4-thiouracil, 8-halogenated, amino, mercapto, thioalkyl, hydroxyl and other 8-substituted adenine and guanine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and acyclic nucleotides. 17. The double-stranded nucleic acid molecule of claim 12, wherein the modification comprises a modification of the phosphodiester backbone. 18. The double-stranded nucleic acid molecule of claim 17, wherein the modification of the phosphodiester backbone is selected from the group consisting of: thiophosphate bond, 3’-deoxy-3’-thio-thiophosphate bond, 5’-deoxy-5’-thio-thiophosphate bond, dithiophosphate bond, selenophosphate bond, 3’-deoxyphosphonite bond, 5’-deoxyphosphonite bond, boron phosphate bond, 3’-deoxy-3’-aminoaminophosphate bond, 5’-deoxy-5’-aminoaminophosphate bond, hydrophosphonate bond, boron phosphate bond, aminophosphate bond, alkyl or aryl phosphonate and phosphate triester or phosphorus bond. 19. The double-stranded nucleic acid molecule of claim 11, having the following structure: The sense strand shown in SEQ ID NO:98 comprises a 2'5' ribonucleotide at positions 15, 16, 17 and 18 or 15, 16, 17, 18 and 19 in the 5' to 3' direction, a nucleotide or non-nucleotide portion covalently linked at the 3' end, and a capped portion covalently linked at the 5' end; and/or, the antisense strand shown in SEQ ID NO:165 comprises a 2'-O-methyl sugar-modified pyrimidine and/or purine, a 2'5' nucleotide at positions 5, 6, 7 or 8 in the 5' to 3' direction, and a nucleotide or non-nucleotide portion covalently linked at the 3' end. 20. The double-stranded nucleic acid molecule of claim 19, wherein the sense strand shown in SEQ ID NO:98 comprises a 2'5' ribonucleotide at positions 15, 16, 17, 18, and 19 in the 5' to 3' direction, a C3Pi or C3OH 3' terminal nonnucleotide portion, and a reverse debased deoxyribonucleotide portion covalently linked at the 5' end; and the antisense strand shown in SEQ ID NO:165 comprises an antisense strand containing: a) 2'-O-methyl sugar-modified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17, and 19 in the 5' to 3' direction, a 2'5' ribonucleotide at position 7, and a C3Pi-C3Pi or C3Pi-C3OH 3' terminal overhang; or b) 2'-O-methyl sugar-modified ribonucleotides and C3Pi-C3Pi or C3Pi-C3OH 3' terminal overhangs at positions 2, 4, 6, 8, 11, 13, 15, 17, and 19 in the 5' to 3' direction; or c) 2'-O-methyl sugar-modified ribonucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, and 19 in the 5' to 3' direction, 2'5' ribonucleotides at position 7, and C3Pi-C3Pi or C3Pi-C3OH 3' terminal overhangs; or d) 2’-O-methyl sugar modified ribonucleotides and C3Pi-C3Pi or C3Pi-C3OH 3’ end protrusions at positions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 in the 5’ to 3’ direction. 21. The double-stranded nucleic acid molecule of claim 20, wherein the sense strand shown in SEQ ID NO:98 comprises a 2'5' ribonucleotide at positions 15, 16, 17, 18, and 19 in the 5' to 3' direction, a C3OH 3' terminal portion, and a reverse debased deoxyribonucleotide portion covalently linked at the 5' end; and the antisense strand shown in SEQ ID NO:165 comprises a 2'-O-methyl sugar-modified ribonucleotide at positions 2, 4, 6, 8, 11, 13, 15, 17, and 19 in the 5' to 3' direction, a 2'5' ribonucleotide at position 7, and a C3Pi-COH 3' terminal overhang. 