Compositions and methods for inhibiting HAO1 (hydroxyacid oxidase 1 (glycolate oxidase)) gene expression

Abstract

The present invention relates to RNAi agents targeting the HAO1 gene, such as double-stranded RNAi agents, and methods of inhibiting HAO1 expression and treating subjects suffering from, for example, PH1 using such RNAi agents. Double-stranded RNAi agents are described herein that inhibit the expression of the HAO1 gene in cells, such as cells in subjects (e.g., mammals, such as humans suffering from HAO1-related disorders), and the use of such double-stranded RNAi agents. In certain aspects of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified.

Description

[0001] The present application is a divisional application of an invention patent application with an application date of October 9, 2015, application number of 2015800666388, and invention name of “Compositions and methods for inhibiting the expression of HAO1 (hydroxyacid oxidase 1 (glycolate oxidase)) gene”.

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 062,751, filed on October 10, 2014, U.S. Provisional Application No. 62 / 147,976, filed on April 15, 2015, and U.S. Provisional Application No. 62 / 214,602, filed on September 4, 2015, each of which is incorporated herein by reference in its entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on October 8, 2015 is named 30864PCT_CRF_sequencelisting.txt and is 735,705 bytes in size. Background of the Invention

[0006] Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder of glyoxylate metabolism. Hepatic glyoxylate detoxification is impaired by mutations in the AGXT gene, which encodes the hepatic peroxisomal alanine-glyoxylate aminotransferase (AGT). AGT1 is the final enzyme in the metabolic breakdown of hydroxyproline. Loss of AGT function, which converts the intermediate metabolite glyoxylate to glycine, can cause glyoxylate to aggregate and be reduced to glycolate, which is oxidized to oxalate by the enzyme glycolate oxidase (GO, also known as hydroxyacid oxidase (HAO1)).

[0007] Regulation of glyoxylate, a key precursor of oxalate, occurs at multiple cellular sites, including mitochondria, peroxisomes, and cytosol. Excess oxalate in PH1 patients cannot be completely excreted by the kidneys, causing the formation and deposition of calcium oxalate crystals in the kidneys and urinary tract. Renal damage is caused by a combination of tubular toxicity from oxalate, renal calcium deposition, and renal obstruction caused by stones. More than 30% of patients develop end-stage renal disease (ESRD).

[0008] The HAO1 gene encodes the enzyme hydroxyacid oxidase 1, also known as glycolate oxidase ("GO"). The HAO1 protein is primarily expressed in the liver and is the most active 2-hydroxyacid oxidase towards glycolate.

[0009] In a mouse model of PH1 in which the AGT1 gene is deleted, urine oxalate levels are reduced when the HAO1 gene is deleted.

[0010] PH1, AGXT and HAO1 are described in the following literature: Angel L. Pey, Armando Albert and Eduardo Salido, "Protein Homeostasis Defects of Alanine-Glyoxylate Aminotransferase: New Therapeutic Strategies in Primary Hyperoxaluria Type I," BioMed Research International, Vol. 2013, Article ID 687658, p. 15, 2013. doi: 10.1155 / 2013 / 687658; Cochat and Rumsby (2013) NEJM 369:7; Salido et al. (2006) PNAS 103:18249; Baker et al. (2004) American Journal of Physiology-Heart and Circulatory Physiology Published 1, October 2004, Volume 287, Issue 4, H1771-H1779 DOI: 10.1152 / ajpheart.00234.2004. SUMMARY OF THE INVENTION

[0011] The present invention provides a composition comprising an RNAi agent targeting HAO1, such as a double-stranded iRNA agent. The present invention also provides a method for inhibiting HAO1 expression and treating HAO1-related disorders (such as PH1) using the composition of the present invention.

[0012] The present invention also relates to the following items:

[0013] 1. A double-stranded RNAi agent capable of inhibiting the expression of HAO1 in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 6, and the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 8;

[0014] wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and

[0015] Wherein the sense strand is bound to a ligand attached at the 3'-end.

[0016] 2. The double-stranded RNAi agent according to item 1, wherein all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0017] 3. The double-stranded RNAi agent of item 1, wherein the sense strand and the antisense strand comprise a complementary region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any of the antisense sequences listed in Table 2.

[0018] 4. A double-stranded RNAi agent as described in any one of items 1 to 3, wherein at least one of the modified nucleotides is selected from the group consisting of: 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, nucleotides containing 5'-thiophosphate groups, nucleotides containing 5' phosphates or 5' phosphate mimetics, and terminal nucleotides connected to a cholesterol derivative or a didecyl amido group of dodecyl.

[0019] 5. The double-stranded RNAi agent of item 1, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0020] 6. The double-stranded RNAi agent of item 1, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0021] 7. A double-stranded RNAi agent capable of inhibiting the expression of HAO1 (hydroxyacid oxidase 1) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of mRNA encoding HAO1, wherein the length of each strand is about 14 to about 30 nucleotides, wherein the double-stranded RNAi agent is represented by formula (III):

[0022] Meaning: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -nq 3'

[0023] Antonym: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)

[0024] in:

[0025] i, j, k and l are each independently 0 or 1;

[0026] p, p', q and q' are each independently 0-6;

[0027] Each N a and N a ' independently refers to an oligonucleotide sequence comprising 0-25 modified or unmodified nucleotides or a combination thereof, each sequence comprising at least two differently modified nucleotides;

[0028] Each N b and N b ' independently represents an oligonucleotide sequence comprising 0-10 modified or unmodified nucleotides or a combination thereof;

[0029] Each n p 、n p '、n q and n q ', each of which may or may not be present and independently represents an overhanging nucleotide;

[0030] XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif having three identical modifications on three consecutive nucleotides;

[0031] N b The modification of N is different from that of Y and b The modification of ' is different from the modification of Y'; and

[0032] The sense strand is bound to at least one ligand.

[0033] 8. The double-stranded RNAi agent according to item 7, wherein i is 0; j is 0; i is 1; j is 1; i and j are both 0; or i and j are both 1.

[0034] 9. The double-stranded RNAi agent according to item 7, wherein k is 0; l is 0; k is 1; l is 1; k and l are both 0; or k and l are both 1.

[0035] 10. The double-stranded RNAi agent of item 7, wherein XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

[0036] 11. The double-stranded RNAi agent according to item 7, wherein the YYY motif is located at or near the cleavage site of the sense strand.

[0037] 12. The double-stranded RNAi agent according to item 7, wherein the Y'Y'Y' motif is located at positions 11, 12 and 13 from the 5' end of the antisense strand.

[0038] 13. The double-stranded RNAi agent as described in item 12, wherein the Y' is 2'-O-methyl.

[0039] 14. The double-stranded RNAi agent according to item 7, wherein formula (III) is represented by formula (IIIa):

[0040] Meaning: 5'n p -N a -YYY-N a -n q 3'

[0041] Antonym: 3'n p' -N a' -Y'Y'Y'-N a' -n q' 5' (IIIa).

[0042] 15. The double-stranded RNAi agent according to item 7, wherein formula (III) is represented by formula (IIIb):

[0043] Meaning: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'

[0044] Antonym: 3'n p' -N a' -Y'Y'Y'-N b' -Z'Z'Z'-N a' -n q' 5' (IIIb)

[0045] Among them, each N b and N b ' independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

[0046] 16. The double-stranded RNAi agent according to item 7, wherein formula (III) is represented by formula (IIIc):

[0047] Meaning: 5'n p -N a -XXX-N b -YYY-N a -n q 3'

[0048] Antonym: 3'n p' -N a' -X'X'X'-N b' -Y'Y'Y'-N a' -n q' 5' (IIIc)

[0049] Among them, each N b and N b ' independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

[0050] 17. The double-stranded RNAi agent according to item 7, wherein formula (III) is represented by formula (IIId):

[0051] Meaning: 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'

[0052] Antonym: 3'n p' -N a' -X'X'X'-N b' -Y'Y'Y'-N b' -Z'Z'Z'-N a' -n q' 5' (IIId)

[0053] Among them, each N b and N b ' independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each N a and N a ' independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides.

[0054] 18. The double-stranded RNAi agent according to item 7, wherein the length of the double-stranded region is 15-30 nucleotide pairs.

[0055] 19. The double-stranded RNAi agent of item 18, wherein the length of the double-stranded region is 17-23 nucleotide pairs.

[0056] 20. The double-stranded RNAi agent of item 18, wherein the length of the double-stranded region is 17-25 nucleotide pairs.

[0057] 21. The double-stranded RNAi agent of item 18, wherein the length of the double-stranded region is 23-27 nucleotide pairs.

[0058] 22. The double-stranded RNAi agent of item 18, wherein the length of the double-stranded region is 19-21 nucleotide pairs.

[0059] 23. The double-stranded RNAi agent of item 18, wherein the length of the double-stranded region is 21-23 nucleotide pairs.

[0060] 24. The double-stranded RNAi agent of item 7, wherein each strand has 15-30 nucleotides.

[0061] 25. The double-stranded RNAi agent of item 1 or 7, wherein each strand has 19-30 nucleotides.

[0062] 26. A double-stranded RNAi agent as described in item 7, wherein the modifications on these nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl and combinations thereof.

[0063] 27. The double-stranded RNAi agent as described in item 26, wherein the modification on these nucleotides is 2'-O-methyl or 2'-fluoro modification.

[0064] 28. The double-stranded RNAi agent of item 1 or 7, wherein the ligand is one or more GalNAc derivatives connected via a divalent or trivalent branched linker.

[0065] 29. The double-stranded RNAi agent of item 1 or 7, wherein the ligand is

[0066]

[0067] 30. The double-stranded RNAi agent of item 1 or 7, wherein the ligand is linked to the 3' end of the sense strand.

[0068] 31. The double-stranded RNAi agent of item 30, wherein the RNAi agent is bound to the ligand, as shown in the following schematic diagram

[0069]

[0070] Wherein X is O or S.

[0071] 32. A double-stranded RNAi agent as described in item 1 or 7, wherein the agent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond.

[0072] 33. The double-stranded RNAi agent of item 32, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 3'-end of one strand.

[0073] 34. The double-stranded RNAi agent as described in item 33, wherein the strand is the antisense strand.

[0074] 35. The double-stranded RNAi agent of item 33, wherein the strand is the sense strand.

[0075] 36. The double-stranded RNAi agent of item 32, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 5'-end of one strand.

[0076] 37. The double-stranded RNAi agent as described in item 36, wherein the strand is the antisense strand.

[0077] 38. The double-stranded RNAi agent of item 36, wherein the strand is the sense strand.

[0078] 39. The double-stranded RNAi agent of item 32, wherein the phosphorothioate or methylphosphonate internucleotide bonds are located at the 5'-end and the 3'-end of one strand.

[0079] 40. The double-stranded RNAi agent of item 39, wherein the strand is the antisense strand.

[0080] 41. The double-stranded RNAi agent of claim 32, wherein the RNAi agent comprises 6-8 phosphorothioate internucleotide bonds.

[0081] 42. The double-stranded RNAi of item 41, wherein the antisense strand comprises two phosphorothioate internucleotide bonds at the 5'-end and two phosphorothioate internucleotide bonds at the 3'-end, and the sense strand comprises at least two phosphorothioate internucleotide bonds at either the 5'-end or the 3'-end.

[0082] 43. The double-stranded RNAi agent of item 1 or 7, wherein the base pair at position 1 of the 5′-end of the antisense strand of the double helix is ​​an AU base pair.

[0083] 44. The double-stranded RNAi agent of item 7, wherein the Y nucleotides contain 2'-fluorine modification.

[0084] 45. The double-stranded RNAi agent of item 7, wherein the Y' nucleotides contain 2'-O-methyl modifications.

[0085] 46. ​​The double-stranded RNAi agent as described in item 7, wherein p'>0.

[0086] 47. The double-stranded RNAi agent according to item 7, wherein p'=2.

[0087] 48. The double-stranded RNAi agent according to item 47, wherein q'=0, p=0, q=0, and the p' overhanging nucleotides are complementary to the target mRNA.

[0088] 49. The double-stranded RNAi agent according to item 47, wherein q'=0, p=0, q=0, and the p' overhanging nucleotide is not complementary to the target mRNA.

[0089] 50. The double-stranded RNAi agent of item 41, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0090] 51. A double-stranded RNAi agent as described in any one of items 46 to 50, wherein at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds.

[0091] 52. A double-stranded RNAi agent as described in item 51, wherein all n p 'Linked to adjacent nucleotides via phosphorothioate bonds.

[0092] 53. The double-stranded RNAi agent of item 1 or 7, wherein the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 1 or 2.

[0093] 54. The double-stranded RNAi agent of item 1, wherein the RNAi agent is selected from the group consisting of AD-62994 and AD-62933.

[0094] 55. The double-stranded RNAi agent according to item 7, wherein the RNAi agent is AD-62994 and AD-62933.

[0095] 56. A double-stranded RNAi agent capable of inhibiting the expression of HAO1 in cells,

[0096] The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region,

[0097] wherein the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 6, and the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 8,

[0098] wherein substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification,

[0099] wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-end,

[0100] wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification,

[0101] wherein the antisense strand comprises two phosphorothioate internucleotide bonds at the 5'-end and two phosphorothioate internucleotide bonds at the 3'-end, and

[0102] The sense strand is bound to one or more GalNAc derivatives, and the one or more GalNAc derivatives are connected via a branched divalent or trivalent linker at the 3'-end.

[0103] 57. The double-stranded RNAi agent of item 56, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise modifications.

[0104] 58. A double-stranded RNAi agent capable of inhibiting the expression of HAO1 (hydroxyacid oxidase 1) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of mRNA encoding HAO1, wherein the length of each strand is about 14 to about 30 nucleotides, wherein the double-stranded RNAi agent is represented by formula (III):

[0105] Meaning: 5'n p- N a- (XXX) i -N b- YYY-N b- (ZZZ) j- N a- n q 3'

[0106] Antonym: 3'n p '-Na '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)

[0107] in:

[0108] i, j, k and l are each independently 0 or 1;

[0109] p, p', q and q' are each independently 0-6;

[0110] Each N a and N a ' independently refers to an oligonucleotide sequence comprising 0-25 modified or unmodified nucleotides or a combination thereof, each sequence comprising at least two differently modified nucleotides;

[0111] Each N b and N b ' independently represents an oligonucleotide sequence comprising 0-10 modified or unmodified nucleotides or a combination thereof;

[0112] Each n p 、n p '、n q and n q ', each of which may or may not be present and independently represents an overhanging nucleotide;

[0113] XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif having three identical modifications on three consecutive nucleotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications;

[0114] N b The modification of N is different from that of Y and b The modification of ' is different from the modification of Y'; and

[0115] The sense strand is bound to at least one ligand.

[0116] 59. A double-stranded RNAi agent capable of inhibiting the expression of HAO1 (hydroxyacid oxidase 1) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of mRNA encoding HAO1, wherein the length of each strand is about 14 to about 30 nucleotides, wherein the double-stranded RNAi agent is represented by formula (III):

[0117] Meaning: 5'np- N a- (XXX) i -N b- YYY-N b- (ZZZ) j- N a- n q 3'

[0118] Antonym: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)

[0119] in:

[0120] i, j, k and l are each independently 0 or 1;

[0121] Each n p 、n q and n q ', each of which may or may not be present and independently represents an overhanging nucleotide;

[0122] p, q and q' are each independently 0-6;

[0123] n p '>0 and at least one n p 'Linked to adjacent nucleotides via phosphorothioate bonds;

[0124] Each N a and N a ' independently refers to an oligonucleotide sequence comprising 0-25 modified or unmodified nucleotides or a combination thereof, each sequence comprising at least two differently modified nucleotides;

[0125] Each N b and N b ' independently represents an oligonucleotide sequence comprising 0-10 modified or unmodified nucleotides or a combination thereof;

[0126] XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif having three identical modifications on three consecutive nucleotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications;

[0127] N b The modification of N is different from that of Y and b The modification of ' is different from the modification of Y'; and

[0128] The sense strand is bound to at least one ligand.

[0129] 60. A double-stranded RNAi agent capable of inhibiting the expression of HAO1 (hydroxyacid oxidase 1) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of mRNA encoding HAO1, wherein the length of each strand is about 14 to about 30 nucleotides, wherein the double-stranded RNAi agent is represented by formula (III):

[0130] Meaning: 5'n p- N a- (XXX) i -N b- YYY-N b- (ZZZ) j- N a- n q 3'

[0131] Antonym: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)

[0132] in:

[0133] i, j, k and l are each independently 0 or 1;

[0134] Each n p 、n q and n q ', each of which may or may not be present and independently represents an overhanging nucleotide;

[0135] p, q and q' are each independently 0-6;

[0136] n p '>0 and at least one n p 'Linked to adjacent nucleotides via phosphorothioate bonds;

[0137] Each N a and N a ' independently refers to an oligonucleotide sequence comprising 0-25 modified or unmodified nucleotides or a combination thereof, each sequence comprising at least two differently modified nucleotides;

[0138] Each N b and N b' independently represents an oligonucleotide sequence comprising 0-10 modified or unmodified nucleotides or a combination thereof;

[0139] XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif having three identical modifications on three consecutive nucleotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications;

[0140] N b The modification of N is different from that of Y and b The modification of ' is different from the modification of Y'; and

[0141] The sense strand is bound to at least one ligand, wherein the ligand is one or more GalNAc derivatives connected via a bivalent or trivalent branched linker.

[0142] 61. A double-stranded RNAi agent capable of inhibiting the expression of HAO1 (hydroxyacid oxidase 1) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of mRNA encoding HAO1, wherein the length of each strand is about 14 to about 30 nucleotides, wherein the double-stranded RNAi agent is represented by formula (III):

[0143] Meaning: 5'n p- N a- (XXX) i -N b- YYY-N b- (ZZZ) j- N a- n q 3'

[0144] Antonym: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)

[0145] in:

[0146] i, j, k and l are each independently 0 or 1;

[0147] Each n p 、n q and n q ', each of which may or may not be present and independently represents an overhanging nucleotide;

[0148] p, q and q' are each independently 0-6;

[0149] n p '>0 and at least one n p 'Linked to adjacent nucleotides via phosphorothioate bonds;

[0150] Each N a and N a ' independently refers to an oligonucleotide sequence comprising 0-25 modified or unmodified nucleotides or a combination thereof, each sequence comprising at least two differently modified nucleotides;

[0151] Each N b and N b ' independently represents an oligonucleotide sequence comprising 0-10 modified or unmodified nucleotides or a combination thereof;

[0152] XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif having three identical modifications on three consecutive nucleotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications;

[0153] N b The modification of N is different from that of Y and b The modification of ' is different from that of Y';

[0154] wherein the sense strand comprises at least one phosphorothioate bond; and

[0155] The sense strand is bound to at least one ligand, wherein the ligand is one or more GalNAc derivatives connected via a bivalent or trivalent branched linker.

[0156] 62. A double-stranded RNAi agent capable of inhibiting the expression of HAO1 (hydroxyacid oxidase 1) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of mRNA encoding HAO1, wherein the length of each strand is about 14 to about 30 nucleotides, wherein the double-stranded RNAi agent is represented by formula (III):

[0157] Meaning: 5'n p- N a- YYY-N a- n q 3'

[0158] Antonym: 3'n p '-N a '-Y'Y'Y'-N a '-n q '5' (IIIa)

[0159] in:

[0160] Each n p 、n q and n q ', each of which may or may not be present and independently represents an overhanging nucleotide;

[0161] p, q and q' are each independently 0-6;

[0162] n p '>0 and at least one n p 'Linked to adjacent nucleotides via phosphorothioate bonds;

[0163] Each N a and N a ' independently refers to an oligonucleotide sequence comprising 0-25 modified or unmodified nucleotides or a combination thereof, each sequence comprising at least two differently modified nucleotides;

[0164] YYY and Y'Y'Y' each independently represent a motif having three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications;

[0165] wherein the sense strand comprises at least one phosphorothioate bond; and

[0166] The sense strand is bound to at least one ligand, wherein the ligand is one or more GalNAc derivatives connected via a bivalent or trivalent branched linker.

[0167] 63. An RNAi agent selected from the group of RNAi agents listed in Table 1 or 2.

[0168] 64. The RNAi agent of item 52, wherein the RNAi agent is selected from the group consisting of AD-62994 and AD-62933.

[0169] 65. A composition comprising a modified antisense polynucleotide agent capable of inhibiting expression of HAO1 in a cell, wherein the agent comprises a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 1 or 2, wherein the polynucleotide is about 14 to about 30 nucleotides in length.

[0170] 66. A vector comprising the double-stranded RNAi agent according to any one of items 1, 7, 56 and 58 to 64.

[0171] 67. A cell containing the double-stranded RNAi agent of any one of items 1, 7, 56, and 58 to 64.

[0172] 68. A pharmaceutical composition comprising the double-stranded RNAi agent of any one of items 1, 7, 56 and 58 to 64, or the modified antisense polynucleotide agent of item 65, or the vector of item 66.

[0173] 69. A pharmaceutical composition as described in claim 68, wherein the RNAi agent is administered in an unbuffered solution.

[0174] 70. The pharmaceutical composition of claim 69, wherein the unbuffered solution is physiological saline or water.

[0175] 71. The pharmaceutical composition of item 68, wherein the siRNA is administered together with a buffer solution.

[0176] 72. A pharmaceutical composition as described in claim 71, wherein the buffer solution comprises acetate, citrate, prolamin, carbonate or phosphate or any combination thereof.

[0177] 73. The pharmaceutical composition of claim 72, wherein the buffer solution is phosphate buffered saline (PBS).

[0178] 74. A method for inhibiting HAO1 expression in a cell, the method comprising:

[0179] (a) contacting the cell with the double-stranded RNAi agent of any one of items 1, 7, 56 and 58 to 64, or the modified antisense polynucleotide agent of item 65, or the vector of item 66, or the pharmaceutical composition of any one of items 68 to 73; and

[0180] (b) maintaining the cells produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the HAO1 gene, thereby inhibiting the expression of the HAO1 gene in the cells.

[0181] 75. The method of claim 74, wherein the cell is located within a subject.