22. The double-stranded nucleic acid molecule of claim 11, having the following structure: The sense strand shown in SEQ ID NO:101 comprises a 2'-O-methyl sugar-modified pyrimidine, optionally a 2'5' ribonucleotide at position 9 or 10, a nucleotide or non-nucleotide portion covalently linked at the 3' end, and optionally a cap portion covalently linked at the 5' end; and/or the antisense strand shown in SEQ ID NO:168 comprises a 2'-O-methyl sugar-modified pyrimidine and/or purine, a 2'5' nucleotide at position 5, 6, 7, or 8 in the 5' to 3' direction, and a nucleotide or non-nucleotide portion covalently linked at the 3' end. 23. The double-stranded nucleic acid molecule of claim 22, wherein the sense strand shown in SEQ ID NO: 101 comprises a pyrimidine modified with a 2'-O-methyl sugar at positions 4, 11, 13, and 17 in the 5' to 3' direction, optionally a 2'5' ribonucleotide at position 9 or 10, a C3Pi or C3OH nonnucleotide portion covalently linked at the 3' end, and a reverse debased deoxyribonucleotide covalently linked at the 5' end; and the antisense strand shown in SEQ ID NO: 168 is selected from antisense strands comprising: a) 2'-O-methyl sugar-modified ribonucleotides at positions 1, 8, and 15 in the 5' to 3' direction, 2'5' ribonucleotides at position 6 or 7, and a C3Pi-C3OH overhang covalently linked at the 3' end; or b) 2'-O-methyl sugar-modified ribonucleotides at positions 1, 4, 8, 13, and 15 in the 5' to 3' direction, 2'5' ribonucleotides at position 6 or 7, and C3Pi-C3OH overhangs covalently linked at the 3' end; or c) 2’-O-methyl sugar-modified ribonucleotides at positions 1, 3, 8, 12, 13 and 15 in the 5’ to 3’ direction, 2’5’ ribonucleotide at position 6 and C3Pi-C3OH moiety covalently linked at the 3’ end. 24. The double-stranded nucleic acid molecule of claim 23, wherein the sense strand shown in SEQ ID NO:101 comprises a 2'-O-methyl sugar-modified ribonucleotide at positions 4, 11, 13, and 17 in the 5' to 3' direction, a 2'5' ribonucleotide at position 9, a C3OH non-nucleotide portion covalently linked at the 3' end, and a reverse debased deoxyribonucleotide portion covalently linked at the 5' end; and the antisense strand shown in SEQ ID NO:168 comprises a 2'-O-methyl sugar-modified ribonucleotide at positions 1, 4, 8, 11, and 15 in the 5' to 3' direction, a 2'5' ribonucleotide at position 6, and a C3Pi-C3OH portion covalently linked at the 3' end. 25. The double-stranded nucleic acid molecule of claim 11, wherein each covalent bond connecting each A, C, G, and U to an adjacent A, C, G, or U is a phosphodiester bond. 26. Use of the double-stranded nucleic acid molecule of claim 1 in the manufacture of a medicament for treating fibrosis in an individual, with an amount sufficient to reduce hsp47 expression. 27. The use according to claim 26, wherein the fibrosis is selected from the group consisting of: liver fibrosis, cirrhosis, pulmonary fibrosis, renal fibrosis, peritoneal fibrosis, chronic liver injury, myocardial fibrosis, retinal fibrosis, posterior orbital fibrosis, lacrimal gland fibrosis, bone marrow fibrosis, intestinal fibrosis, vocal cord mucosal fibrosis, laryngeal fibrosis, renal systemic fibrosis, congenital liver fibrosis, and oral submucosal fibrosis. 28. The use of claim 27, wherein the fibrosis includes cirrhosis, liver fibrosis, pulmonary fibrosis, renal fibrosis, myelofibrosis, and intestinal fibrosis. 29. The use according to claim 26, wherein the fibrosis is fibrosis of the skin, peritoneum, liver, pancreas, kidney, heart, lungs, bone marrow, eyes, intestines, vocal cords and/or vascular system. 30. A composition comprising the double-stranded nucleic acid molecule of any one of claims 1-25; and a pharmaceutically acceptable carrier.