[0182] 76. The method of claim 75, wherein the subject is a human.

[0183] 77. The method of any one of items 74 to 76, wherein the HAO1 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

[0184] 78. A method for treating a subject suffering from an HAO1-related disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent as described in any one of items 1, 7, 56 and 58 to 64, or a modified antisense polynucleotide agent as described in item 65, or a vector as described in item 66, or a pharmaceutical composition as described in any one of items 68 to 73, thereby treating the subject.

[0185] 79. A method for treating a subject suffering from a HAO1-related disorder, comprising subcutaneously administering to the subject a therapeutically effective amount of a double-stranded RNAi agent,

[0186] The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region,

[0187] wherein the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 6, and the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 8,

[0188] wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification,

[0189] wherein the antisense strand comprises two phosphorothioate internucleotide bonds at the 5'-end and two phosphorothioate internucleotide bonds at the 3'-end,

[0190] wherein substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification,

[0191] wherein the sense strand comprises two phosphorothioate internucleotide bonds at the 5'-end and,

[0192] wherein the sense strand is conjugated to one or more GalNAc derivatives, the one or more GalNAc derivatives being linked via a branched divalent or trivalent linker at the 3' end, thereby treating the subject.

[0193] 80. The method of item 79, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise modifications.

[0194] 81. The method of item 78 or 79, wherein the subject is human.

[0195] 82. The method of claim 81, wherein the human suffers from PH1.

[0196] 83. The method of item 78 or 79, wherein the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 1 mg / kg to about 10 mg / kg.

[0197] 84. The method of claim 83, wherein the double-stranded RNAi agent is administered at a dose of about 0.1 mg / kg, about 1.0 mg / kg, or about 3.0 mg / kg.

[0198] 85. The method of claim 83, wherein the double-stranded RNAi agent is administered at a dose of about 1 mg / kg to about 10 mg / kg.

[0199] 86. A method as described in item 83, wherein the double-stranded RNAi agent is administered subcutaneously.

[0200] 87. The method of claim 83, wherein the double-stranded RNAi agent is administered intravenously.

[0201] 88. The method of claim 83, wherein the RNAi agent is administered in two or more doses.

[0202] 89. A method as described in item 88, wherein the RNAi agent is administered at an interval selected from the group consisting of once every about 12 hours, once every about 24 hours, once every about 48 hours, once every about 72 hours, and once every about 96 hours.

[0203] 90. The method of claim 88, wherein the RNAi agent is administered once a week for up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 5 weeks or longer. BRIEF DESCRIPTION OF THE DRAWINGS

[0204] Figure 1 The nucleotide sequence of Homo sapiens HAO1 mRNA is shown (SEQ ID NO: 1).

[0205] Figure 2 The nucleotide sequence of mouse (Mus musculus) HAO1 mRNA is shown (SEQ ID NO: 2).

[0206] Figure 3A A graph showing the results of in vitro screening of GO(HAO)GalNac-siRNA conjugates in primary Macaca mulatta hepatocytes.

[0207] Figure 3BGraph showing the dose response curve of GO(HAO)GalNac-siRNA conjugate in primary cynomolgus monkey hepatocytes.

[0208] Figure 4A Graph showing the results of in vivo evaluation of GO(HAO)GalNac-siRNA conjugates in C57B6 mice after a single administration.

[0209] Figure 4B Graphs showing the results of in vivo evaluation of GO(HAO)GalNac-siRNA conjugates in C57B6 mice after repeated dosing.

[0210] Figure 5A Graph showing urinary oxalate levels in AGXT knockout (KO) mice following treatment with GO(HAO)GalNac-siRNA conjugate.

[0211] Figure 5B Figure 2 shows urinary glycolate levels in AGXT KO mice after treatment with GO(HAO)GalNac-siRNA conjugate.

[0212] Fig. 6A Figure 2 is a graph showing AGXT mRNA levels in the rat model of PH1 72 hours after a single administration of AGXT siRNA.

[0213] Figure 6B Figure 2 shows urinary oxalate levels in a rat model of PH1 72 hours after treatment with GO(HAO)GalNac-siRNA conjugate.

[0214] Figure 6C Graph showing urinary oxalate levels in the rat model of PH1 following continued weekly dosing of AF-011-63102 and AD-62994 for 49 days with 24 hour urine collection as indicated on days 14 and 21.

[0215] Fig.6D Figure 2 is a graph showing the duration of HAO1 knockdown in rats. Figure 6C mRNA levels were measured one or four weeks after incubation with PBS (days 28 and 49 in PBS-treated rats) and are expressed relative to levels found in rats treated with PBS.

[0216] Figure 7 The reverse complement of the nucleotide sequence of Homo sapiens HAO1 mRNA (SEQ ID NO: 3) is shown.

[0217] Figure 8 The reverse complement of the nucleotide sequence of mouse HAO1 mRNA (SEQ ID NO: 4) is shown.

[0218] Fig. 9 The nucleotide sequence of cynomolgus monkey (Macaca fascicularis) HAO1 mRNA is shown (SEQ ID NO: 5).

[0219] Fig.10 The nucleotide sequence of Rattus norvegicus HAO1 mRNA is shown (SEQ ID NO: 6).

[0220] Fig.11 The reverse complement of the nucleotide sequence of cynomolgus monkey HAO1 mRNA (SEQ ID NO: 7) is shown.

[0221] Fig.12 The reverse complement of the nucleotide sequence of Rattus norvegicus HAO1 mRNA (SEQ ID NO: 8) is shown.

[0222] Fig.13 In vivo screening of GO GalNAc conjugates is demonstrated.

[0223] Fig.14 is a graph showing in vivo evaluation of GO-GalNAc conjugates in mice.

[0224] Fig.15 Figure 2 is a graph showing dose response evaluation of GO-GalNAc conjugate in mice.

[0225] Fig.16 Figure 2 is a graph showing dose response evaluation of GO-GalNAc conjugate in mice.

[0226] Fig.17 Figure 2 is a graph showing dose response evaluation in mice.

[0227] Fig.18 Two curves showing the relationship between mRNA knockdown and serum glycolate levels in mice.

[0228] Fig.19 Two graphs showing the relationship between mRNA knockdown and serum glycolate levels in rats.

[0229] Fig. 20 Graph showing dose-dependent inhibition of HAO1 mRNA by ALN-65585 in primary cynomolgus monkey hepatocytes.

[0230] Fig.21 Two graphs showing HAO1 mRNA and serum glycolate levels in mice after a single dose treatment with ALN-GO1.

[0231] Fig. 22is a graph showing the duration of HAO1 mRNA silencing in mice following a single dose treatment with ALN-GO1.

[0232] Fig.23 is a graph showing HAO1 mRNA and serum glycolate levels in rats after a single dose treatment with ALN-GO1.

[0233] Fig.24 Two graphs showing urinary oxalate and glycolate levels in a mouse model of primary type I hyperoxaluria following a single administration of ALN-GO1.

[0234] Fig.25A A graph showing HAO1 mRNA levels in a rat model of primary type I hyperoxaluria after a single administration of ALN-GO1.

[0235] Fig.25B Graph showing urinary oxalate levels in a rat model of primary type I hyperoxaluria following a single administration of ALN-GO1.

[0236] Fig.26 Two graphs showing HAO1 mRNA and urinary oxalate levels in a rat model of primary type I hyperoxaluria following repeated administration of ALN-GO1.

[0237] Fig. 27 Two graphs showing HAO1 mRNA and serum glycolate levels following repeated dosing in non-human primates.

[0238] Detailed description of the invention

[0239] The present invention provides a composition comprising an RNAi agent targeting HAO1, such as a double-stranded RNAi agent. The present invention also provides a method of using the composition of the present invention to inhibit HAO1 expression and treat HAO1-related disorders.

[0240] I. Definitions

[0241] In order to make the present invention more easily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values ​​for a parameter is stated, it is intended that the value and intermediate values ​​within the range of values ​​are also intended to be part of the present invention.

[0242] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. As an example, "an element" means one element or more than one element, such as a plurality of elements.

[0243] The term "comprising" is used herein to mean, and is interchangeable with, the phrase "including, but not limited to."

[0244] The term "or" is used herein to mean, and is interchangeable with, the term "and / or," unless the context clearly indicates otherwise.

[0245] As used herein, "HAO1" refers to the gene encoding the enzyme hydroxyacid oxidase 1. Other gene names include GO, GOX, GOX1, and HAOX1. The protein is also known as glycolate oxidase and (S)-2-hydroxyacid oxidase. The GenBank accession number for human HAO1 mRNA is NM_017545.2; cynomolgous monkey (Macacafascicularis) HAO1 mRNA is XM_005568381.1; Mouse (Mouse / Mus musculus) HAO1 mRNA is NM_010403.2; and rat (Rattus norvegicus) HAO1 mRNA is XM_006235096.1.

[0246] As used herein, the term "HAO1" also refers to naturally occurring DNA sequence variants of the HAO1 gene, such as single nucleotide polymorphisms (SNPs) in the HAO1 gene. Exemplary SNPs can be found in the NCBI dbSNP short gene variant database available at www.ncbi.nlm.nih.gov / projects / SNP.

[0247] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the HAO1 gene, including mRNA that is an RNA processing product of the primary transcription product.

[0248] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

[0249] "G", "C", "A" and "U" generally represent nucleotides containing guanine, cytosine, adenine and uracil as bases, respectively. "T" and "dT" are used interchangeably herein and refer to deoxyribonucleotides in which the nucleobase is thymine, such as deoxyribothymine, 2'-deoxythymidine or thymidine. However, it should be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" may also refer to modified nucleotides, as further described in detail below, or to alternative replacement moieties. It is well known to those of ordinary skill in the art that guanine, cytosine, adenine and uracil can be replaced with other moieties without substantially changing the base pairing properties of oligonucleotides comprising nucleotides carrying such replacement moieties. For example, but not limited to, a nucleotide comprising inosine as its base can base pair with a nucleotide containing adenine, cytosine or uracil. Therefore, a nucleotide containing uracil, guanine or adenine can be replaced in the nucleotide sequence of the present invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.

[0250] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interfering agent", as used interchangeably herein, refer to agents containing RNA as that term is defined herein, and which mediate targeted cleavage of RNA transcripts via an RNA induced silencing complex (RISC) pathway. iRNA directs sequence-specific degradation of mRNA via a process known as RNA interference (RNAi). iRNA regulates, for example, inhibits expression of HAO1 in a cell, for example, a cell within a subject (such as a mammalian subject).

[0251] In one embodiment, the RNAi agent of the present invention includes a single-stranded RNA that interacts with a target RNA sequence (e.g., a HAO1 target mRNA sequence) to guide the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that the long double-stranded RNA introduced into the cell is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al. (2001) Genes Dev. 15: 485). Dicer is a ribonuclease-III-like enzyme that processes dsRNA into short interfering RNAs of 19-23 base pairs characterized by two base 3' overhangs (Bernstein et al., (2001) Nature 409: 363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unfold the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107: 309). When bound to an appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15: 188). Therefore, in one aspect, the present invention relates to single-stranded RNA (siRNA) produced in a cell and which promotes the formation of a RISC complex to achieve silencing of a target gene (i.e., the HAO1 gene). Therefore, the term "siRNA" is also used herein to refer to RNAi as described above.

[0252] In another embodiment, the RNAi agent can be a single-stranded siRNA introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which in turn cleaves the target mRNA. Single-stranded siRNAs are typically 15-30 nucleotides and chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, each of which is incorporated herein by reference in its entirety. Any of the antisense nucleotide sequences described herein can be used as a single-stranded siRNA as described herein or chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0253] In another embodiment, the present invention provides a single-stranded antisense oligonucleotide molecule targeting HAO1. "Single-stranded antisense oligonucleotide molecule" is complementary to the sequence in the target mRNA (i.e., HAO1). Single-stranded antisense oligonucleotide molecules can inhibit translation in a stoichiometric manner by base pairing with mRNA and physically hindering the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Alternatively, single-stranded antisense oligonucleotide molecules inhibit target mRNA by hybridizing with the target and cleaving the target via an RNaseH cleavage event. Single-stranded antisense oligonucleotide molecules can be about 10 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, a single-stranded antisense oligonucleotide molecule may comprise a sequence in which at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides are from any of the antisense nucleotide sequences described herein, such as the sequences provided in any of Tables 1 or 2, or bind to any of the target sites described herein. The single-stranded antisense oligonucleotide molecule may comprise modified RNA, DNA, or a combination thereof.

[0254] In another embodiment, the "iRNA" used in the compositions, uses and methods of the present invention is a double-stranded RNA and is referred to herein as a "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent" or "dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double helix structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to the target RNA (i.e., HAO1 gene). In some embodiments of the present invention, the double-stranded RNA (dsRNA) causes the degradation of the target RNA (e.g., mRNA) via a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

[0255] Typically, most of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. In addition, as used in this specification, "RNAi agents" may include ribonucleotides with chemical modifications; RNAi agents may include substantial modifications at multiple nucleotides. Such modifications may include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications (as used in siRNA-type molecules) are covered by "RNAi agents".

[0256] The two chains forming the double helix structure may be different parts of a larger RNA molecule, or they may be separate RNA molecules. When the two chains are parts of a larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3'-end of one chain and the 5'-end of the respective other chains forming the double helix structure, the connecting RNA chain is referred to as a "hairpin loop". When the two chains are covalently connected by means other than an uninterrupted chain of nucleotides between the 3'-end of one chain and the 5'-end of the respective other chains forming the double helix structure, the connecting structure is referred to as a "connector". The RNA chains may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the double helix. In addition to the double helix structure, the RNAi agent may include one or more nucleotide overhangs.

[0257] In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence (e.g., a HAO1 target mRNA sequence) to guide the cleavage of the target RNA. Without wishing to be bound by theory, the long double-stranded RNA introduced into the cell is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al., Genes Dev. 2001, 15: 485). Dicer is a ribonuclease-III-like enzyme that processes dsRNA into short interfering RNAs of 19-23 base pairs characterized by two base 3' overhangs (Bernstein et al., (2001) Nature 409: 363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unfold the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107: 309). When bound to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0258] As used herein, "nucleotide overhang" refers to unpaired nucleotides that protrude from the double helical structure of the RNAi agent when the 3'-end of one strand of the RNAi agent extends beyond the 5'-end of the other strand, or vice versa. "Blunt" or "blunt end" means that there are no unpaired nucleotides at the ends of the double-stranded RNAi agent, that is, there are no nucleotide overhangs. "Blunt-ended" RNAi agents are dsRNAs that are double-stranded throughout their length, that is, there are no nucleotide overhangs at either end of the molecule. The RNAi agents of the present invention include RNAi agents having a nucleotide overhang at one end (that is, an agent having an overhang and a blunt end) or having nucleotide overhangs at both ends.

[0259] The term "antisense strand" refers to a strand of a double-stranded RNAi agent that includes a region that is substantially complementary to a target sequence (e.g., human HAO1 mRNA). As used herein, the term "region that is complementary to a portion of an mRNA encoding HAO1" refers to a region on the antisense strand that is substantially complementary to a portion of the HAO1 mRNA sequence. In the case where the complementary region is not completely complementary to the target sequence, mismatches are most tolerated to occur in the terminal region and, if present, typically occur in the terminal region, such as within 6, 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends.

[0260] As used herein, the term "sense strand" refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

[0261] As used herein, the term "cleavage region" refers to a region located adjacent to a cleavage site. A cleavage site is a site on a target where cleavage occurs. In some embodiments, the cleavage region comprises three bases located on either end of the cleavage site and adjacent to the cleavage site. In some embodiments, the cleavage region comprises two bases located on either end of the cleavage site and adjacent to the cleavage site. In some embodiments, the cleavage site is specifically present at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0262] As used herein and unless otherwise indicated, as will be understood by one of ordinary skill in the art, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a double helical structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Such conditions may, for example, be stringent conditions, wherein stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours, followed by washing. Other conditions may be applied, such as physiologically relevant conditions that may be encountered inside an organism. For example, complementary sequences are sufficient to allow the relevant functions of nucleic acids to occur, such as RNAi. One of ordinary skill in the art will be able to determine the set of conditions that are most suitable for testing the complementarity of two sequences based on the final application of the hybridized nucleotides.

[0263] When there is base pairing of nucleotides of a first nucleotide sequence with nucleotides of a second nucleotide sequence over the entire length of the first and second nucleotide sequences, the sequences may be "fully complementary" with respect to each. However, where a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but typically no more than 4, 3, or 2 mismatched base pairs upon hybridization while retaining the ability to hybridize under conditions most relevant to their ultimate application. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches with respect to complementarity determinations. For example, for the purposes described herein, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may still be referred to as "fully complementary".

[0264] As used herein, "complementary" sequences may also include non-Watson-Crick base pairs and / or base pairs formed by non-natural and modified nucleotides or may be formed entirely by these base pairs, as long as the above requirements for their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairings.

[0265] The terms "complementary", "fully complementary" and "substantially complementary" herein may be used in conjunction with base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as understood according to the context of their use.

[0266] As used herein, a polynucleotide that is "substantially complementary to at least a portion of a messenger RNA (mRNA)" refers to a polynucleotide that is substantially complementary to a contiguous portion of a relevant mRNA (e.g., an mRNA encoding HAO1) ​​including a 5'UTR, an open reading frame (ORF), or a 3'UTR. For example, if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding HAO1, then the polynucleotide is complementary to at least a portion of the HAO1 mRNA.

[0267] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any degree of inhibition.

[0268] As used herein, the phrase "inhibiting the expression of HAO1" includes inhibiting the expression of any HAO1 gene (such as mouse HAO1 gene, rat HAO1 gene, monkey HAO1 gene or human HAO1 gene) and variants (e.g. naturally occurring variants), or mutations of the HAO1 gene. Therefore, the HAO1 gene may be a wild-type HAO1 gene, a mutant HAO1 gene or a transgenic HAO1 gene (in the case of genetically manipulated cells), a group of cells or an organism.

[0269] “Inhibiting the expression of the HAO1 gene” includes any degree of inhibition of the HAO1 gene, for example, at least partially inhibiting the expression of the HAO1 gene, such as inhibiting at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%.

[0270] The expression of the HAO1 gene can be assessed based on the level of any variable associated with HAO1 gene expression (e.g., HAO1 mRNA level or HAO1 protein level in tissue and / or urinary oxalate level). Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a baseline level before administration or a level measured by a similar subject, cell or sample that is untreated or treated with a control (such as a buffer-only control or an inactive agent control).

[0271] As used herein, the phrase "contacting a cell with a double-stranded RNAi agent" includes contacting the cell by any possible means. Contacting a cell with a double-stranded RNAi agent includes contacting the cell with the RNAi agent in vitro or contacting the cell with the RNAi agent in vivo. The contact may be made directly or indirectly. Thus, for example, the RNAi agent may be brought into physiological contact with the cell by a subject performing the method, alternatively, the RNAi agent may be placed in a situation that permits or causes its subsequent contact with the cell.

[0272] Contacting cells in vitro can be performed, for example, by incubating the cells with an RNAi agent. Contacting cells in vivo can be performed, for example, by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another area, the bloodstream, or the subcutaneous space so that the agent will then reach the tissue where the cells to be contacted are located. For example, the RNAi agent may contain a ligand (e.g., a GalNAc3 ligand) and / or be coupled to a ligand that directs the RNAi agent to a relevant site, such as the liver. Combinations of in vitro and in vivo contact methods are also possible. With respect to the methods of the present invention, cells may also be contacted with RNAi agents in vitro and then transplanted into a subject.

[0273] As used herein, "subject" includes a human or non-human animal, preferably a vertebrate and more preferably a mammal. The subject may include a transgenic organism. The subject is most preferably a human, such as a human suffering from or prone to developing a HAO1-related disorder.

[0274] As used herein, "patient" or "subject" is intended to include human or non-human animals, preferably mammals, such as humans or monkeys. The subject or patient is most preferably a human.

[0275] As used herein, "HAO1-associated disorders" are intended to include disorders that can be treated or prevented by inhibiting the expression of HAO1, or symptoms thereof that can be alleviated. Examples include, but are not limited to, primary hyperoxaluria 1 (PH1).

[0276] As used herein, "therapeutically effective amount" is intended to include an amount of RNAi agent sufficient to achieve treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease) when administered to a patient for the treatment of an HAO1-related disease. The "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and history, age, weight, family history, genetic makeup, the state of the pathological process mediated by HAO1 expression, the type of previous or concomitant treatment, and other individual characteristics of the patient being treated, if any.

[0277] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent sufficient to prevent or improve a disease or one or more symptoms of the disease when administered to a subject who does not experience or show symptoms of a HAO1-related disease but may be susceptible to the disease. Improving a disease includes slowing the course of the disease or reducing the severity of a subsequently developed disease. A "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of disease risk and medical history, age, weight, family history, genetic makeup, type of previous or concomitant treatment, and other individual characteristics of the patient being treated, if any.

[0278] "Therapeutically effective amount" or "prophylactically effective amount" also includes the amount of RNAi agent that produces a desired local or systemic effect applicable to any treatment at a reasonable benefit / risk ratio. The RNAi agent used in the method of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0279] As used herein, the term "sample" includes a collection of similar body fluids, cells or tissues separated from a subject, as well as body fluids, cells or tissues in a subject. Examples of biological fluids include blood, serum and serosal fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva and the like. Tissue samples may include samples from tissues, organs or local areas. For example, a sample may be derived from a body fluid or cell in a specific organ, a part of an organ or these organs. In certain embodiments, a sample may be derived from the liver (e.g., a complete liver or certain fragments of the liver or certain types of cells in the liver, such as hepatocytes). In certain embodiments, "sample derived from a subject" refers to blood or plasma extracted from a subject. In other embodiments, "sample derived from a subject" refers to liver tissue (or its subcomponent) derived from a subject.

[0280] II. dsRNA iRNA Agents of the Present Invention

[0281] Described herein are double-stranded RNAi agents that inhibit expression of the HAO1 gene in a cell, such as a cell within a subject (eg, a mammal, such as a human suffering from an HAO1-related disorder), and uses of such double-stranded RNAi agents.

[0282] Therefore, the present invention provides a chemically modified double-stranded RNAi agent capable of inhibiting the expression of a target gene (ie, HAO1 gene) in vivo.

[0283] As described in more detail below, in certain aspects of the invention, substantially all of the nucleotides of the iRNA of the invention are modified. In other embodiments of the invention, the nucleotides of all of the iRNA of the invention are modified. The iRNA of the invention wherein "substantially all of the nucleotides are modified" is mostly but not completely modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0284] The RNAi agent comprises a sense strand and an antisense strand. The length of each strand of the RNAi agent may be in the range of 12-30 nucleotides. For example, the length of each strand may be between 14-30 nucleotides, 17-30 nucleotides, 19-30 nucleotides, 25-30 nucleotides, 27-30 nucleotides, 17-23 nucleotides, 17-21 nucleotides, 17-19 nucleotides, 19-25 nucleotides, 19-23 nucleotides, 19-21 nucleotides, 21-25 nucleotides, or 21-23 nucleotides.

[0285] Each strand can be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Each strand of the RNAi agent can be the same length or can be of different lengths.

[0286] The sense strand and the antisense strand typically form a double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent". The length of the double-stranded region of the RNAi agent may be 12-30 nucleotide pairs. For example, the length of the double-stranded region may be between 14-30 nucleotide pairs, 17-30 nucleotide pairs, 27-30 nucleotide pairs, 17-23 nucleotide pairs, 17-21 nucleotide pairs, 17-19 nucleotide pairs, 19-25 nucleotide pairs, 19-23 nucleotide pairs, 19-21 nucleotide pairs, 21-25 nucleotide pairs, or 21-23 nucleotide pairs. In another example, the length of the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs.

[0287] In one embodiment, the RNAi agent may contain one or more overhanging regions and / or capping groups at the 3'-end, 5'-end or both ends of one or both chains. The length of the overhang may be 1-6 nucleotides, for example, 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides or 1-2 nucleotides in length. The overhang may be a product in which one chain is longer than the other, or a product in which two chains of the same length are staggered. The overhang may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence or may be another sequence. The first and second chains may also be joined, for example, by other bases to form a hairpin, or by a non-basic connector.

[0288] In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide, including but not limited to a 2' sugar modification such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be an overhang sequence at either end on either strand. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence or can be another sequence.

[0289] The 5'-overhang or 3'-overhang at the sense strand, antisense strand, or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, wherein the two nucleotides may be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0290] RNAi agents may contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability. For example, a single-stranded overhang may be located at the 3'-end of the sense strand or the 3'-end of the antisense strand. RNAi may also have a blunt end, which is located at the 5'-end of the antisense strand (or the 3'-end of the sense strand) or vice versa. Typically, the antisense strand of RNAi has a nucleotide overhang at the 3'-end and a blunt end at the 5'-end. Although not wishing to be bound by theory, the asymmetric blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand help guide the strand load into the RISC process.

[0291] Synthesis and modification

[0292] Any of the nucleic acids characterized by the invention (e.g., RNAi) can be synthesized and / or modified by methods well recognized in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse linkage) or 3'-terminal modifications (conjugation, DNA nucleotide, reverse linkage, etc.); base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that pair with an expanded antibody library of a partner, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2'-position or 4'-position) or substitution of sugars; and / or backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of iRNA compounds suitable for use in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or non-natural internucleoside bonds. RNAs with modified backbones particularly include those without a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes mentioned in the art, modified RNAs without a phosphorus atom in the intranucleoside backbone may also be considered oligonucleosides. In some embodiments, modified iRNAs will have a phosphorus atom in their internucleoside backbone.

[0293] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates with normal 3'-5' bonds, 2'-5'-linked analogs of each of the above, and backbones with inverted polarity, wherein adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0294] Representative U.S. patents that teach the preparation of the above phosphorus-containing bonds include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,3 No. 21,131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5 ,587,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172,209; No. 6,239,265; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,444,423; No. 6,531,590; No. 6,534,639; No. 6,608,035; No. 6,683,167; No. 6,858,715; No. 6,867,294; No. 6,878,805; No. 7,015,315; No. 7,041,816; No. 7,273,933; No. 7,321,029; and U.S. Patent RE39464, the entire contents of each of which are incorporated herein by reference.

[0295] Modified RNA backbones that do not include phosphorus atoms have backbones formed from short-chain alkyl or cycloalkyl internucleoside bonds, mixed heteroatom and alkyl or cycloalkyl internucleoside bonds, or one or more short-chain heteroatom or heterocyclic internucleoside bonds. These backbones include backbones with morpholino bonds (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfenyl, sulfoxide and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; backbones containing olefins; sulfamate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S and CH2 components.

[0296] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5, No. 5,677,437; and No. 5,677,439, each of which is incorporated herein by reference in its entirety.

[0297] In other embodiments, suitable RNA mimics are contemplated for use in iRNAs, wherein the sugar and internucleoside bonds (i.e., the backbone) of the nucleotide units are replaced by new groups. The base units remain hybridized with appropriate nucleic acid target compounds. One such oligomeric compound (an RNA mimic that has been shown to have excellent hybridization properties) is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, specifically an aminoethylglycine backbone. The nucleobase is retained and the aza nitrogen atom is directly or indirectly bound to the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference in its entirety. Other compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0298] Some embodiments characterized by the present invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular, --CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- [referred to as methylene (methylimino) or MMI backbones], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [where the primary phosphodiester backbone is represented as --O--P--O--CH2--] of the above-mentioned U.S. Patent No. 5,489,677, and the amide backbone of the above-mentioned U.S. Patent No. 5,602,240. In some embodiments, the RNA characterized herein has a morpholino backbone structure of the above-mentioned U.S. Patent No. 5,034,506.

[0299] The modified RNA may also contain one or more substituted sugar moieties. The iRNAs (e.g., dsRNAs) characterized herein may include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n C3)]2, wherein n and m are 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: C1 to C 10Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA or group for improving the pharmacodynamic properties of iRNA and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), i.e., alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the Examples below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2.

[0300] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of the 5' terminal nucleotide. The iRNA can also have sugar mimetics, such as a cyclobutyl moiety replacing the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patents Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5, No. 5,670,633; and No. 5,700,920, some of which are commonly owned with the present application. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0301] iRNAs may also include modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxythymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine substituted uracil and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, and other substituted adenine and guanine at the 8-position, 5-halo (especially 5-bromo, 5-trifluoromethyl and other substituted uracil and cytosine at the 5-position), 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Other nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P., ed., Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, J. L., ed., John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, S. T. and Lebleu, B., eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds characterized by the present invention. These nucleobases include pyrimidines substituted at position 5, 6-azapyrimidines, and purines substituted at positions N-2, N-6, and 0-6, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6°C to 1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and is an exemplary base substitution, more particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0302] Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; No. 5,594,121, 5,596,091; No. 5,614,617; No. 5,681,941; No. 5,750,692; No. 6,015,886; No. 6,147,200; No. 6,166,197; No. 6,222,025; No. 6,235,887; No. 6,380,368; No. 6,528,640; No. 6,639,062; No. 6,617,438; No. 7,045,610; No. 7,427,672; and No. 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0303] The RNA of the iRNA can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with a modified ribose moiety, wherein the ribose moiety includes an external bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose into a 3'-endo configuration. It has been shown that adding locked nucleic acids to siRNA can increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0304] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is incorporated herein by reference in its entirety.

[0305] Potential stabilizing modifications at the ends of RNA molecules may include N-(acetylaminohexanoyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT) and other modifications. Disclosure of such modifications may be found in PCT Publication No. WO2011 / 005861.

[0306] Modified iRNA comprising the motif of the invention

[0307] In certain aspects of the invention, the double-stranded RNAi agent of the invention includes an agent with a chemical modification as disclosed in, for example, U.S. Provisional Application No. 61 / 561,710, filed on November 18, 2011, or PCT / US2012 / 065691, filed on November 16, 2012 and published as WO2013075035A1, the entire contents of each of which are incorporated herein by reference.

[0308] As shown herein and in provisional application No. 61 / 561,710, superior results can be obtained by introducing one or more motifs having three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand of an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the RNAi agent may be completely modified in other ways. The introduction of these motifs may interrupt the modification pattern of the sense and / or antisense strands, if present. The RNAi agent may optionally be combined with a GalNAc-derived ligand (e.g., on the sense strand). The resulting RNAi agent exhibits superior gene silencing activity.

[0309] More specifically, it was unexpectedly discovered that when the sense and antisense strands of a double-stranded RNAi agent are modified to have one or more motifs having three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent, the gene silencing activity of the RNAi agent is significantly enhanced.

[0310] In one embodiment, the RNAi agent is a double-terminated blunt of 19 nucleotides in length, wherein the sense strand contains at least one motif having three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 of the 5' end. The antisense strand contains at least one motif having three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end.

[0311] In another embodiment, the RNAi agent is a 20 nucleotide long double-terminated blunt, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 of the 5' end. The antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end.

[0312] In another embodiment, the RNAi agent is a double-terminated blunt of 21 nucleotides in length, wherein the sense strand contains at least one motif having three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 of the 5' end. The antisense strand contains at least one motif having three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end.

[0313] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif having three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 at the 5' end; the antisense strand contains at least one motif having three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 at the 5' end, wherein one end of the RNAi agent is blunt and the other end comprises a 2-nucleotide overhang. The 2-nucleotide overhang is preferably located at the 3' end of the antisense strand. When the 2-nucleotide overhang is located at the 3' end of the antisense strand, two phosphorothioate internucleotide bonds may exist between the terminal three nucleotides, wherein two of the three nucleotides are overhang nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide bonds between the terminal three nucleotides at the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, each nucleotide in the sense and antisense strands of the RNAi agent (including nucleotides that are part of the motif) is a modified nucleotide. In one embodiment, each residue is independently 2'-O-methyl or 3'-fluoro modified, for example in an alternative motif. The RNAi agent optionally further comprises a ligand (preferably GalNAc3).

[0314] In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the RNAi agent comprises a first strand of at least 25 and at most 29 nucleotides in length and a second strand of at most 30 nucleotides in length, which has at least one motif having three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer than the first strand at its 3' end, wherein the length of the duplex region is at least 25 nucleotides, and when the RNAi agent is introduced into a mammalian cell, the second strand is sufficient to be complementary to a target mRNA along at least 19 nucleotides of the length of the second strand to reduce target gene expression, and wherein dicer cleavage of the RNAi agent preferably generates an siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in a mammal. The RNAi agent optionally further comprises a ligand.

[0315] In one embodiment, the sense strand of the RNAi agent contains at least one motif having three identical modifications on three consecutive nucleotides, wherein one of the motifs is located at the cleavage site in the sense strand.

[0316] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif having three identical modifications on three consecutive nucleotides, wherein one of the motifs is located at or near the cleavage site in the antisense strand.

[0317] For RNAi agents with a duplex region of 17-23 nucleotides in length, the cleavage site of the antisense strand is usually around positions 10, 11, and 12 from the 5' end. Therefore, three identical modified motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, the count starting from the first nucleotide at the 5' end of the antisense strand, the count starting from the first paired nucleotide in the duplex region at the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the RNAi duplex region at the 5' end.

[0318] The sense strand of the RNAi agent may contain at least one motif having three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif having three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA double helix, the sense strand and the antisense strand may be arranged so that one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0319] In one embodiment, the sense strand of an RNAi agent may contain more than one motif having three identical modifications on three consecutive nucleotides. The first motif may be located at or near the cleavage site of the chain, and the other motif may be a wing modification. The term "wing modification" herein refers to a motif at another part of the chain that is separated from a motif at or near the cleavage site of the same chain. The wing modification is adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are adjacent to each other, the chemical properties of the motifs are different from each other, and when the motifs are separated by one or more nucleotides, the chemical properties may be the same or different. Two or more wing modifications may be present. For example, when there are two wing modifications, each wing modification may be located at one end relative to the first motif, which is located at or near the cleavage site or on either side of the main motif.

[0320] Similar to the sense strand, the antisense strand of the RNAi agent may contain one or more motifs having three identical modifications on three consecutive nucleotides, wherein at least one motif is located at or near the cleavage site of the strand. The antisense strand may also contain one or more wing modifications arranged in a similar manner to the wing modifications that may be present on the sense strand.

[0321] In one embodiment, wing modifications on the sense or antisense strand of the RNAi agent typically exclude the first one or two terminal nucleotides at the 3 '-end, 5 '-end, or both ends of the strand.

[0322] In another embodiment, the wing modifications on the sense or antisense strand of the RNAi agent typically exclude the first one or two paired nucleotides within the duplex region at the 3'-end, 5'-end, or both ends of the strand.

[0323] When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may be located at the same end of the duplex region and have an overlap of one, two or three nucleotides.

[0324] When the sense and antisense strands of the RNAi agent each contain at least two wing modifications, the sense and antisense strands can be arranged so that the two modifications each from one strand are located on one end of the duplex region with an overlap of one, two, or three nucleotides; the two modifications each from one strand are located on the other end of the duplex region with an overlap of one, two, or three nucleotides; the two modifications from one strand are located on each side of the main motif with an overlap of one, two, or three nucleotides in the duplex region.

[0325] In one embodiment, each nucleotide in the sense and antisense strands of the RNAi agent (including nucleotides that are part of the motif) can be modified. Each nucleotide can be modified with the same or different modifications, which can include one or more changes in one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens; changes in the composition of the ribose (e.g., the 2' hydroxyl group on the ribose); large-scale replacement of the phosphate moiety by a "dephospho" linker; modification or replacement of naturally occurring bases; and replacement or modification of the ribose-phosphate backbone.

[0326] Because nucleic acid is the polymer of subunit, many modifications are present in the position repeated in nucleic acid, such as the modification of non-connected O of base or phosphate moiety or phosphate moiety.In some cases, modification will be present in all target positions in nucleic acid, but in many cases it is not so.As an example, modification may be present only in 3' or 5' terminal position, may be present only in terminal region, such as at the position on terminal nucleotide or in the last 2,3,4,5 or 10 nucleotides of chain.Modification may be present in double-stranded region, single-stranded region or both thereof.Modification may be present only in the double-stranded region of RNA or may be present only in the single-stranded region of RNA.For example, phosphorothioate modification at non-connected O position may be present only in one or two ends, may be present only in a terminal region, such as at the position on terminal nucleotide or in the last 2,3,4,5 or 10 nucleotides of chain, or may be present in double-stranded and single-stranded regions, especially at the end.5' end can be phosphorylated.

[0327] It may, for example, enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang (e.g., in the 5' or 3' overhang or both). For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified (e.g., with modifications described herein). Modifications may include, for example, modification at the 2' position of the ribose using modifications known in the art, such as using modified deoxyribonucleotides, 2' to deoxy-2'-fluoro (2'-F) or 2'-O-methyl to replace the ribose of the nucleobase, and modifications in the phosphate group, such as phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0328] In one embodiment, each residue in the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. A strand may contain more than one modification. In one embodiment, each residue in the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0329] There are usually at least two different modifications on the sense strand and the antisense strand. These two modifications can be 2'-O-methyl or 2'-fluoro modifications, or other modifications.

[0330] In one embodiment, N a and / or N b Modifications comprising alternating patterns. As used herein, the term "alternating motif" refers to a motif having one or more modifications, wherein each modification is present on alternating nucleotides of a chain. Alternating nucleotides may refer to every other nucleotide or every third nucleotide, or a similar pattern. For example, if A, B, and C each represent a modification to a nucleotide, the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0331] The types of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent a modification to a nucleotide, the alternating pattern, i.e., the modification on every other nucleotide may be the same, but each of the sense strand or antisense strand may be selected from several modification possibilities within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...", etc.

[0332] In one embodiment, the RNAi agent of the present invention comprises a modification pattern of an alternating motif on the sense strand that is offset relative to the modification pattern of an alternating motif on the antisense strand. The offset can cause the modified nucleotide groups of the sense strand to correspond to the differently modified nucleotide groups of the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in the dsRNA double helix, the alternating motif in the sense strand can start with "ABABAB" at 5'-3' of the strand, and the alternating motif in the antisense strand can start with "BABABA" at 5'-3' of the strand in the double helix region. As another example, the alternating motif in the sense strand can start with "AABBAABB" at 5'-3' of the strand and the alternating motif in the antisense strand can start with "BBAABBAA" at 5'-3' of the strand in the double helix region, so that there is a complete or partial offset of the modification pattern between the sense strand and the antisense strand.

[0333] In one embodiment, the RNAi agent comprises a pattern of alternating motifs of 2'-O-methyl modifications and the 2'-F modifications on the sense strand are initially offset relative to the pattern of alternating motifs of 2'-O-methyl modifications, and the 2'-F modifications on the antisense strand are initially (i.e., 2'-O-methyl modified nucleotides on the sense strand) base paired with 2'-F modified nucleotides on the antisense strand, and vice versa. Position 1 of the sense strand may start with a 2'-F modification, and position 1 of the antisense strand may start with a 2'-O-methyl modification.

[0334] The introduction of one or more motifs having three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand can interrupt the initial modification pattern in the sense strand and / or antisense strand. This interruption of the modification pattern of the sense and / or antisense strand by introducing one or more motifs having three identical modifications on three consecutive nucleotides into the sense and / or antisense strand unexpectedly promotes gene silencing activity on the target gene.

[0335] In one embodiment, when a motif having three identical modifications on three consecutive nucleotides is introduced into any chain, the modification of the nucleotides next to the motif is a modification different from the modification of the motif. For example, the portion of the sequence containing the motif is "... N a YYYN b …”, where “Y” represents a modification with a motif of three identical modifications on three consecutive nucleotides, and “N a " and "N b " indicates a modification of the nucleotide immediately adjacent to the motif "YYY" that is different from the modification of Y, and wherein N a and N b Can be the same or different modifications. Alternatively, when a wing modification is present, N may or may not be present. a and / or N b .

[0336] The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may be present on any nucleotide of the sense strand or the antisense strand or both strands at any position of the strand. For example, the internucleotide bond modification may be present on each nucleotide on the sense strand and / or the antisense strand; each internucleotide bond modification may be present on the sense strand and / or the antisense strand in an alternating pattern; or the sense strand or the antisense strand may contain two internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide bond modification on the sense strand may have an offset relative to the alternating pattern of the internucleotide bond modification on the antisense strand.

[0337] In one embodiment, RNAi comprises phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region may contain two nucleotides, which have a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. The internucleotide bond modification may also connect the overhang nucleotide to the terminal paired nucleotide in the double helix region. For example, at least 2, 3, 4 or all overhang nucleotides may be connected via phosphorothioate or methylphosphonate internucleotide bonds, and other phosphorothioate or methylphosphonate internucleotide bonds connecting the overhang nucleotide to the paired nucleotide close to the overhang nucleotide may be present as appropriate. For example, at least two phosphorothioate internucleotide bonds may be present between the terminal three nucleotides, two of which are overhang nucleotides, and the third is a paired nucleotide close to the overhang nucleotide. The terminal three nucleotides may be located at the 3'-end of the antisense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, and / or the 5'-end of the antisense strand.

[0338] In one embodiment, a 2-nucleotide overhang is located at the 3'-end of the antisense strand, and there are two phosphorothioate internucleotide bonds between the terminal three nucleotides, wherein two of the three nucleotides are overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. The RNAi agent may optionally have two additional phosphorothioate internucleotide bonds between the terminal three nucleotides at the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0339] In one embodiment, the RNAi agent comprises a mismatch or combination thereof with a target within the duplex. Mismatches may be present in overhanging regions or in duplex regions. Base pairs may be ranked based on their propensity to promote resolution or fusion (e.g., based on the free energy of association or resolution of a particular pairing, the simplest approach being to examine pairing on individual pairs of bases, but then adjacent or similar analyses may also be used). A:U is preferred over G:C in promoting resolution; G:U is preferred over G:C; and I:C is preferred over G:C (I = inosine). Mismatches, e.g., atypical or in addition to typical pairings (as described elsewhere herein) are preferred over typical (A:T, A:U, G:C) pairings; and pairings including universal bases are preferred over typical pairings.

[0340] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4 or 5 base pairs within the duplex region at the 5' end of the antisense strand, independently selected from the group consisting of A:U, G:U, I:C and mismatch pairings, such as atypical or in addition to typical pairings or pairings including universal bases, to promote disassembly of the antisense strand at the 5' end of the duplex.

[0341] In one embodiment, the nucleotide at position 1 in the double helix region at the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT. Alternatively, at least one of the first 1, 2 or 3 base pairs in the double helix region at the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double helix region at the 5' end of the antisense strand is an AU base pair.

[0342] In one embodiment, the sense strand sequence can be represented by formula (I):

[0343] 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' (I)

[0344] in:

[0345] i and j are each independently 0 or 1;

[0346] p and q are each independently 0-6;

[0347] Each N a independently represent oligonucleotide sequences comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

[0348] Each N b independently represent an oligonucleotide sequence comprising 0-10 modified nucleotides;

[0349] Each n p and n q Overhanging nucleotides are represented independently;

[0350] Where N b and Y does not have the same modification; and

[0351] XXX, YYY and ZZZ each independently represent a motif having three identical modifications on three consecutive nucleotides. YYY is preferably a nucleotide that is all 2'-F modified.

[0352] In one embodiment, N a and / or N b Contains alternating patterns of modifications.

[0353] In one embodiment, the YYY motif is present at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif may be present at or near the cleavage site of the sense strand (e.g., may be present at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12 or 11, 12, 13), counting from the first nucleotide at the 5' end; or counting from the first pair of nucleotides in the duplex region at the 5' end as appropriate.

[0354] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. The sense strand can thus be represented by the formula:

[0355] 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib);

[0356] 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or

[0357] 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0358] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. a An oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides may independently be represented.

[0359] When the sense strand is represented as (Ic), N b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. a An oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides may independently be represented.

[0360] When the sense strand is represented by formula (Id), each N bN independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. b Preferably, it is 0, 1, 2, 3, 4, 5 or 6. Each N a An oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides may independently be represented.

[0361] Each of X, Y, and Z may be the same as or different from each other.

[0362] In other embodiments, i is 0 and j is 0, and the sense strand can be represented by the formula:

[0363] 5'n p -N a -YYY-N a -n q 3'(Ia).

[0364] When the sense strand is represented by formula (Ia), each N a An oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides may independently be represented.

[0365] In one embodiment, the antisense strand sequence of RNAi can be represented by formula (II):

[0366] 5'n q' -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3' (II)

[0367] in:

[0368] k and l are each independently 0 or 1;

[0369] p' and q' are each independently 0-6;

[0370] Each N a ' independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

[0371] Each N b ' independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

[0372] Each n p ' and n q 'Independently represent the overhanging nucleotides;

[0373] Where N b ' and Y' do not have the same modification;

[0374] and

[0375] X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif having three identical modifications on three consecutive nucleotides.

[0376] In one embodiment, N a ' and / or N b 'Contains alternating pattern modifications.

[0377] The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the Y'Y'Y' motif may be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide at the 5' end; or counting from the first pair of nucleotides at the 5' end in the duplex region as appropriate. The Y'Y'Y' motif is preferably present at positions 11, 12, 13.

[0378] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0379] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0380] The antisense strand can therefore be represented by the following formula:

[0381] 5'n q' -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p' 3'(IIb);

[0382] 5'n q' -N a '-Y'Y'Y'-N b '-X'X'X'-n p' 3'(IIc); or

[0383] 5'n q' -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p' 3'(IId).

[0384] When the antisense strand is represented by formula (IIb), N b ' represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0385] When the antisense strand is represented as (IIc), N b ' represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0386] When the antisense strand is represented by formula (IId), each N b ' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides. N b Preferably it is 0, 1, 2, 3, 4, 5 or 6.

[0387] In other embodiments, k is 0 and l is 0, and the antisense strand can be represented by the formula:

[0388] 5'n p' -N a' -Y'Y'Y'-N a' -n q' 3'(Ia).

[0389] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0390] Each of X', Y' and Z' may be the same as or different from each other.

[0391] Each nucleotide of the sense strand and the antisense strand can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro. For example, each nucleotide of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. Each of X, Y, Z, X', Y' and Z' can particularly represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0392] In one embodiment, when the duplex region is 21 nt, the sense strand of the RNAi agent may contain a YYY motif present at positions 9, 10, and 11 of the strand, counting from the first nucleotide at the 5' end; or counting from the first pair of nucleotides in the duplex region from the 5' end as appropriate; and Y represents a 2'-F modification. The sense strand may additionally contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0393] In one embodiment, the antisense strand may contain a Y'Y'Y' motif present at positions 11, 12, 13 of the strand, counting starting from the first nucleotide at the 5' end, or counting starting from the first pair of nucleotides at the 5' end in the duplex region as appropriate; and Y' represents a 2'-O-methyl modification. The antisense strand may additionally contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region; and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0394] The sense strand represented by any one of the above formulae (Ia), (Ib), (Ic) and (Id) forms a double helix with the antisense strand represented by any one of the formulae (IIa), (IIb), (IIc) and (IId).

[0395] Therefore, the RNAi agent used in the method of the present invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi double helix is ​​represented by formula (III):

[0396] Meaning: 5'n p- N a -(XXX) i- N b -YYY-N b- (ZZZ) j -N a -n q 3'

[0397] Antonym: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'

[0398] (III)

[0399] in:

[0400] i, j, k and l are each independently 0 or 1;

[0401] p, p', q and q' are each independently 0-6;

[0402] Each N a and N a ' independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

[0403] Each N b and N b ' independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

[0404] in

[0405] Each n p '、n p 、n q ' and n q , each of which may or may not be present, independently represents an overhanging nucleotide; and

[0406] XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif having three identical modifications on three consecutive nucleotides.

[0407] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or i and j are both 0; or i and j are both 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or k and l are both 0; or k and l are both 1.

[0408] Exemplary combinations of sense and antisense strands that form an RNAi duplex include the following:

[0409] 5'n p- N a- YYY-N a -n q 3'

[0410] 3'n p '-N a '-Y'Y'Y'-N a 'n q '5'

[0411] (IIIa)

[0412] 5'n p- N a- YYY-N b- ZZZ-N a -n q 3'

[0413] 3'n p '-N a '-Y'Y'Y'-N b '-Z'Z'Z'-N a 'n q '5'

[0414] (IIIb)

[0415] 5'n p -N a -XXX-N b- YYY-N a -n q 3'

[0416] 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N a '-n q '5'

[0417] (IIIc)

[0418] 5'n p- N a- XXX-N b -YYY-N b -ZZZ-N a -n q 3'

[0419] 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N b '-Z'Z'Z'-N a -n q '5'

[0420] (IIId)

[0421] When the RNAi agent is represented by formula (IIIa), each N a Independently refers to an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0422] When the RNAi agent is represented by formula (IIIb), each N b N independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. a Independently refers to an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0423] When the RNAi agent is represented by formula (IIIc), each N b 、N b ' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a Independently refers to an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0424] When the RNAi agent is represented by formula (IIId), each N b 、N b ' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a 、N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides. a 、N a '、N b and N b 'Independently contains alternating patterns of modifications.

[0425] Each of X, Y and Z in formula (III), (IIIa), (IIIb), (IIIc) and (IIId) may be the same as or different from each other.

[0426] When the RNAi agent is represented by Formula (III), (IIIa), (IIIb), (IIIc) and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides form a base pair with the corresponding Y' nucleotide; or all three Y nucleotides form a base pair with the corresponding Y' nucleotide.

[0427] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can form a base pair with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides form a base pair with the corresponding Z' nucleotide; or all three Z nucleotides form a base pair with the corresponding Z' nucleotide.

[0428] When the RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can form a base pair with one of the X' nucleotides. Alternatively, at least two of the X nucleotides form a base pair with the corresponding X' nucleotide; or all three X nucleotides form a base pair with the corresponding X' nucleotide.

[0429] In one embodiment, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide and / or the modification on the X nucleotide is different from the modification on the X' nucleotide.

[0430] In one embodiment, when the RNAi agent is represented by formula (IIId), N a In another embodiment, when the RNAi agent is represented by formula (IIId), N a Modification is 2'-O-methyl or 2'-fluoro, and n p '>0 and at least one n p ' is linked to the adjacent nucleotide via a phosphorothioate bond. In another embodiment, when the RNAi agent is represented by formula (IIId), N a Modified to 2'-O-methyl or 2'-fluoro, n p '>0 and at least one n p ' is linked to the adjacent nucleotide via a phosphorothioate bond, and the sense strand is bound to one or more GalNAc derivatives connected via a divalent or trivalent branched linker. In another embodiment, when the RNAi agent is represented by formula (IIId), N a Modified to 2'-O-methyl or 2'-fluoro, n p '>0 and at least one n p ' is linked to the adjacent nucleotide via a phosphorothioate bond, the sense strand comprises at least one phosphorothioate bond, and the sense strand is bound to one or more GalNAc derivatives connected via a divalent or trivalent branched linker.

[0431] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a Modified to 2'-O-methyl or 2'-fluoro, n p '>0 and at least one n p ' is linked to the adjacent nucleotide via a phosphorothioate bond, the sense strand comprises at least one phosphorothioate bond, and the sense strand is bound to one or more GalNAcs connected via a bivalent or trivalent branched linker.

[0432] In one embodiment, the RNAi agent is a polymer containing at least two double helices represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the double helices are connected by a linker. The linker may be cleavable or non-cleavable. The polymer further comprises a ligand as appropriate. Each of the double helices may target the same gene or two different genes; or each of the double helices may target two different target locations of the same gene.

[0433] In one embodiment, the RNAi agent is a polymer containing three, four, five, six or more double helices represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the double helices are connected by a linker. The linker may be cleavable or non-cleavable. The polymer further comprises a ligand as appropriate. Each of the double helices may target the same gene or two different genes; or each of the double helices may target two different target locations of the same gene.

[0434] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId) are linked to each other at the 5' end, and one or both of the 3' ends are optionally bound to a ligand. Each agent can target the same gene or two different genes; or each agent can target two different target locations of the same gene.

[0435] Many publications describe poly RNAi agents that can be used in the methods of the invention. Such publications include WO 2007 / 091269, U.S. Pat. No. 7,858,769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520, the entire contents of each of which are incorporated herein by reference.

[0436] RNAi agents containing one or more carbohydrate moieties combined with RNAi agents can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be connected to the modified subunit of the RNAi agent. For example, the ribose of one or more ribonucleotide subunits of the dsRNA agent can be replaced by another part, such as a non-carbohydrate (preferably cyclic) carrier connected to a carbohydrate ligand. The ribonucleotide subunit in which the ribose of the subunit has been replaced is referred to herein as a ribose replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic system, that is, all ring atoms are carbon atoms, or a heterocyclic system, that is, one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic carrier can be a monocyclic system, or can contain two or more rings, such as a fused ring. The cyclic carrier can be a fully saturated ring system, or it can contain one or more double bonds.

[0437] The ligand can be connected to the polynucleotide via the carrier. The carrier includes (i) at least one "main chain connection point", preferably two "main chain connection points" and (ii) at least one "tether connection point". As used herein, "main chain connection point" refers to a functional group, such as a hydroxyl group, or a bond that is generally available and suitable for incorporating the carrier into the main chain, such as a phosphate group, or a modified phosphate group, such as a sulfur-containing main chain of ribonucleic acid. In some embodiments, a "tether connection point" (TAP) refers to a constituent ring atom of a cyclic carrier, such as a carbon atom or a heteroatom (different from the atom providing the main chain connection point), which connects a selected portion. The portion can be, for example, a carbohydrate, such as a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide. The selected portion is optionally connected to the cyclic carrier by an intervening tether. Therefore, the cyclic carrier will generally include a functional group, such as an amino group, or generally provide a bond suitable for incorporating or tethering another chemical subject, such as incorporating or connecting a ligand into a constituent ring.

[0438] The RNAi agent can be bound to the ligand via a carrier, wherein the carrier can be a cyclic group or a non-cyclic group; the cyclic group is preferably selected from pyrrolidinyl, pyrazolyl, pyrazolidinyl, dihydroimidazolyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene; the non-cyclic group is preferably selected from a serinol backbone or a diethanolamine backbone.

[0439] In certain specific embodiments, the RNAi agent used in the methods of the present invention is an agent selected from the group of agents listed in any one of Tables 1 and 2. In one embodiment, when the agent is an agent listed in Table 1, the agent may not contain a terminal dT.

[0440] The present invention further includes a double-stranded RNAi agent comprising any of the sequences listed in any one of Tables 1 or 2, the sequence comprising a 5' phosphate or phosphate mimetic on the antisense strand (see, e.g., PCT Publication No. 2011005860). In addition, the present invention includes a double-stranded RNAi agent comprising any of the sequences listed in any one of Tables 1 or 2, the sequence comprising a 2'-fluoro group at the 5' end of the sense strand instead of a 2'-OMe group.

[0441] Other motifs

[0442] In certain aspects, a double-stranded RNAi agent described herein comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise less than eleven, ten, nine, eight, seven, six, or five 2'-deoxyfluorines.

[0443] In certain aspects, a double-stranded RNAi agent described herein comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise less than ten, nine, eight, seven, six, five, four phosphorothioate internucleotide linkages.

[0444] In certain aspects, a double-stranded RNAi agent described herein comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise less than ten 2'-deoxyfluorines and less than six phosphorothioate internucleotide linkages.

[0445] In certain aspects, a double-stranded RNAi agent described herein comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise less than eight 2'-deoxyfluorine and less than six phosphorothioate internucleotide linkages.

[0446] In certain aspects, a double-stranded RNAi agent described herein comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise less than nine 2'-deoxyfluorines and less than six phosphorothioate internucleotide linkages.

[0447] Ligand

[0448] The double-stranded RNAi agent of the present invention may be optionally bound to one or more ligands. The ligand may be attached to the sense strand, the antisense strand, or both strands at the 3'-end, the 5'-end, or both ends. For example, the ligand may be bound to the sense strand. In some embodiments, the ligand is bound to the 3'-end of the sense strand. In one embodiment, the ligand is a GalNAc ligand. In some specific embodiments, the ligand is GalNAc3. The ligand is coupled directly or indirectly (preferably covalently) via an intervening tether.

[0449] In some embodiments, a ligand alters the distribution, targeting, or lifetime of a molecule into which it is incorporated. In some embodiments, a ligand provides enhanced affinity for a selected target, such as a molecule, cell or cell type, compartment, receptor, such as a cell or organ compartment, tissue, organ, or region of the body, compared to a substance in the absence of such ligand. Ligands that provide enhanced affinity for a selected target are also referred to as targeting ligands.

[0450] Some ligands may have endocytic properties. Endococtic ligands promote the dissolution of endosomes and / or the delivery of the compositions of the invention or their components from endosomes to the cytoplasm of cells. Endococtic ligands may be multivalent anionic peptides or peptide mimetics that exhibit pH-dependent cell membrane activity and fusion. In one embodiment, the endocytic ligand assumes its active conformation at endosomal pH. An "active" conformation is one in which the endocytic ligand promotes the dissolution of endosomes and / or the delivery of the compositions of the invention or their components from endosomes to the cytoplasm of cells. Exemplary endocytic ligands include GALA peptides (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), EALA peptides (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586) and derivatives thereof (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, the endoplasmic reticulum component may contain chemical groups (eg, amino acids) that will undergo a change in charge or protonation in response to a change in pH. The endoplasmic reticulum component may be linear or branched.

[0451] Ligands can improve delivery, hybridization, and specificity properties and can also improve the nuclease resistance of the resulting natural or modified oligoribonucleotides, or polymeric molecules comprising any combination of monomers and / or natural or modified ribonucleotides described herein.

[0452] Ligands may generally include therapeutic modulators, e.g., for enhancing absorption; diagnostic compounds or reporter groups, e.g., for monitoring distribution; cross-linking agents; and moieties that confer nuclease resistance. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0453] The ligand may include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); carbohydrates (e.g., polydextrose, pullulan, chitin, polyglucosamine, inulin, cyclodextrin, or hyaluronic acid); or lipids. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid, an oligonucleotide (e.g., an aptamer). Examples of polyamino acids include polyamino acids such as polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0454] The ligand may also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid or protein (e.g., an antibody) that binds to a specific cell type, such as a kidney cell. The targeting group may be thyrotropin, melanocyte stimulating hormone, a lectin, a glycoprotein, surfactant protein A, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-galactosamine multivalent mannose, a multivalent trehalose, a glycosylated polyamino acid, a multivalent galactose, transferrin, a bisphosphonate, a polyglutamate, a polyaspartic acid, a lipid, cholesterol, a steroid, bile acid, folic acid, vitamin B12, biotin or an RGD peptide or RGD peptide mimetic or aptamer.

[0455] Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelators (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, tetraprenyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl) cholecalciferol, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., tentacle peptides, Tat peptides), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEGs (e.g., PEG-40K), MPEG, [MPEG]2, polyamino groups, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), delivery / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazoles, bisimidazoles, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl groups, HRP or AP.

[0456] The ligand may be a protein, such as a glycoprotein, or a peptide, such as a molecule with a specific affinity for a co-ligand, or an antibody, such as an antibody that binds to a specific cell type, such as a cancer cell, an endothelial cell, or a bone cell. The ligand may also include hormones and hormone receptors. It may also include non-peptide substances, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-galactosamine multivalent mannose, multivalent trehalose, or an aptamer. The ligand may be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0457] The ligand can be a substance, such as a drug, which can enhance the uptake of an iRNA agent in a cell, for example, by disrupting the cell's cytoskeleton, for example, by disrupting the cell's microtubules, microfilaments, and / or medium filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0458] The ligand can increase the uptake of the oligonucleotide in the cell by, for example, activating an inflammatory response. Exemplary ligands that would have this effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or interferon gamma.

[0459] In one aspect, the ligand is a lipid or lipid-based molecule. Such lipids or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA binding ligands allow the conjugate to be distributed to target tissues, such as non-kidney target tissues of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipids or lipid-based ligands can (a) increase the resistance of the conjugate to degradation, (b) improve targeting or delivery in target cells or cell membranes, and / or (c) can be used to regulate binding to serum proteins (such as HSA).

[0460] Lipid-based ligands can be used to modulate (e.g., control) the binding of a conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds more tightly to HSA will be less likely to be targeted to the kidneys and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds less tightly to HSA can be used to target a conjugate to the kidneys.

[0461] In one embodiment, the lipid-based ligand binds to HSA. It preferably binds to HSA with sufficient affinity such that the conjugate will preferentially distribute to non-renal tissues. In one embodiment, the affinity is such that the HSA-ligand binding is reversible. In another embodiment, the lipid-based ligand binds to HSA weakly or not at all such that the conjugate will preferentially distribute to the kidney. Other moieties that target kidney cells may also be used in place of or in addition to lipid-based ligands.

[0462] In another aspect, the ligand is a portion of, for example, a vitamin that is taken up by a target cell, for example, a proliferating cell. It is particularly useful for treating conditions characterized by unwanted cell proliferation, for example, malignant or non-malignant types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. HAS, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) are also included.

[0463] In another aspect, the ligand is a cell permeating agent, preferably a helical cell permeating agent. The agent is preferably amphoteric. Exemplary agents are peptides, such as tat or tentacles. If the agent is a peptide, it may be modified to include peptide mimetics, invertomers, non-peptide or pseudo-peptide bonds, and use D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and oleophobic phase.

[0464] The ligand may be a peptide or a peptide mimetic. A peptide mimetic (also referred to herein as an oligopeptide mimetic) is a molecule that can fold into a defined three-dimensional structure similar to a natural peptide. The length of the peptide or peptide mimetic portion may be about 5-50 amino acids, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length. The peptide or peptide mimetic may be, for example, a cell permeable peptide, a cationic peptide, an amphipathic peptide or a hydrophobic peptide (e.g., mainly composed of Tyr, Trp or Phe). The peptide portion may be a dendritic peptide, a defined peptide or a cross-linked peptide. In another alternative, the peptide portion may include a hydrophobic membrane translocation sequence (MTS). An exemplary peptide containing a hydrophobic MTS is an RFGF having an amino acid sequence of AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analog containing a hydrophobic MTS (e.g., an amino acid sequence of AALLPVLLAAP (SEQ ID NO: 10)) may also be a targeting portion. The peptide portion may be a "delivery" peptide that can carry large polar molecules (including peptides, oligonucleotides, and proteins) across the cell membrane. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the Drosophila antenna foot protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been found to be useful as delivery peptides. Peptides or peptide mimetics may be encoded by random DNA sequences, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354: 82-84, 1991). Peptides or peptide mimetics tethered to the iRNA agent via an incorporated monomer unit are cell targeting peptides, such as arginine-glycine-aspartic acid (RGD)-peptides or RGD mimetics. The length of the peptide portion may range from about 5 amino acids to about 40 amino acids. The peptide portion may have structural modifications, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be used. The RGD peptide portion can be used to target tumor cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can promote iRNA agents to target tumors of many other tissues, including the lungs, kidneys, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). The RGD peptide will preferably promote the targeting of iRNA agents to the kidneys. The RGD peptide can be linear or cyclic and can be modified (e.g., glycosylated or methylated) to facilitate targeting to specific tissues. For example, glycosylated RGD peptides can deliver iRNA agents to tumor cells expressing α Vβ3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides targeting markers enriched in proliferative cells can be used. For example, peptides and peptide mimetics containing RGD can target cancer cells, especially cells presenting integrins. Thus, RGD peptides, cyclic peptides containing RGD, RGD peptides including D-amino acids, and synthetic RGD mimetics can be used. In addition to RGD, other parts of integrin targeting ligands can be used. In general, such ligands can be used to control proliferative cells and angiogenesis. Some conjugates of this type of ligand target PECAM-1, VEGF, or other cancer genes, such as the cancer genes described herein.

[0465] "Cell penetrating peptides" are capable of penetrating cells, e.g., microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell penetrating peptides may be, for example, α-helical linear peptides (e.g., LL-37 or CeropinP1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two primary amino acids (e.g., PR-39 or indolicidin). Cell penetrating peptides may also include a nuclear localization signal (NLS). For example, a cell penetrating peptide may be a bipartite amphipathic peptide (such as MPG), which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the large T antigen of SV40 (Simeoni et al., Nucl. Acids Res. 31: 2717-2724, 2003).

[0466] In one embodiment, the targeting peptide may be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, BLP, cathelicidins, ceratotoxins, S. clava peptides, HFIAP, magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus peptides, esculentinis-1, and caerins. Many factors will be preferred to maintain the integrity of the helical stability. For example, the greatest number of helix stabilizing residues (e.g., leu, ala, or lys) will be used, and the least number of helix destabilizing residues (e.g., proline or cyclic monomer units) will be used. To account for terminal residues, for example, Gly is an exemplary N-terminal residue and / or C-terminal amidation can be used to provide additional H binding to stabilize the helix. Formation of salt bridges between residues and opposite charges separated by i±3 or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homoarginine, ornithine, or histidine can form salt bridges with anionic residues glutamic acid or aspartic acid.

[0467] Peptide and peptidomimetic ligands include ligands having naturally occurring modified peptides, such as D or L peptides; α, β or γ peptides; N-methyl peptides; aza peptides; peptides having one or more amides, i.e., peptide bonds replaced by one or more urea, thiourea, carbamate or sulfonylurea bonds; or cyclic peptides.

[0468] The targeting ligand can be any ligand capable of targeting a specific receptor. Examples are: folic acid, GalNAc, galactose, mannose, mannose-6P, clusters of sugars, such as GalNAc clusters, mannose clusters, galactose clusters, or aptamers. A cluster is a combination of two or more sugar units. Targeting ligands also include integrin receptor ligands, chemocytokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. Ligands can also be based on nucleic acids, such as aptamers. Aptamers can be unmodified or have any combination of modifications disclosed herein.

[0469] Endosomal release agents include imidazoles, poly- or oligoimidazoles, PEI, peptides, membrane fusogenic peptides, polycarboxylates, polyanions, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.

[0470] PK modulators refer to pharmacokinetic modulators. PK modulators include lipophiles, bile acid, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, bile acid, lithocholic acid, dialkyl glycerides, diacetyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. It is also known that oligonucleotides containing many phosphorothioate bonds bind to serum proteins, so short oligonucleotides containing multiple phosphorothioate bonds in the backbone (e.g., oligonucleotides having about 5 bases, 10 bases, 15 bases, or 20 bases) are also ligands of the present invention (e.g., as PK modulating ligands).

[0471] In addition, aptamers that bind to serum components (eg, serum proteins) are also used as PK modulating ligands in the present invention.

[0472] Other ligand binders covered by the present invention are described in U.S. patent applications USSN: 10 / 916,185 filed on August 10, 2004; USSN: 10 / 946,873 filed on September 21, 2004; USSN: 10 / 833,934 filed on August 3, 2007; USSN: 11 / 115,989 filed on April 27, 2005 and USSN: 11 / 944,227 filed on November 21, 2007, which are incorporated herein by reference in their entirety for all purposes.

[0473] When two or more ligands are present, the ligands may all have the same properties, all have different properties, or some ligands may have the same properties while others may have different properties. For example, the ligands may have targeting properties, have endocytic activity, or have PK modulating properties. In one embodiment, all ligands have different properties.

[0474] The ligand can be coupled to the oligonucleotide at many positions, such as the 3'-end, the 5'-end and / or an internal position. In some embodiments, the ligand is attached to the oligonucleotide via an intervening tether, such as a carrier described herein. The ligand or tethered ligand may be present on the monomer as it is incorporated into the growing chain. In some embodiments, the ligand may be incorporated via coupling to the "precursor" monomer after the "precursor" monomer is incorporated into the growing chain. For example, a monomer with, for example, an amino-terminal tether (i.e., without an associated ligand), such as TAP-(CH2) n NH2 can be incorporated into the growing oligonucleotide chain. In a subsequent operation, that is, after the precursor monomer is incorporated into the chain, a ligand having an electrophilic group (e.g., a pentafluorophenyl ester or aldehyde group) can then be attached to the precursor monomer by coupling the electrophilic group of the ligand to the terminal nucleophilic group of the tether of the precursor monomer.

[0475] In another example, a monomer having a chemical group suitable for participating in a click chemistry reaction, such as an azide or alkyne-terminated tether / linker, can be incorporated. In a subsequent operation, i.e., after the precursor monomer is incorporated into the chain, a ligand having a complementary chemical group (e.g., an alkyne or an azide group) can be attached to the precursor monomer by coupling the alkyne and azide groups together.

[0476] In some embodiments, the ligand may be bound to a nucleobase, a sugar moiety, or an internucleoside bond of a nucleic acid molecule. Binding to a purine nucleobase or a derivative thereof may occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is connected to a binder moiety. Binding to a pyrimidine nucleobase or a derivative thereof may also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase may be substituted by a binder moiety. Binding to the sugar moiety of a nucleoside may occur at any carbon atom. Example carbon atoms of the sugar moiety that may be connected to a binder moiety include 2', 3', and 5' carbon atoms. The 1' position may also be connected to a binder moiety, such as in a baseless residue. An internucleoside bond may also have a binder moiety. For phosphorus-containing bonds (e.g., phosphodiester, phosphorothioate, phosphorothioate, phosphoamidoamine, and the like), the binder moiety may be directly connected to the phosphorus atom or to an O, N, or S atom that is bound to the phosphorus atom. For amine-containing or amide-containing internucleoside linkages (eg, PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0477] GalNAc ligands and linkers

[0478] In some embodiments, the siRNA targeting the HAO1 gene is bound to a carbohydrate, such as a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, a polysaccharide. In some embodiments, the siRNA is bound to an N-acetylgalactosamine (GalNAc) ligand. The efficacy of delivery to hepatocytes after subcutaneous administration is promoted. The method of combining carbohydrates (such as N-acetylgalactosamine) with, for example, siRNA is well known to those of ordinary skill in the art. Examples can be found in US 8,106,022 and WO 2014 / 025805.

[0479] In some embodiments, the siRNA targeting the HAO1 gene is bound to a ligand, such as GalNac, via a linker. For example, the ligand may be one or more GalNAc (N-acetylglucosamine) derivatives connected via a divalent or trivalent branched linker.

[0480] In one embodiment, the dsRNA of the invention is bound to a divalent and trivalent branched linker, including the structure shown in any one of Formulas (IV)-(VII):

[0481]

[0482]

[0483] in:

[0484] q 2A ,q 2B ,q 3A ,q 3B 、q4 A ,q 4B ,q 5A ,q 5B and q 5C represents independently at each occurrence 0-20 and wherein the repeating units may be the same or different;

[0485] P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5Cis independently absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CHO at each occurrence;

[0486] Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently absent at each occurrence, alkylene, substituted alkylene, wherein one or more of the methylene groups may be substituted with O, S, S(O), SO2, N(R N ), C(R')=C(R"), C≡C or C(O) or terminated by one or more of them;

[0487] R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C Each occurrence is independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, or a heterocyclic group;

[0488] L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; that is, each independently at each occurrence is a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide or a polysaccharide; and

[0489] R a is H or an amino acid side chain.

[0490] Trivalent GalNAc-binding derivatives are particularly suitable for RNAi agents for inhibiting the expression of target genes, such as derivatives of formula (VII):

[0491]

[0492] Where L 5A , L 5B and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0493] Examples of suitable divalent and trivalent branched linking groups for conjugating GalNAc derivatives include, but are not limited to, the following compounds:

[0494]

[0495]

[0496]

[0497] Other ligands

[0498] In some embodiments, the ligand is selected from one of the following:

[0499]

[0500]

[0501] III. Delivery of iRNA of the Invention

[0502] Delivery of an iRNA agent of the invention to a cell, such as a cell within a subject, such as a human subject (e.g., a subject in need, such as a subject with an HAO1-related disorder) can be achieved in many different ways. For example, delivery can be performed by contacting a cell with an iRNA of the invention in vitro or in vivo. In vivo delivery can also be performed directly by administering a composition comprising an iRNA (e.g., dsRNA) to a subject. Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are further discussed below.

[0503] In general, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the invention (see, e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5): 139-144 and WO 94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, considerations associated with delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and aggregation of the delivered molecule in the target tissue. Nonspecific effects of iRNAs can be minimized by local administration, such as by direct injection or implantation into a tissue or by local administration of a formulation. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may otherwise be damaged by the agent or that may decompose the agent, and allows for a lower total dose of the iRNA molecule to be administered. Several studies have demonstrated successful blocking of gene expression of a gene product when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ et al. (2004) Retina 24: 132-138) and subretinal injection in mice (Reich, SJ et al. (2003) Mol. Vis. 9: 210-216) has been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther. 11: 267-274) and can prolong the survival of tumor-bearing mice (Kim, WJ et al. (2006) Mol. Ther. 14: 343-350; Li, S. et al. (2007) Mol. Ther. 15: 515-523).RNA interference has also been shown to be successfully delivered locally to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al., (2005) Gene Ther. 12:59-66; Makimura, H. et al., (2002) BMC Neurosci. 3:18; Shishkina, GT. et al., (2004) Neuroscience 129:521-528; Thakker, ER. et al., (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y. et al., (2005) J. Neurophysiol. 93:594-602) and delivered to the lungs by intranasal administration (Howard, KA. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For systemic administration of iRNA to treat disease, the RNA can be modified or delivered using a drug delivery system; both methods are used to prevent rapid degradation of dsRNA by endo- and exonucleases in vivo. Modification of RNA or pharmaceutical carriers can also allow iRNA compositions to be targeted to target tissues and avoid unwanted off-target effects. iRNA molecules can be modified by chemical binding to lipophilic groups (such as cholesterol) to enhance cellular uptake and prevent degradation. For example, iRNAs directed against ApoB bound to the lipophilic cholesterol portion were systemically injected into mice and resulted in blocking apoB mRNA gene expression in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432: 173-178). iRNA binding to aptamers has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO. et al., (2006) Nat. Biotechnol. 24: 1005-1015). In alternative embodiments, iRNAs can be delivered using drug delivery systems (such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems). Positively charged cationic delivery systems promote the binding of iRNA molecules (negatively charged) and enhance interactions at negatively charged cell membranes to allow cells to efficiently absorb iRNA. Cationic lipids, dendrimers or polymers can bind to iRNA or induce the formation of vesicles or micelles that enclose iRNA (see, e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116).Formation of vesicles or micelles further prevents iRNA decomposition when administered systemically. Methods for preparing and administering cation-iRNA complexes are well within the capabilities of those of ordinary skill in the art (see, e.g., Sorensen, DR. et al., (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al. (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems suitable for systemic delivery of iRNA include DOTAP (Sorensen, DR. et al., (2003), supra; Verma, UN. et al., (2003), supra), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al., (2006) Nature 441: 111-114), cardiolipin (Chien, PY. et al., (2005) Cancer Gene Ther. 12: 321-328; Pal, A. et al., (2005) Int J. Oncol. 26: 1087-1091), polyethyleneimine (Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3: 472-487) and polyamidoamines (Tomalia, DA. et al., (2007) Biochem. Soc. Trans. 35: 61-67; Yoo, H. et al., (1999) Pharm. Res. 16: 1799-1804). In some embodiments, the iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNA and cyclodextrin can be found in U.S. Pat. No. 7,427,605, which is incorporated herein by reference in its entirety.

[0504] Vector encoding the iRNA of the present invention

[0505] iRNA targeting the HAO1 gene can be expressed from a transcription unit inserted into a DNA or RNA vector (see, e.g., Couture, A et al., TIG. (1996), 12: 5-10; Skillern, A et al., International Publication No. WO 00 / 22113, Conrad, International PCT Publication No. WO 00 / 22114, and Conrad, U.S. Pat. No. 6,054,299). Depending on the specific construct used and the target tissue or cell type, expression can be transient (about hours to weeks) or persistent (weeks to months or longer). These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plastids (Gassmann et al., Proc. Natl. Acad. Sci. USA (1995) 92: 1292).

[0506] Individual chains or chains of iRNA can be transcribed from promoters on expression vectors. If two separate chains are to be expressed to generate, for example, dsRNA, the two separate expression vectors can be introduced together (e.g., by transfection or infection) into the target cell. Alternatively, each subject chain of the dsRNA can be transcribed by a promoter, all of which are located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence, so that the dsRNA has a backbone and loop structure.

[0507] The iRNA expression vector is usually a DNA plasmid or a viral vector. An expression vector compatible with eukaryotic cells, preferably compatible with vertebrate cells, can be used to produce a recombinant construct for the expression of iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from many commercial sources. Such vectors provided generally contain convenient restriction sites for the insertion of the desired nucleic acid fragment. The delivery of the iRNA expression vector can be systemic delivery, such as by intravenous or intramuscular administration, by administering to a target cell that is subsequently introduced into the patient after ex vivo planting from the patient, or by any other means that allows introduction into the desired target cell.

[0508] iRNA expression plasmids can be combined with cationic lipid vectors (such as oligophenamine) or non-cationic lipid-based vectors (such as Transit-TKO TM) complex into the target cell. The present invention also encompasses a variety of lipid transfections that target different regions of the target RNA for a period of one week or more. The successful introduction of the vector into the host cell can be monitored using a variety of known methods. For example, transient transfections can use reporters, such as fluorescent markers, such as green fluorescent protein (GFP) to signal. Markers that provide resistance to specific environmental factors (e.g., antibiotics and drugs) to the transfected cells (such as hygromycin B resistance) can be used to ensure stable transfection of cells in vitro.

[0509] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including, but not limited to, lentiviral vectors, moloneymurine leukemia virus, etc.; (c) adeno-associated viral vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, e.g., vaccinia virus vectors or avian poxviruses, e.g., canarypox or fowlpox; and (j) helper virus-dependent or naked adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors will or will not be incorporated into the cell genome. If desired, the construct may include viral sequences for transfection. Alternatively, the construct may be incorporated into a vector capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNAs will generally require regulatory elements (eg, promoters, enhancers, etc.) to ensure expression of the iRNA in the target cells. Other aspects of vectors and constructs to be considered are further described below.

[0510] Vectors suitable for delivering iRNA will include regulatory elements (promoters, enhancers, etc.) sufficient to express the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide constitutive or regulated / inducible expression.

[0511] The expression of iRNA can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to certain physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J. 8: 20-24). Such inducible expression systems suitable for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, dimerization chemical inducers, and isopropyl-β-D1-thiogalactoside (IPTG). One of ordinary skill in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0512] Viral vectors containing nucleic acid sequences encoding iRNA can be used. For example, retroviral vectors (see Miller et al., Meth. Enzymol. 217: 581-599 (1993)) can be used. These retroviral vectors contain the correct packaging of the viral genome and the necessary elements for integration into the host cell DNA. The nucleic acid sequence encoding the iRNA is cloned into one or more vectors, which promotes the delivery of nucleic acids to the patient. More details about retroviral vectors can be found in, for example, Boesen et al., Biotherapy 6: 291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make stem cells more resistant to chemotherapy. Other references describing the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, HIV-based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557 and 5,981,276, which are incorporated herein by reference.

[0513] Adenoviruses are also contemplated for delivery of the iRNAs of the present invention. Adenoviruses are particularly attractive vehicles, for example, for delivering genes to the respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium, where they cause mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrate the use of adenovirus vectors to transfer genes into respiratory epithelial cells of rhesus monkeys. Other cases of using adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO 94 / 12649; and Wang et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing the iRNA characterized by the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0514] Adeno-associated virus (AAV) vectors can also be used to deliver the iRNA of the present invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204: 289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, a U6 or H1 RNA promoter or a cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the characterized dsRNA of the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al., (1987), J. Virol. 61:3096-3101; Fisher KJ et al., (1996), J. Virol, 70:520-532; Samulski R et al., (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94 / 13788; and International Patent Application No. WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0515] Another viral vector suitable for delivering the iRNA of the invention is a poxvirus, such as vaccinia virus, for example, inactivated vaccinia, such as Modified Virus Ankara (MVA) or NYVAC, avian pox, such as fowlpox or canarypox.

[0516] The tropism of viral vectors can be modified by pseudopatterning the vectors with envelope proteins or other surface antigens from other viruses or by substituting different viral capsid proteins as desired. For example, lentiviral vectors can be pseudopatterned with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be prepared to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz JE et al., (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0517] The pharmaceutical preparation of the vector may include the vector in an acceptable diluent, or may include a slow release matrix embedded with the gene delivery vehicle. Alternatively, if the entire gene delivery vector can be manufactured intact from recombinant cells (e.g., retroviral vectors), the pharmaceutical preparation may include one or more cells that produce the gene delivery system.

[0518] IV. Pharmaceutical Compositions of the Present Invention

[0519] The present invention also includes pharmaceutical compositions and formulations comprising the iRNA of the present invention. In one embodiment, provided herein are pharmaceutical compositions containing the iRNA as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing the iRNA are suitable for treating HAO1-related diseases or conditions. Such pharmaceutical compositions are formulated based on the delivery mode.

[0520] Pharmaceutical compositions comprising RNAi agents of the invention may be, for example, solutions with or without buffers, or compositions containing pharmaceutically acceptable carriers. Such compositions include, for example, aqueous or crystalline compositions, lipid formulations, micelle formulations, emulsions, and gene therapy vectors.

[0521] In the method of the present invention, the RNAi agent can be administered in a solution. The free RNAi agent can be administered in an unbuffered solution, such as in saline or water. Alternatively, the free siRNA can also be administered in a suitable buffer solution. The buffer solution can contain acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer solution containing the RNAi agent can be adjusted to make it suitable for administration to a subject.

[0522] In some embodiments, the buffer solution further comprises an agent for controlling the osmotic concentration of the solution so that the osmotic concentration is maintained at a desired value, such as a physiological value in human plasma. Solutes that can be added to the buffer solution to control the osmotic concentration include, but are not limited to, proteins, peptides, amino acids, non-metabolizable polymers, vitamins, ions, carbohydrates, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmotic concentration of the solution is a salt. In certain embodiments, the agent for controlling the osmotic concentration of the solution is sodium chloride or potassium chloride.

[0523] The pharmaceutical composition of the present invention can be administered at a dose sufficient to inhibit the expression of the HAO1 gene.

[0524] dose

[0525] Typically, suitable dosages of iRNAs of the invention will be within the range of about 0.001 to about 200.0 mg per kg of recipient body weight per day, typically within the range of about 0.1 to 10 mg or 1 to 50 mg per kg of body weight per day. For example, dsRNAs can be administered at about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per single dose.

[0526] In another embodiment, the RNAi agent, e.g., dsRNA, is administered at a dose of about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / mg, about 1.5 to about 50 mg / kg, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.1 to about 45 mg / kg, about 0.25 to about 45 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / kg, about 1.5 to about 45 mg / kg, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg , about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / kg, about 1.5 to about 40 mg / kg, about 2 to about 40 mg / kg, mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0. 1 to about 20 mg / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / mg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and ranges intermediate to these values ​​are also intended to be part of this invention.

[0527] For example, an RNAi agent, such as a dsRNA, can be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17. 48, 49, or about 50 mg / kg. Values ​​and ranges intermediate to these values ​​are also intended to be part of this invention.

[0528] In certain embodiments of the invention, for example, when a double-stranded RNAi agent comprises a modification (e.g., one or more motifs having three identical modifications on three consecutive nucleotides, including one such motif at or near the cleavage site of the agent), six phosphorothioate bonds, and a ligand, such an agent is administered at a dose of about 0.01 to about 0.5 mg / kg, about 0.01 to about 0.4 mg / kg, about 0.01 to about 0.3 mg / kg, about 0.01 to about 0.2 mg / kg, about 0.01 to about 0.1 mg / kg, about 0.01 mg / kg to about 0.09 mg / kg, about 0.01 mg / kg to about 0.08 mg / kg, about 0.01 mg / kg to about 0.07 mg / kg. g / kg, about 0.01 mg / kg to about 0.06 mg / kg, about 0.01 mg / kg to about 0.05 mg / kg, about 0.02 to about 0.5 mg / kg, about 0.02 to about 0.4 mg / kg, about 0.02 to about 0.3 mg / kg, about 0.02 to about 0.2 mg / kg, about 0.02 to about 0.1 mg / kg, about 0.02 mg / kg to about 0.09 mg / kg, about 0.02 mg / kg to about 0.08 mg / kg, about 0.02 mg / kg to about 0.07 mg / kg, about 0.02 mg / kg to about 0.06 mg / kg, about 0.02 mg / kg to about 0.05 mg / kg, about 0.03 to about 0. 5mg / kg, about 0.03 to about 0.4mg / kg, about 0.03 to about 0.3mg / kg, about 0.03 to about 0.2mg / kg, about 0.03 to about 0.1mg / kg, about 0.03mg / kg to about 0.09mg / kg, about 0.03mg / kg to about 0.08mg / kg, about 0.03mg / kg to about 0.07mg / kg, about 0.03mg / kg to about 0.06mg / kg, about 0.03mg / kg to about 0.05mg / kg, about 0.04 to about 0.5mg / kg, about 0.04 to about 0.4mg / kg, about 0.04 to about 0.3mg / kg, about 0.04 to about 0.2mg / kg, about 0.03mg / kg to about 0.1mg / kg, about 0.03mg / kg to about 0.09mg / kg, about 0.03mg / kg to about 0.08mg / kg, about 0.03mg / kg to about 0.07mg / kg, about 0.03mg / kg to about 0.06mg / kg, about 0.03mg / kg to about 0.05mg / kg, about 0.04 to about 0.5mg / kg, about 0.04 to about 0.4mg / kg, about 0.04 to about 0.3mg / kg, about 0.04 to about 0.2mg / kg, about 0 In some embodiments, the present invention relates to a method for preparing an aqueous solution of at least one embodiment of the present invention, wherein the aqueous solution contains at least one iodine, at least one iodine and at least one iodine. In some embodiments, the aqueous solution contains at least one iodine, at least one iodine and at least one iodine. In some embodiments, the aqueous solution contains at least one iodine, at least one iodine and at least one iodine. In some embodiments, the aqueous solution contains at least one iodine, at least one iodine and at least one iodine.Values ​​and ranges intermediate to the aforementioned values ​​are also intended to be part of the invention, for example, the RNAi agent can be administered to a subject at a dosage of about 0.015 mg / kg to about 0.45 mg / mg.

[0529] For example, an RNAi agent, such as an RNAi agent in a pharmaceutical composition, can be administered at a dose of about 0.01 mg / kg, 0.0125 mg / kg, 0.015 mg / kg, 0.0175 mg / kg, 0.02 mg / kg, 0.0225 mg / kg, 0.025 mg / kg, 0.0275 mg / kg, 0.03 mg / kg, 0.0325 mg / kg, 0.035 mg / kg, 0.0375mg / kg, 0.04mg / kg, 0.0425mg / kg, 0.045mg / kg, 0.0475mg / kg, 0.05mg / kg, 0.05 25mg / kg, 0.055mg / kg, 0.0575mg / kg, 0.06mg / kg, 0.0625mg / kg, 0.065mg / kg, 0.0675mg / kg, 0 .07mg / kg, 0.0725mg / kg, 0.075mg / kg, 0.0775mg / kg, 0.08mg / kg, 0.0825mg / kg, 0.085mg / kg, 0.0875mg / kg, 0.09mg / kg, 0.0925mg / kg, 0.095mg / kg, 0.0975mg / kg, 0.1mg / kg, 0.125mg / kg, 0.15 mg / kg, 0.175 mg / kg, 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg, 0.325 mg / kg, 0.35 mg / kg, 0.375 mg / kg, 0.4 mg / kg, 0.425 mg / kg, 0.45 mg / kg, 0.475 mg / kg or about 0.5 mg / kg. Values ​​and ranges intermediate to the above recited values ​​are also intended to be part of this invention.

[0530] Treatment options

[0531] The pharmaceutical composition can be given once a day, or the iRNA can be given in two, three or more sub-doses at appropriate intervals throughout the day or even continuously infused or delivered using a controlled release formulation. In this case, the iRNA contained in each sub-dose must be correspondingly less to reach the total daily dose. The dosage unit can also be compounded to deliver over several days, for example, using a conventional sustained release formulation that provides sustained release of the iRNA over several days. Sustained release formulations are well known in the art and are particularly suitable for delivering agents to specific sites, such as agents that can be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding plurality of daily doses.

[0532] In other embodiments, a single administration of the pharmaceutical composition may be sustained, with subsequent doses being administered at intervals of no more than 3, 4, or 5 days, or at intervals of no more than 1, 2, 3, or 4 weeks. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once a week. In other embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once every two months.

[0533] Generally, one of ordinary skill in the art will appreciate that certain factors may affect the dosage and timing of effective treatment of a subject, including, but not limited to, the severity of the disease or condition, previous treatment, the general health and / or age of the subject, and other diseases present. In addition, treatment of a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. The assessment of the effective dosage and in vivo half-life of individual iRNAs encompassed by the present invention can be performed using conventional methods.

[0534] The evaluation of the effective dose and in vivo half-life of the individual iRNAs encompassed by the present invention can also be performed based on in vivo testing using suitable animal models. For example, the development of mouse genetics has produced many mouse models for the study of a variety of human diseases (such as disorders associated with the expression of HAO1). Such models can be used for in vivo testing of iRNAs, and for determining therapeutically effective doses. Suitable mouse models are known in the art and include, for example, animal models described herein.

[0535] Giving Methods

[0536] The pharmaceutical compositions of the present invention may be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (e.g., by a skin patch), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including by a nebulizer), intratracheal, intranasal, epidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, for example, via an implant device; or intracranial, for example, by intraparenchymal, intrathecal, or intraventricular administration.

[0537] iRNA can be delivered in a manner that targets specific tissues, such as the liver.

[0538] Preparations

[0539] The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and formulations containing liposomes. These compositions can be produced from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids.

[0540] The pharmaceutical formulations of the present invention, which are preferably presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing the active ingredient into association with a pharmaceutical carrier or excipient. In general, the formulation is prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely powdered solid carrier or both, and then, if necessary, shaping the product.

[0541] The compositions of the present invention may be formulated into any of a number of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid pastes, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or polydextrose. The suspension may also contain a stabilizer.

[0542] The compositions of the invention may be formulated for oral administration, parenteral, intraparenchymal (to the brain), intrathecal, intraventricular or intrahepatic administration, and / or topical administration.

[0543] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or microtablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or adhesives may be required. In some embodiments, oral formulations are formulations administered in combination with one or more penetration enhancing surfactants and chelating agents characterized by the present invention. Suitable surfactants include fatty acids and / or their esters or salts, bile acids and / or their salts. Suitable bile acids / salts include chenodeoxychenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glutamic acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, tauro-24,25-dihydro-sodium fusidate and sodium glycodihydrofusidate. Suitable fatty acids include eicosatetraenoic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicapric acid, tricapric acid, monoolein, dilaurin, 1-monocapric glyceride, 1-dodecylazacycloheptane-2-ketone, acylcarnitines, acylcholines or monoglycerides, diglycerides or their pharmaceutically acceptable salts (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as a combination of fatty acids / salts and bile acids / salts. An exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Other penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The DsRNA characterized by the present invention can include a granular form of spray-dried particles or a granular form of a composite micron or nanoparticle for oral delivery. DsRNA complexes include polyamino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkyl cyanoacrylates; cationic gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkyl cyanoacrylates; DEAE-derivatized polyimines, pullulan, cellulose and starch. Suitable complexing agents include polyglucosamine, N-trimethyl polyglucosamine, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polydiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), poly(isohexyl cyanoacrylate), DEAE-methacrylate, DEAE-hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-polydextrose, polymethyl acrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid)-co-glycolic acid (PLGA), alginate and polyethylene glycol (PEG).Oral formulations of dsRNA and their preparation are described in detail in US Pat. No. 6,887,906, US Publication No. 20030027780, and US Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0544] Compositions and formulations for parenteral, intraparenchymal (to the brain), intrathecal, intraventricular, or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0545] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be applicable. Suitable topical formulations include iRNAs characterized by the present invention mixed with surface delivery agents (such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants). Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA characterized by the present invention can be encapsulated in liposomes or can form complexes thereon (especially for cationic liposomes). Alternatively, the iRNA can be complexed with lipids, especially cationic lipids. Suitable fatty acids and esters include, but are not limited to, eicosatetraenoic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, 1-monocaprin, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines or C 1-20 Alkyl esters (eg, isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof.Topical formulations are described in detail in US Pat. No. 6,747,014, which is incorporated herein by reference.

[0546] iRNA formulations containing membrane-bound molecular assemblies

[0547] The iRNA used in the compositions and methods of the present invention can be formulated for delivery in a membrane molecular assembly, such as a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in the form of at least one bilayer (e.g., one bilayer or a plurality of bilayers). Liposomes include single-layer and multi-layer vesicles having a membrane formed by a lipophilic substance and an aqueous internal substance. The aqueous portion contains an iRNA composition. The lipophilic substance separates the aqueous internal substance from the aqueous external substance, which generally does not include an iRNA composition, but in some instances it may include an iRNA composition. Liposomes are suitable for transferring and delivering active ingredients to the site of action. Because the lipid membrane is structurally similar to a biological membrane, when the liposome is applied to a tissue, the lipid bilayer fuses with the bilayer of the cell membrane. As the liposome and cell merge, the internal aqueous contents including the iRNA are delivered to the cell, where the iRNA can specifically bind to the target RNA and can mediate RNAi. In some cases, liposomes are also specifically targeted, for example, to guide the iRNA to a specific cell type.

[0548] Liposomes containing RNAi agents can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed by the lipid component. For example, the lipid component can be amphoteric cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles including the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example by dialysis, to produce a lipid preparation of the RNAi agent.

[0549] If desired, a carrier compound that aids condensation may be added during the condensation reaction, for example by controlled addition. For example, the carrier compound may be a polymer other than a nucleic acid (such as spermine or spermidine). The pH may also be adjusted to promote condensation.

[0550] Methods for producing stable polynucleotide delivery vehicles (which incorporate polynucleotide / cationic lipid complexes as structural components of the delivery vehicle) are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation may also include one or more aspects of the exemplary methods described in the following references: Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Common techniques for preparing lipid aggregates of a suitable size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., Biochim. Biophys. Acta 858:161, 1986). When consistently small (50 to 200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew et al., Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to encapsulate RNAi agent formulations into liposomes.

[0551] Liposomes are divided into two major categories. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complex binds to the negatively charged cell surface and is internalized in the endosome. Due to the acidic pH in the endosome, the liposomes rupture and release their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0552] pH-sensitive or negatively charged liposomes capture nucleic acids rather than complexing with them. Because nucleic acids and lipids are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acids are encapsulated in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of exogenous genes was detected in target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0553] The lipid composition of a kind of main type comprises the phospholipid except the choline phospholipid of natural derivation.Neutral liposome composition can be formed by dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) for example.Anionic liposome composition is usually formed by dimyristoylphosphatidylglycerol, and anionic membrane fusion liposome is mainly formed by dioleoylphosphatidylethanolamine (DOPE).Another type of lipid composition is formed by phosphatidylcholine (PC) (such as soybean PC and egg PC).Another type is formed by the mixture of phospholipid and / or phosphatidylcholine and / or cholesterol.

[0554] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0555] Nonionic lipid systems, particularly those containing nonionic surfactants and cholesterol, have also been examined for their effectiveness in delivering drugs to the skin. TM I (Dilaurin / Cholesterol / Polyoxyethylene-10-stearyl ether) and Novasome TM A nonionic lipid formulation of II (distearin / cholesterol / polyoxyethylene-10-stearyl ether) was used to deliver cyclosporine A into the dermis of mouse skin. The results indicate that such a nonionic lipid system effectively promotes the deposition of cyclosporine A into different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4(6), 466).

[0556] Liposomes also include "sterically stabilized" liposomes, as used herein, meaning that the liposomes contain one or more special lipids that, when incorporated into the liposomes, result in an extended circulation life relative to liposomes lacking such special lipids. Examples of sterically stabilized liposomes are liposomes in which the portion (A) that forms the vesicle portion of the liposomes contains one or more glycolipids, such as monosialoganglioside G M1 Or (B) derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. While not wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelins, or PEG-derivatized lipids, the extended circulation half-life of these sterically stabilized liposomes is due to reduced uptake in cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0557] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) reported monosialoganglioside G M1 , galactocerebroside sulfate, and phosphatidyl cyclohexane hexol improve the blood half-life of liposomes. These results are described in detail by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and WO 88 / 04924 issued to Allen et al. disclose liposomes comprising: (1) sphingomyelin and (2) ganglioside G M1 or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0558] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes (although not as effectively fused with the plasma membrane) are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

[0559] Other advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a variety of water- and lipid-soluble drugs; liposomes can protect encapsulated RNAi agents from metabolism and degradation in their intrinsic compartments (Rosoff, "Pharmaceutical Dosage Forms", Lieberman, Rieger and Banker (eds.), 1988, Vol. 1, p. 245). Important considerations in preparing liposome formulations are the lipid surface charge, vesicle size and aqueous volume of the liposomes.

[0560] The positively charged synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-to-nucleic acid complexes that are able to fuse with negatively charged lipids of the cell membranes of tissue culture cells, leading to the delivery of RNAi agents (for a description of DOTMA and its use with DNA, see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355).

[0561] The DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylamino)propane (DOTAP) can be used in combination with phospholipids to form DNA complexed vesicles. TM (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells, which contain positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When sufficient positively charged liposomes are used, the net charge on the resulting complex is also positive. The positively charged complex prepared in this way spontaneously attaches to the negatively charged cell surface, fuses with the plasma membrane and effectively delivers functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylamino)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in that the oleoyl moiety is linked by an ester rather than an ether bond.

[0562] Other reported cationic lipid compounds include compounds that have been conjugated to a variety of moieties, including, for example, carboxyspermine, which is conjugated to one of the two lipids and includes compounds such as 5-carboxyarginylglycine dioctadecyloleylamide ("DOGS") (Transfectam TM, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyarginylamide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0563] Another cationic lipid conjugate includes the derivatization of lipids with cholesterol ("DC-Chol"), which has been formulated into a combination of liposomes and DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, prepared by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more effective transfection than compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and DOSPA (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0564] Lipid formulations are particularly suitable for topical administration, and liposomes have several advantages over other formulations. Such advantages include reducing the side effects associated with the high systemic absorption of the administered drug, increasing the aggregation of the drug administered at the desired target, and the ability to administer RNAi agents to the skin. In some implementations, liposomes are used to deliver RNAi agents to epidermal cells and enhance the penetration of RNAi agents into transdermal tissues (e.g., skin). For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been demonstrated (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, Vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855,1987).

[0565] Nonionic lipid systems (particularly systems comprising nonionic surfactants and cholesterol) have also been tested to determine their effectiveness in delivering drugs to the skin. Nonionic lipid formulations comprising Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-octadecanoyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-octadecanoyl ether) were used to deliver drugs to the dermis of mouse skin. Such formulations of RNAi agents are suitable for treating skin diseases.

[0566] Liposomes including iRNA can be made highly deformable. Such deformability can enable liposomes to penetrate via holes less than the average radius of liposomes. For example, transfer bodies are a kind of deformable liposomes. Transfer bodies can be prepared by adding surface edge activators (usually surfactants) to standard lipid compositions. Transfer bodies including RNAi medicaments can be delivered subcutaneously, for example, by infection, to deliver RNAi medicaments to the keratinocytes in the skin. In order to pass through complete mammalian skin, lipid vesicles must pass through a series of pores less than 50nm in diameter under the influence of a suitable transdermal gradient. In addition, due to lipid properties, these transfer bodies can be self-optimized (adapting to the shape of holes in the skin, for example), self-repaired and can usually reach their targets without segmentation, and usually self-loaded.

[0567] Other formulations encompassed by the present invention are described in U.S. Provisional Application Nos. 61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008; and 61 / 051,528, filed May 8, 2008. PCT Application No. PCT / US2007 / 080331, filed October 3, 2007, also describes formulations encompassed by the present invention.

[0568] Transfer body is another type of liposome, and is the highly deformable lipid aggregate as the attractive candidate for drug delivery medium. Transfer body can be described as lipid droplet, and its highly deformable makes it possible to easily pass through the hole that is less than droplet. Transfer body is suitable for its use environment, for example, its self optimization (adaptation to the hole shape in the skin), self repair, usually arrives at its target without segmentation and usually self loading. In order to prepare transfer body, can add surface edge activator, usually surfactant to standard lipid composition. Transfer body has been used to transmit serum albumin to skin. It is effective that the serum albumin transmission of transfer body mediation has shown that subcutaneous injection contains serum albumin solution.

[0569] Surfactants find widespread use in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of the many different types of surfactants (natural and synthetic) is by using the hydrophile / lipophile balance (HLB). The nature of the hydrophilic group (also known as the "IR") provides the most useful way to classify the different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0570] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and decorative products and can be used over a wide pH range. Typically, their HLB values ​​range from 2 to about 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glycerol esters, polyglycerol esters, sorbitan esters, sucrose esters, and ethoxylated esters. Also included in this category are nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0571] If the surfactant molecule carries a negative charge, when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates, such as soaps; acyl lactylates; amides of amino acids; sulfates, such as alkyl sulfates and ethoxylated alkyl sulfates; sulfonates, such as alkyl benzenesulfonates, acyl isethionates, acyl taurates, and sulfosuccinates; and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0572] If the surfactant molecule carries a positive charge, when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0573] If the surfactant molecule is capable of carrying either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkyl amides, N-alkyl betaines, and phospholipids.

[0574] The use of surfactants in pharmaceuticals, formulations, and emulsions has been reviewed (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0575] The iRNA used in the method of the present invention can also be provided in the form of micelle preparation. "Micelle" is defined herein as a specific type of molecular assembly in which the amphiphilic molecules are arranged in a spherical structure so that all the hydrophobic parts of the molecules are directed inwardly, retaining the hydrophilic parts in contact with the surrounding water. If the environment is hydrophobic, then there is an opposite arrangement.

[0576] Mixed micelle formulations suitable for transdermal delivery can be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C8 to C 22The micelle-forming compound is prepared by adding alkali metal alkyl sulfate and micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoenal, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxy choline glycine and pharmaceutically acceptable salts thereof, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ether and analogs thereof, polyether alcohol alkyl ether and analogs thereof, chenodeoxycholate, deoxycholate and mixtures thereof. The micelle-forming compound can be added simultaneously with or after the alkali metal alkyl sulfate. Mixed micelles can be formed by mixing ingredients of substantially any kind, but vigorous mixing is performed to provide micelles of smaller size.

[0577] In one method, a first micelle composition is prepared containing an siRNA composition and at least an alkali metal alkyl sulfate. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, a micelle composition is prepared by mixing an siRNA composition, an alkali metal alkyl sulfate, and at least one of the micelle-forming compounds, followed by adding the remaining micelle-forming compounds and vigorously mixing.

[0578] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added with the micelle forming ingredients. Isotonic agents, such as glycerol, may also be added after the mixed micelle composition is formed.

[0579] For delivery of micellar formulations in the form of a spray, the formulation can be placed in an aerosol dispenser and the propellant can be loaded into the dispenser. The propellant under pressure is in liquid form in the dispenser. The ratio of the ingredients is adjusted so that the aqueous phase and the propellant phase become one phase, that is, there is one phase. If there are two phases, the dispenser must be shaken before dispensing a portion of the contents (for example, via a metering valve). The dispensed amount of the medicament is propelled by the metering valve in the form of a fine spray.

[0580] Propellants may include hydrochlorofluorocarbons, hydrofluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0581] The specific concentration of the essential ingredient can be determined by relatively simple experiments.For absorption via the oral cavity, an increased (eg at least two or three times) dose is usually required compared to administration via injection or via the gastrointestinal tract.

[0582] Lipid particles

[0583] An iRNA (eg, dsRNA) of the invention can be fully encapsulated in a lipid formulation (eg, LNP) or other nucleic acid-lipid particle.

[0584] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNP contains cationic lipids, non-cationic lipids and lipids that prevent particles (e.g., PEG-lipid conjugates) from gathering. LNP is extremely suitable for systemic administration because it presents a long circulation life after intravenous (iv) injection and accumulates at terminal sites (e.g., a site physically separated from the site of administration). LNP includes "pSPLP", which includes an encapsulated condensing agent-nucleic acid complex as described in PCT Publication No. WO00 / 03683. The particles of the present invention typically have an average diameter of about 50nm to about 150nm, more typically about 60nm to about 130nm, more typically about 70nm to about 110nm, most typically about 70nm to about 90nm and substantially nontoxic. In addition, nucleic acids, when present in nucleic acid-lipid particles of the present invention, are resistant to nuclease decomposition in aqueous solutions. Nucleic acid-lipid particles and methods for preparing them are disclosed in, for example, U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010 / 0324120 and PCT Publication No. WO 96 / 40964.

[0585] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of about 1: 1 to about 50: 1, about 1: 1 to about 25: 1, about 3: 1 to about 15: 1, about 4: 1 to about 10: 1, about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1. Ranges intermediate to the above-recited ranges are also contemplated as part of the invention.

[0586] The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyl-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyl-N,N-dimethylaminopropane (DLenDMA), )、1,2-Dilinoleylcarbonyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyl-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyl-3-morpholinylpropane (DLin-MA), 1,2-Dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyl-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyl-3-trimethylaminopropane 2-Dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyl-3-(N-methylpiperazinyl)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyl-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyl-N,N-dimethylaminopropane (DLinDMA), 2,2-linoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin- In some embodiments, the present invention relates to a cationic lipid, a cationic lipid having a molecular weight of 400 μm, a molecular weight of 400 μm, a molecular weight of 400 μm, a molecular weight of 2 ...

[0587] In another embodiment, the compound 2,2-linoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. The synthesis of 2,2-linoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in International Application No. PCT / US 2009 / 061897, which is published as WO / 2010 / 048536 and is incorporated herein by reference.

[0588] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-linoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (mol %), with a particle size of 63.0±20 nm and a siRNA / lipid ratio of 0.027.

[0589] The ionizable / non-cationic lipids may be anionic lipids or neutral lipids, including but not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POP E), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or a mixture thereof. If cholesterol is included, the non-cationic lipid may be about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % of the total lipids present in the particle.

[0590] The bound lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (Ci8). The bound lipid that prevents particle aggregation can be about 0 mol% to about 20 mol% or about 2 mol% of the total lipid present in the particle.

[0591] In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, e.g., from about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0592] In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230 filed March 26, 2008, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions can then be combined, for example, in a 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35%-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles are usually formed spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a Thermobarrel extruder, such as a Lipex extruder (Northern Lipids, Inc). In some cases, the extrusion step may be omitted. Ethanol removal and simultaneous buffer exchange can be achieved by, for example, dialysis or tangential flow filtration. The buffer may be replaced with, for example, phosphate buffered saline (PBS) at about pH 7, such as about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. LNP01 formulations are described, for example, in International Application Publication No. WO2008 / 042973, which is incorporated herein by reference.

[0593] Other exemplary lipid-dsRNA formulations are described in Table A.

[0594] Table A. Exemplary lipid dsRNA formulations

[0595]

[0596]

[0597]

[0598] The abbreviations in Table A include the following: DSPC: distearoylphosphatidylcholine; DPPC: dipalmitoylphosphatidylcholine; PEG-DMG: PEG-dimyristoylglycerol (C14-PEG, or PEG-C14) (PEG with an average molar weight of 2000); PEG-DSG: PEG-distyrylglycerol (C18-PEG, or PEG-C18) (PEG with an average molar weight of 2000); PEG-cDMA: PEG to carbamoyl-1,2-ditetradecanoylpropylamine (PEG with an average molar weight of 2000).

[0599] Formulations comprising DLinDMA (1,2-dilinolenyl-N,N-dimethylaminopropane) are described in International Publication No. WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0600] Formulations containing XTC are described, for example, in U.S. Provisional No. 61 / 148,366, filed January 29, 2009; U.S. Provisional No. 61 / 156,851, filed March 2, 2009; U.S. Provisional No. 61 / 228,373, filed July 24, 2009; U.S. Provisional No. 61 / 239,686, filed September 3, 2009, and International Application No. PCT / US2010 / 022614, filed January 29, 2009, which are incorporated herein by reference.

[0601] Formulations comprising MC3 are described, for example, in U.S. Publication No. 2010 / 0324120, filed June 10, 2010, which is incorporated herein by reference in its entirety.

[0602] Formulations comprising ALNY-100 are described, for example, in International Patent Application No. PCT / US 09 / 63933, filed November 10, 2009, which is incorporated herein by reference.

[0603] Formulations comprising C12-200 are described in U.S. Provisional No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US 10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0604] Other preparations

[0605] i. Lotion

[0606] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets (usually greater than 0.1 μm in diameter) (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 199; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 245). Dekker, Inc., New York, NY, Vol. 2, p. 335; Higuchi et al., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). An emulsion is generally a two-phase system comprising two immiscible liquid phases that are intimately mixed and dispersed with each other. Typically, emulsions can be water-in-oil (w / o) or oil-in-water (o / w) variants. When the aqueous phase is subdivided into tiny droplets and dispersed into the bulk oil phase as tiny droplets, the resulting composition is referred to as a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is subdivided into tiny droplets and dispersed into the bulk aqueous phase as tiny droplets, the resulting composition is referred to as an oil-in-water (o / w) emulsion. The emulsion may additionally contain components other than the dispersed phase, and an active drug that may be present in the form of an aqueous phase, an oil phase, or a solution that is itself a separated phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed. Pharmaceutical emulsions can also be many emulsions containing more than two phases, such as in the case of oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer certain advantages that simple binary emulsions cannot offer. Multiple emulsions of individual oil droplets enclosing water droplets of o / w emulsions constitute w / o / w emulsions.Likewise, a system of small oil droplets enclosed in stable aqueous globules in an oily continuous phase provides an o / w / o emulsion.

[0607] Emulsions are characterized by very low or no thermodynamic stability. Typically, the dispersed or discontinuous phase of an emulsion is well dispersed in an external or continuous phase and maintained in this form by means of an emulsifier or the viscosity of the formulation. As in the case of emulsion-type ointment bases and creams, either phase of the emulsion may be semisolid or solid. Other ways to stabilize emulsions require the use of an emulsifier that can be incorporated into either phase of the emulsion. Emulsifiers can be generally divided into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely divided solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 199).

[0608] Synthetic surfactants, also known as surfactants, have found wide applicability in emulsion formulation and have been reviewed in the literature (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 285; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, page 199). Surfactants are generally amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilicity to the hydrophobicity of a surfactant is called the hydrophilic / lipophilic balance (HLB) and is an important tool for classifying and selecting surfactants in formulation preparation. Surfactants can be divided into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic, and amphoteric (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 285).

[0609] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and gum arabic. Absorbent bases have hydrophilic properties such that they can absorb water to form w / o emulsions but still retain their semisolid consistency, such as anhydrous lanolin and hydrophilic paraffin fats. Finely powdered solids have also been used as good emulsifiers, especially in combination with surfactants and in viscous preparations. These include polar inorganic solids such as heavy metal hydroxides; non-swelling clays such as bentonite, attapulgite, laponite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal magnesium aluminum silicate; pigments and non-polar solids such as carbon or tristearin.

[0610] A variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 335; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 199).

[0611] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., gum arabic, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxymethylpropylcellulose), and synthetic polymers (e.g., carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming a strong interfacial film around the dispersed phase liquid and by increasing the viscosity of the external state.

[0612] Because emulsions usually contain many components that easily support microbial growth, such as carbohydrates, proteins, sterols and phospholipids, these formulations are usually incorporated with preservatives. Common preservatives included in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, parabens and boric acid. Antioxidants are also usually added to emulsion formulations to prevent formulation deterioration. The antioxidant used can be a free radical scavenger, such as tocopherol, alkyl gallate, butylated hydroxymethoxybenzene, butylated hydroxytoluene or a reducing agent (such as ascorbic acid and sodium metabisulfite), and an antioxidant synergist (such as citric acid, tartaric acid and lecithin).

[0613] Emulsion formulations for administration via dermal, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation and from an absorption and bioavailability standpoint (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams and Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 245; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutritional preparations are substances that are commonly administered orally as o / w emulsions.

[0614] ii. Microemulsion

[0615] In one embodiment of the invention, the iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and an amphiphilic agent that is a single optically isotropic and thermodynamically stable liquid solution (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams and Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 245). A microemulsion is typically a system prepared by first dispersing an oil in an aqueous surfactant solution, followed by the addition of a sufficient amount of a fourth component (generally a medium chain length alcohol) to form a transparent system. Therefore, microemulsions are also described as thermodynamically stable isotropic clear dispersions of two immiscible liquids, which are stabilized by an interfacial film of surfactant molecules (Leung and Shah, Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are usually prepared by combining three to five components including oil, water, surfactant, co-surfactant and electrolyte. Microemulsions are determined to be water-in-oil (w / o) or oil-in-water (o / w) types depending on the characteristics of the oil and surfactant used and the structure and geometric packing of the polar top and hydrocarbon tail of the surfactant molecule (Schott, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0616] Phenomenological methods utilizing phase diagrams have been well studied and have resulted in a comprehensive knowledge of how to formulate microemulsions for those of ordinary skill in the art (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams and Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 245; Block, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 335). Microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of a thermodynamically stable liquid that forms spontaneously, compared to conventional emulsions.

[0617] Surfactants used to prepare microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglyceryl fatty acids, tetraglyceryl monolaurate (ML310), tetraglyceryl monooleate (MO310), hexaglyceryl monooleate (PO310), hexaglyceryl pentaoleate (PO500), decaglyceryl monocaprate (MCA750), decaglyceryl monooleate (MO750), decaglyceryl sesquioleate (SO750), decaglyceryl decaoleate (DAO750), either alone or in combination with a co-surfactant. Co-surfactants are typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, which are used to increase interfacial fluidity by penetrating into the surfactant film and thereby creating a disordered film (because voids are created between surfactant molecules). However, microemulsions can be prepared without the use of co-surfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may generally be (but not limited to) water, aqueous drug solution, glycerol, PEG300, PEG400, polyglycerol, propylene glycol and ethylene glycol derivatives. The oil phase may include (but not limited to) Captex 300, Captex355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di- and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oils.

[0618] Microemulsions are of particular interest from the perspective of drug solubilization and improved drug absorption. Lipid-based microemulsions (o / w and w / o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the advantages of improved drug solubility, protection of the drug from enzymatic hydrolysis, increased drug absorption, possibly due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, easier oral administration than solid dosage forms, increased clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions are usually formed spontaneously when the components of the microemulsions are brought together at ambient temperature. This is particularly advantageous when formulating thermolabile drugs (peptides or iRNAs). Microemulsions have also been effective for transdermal delivery of active ingredients in cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate enhanced systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as enhance local cellular uptake of iRNAs and nucleic acids.

[0619] The microemulsions of the present invention may also contain other components and additives (such as sorbitan monostearate (Grill 3), caprylate and penetration enhancers) to improve formulation properties and enhance the absorption of the iRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92). Each of these categories has been discussed above.

[0620] iii. Microparticles

[0621] The RNAi agents of the invention can be incorporated into particles (eg, microparticles). Microparticles can be produced by spray drying, but can also be produced by other methods, including freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

[0622] iv. Penetration enhancers

[0623] In one embodiment, the present invention uses a variety of penetration enhancers to effectively deliver nucleic acids, especially iRNA, to animal skin. Most drugs exist in solution in ionized and non-ionized forms. However, generally only lipid-soluble or lipophilic drugs easily penetrate cell membranes. It has been found that even non-lipophilic drugs can penetrate cell membranes if the membrane to be penetrated is treated with a penetration enhancer. In addition to helping non-lipophilic drugs diffuse through cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

[0624] Penetration enhancers can be classified as belonging to one of five broad categories, namely surfactants, fatty acids, bile salts, chelators, and non-chelating non-surfactants (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above classes of penetration enhancers is described in more detail below.

[0625] Surfactants (or "surfactants") are chemical entities that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, such that iRNA absorption through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-dodecyl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0626] Various fatty acids and their derivatives that are useful as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, eicosatetraenoic acid, 1-monocaprin, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C-1-1-hydroxyethylcarnitines, and the like. 1-20 Alkyl esters (e.g., methyl, isopropyl, and t-butyl), and mono- and diglycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, e.g., Touitou, E. et al., Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0627] The physiological role of bile includes promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th edition, Hardman et al., ed., McGraw-Hill, New York, 1996, pp. 934-935). A variety of natural bile salts and their synthetic derivatives are used as penetration enhancers. The term "bile salt" therefore includes any naturally occurring component of bile as well as any synthetic derivative thereof. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glutamic acid (sodium glutamic acid), glycocholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Swinyard, Chapter 39, Remington's Pharmaceutical Sciences, 18th ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pp. 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0628] Chelating agents used in conjunction with the present invention can be defined as compounds that remove metal ions from solution by forming a complex with the solution, increasing the absorption of iRNA through the mucosa. With regard to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also acting as DNase inhibitors, since most characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanillate), N-acyl derivatives of collagen, lauryl-9, and N-aminoacyl derivatives (enamines) of β-diketones (see, e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0629] As used herein, non-chelating non-surfactant type penetration enhancing compounds can be defined as compounds that do not exhibit the same significant activity as chelators or surfactants, but still enhance iRNA absorption through the digestive mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Such penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenyl azacyclic-aliphatic ketone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0630] Agents that increase the uptake of iRNA at the cellular level may also be added to the pharmaceutical compositions and other compositions of the present invention. For example, cationic lipids, such as liposomes (Junichi et al., U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97 / 30731), are also known to increase the cellular uptake of dsRNA. Examples of commercially available transfection agents include, for example, Lipofectamine TM (Invitrogen; Carlsbad, CA), Lipofectamine 2000 TM (Invitrogen; Carlsbad, CA), 293fectin TM (Invitrogen; Carlsbad, CA), Cellfectin TM (Invitrogen; Carlsbad, CA), DMRIE-C TM (Invitrogen; Carlsbad, CA), FreeStyle TM MAX (Invitrogen; Carlsbad, CA), Lipofectamine TM 2000CD (Invitrogen; Carlsbad, CA), Lipofectamine TM (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine TM (Invitrogen; Carlsbad, CA), Optifect TM (Invitrogen; Carlsbad, CA), Reagent (Promega; Madison, WI), TransFastTM 转染试剂(普洛麦格公司;威斯康星州麦迪逊)、Tfx TM -20试剂(普洛麦格公司;威斯康星州麦迪逊)、Tfx TM -50试剂(普洛麦格公司;威斯康星州麦迪逊)、DreamFect TM (奥兹生物科学公司;法国马赛)、EcoTransfect(奥兹生物科学公司;法国马赛)、TransPass a D1转染试剂(新英格兰生物实验室;美国马萨诸塞州伊普斯威奇)、LyoVec TM / LipoGen TM (英杰公司;美国加利福尼亚州圣地亚哥)、PerFectin转染试剂(Genlantis公司;美国加利福尼亚州圣地亚哥)、NeuroPORTER转染试剂(Genlantis公司;美国加利福尼亚州圣地亚哥)、GenePORTER转染试剂(Genlantis公司;美国加利福尼亚州圣地亚哥)、GenePORTER 2转染试剂(Genlantis公司;美国加利福尼亚州圣地亚哥)、Cytofectin转染试剂(Genlantis公司;美国加利福尼亚州圣地亚哥)、BaculoPORTER转染试剂(Genlantis公司;美国加利福尼亚州圣地亚哥)、TroganPORTER TMtransfection Reagent (Genlantis; San Diego, CA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA) or HiFect TM (B-Bridge International, Mountain View, CA, USA).

[0631] Other agents that can be used to enhance penetration of an administered nucleic acid include glycols, such as ethylene glycol and propylene glycol; pyrroles, such as 2-pyrrole; azones, and terpenes, such as limonene and menthone.

[0632] v. Carrier

[0633] Certain compositions of the invention also incorporate a carrier compound in the formulation. As used herein, a "carrier compound" or "carrier" may refer to a nucleic acid or an analog thereof that is inert (i.e., not biologically active itself) but is recognized as a nucleic acid by an in vivo process that reduces the bioavailability of biologically active nucleic acids by, for example, breaking down the biologically active nucleic acid or promoting its removal from circulation. Co-administration of a nucleic acid and a carrier compound (usually in excess) may result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidneys, or other extracirculatory reservoir organs, possibly due to competition between the carrier compound and the nucleic acid for shared receptors. For example, the recovery of some phosphorothioate dsRNAs in liver tissue can be reduced when co-administered with polyfibronic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'isothiocyanato-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0634] vi. Excipients

[0635] In contrast to carrier compounds, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients may be liquid or solid and are selected by the planned mode of administration so as to provide the desired body, consistency, etc. when combined with the nucleic acid and another component of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0636] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration and that do not adversely react with nucleic acids may also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, lactose, straight-chain starch, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0637] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents (such as alcohols), or solutions of nucleic acids in liquid or solid oil bases. The solution may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration and that do not adversely react with nucleic acids may be used.

[0638] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone and the like.

[0639] vii. Other components

[0640] The composition of the present invention may additionally contain other adjuvant components conventionally present in the pharmaceutical composition of the technically determined amount of use. Therefore, for example, the composition may contain other compatible pharmaceutically active materials, such as antipruritic agents, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials suitable for physically preparing the various dosage forms of the composition of the present invention, such as dyes, flavorings, preservatives, antioxidants, sunscreens, thickeners and stabilizers. However, such materials should not inappropriately interfere with the biological activity of the components of the composition of the present invention when added. The preparation can be sterilized and, when necessary, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, colorants, flavorings and / or aromatic substances and the like, which do not interact harmfully with the nucleic acids of the preparation.

[0641] Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or polydextrose. The suspension may also contain stabilizers.

[0642] In some embodiments, pharmaceutical compositions characterized by the invention include (a) one or more iRNA compounds and (b) one or more agents that act via non-RNAi mechanisms and are useful for treating, for example, PH1.

[0643] Composition Testing

[0644] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred.

[0645] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for humans. The dosage of the composition characterized by the present invention is generally within a circulating concentration range that includes an ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For any compound used in the method characterized by the present invention, the therapeutically effective dose can be initially estimated from the cell culture assay. The dosage can be formulated in an animal model to obtain a circulating plasma concentration range of the compound, or, when appropriate, to obtain a circulating plasma concentration range of the polypeptide product of the target sequence (e.g., to obtain a reduced polypeptide concentration), which includes the IC as determined in cell culture. 50 (ie, the concentration of the test compound that achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine suitable doses for humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0646] In addition to its administration, as discussed above, the iRNA characterized by the present invention can be administered in combination with other known agents that are effective in treating pathological processes mediated by iron overload and treatable by inhibiting HAO1 expression. In any case, the administering physician can adjust the amount and timing of iRNA administration based on the results observed using standard efficacy measurements known in the art or described herein.

[0647] V. Methods for Inhibiting HAO1 Expression

[0648] The present invention provides methods for inhibiting the expression of HAO1 (hydroxyacid oxidase 1) in cells. These methods include contacting cells with an RNAi agent (eg, a double-stranded RNAi agent) in an amount effective to inhibit the expression of HAO1 in cells, thereby inhibiting the expression of HAO1 in cells.

[0649] Contact of cells with double-stranded RNAi agents can be performed in vitro or in vivo. Contact of cells with RNAi agents in vivo includes contacting cells or cell populations in a subject (e.g., a human subject) with an RNAi agent. Combinations of in vitro and in vivo contact methods are also possible. As discussed above, contact may be direct or indirect. In addition, contacting cells may be achieved via targeting ligands (including any ligands described herein or known in the art). In some embodiments, the targeting ligand is a carbohydrate moiety, such as a GalNAc3 ligand, or any other ligand that directs the RNAi agent to a relevant site (e.g., the liver of a subject).

[0650] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," and other similar terms, and includes any degree of inhibition.

[0651] The phrase "inhibits the expression of HAO1" means the inhibition of any HAO1 gene (such as mouse HAO1 gene, rat HAO1 gene, monkey HAO1 gene or human HAO1 gene) and variants or mutants of the HAO1 gene. Therefore, the HAO1 gene may be a wild-type HAO1 gene, a mutant HAO1 gene or a transgenic HAO1 gene (in the case of genetically manipulated cells), a group of cells or an organism.

[0652] "Inhibiting the expression of the HAO1 gene" includes any degree of inhibition of the HAO1 gene, such as at least partial inhibition of the expression of the HAO1 gene. The expression of the HAO1 gene can be assessed based on the content or change in the content of any variable related to the expression of the HAO1 gene (e.g., HAO1 mRNA content, HAO1 protein content). This content can be assessed in individual cells or in a cell population, including, for example, a sample derived from a subject.

[0653] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with HAO1 expression compared to a control level. The control level can be any type of control level used in the art, such as a baseline level before administration or a level measured by a similar subject, cell or sample that is untreated or treated with a control (such as a buffer only control or an inactive agent control).

[0654] In some embodiments of the methods of the present invention, the expression of the HAO1 gene is suppressed by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0655] Inhibition of expression of the HAO1 gene can be demonstrated by a decrease in the amount of mRNA expressed by a first cell or cell population (such cells can be present, for example, in a sample derived from a subject), wherein the HAO1 gene is transcribed and treated (e.g., by contacting the cell with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which these cells are present) such that expression of the HAO1 gene is inhibited compared to a second cell or cell population (control cells) that is substantially identical to the first cell or cell population but has not been so treated. In some embodiments, inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells using the following formula:

[0656]

[0657] Alternatively, inhibition of HAO1 gene expression can be assessed based on a reduction in a parameter functionally associated with HAO1 gene expression (e.g., HAO1 protein expression). HAO1 gene silencing can be determined constitutively or by genome engineering in any cell expressing HAO1 and by any assay known in the art. The liver is the primary site of HAO1 expression. Other significant expression sites include the kidney and uterus.

[0658] The inhibition of the expression of HAO1 protein can be demonstrated by a decrease in the amount of HAO1 protein expressed by a cell or cell population (e.g., the amount of protein expressed in a sample from a subject). As described above for the assessment of mRNA inhibition, the inhibition of protein expression in treated cells or cell populations can be similarly expressed as a percentage of the protein content in control cells or cell populations.

[0659] Control cells or cell groups that can be used to evaluate the inhibition of HAO1 gene expression include cells or cell groups that have not been contacted with the RNAi agent of the present invention. For example, the control cells or cell groups can be derived from individual subjects (eg, human or animal subjects) before the subject is treated with the RNAi agent.

[0660] The content of HAO1 mRNA expressed by a cell or cell group can be measured using any method known in the art for evaluating mRNA expression. In one embodiment, the expression of HAO1 in a sample is measured by detecting a transcribed polynucleotide or a portion thereof (e.g., mRNA of the HAO1 gene). RNA can be extracted from cells using RNA extraction techniques, including, for example, extraction using acid phenol / guanidine isothiocyanate (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical analysis methods using RNA hybridization include cell nucleus analysis, RT-PCR, RNase protection analysis (Melton et al., Nuc. Acids Res. 12: 7035), Northern blotting, field hybridization, and microarray analysis.

[0661] In one embodiment, the expression of HAO1 is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule that can selectively bind to a specific HAO1. The probe can be synthesized by a person of ordinary skill in the art, or derived from a suitable biological preparation. The probe can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0662] The isolated mRNA can be used in hybridization or amplification analysis methods, including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. A method for determining mRNA content involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize with HAO1 mRNA. In one embodiment, the mRNA is fixed on a solid surface and contacted with the probe, for example, by flowing the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane (such as nitrocellulose). In an alternative embodiment, the probe is fixed to a solid surface and the mRNA is contacted with the probe (e.g., in an Affymetrix gene chip array). Those of ordinary skill in the art can easily adapt known mRNA detection methods for determining the content of HAO1 mRNA.

[0663] Alternative methods for determining the expression level of HAO1 in a sample involve amplification of nucleic acids such as mRNA in a sample and / or reverse transcriptase (to prepare cDNA), such as by RT-PCR (Mullis, 1987, experimental examples described in U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-β replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those of ordinary skill in the art. These detection schemes are particularly useful for detecting nucleic acid molecules if they are present in very low numbers. In a particular aspect of the invention, quantitative fluorescent RT-PCR (i.e., TaqMan TM The expression level of HAO1 was determined by the PCR amplification system.

[0664] The expression of HAO1 mRNA is monitored using membrane blots (such as for hybridization analysis, such as Northern blots, Southern blots and similar methods) or microwells, sample tubes, gels, beads or fibers (or any solid support containing bound nucleic acids). See U.S. Patents 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of HAO1 expression may also include the use of nucleic acid probes in solution.

[0665] In some embodiments, branched DNA (bDNA) analysis or real-time PCR (qPCR) is used to assess the expression of mRNA. The use of these methods is described and exemplified in the examples presented herein.

[0666] The expression level of HAO1 protein can be determined using any method known in the art for measuring protein content. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), high diffusion chromatography, fluid or gel precipitation reaction, absorption spectroscopy, colorimetric analysis, spectrophotometric analysis, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence analysis, electrochemiluminescence analysis and the like.

[0667] As used herein, the term "sample" refers to a collection of similar body fluids, cells or tissues separated from a subject, as well as body fluids, cells or tissues in a subject. Examples of biological fluids include blood, serum and serosal fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, ocular fluid and the like. Tissue samples may include samples from tissues, organs or local areas. For example, a sample may be derived from a body fluid or cell in a specific organ, a part of an organ or these organs. In certain embodiments, a sample may be derived from the liver (e.g., a complete liver or certain fragments of the liver or certain types of cells in the liver, such as hepatocytes). In certain embodiments, "sample derived from a subject" refers to blood or plasma extracted from a subject. In other embodiments, "sample derived from a subject" refers to liver tissue derived from a subject.

[0668] In some embodiments of the methods of the present invention, the RNAi agent is administered to the subject so that the RNAi agent is delivered to a specific site in the subject. The inhibition of the expression of HAO1 can be evaluated using the measurement results of the content of HAO1 mRNA or HAO1 protein in the sample or the change in the content, and the sample is derived from a body fluid or tissue from a specific site in the subject. In some embodiments, the site is the liver. The site may also be a sub-portion or sub-group of cells from any of the aforementioned sites. The site may also include cells expressing a specific type of receptor.

[0669] VI. Methods for treating or preventing HAO1-related disorders

[0670] The present invention also provides methods for treating or preventing diseases and conditions that can be modulated by HAO1 gene expression. For example, the compositions described herein can be used to treat any PH1-related disorder.

[0671] The efficacy of the treatment or prevention of a disease can be evaluated, for example, by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, drug dosage required to maintain therapeutic effect, levels of disease markers, or any other measurable parameter suitable for the prevention of a given disease being treated or targeted. It is within the capabilities of those of ordinary skill in the art to monitor the efficacy of treatment or prevention by measuring any one or any combination of such parameters.

[0672] The therapeutic or preventive effect is significant when there is a statistically significant improvement in one or more disease condition parameters or no worsening or otherwise expected symptoms. As an example, a favorable change of at least 10% and preferably at least 20%, 30%, 40%, 50% or more in a measurable parameter of the disease can indicate effective treatment. The efficacy of a given iRNA drug or formulation of that drug can also be judged using experimental animal models for a given disease known in the art. When using an experimental animal model, a statistically significant reduction in a marker or symptom is observed indicating therapeutic efficacy.

[0673] Alternatively, efficacy may be measured by a reduction in the severity of disease, as determined by a skilled diagnostician based on a clinically accepted disease severity rating scale.

[0674] In some embodiments of the methods of the present invention, HAO1 expression is reduced over a long period of time, such as at least one week, two weeks, three weeks or four weeks or longer. For example, in certain instances, the expression of the HAO1 gene is suppressed by administering an iRNA agent described herein to at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100%. In some embodiments, the HAO1 gene is suppressed by administering an iRNA agent to at least about 60%, 70% or 80%. In some embodiments, the HAO1 gene is suppressed by administering a double-stranded oligonucleotide to at least about 85%, 90% or 95%. In another embodiment, the HAO1 gene is suppressed for 7 days, 10 days, 20 days, 30 days or longer after administration.

[0675] give

[0676] Any mode of administration known in the art can be used to administer the RNAi agents of the present invention to a subject, including, but not limited to, subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof. In some embodiments, the agent is administered subcutaneously.

[0677] In some embodiments, the reservoir injection is administered. The reservoir injection can release the RNAi agent in a constant manner over an extended period of time. Therefore, the reservoir injection can reduce the frequency of administration required to obtain the desired effect, such as the desired HAO1 inhibition or treatment or prevention effect. The reservoir injection can also provide a more constant serum concentration. The reservoir injection may include a subcutaneous injection or an intramuscular injection. In some embodiments, the reservoir injection is a subcutaneous injection.

[0678] In some embodiments, administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is an osmotic pump implanted subcutaneously. In other embodiments, the pump is an infusion pump. The infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusion. In some embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi agent to the liver.

[0679] Other modes of administration include epidural, intracerebral, intraventricular, nasal administration, intraarterial, intracardiac, intraosseous infusion, intrathecal, and intravitreal and pulmonary. The mode of administration can be selected based on whether local or systemic treatment is desired and based on the area being treated. The route and site of administration can be selected to enhance targeting.

[0680] The method comprises administering an iRNA agent, e.g., a dose sufficient to inhibit the level of HAO1 mRNA for at least 5 days, more preferably 7, 10, 14, 21, 25, 30 or 40 days; and optionally administering a second single dose of dsRNA, wherein the second single dose is administered at least 5 days, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose, thereby inhibiting the expression of the HAO1 gene in the subject.

[0681] In one embodiment, the dosage of the iRNA medicament of the present invention is administered no more than once every four weeks, no more than once every three weeks, no more than once every two weeks, or no more than once a week. In another embodiment, administration can be maintained for one month, two months, three months, or six months, or one year or longer. In another embodiment, the dosage of the iRNA medicament of the present invention is administered once a week for three weeks.

[0682] Typically, the iRNA agent does not activate the immune system, e.g., it does not increase cytokine levels, such as TNF-α or IFN-α levels. For example, when measured by an assay such as described herein, such as an in vitro PBMC assay, the increase in TNF-α or IFN-α levels is less than 30%, 20%, or 10% of control cells treated with a control dsRNA (such as a dsRNA that does not target HAO1).

[0683] For example, a subject may be given a therapeutic amount of an iRNA agent, such as 0.3 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg or 3 mg / kg of dsRNA. The iRNA agent may be given by intravenous infusion over a period of time (such as a 5 minute, 10 minute, 15 minute, 20 minute or 25 minute period). Repeated administration, for example, according to routine, such as once every two weeks (i.e., once every two weeks) for one month, two months, three months, four months or longer. After the initial treatment regimen, treatment may be given at a lower frequency. For example, after giving for three months every two weeks, administration may be repeated monthly for six months or one year or longer. Administration of an iRNA agent can reduce the level of HAO1 in, for example, a patient's cells, tissues, blood, urine or other compartments by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0684] Prior to administering a full dose of the iRNA agent, a smaller dose, such as a dose that causes less than 5% perfusion response, may be administered to the patient and side effects, such as an allergic reaction, or elevated lipid levels or blood pressure, may be monitored. In another example, the patient may be monitored for undesirable immune stimulation, such as elevated levels of cytokines (e.g., TNF-α or INF-α).

[0685] Patients in need of HAO1 RNAi agents can be identified by obtaining a family medical history. A care provider, such as a doctor, nurse, or family member can obtain a family medical history before prescribing or administering HAO1 dsRNA. Before administering the HAO1 RNAi agent to a patient, a DNA test can also be performed on the patient to identify mutations in the AGT1 gene. The diagnosis of PH1 can be confirmed by any test known to those of ordinary skill in the art.

[0686] The therapeutic or preventive effect is significant when there is a statistically significant improvement in one or more disease condition parameters or no worsening or otherwise expected symptoms. As an example, at least 10% and preferably at least 20%, 30%, 40%, 50% or more favorable changes in the measurable parameters of the disease can indicate effective treatment. The efficacy of a given iRNA medicament of the present invention or a formulation of the iRNA medicament can also be determined using experimental animal models known in the art for a given disease. When using an experimental animal model, a statistically significant reduction in a marker or symptom is observed to indicate therapeutic efficacy.

[0687] The dosage of the RNAi agent administered to a subject can be tailored to balance the risks and benefits of a particular dosage, e.g., to obtain a desired degree of HAO1 gene inhibition (as assessed, e.g., based on HAO1 mRNA inhibition, HAO1 protein expression, or reduction in oxalate levels) or a desired therapeutic or prophylactic effect, while avoiding undesirable side effects.

[0688] In some embodiments, RNAi medicament is divided into two or more doses. If it is necessary to promote repeated or frequent infusion, implanting a transfer device may be desirable, such as a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular) or a reservoir. In some embodiments, the number or amount of subsequent doses depends on the achievement of the desired effect (e.g., suppressing the HAO1 gene), or the achievement of the treatment or preventive effect (e.g., reducing iron overload). In some embodiments, RNAi medicament is given according to the time course. For example, RNAi medicament can be given once a week, twice a week, three times a week, four times a week, or five times a week. In some embodiments, the time course involves giving at regular intervals, such as per hour, per four hours, per six hours, per eight hours, per twelve hours, every day, per 2 days, per 3 days, per 4 days, per 5 days, weekly, every two weeks, or monthly. In other embodiments, the time course involves giving at close intervals, and then the medicament is not given for a long period of time. For example, the time course may involve administering the initial dose group in a relatively short period of time (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours), followed by a longer period of time without administering the RNAi agent (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks). In one embodiment, the RNAi agent is initially administered every hour and then administered at longer intervals (e.g., daily, weekly, biweekly, or monthly). In another embodiment, the RNAi agent is initially administered daily and then administered at longer intervals (e.g., weekly, biweekly, or monthly). In certain embodiments, longer intervals increase over time or are determined based on the achievement of the desired effect. In a specific embodiment, the RNAi agent is administered once a day during the first week, followed by administration starting once a week on the eighth day. In another specific embodiment, the RNAi agent is administered every other day in the first week, followed by administration starting once a week on the eighth day.

[0689] In some embodiments, the RNAi agent is administered with a dosing regimen that includes closely spaced "loading phases" (which may be followed by "maintenance phases"), wherein the RNAi agent is administered at longer intervals. In one embodiment, the loading phase comprises five daily administrations of the RNAi agent during the first week. In another embodiment, the maintenance phase comprises one or two weekly administrations of the RNAi agent. In another embodiment, the maintenance phase lasts for 5 weeks.

[0690] Any of these schedules may be repeated one or more times as appropriate. The number of iterations may depend on the achievement of a desired effect (e.g., inhibition of the HAO1 gene), and / or the achievement of a therapeutic or preventive effect (e.g., reduction of oxalate levels or reduction of symptoms of PH1).

[0691] In another aspect, the present invention features a method of instructing an end user (e.g., a caregiver or subject) on how to administer an iRNA agent as described herein. The method includes providing one or more doses of an iRNA agent to the end user as appropriate, and instructing the end user to administer the iRNA agent according to the protocol described herein, thereby instructing the end user.

[0692] VII. Kit

[0693] The present invention also provides a kit for using any of the iRNA agents of the present invention and / or performing any of the methods of the present invention. Such kits include one or more RNAi agents and instructions for use, such as instructions for inhibiting the expression of HAO1 in cells by contacting cells with an amount of RNAi agents that can effectively inhibit the expression of HAO1. The kit may optionally further include a component (such as an injection device) for contacting cells with RNAi agents, or a component for measuring the inhibition of HAO1 (such as a component for measuring the inhibition of HAO1 mRNA or protein). Such components for measuring the inhibition of HAO1 may include components for obtaining samples (such as plasma samples) from subjects. The kit of the present invention may optionally further include a component for administering an RNAi agent to a subject or a component for determining a therapeutically effective or preventively effective amount.

[0694] VII. Diagnostic markers for PH1 and related conditions

[0695] Also described herein are markers and methods for the diagnosis of disease conditions resulting from overproduction of oxalate, in particular, PH1 and related conditions, as well as agents for the treatment of such conditions.

[0696] According to another aspect, the present invention relates to a method for treating a PH1 condition (stone forming disease, in particular PH1) in a subject. The diagnostic method comprises the following steps: (a) eliminating HAO1 expression in a subject, (b) obtaining biological serum from the subject; and (c) determining the glycolate content in the serum. It should be understood that an increase in the glycolate content in the serum compared to a negative control indicates inhibition of glycolate oxidase to prevent oxalate production that can cause PH1 conditions.

[0697] In one embodiment, described herein is a kit for diagnosing a PH1 condition, the kit comprising the following: (a) a reagent for determining the presence of a relevant analyte in serum, wherein the relevant analyte is a glycolate; and (b) a calibration component. For example, the relevant analyte is glycolate and the reagent is a siRNA targeting HAO1.

[0698] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar to or equivalent to the methods and materials described herein can be used to implement or test the iRNA and methods of the present invention, suitable methods and materials are described below. All public cases, patent applications, patents and other references mentioned are incorporated herein by reference in their entirety. If there is a conflict, this specification (including definitions) will dominate. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0699] Examples

[0700] Materials and Methods

[0701] The following materials and methods were used in the Examples. As used herein, "HAO" and "GO" are used interchangeably.

[0702] siRNA synthesis

[0703] The synthesis was performed using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, Proligo Biochemie GmbH, Hamburg, Germany), single-stranded RNA is produced by solid phase synthesis at a scale of 1 μmole. The corresponding phosphoramidate and 2'-O-methyl phosphoramidate (Proligo Biochemie GmbH, Hamburg, Germany) are used respectively to produce RNA and RNA containing 2'-O-methyl nucleotides by solid phase synthesis. Using standard nucleoside phosphoramidate chemistry, these building blocks are incorporated at selected sites within the sequence of the oligoribonucleotide chain, such as Current protocols innucleic acid chemistry, Beaucage, SL et al. (ed.), John Wiley & Sons, Inc., New York, NY, USA. Phosphorothioate bonds are introduced by replacing the iodine oxidation solution with a solution of Becage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Other auxiliary reaction agents are obtained from Mallinckrodt Baker (Griesheim, Germany).

[0704] Deprotection and purification of crude oligoribonucleotides were performed by anion exchange HPLC according to recognized procedures. Yield and concentration were determined by UV absorption of the respective RNA solution at a wavelength of 260 nm using a spectrophotometer (DU 640B, Beckman Coulter GmbH, Unterschleißheim, Germany).

[0705] Double-stranded RNA was generated by mixing equimolar solutions of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated at 85°C-90°C in a water bath for 3 minutes and cooled to room temperature over a period of 3-4 hours. Annealed RNA solutions were stored at -20°C until use.

[0706] In some cases, a duplex (dsRNA) is synthesized more than once. Different batches are labeled with different extensions. For example, AD-62933.1 and AD-62933.2 are different batches of the same duplex.

[0707] Cell culture and transfection

[0708] Primary cynomolgus monkey hepatocytes (PCH) and primary mouse hepatocytes (PMH) were used. PCH (Celsis catalog number M003055, lot number CBT) or PMH (freshly isolated) were transfected by adding 14.8 μl Opti-MEM plus 0.2 μl Lipofectamine RNAiMax / well (Invitrogen, Carlsbad CA., catalog number 13778-150) to 5 μl siRNA duplex / well in a 96-well plate and incubated at room temperature for 15 minutes. Approximately 2×10 4 80 μl InVitroGRO CP rat medium (InVitro Technologies) per PCH or PMH cell. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 or 20 nM and 0.1 or 0.2 nM final duplex concentrations and dose response experiments were performed over a dose range of 10 nM to 36 fM final duplex concentrations (8, 6-fold dilutions).

[0709] Complete RNA Isolation

[0710] Complete RNA isolation was performed using the DYNABEADS mRNA Isolation Kit (Invitrogen, item number: 610-12). Cells were collected and dissolved in 150 μl of lysis / binding buffer, then mixed at 850 rpm using an Eppendorf Thermomixer for 5 minutes (mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl of lysis / binding buffer mixture were added to the round bottom plate and mixed for 1 minute. The magnetic beads were captured using a magnetic stand and the supernatant was removed without disturbing the beads. After removing the supernatant, the dissolved cells were added to the remaining beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed twice with 150 μl of wash buffer A and mixed for 1 minute. The beads were captured again and the supernatant was removed. The beads were then washed with 150 μl of wash buffer B, captured and the supernatant was removed. The beads were then washed with 150 μl of elution buffer, captured and the supernatant was removed. The beads were dried for 2 minutes. After drying, add 50 μl of elution buffer and mix for 5 minutes at 70°C. Capture beads on magnet for 5 minutes. Remove 40 μl of supernatant and add to another 96-well plate.

[0711] cDNA synthesis

[0712] cDNA synthesis was performed using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, catalog number 4368813).

[0713] For each reaction, 2 μl of 10X buffer, 0.8 μl of 25X dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNase inhibitor, and 3.2 μl of H O were added to 10 μl of complete RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) via the following steps: 25° C. for 10 min, 37° C. for 120 min, 85° C. for 5 sec, 4° C. hold.

[0714] Real-time PCR

[0715] In 50 384-well plates (Roche, catalogue number 04887301001), 2 μl of cDNA was added to a master mix containing 0.5 μl of mouse GAPDH (catalogue number 4352339E, Life Technologies) or custom designed cynomolgus GAPDH TaqMan probe: (F-GCATCCTGGGCTACACTGA, (SEQ ID NO: 13) R-TGGGTGTCGCTGTTGAAGTC (SEQ ID NO: 14), probe-CCAGGTGGTCTCCTCC (SEQ ID NO: 15)), 0.5 μl of human or mouse HAO1 (HS00213909_M1, which is cross-reactive with cynomolgus HOA1, Mm 00439249_m1 (life technologies) for mouse analysis) and 5 μl of Lightcycler in each well. 480 probe master mix (Roche, catalog number 04887301001). Real-time PCR was performed in a LightCycler 480 Real-Time PCR System (Roche) using the ΔΔCt (RQ) assay. Unless otherwise specified in the summary table, each duplex was tested in two independent transfections and transfections were analyzed in duplicate.

[0716] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells. IC50s were calculated using a 4-parameter fitting model using XLFit and normalized to cells transfected with AD-1955 or primary cells.

[0717] The sense and antisense sequences of AD-1955 are: sense: 5'-cuuAcGcuGAGuAcuucGAdTsdT-3' (SEQ ID NO: 16); and antisense: 5'-UCGAAGuACUcAGCGuAAGdTsdT-3' (SEQ ID NO: 17).

[0718] Table B: Abbreviations of nucleotide monomers used in nucleic acid sequence representation

[0719]

[0720]

[0721]

[0722] Example 1. Design, specificity and efficacy prediction of siRNA

[0723] siRNA design was performed to identify siRNAs targeting human, cynomolgus monkey, mouse and rat HAO1 transcripts annotated in the NCBI gene database (http: / / www.ncbi.nlm.nih.gov / gene / ).

[0724] The design used the following transcripts from the NCBI RefSeq collection: human (Homo sapiens) HAO1 mRNA is NM_017545.2; cynomolgus monkey (Macaca fascicularis) HAO1 mRNA is XM_005568381.1; mouse (Mus musculus) HAO1 mRNA is NM_010403.2; rat (Rattus norvegicus) HAO1 mRNA is XM_006235096.1.

[0725] Due to high primate / rodent sequence divergence, siRNA duplexes were designed in several separate batches, including, but not limited to, duplexes containing only matches to human and cynomolgus monkey transcripts; only human, cynomolgus monkey, mouse, and rat transcripts; and only mouse and rat transcripts. All siRNA duplexes were designed to share 100% identity between the listed human transcripts and transcripts from other species considered in each design batch (described above).

[0726] The specificity of all possible 19mers was predicted from each sequence. Candidate 19mers that did not contain repetitions longer than 7 nucleotides were then selected. These 1069 candidate human / cynomolgus macaques, 184 humans / cynomolgus macaques / mouse / rat and 579 mice / rat siRNAs were used for a comprehensive study of appropriate transcripts (defined as a collection of NM_ and XM_ records in the NCBI Refseq group of humans, cynomolgus macaques, mice or rats) using a detailed "brute force" algorithm constructed in the python script 'BruteForce.py'. The script then parses transcript-oligo-alignment, generating a score based on the position and number of mismatches between siRNA and any potential 'off-target' transcripts. Off-target scores are weighted to emphasize the differences in the 'seed' region of siRNA in the position 2-9 from the 5' end of the molecule. Mismatch scores for each oligo-transcript pair from the brute force study were given by summing the individual mismatch scores; mismatches in positions 2-9 were considered 2.8, mismatches in cleavage site positions 10-11 were considered 1.2, and mismatches in regions 12-19 were considered 1.0. Additional off-target predictions were made by comparing the frequencies of heptamers and octamers, which are derived from hexamers derived from 3 unique seeds of each oligo. Hexamers at positions 2-7 relative to the 5' start were used to form 2 heptamers and one octamer. Heptamer 1 was formed by adding 3'A to the hexamer, and heptamer 2 was formed by adding 5'A to the hexamer; octamers were formed by adding A to the 5' and 3' ends of the hexamer. Octamer and heptamer frequencies in the 3'UTRome of human, cynomolgus monkey, mouse or rat (defined as subsequences of transcripts from NCBI's Refseq database, where the terminal 'CDS' of the coding region is well defined) were pre-calculated. The octamer frequency was normalized to the heptamer frequency using the median value from the range of octamer frequencies. The 'mirSeedScore' was then calculated by calculating the sum of ((3×normalized octamer counts)+(2×heptamer 2 counts)+(1×heptamer 1 counts)).

[0727] The two siRNA chains are divided into specificity categories according to the calculated scores: scores above 3 are qualified as highly specific, equal to 3 are qualified as specific, and 2.2 to 2.8 are qualified as medium specific. siRNAs are sorted by the specificity of the antisense strand. Then double helices from human / cynomolgus macaque and mouse / rat collections are selected, whose antisense oligonucleotides do not contain GC at the first position, do not contain G at positions 13 and 14, and have 3 or more U or A in the seed region (features of double helices with high predictive efficacy). Similarly, double helices from human / cynomolgus macaque / mouse and human / cynomolgus macaque / mouse / rat collections with 3 or more U or A in the seed region are selected.

[0728] Candidate GalNac-bound duplexes were designed to be 21 and 23 nucleotides long on the sense and antisense strands, respectively, by extending the antisense 19mer by 4 additional nucleotides in the 3' direction (maintaining perfect complementarity to the target transcript). The sense strand was defined as the reverse complement of the first 21 nucleotides of the antisense 23mer. Duplexes that perfectly matched all 23 nucleotides of transcripts from all selected species were selected.

[0729] Antisense strands containing a C or G at the first 5' position are modified to have a U at the first 5' position, unless this would introduce 4 or more consecutive Us (5'→3'), in which case they are modified to have an A at the first 5' position. The sense strands to be paired into the duplex with these "UA swap" antisense strands are modified accordingly to preserve complementarity. Examples described below include AD-62989 and AD-62993.

[0730] A total of 31 sense and 31 antisense derived human / cynomolgus monkey, 19 sense and 19 antisense derived human / cynomolgus monkey / mouse / rat and 48 sense and 48 antisense derived mouse / rat 21 / 23mer oligonucleotides were synthesized and formed as GalNAc-bound duplexes.

[0731] The sense and antisense strand sequences of the modified duplex are shown in Table 1, and the sense and antisense strand sequences of the unmodified duplex are shown in Table 2.

[0732] Table 1a. Sequences modified by HAO1

[0733]

[0734]

[0735]

[0736] Table 1b: Other sequences modified by HAO1

[0737]

[0738]

[0739]

[0740]

[0741]

[0742]

[0743] Table 2a. Sequences without HAO1 modification (human and human / mouse)

[0744]

[0745]

[0746] Table 2b. Sequences without HAO1 modification (mouse)

[0747]

[0748]

[0749] Table 2c: Other sequences not modified by HAO1

[0750]

[0751]

[0752]

[0753]

[0754]

[0755]

[0756] Example 2. In vitro single-dose screening in primary monkey hepatocytes

[0757] The efficacy of modified and conjugated HAO1 siRNA duplexes was evaluated by transfection analysis in primary monkey hepatocytes. HAO1 siRNA was transfected at two doses: 10 nM and 0.1 nM. The results of these analyses are shown in Table 3 and the data are expressed as the fraction of the remaining message in cells transfected with siRNA targeting HAO1 relative to cells transfected with negative control siRNA (AD-1955 ± standard deviation (SD)).

[0758] The results are also shown in Figure 3A middle. Figure 3B The dose response of one of the most active binders (no. 31) (AD-62933) from the main two dose screen is shown; IC50 is approximately 19 pM.

[0759] Table 3a. Single-dose screening of HAO1 in monkey hepatocytes

[0760]

[0761]

[0762]

[0763] Table 3b. Other HAO1 single dose screens in primary monkey hepatocytes

[0764]

[0765]

[0766]

[0767] Example 3. In vitro single-dose screening in primary mouse hepatocytes

[0768] The efficacy of modified and conjugated HAO1 siRNA duplexes was evaluated by transfection analysis in primary mouse hepatocytes. HAO1 siRNA was transfected at two doses: 20 nM and 0.2 nM. The results of these analyses are shown in Table 4 and the data are expressed as the fraction of the remaining message in cells transfected with siRNA targeting HAO1 relative to cells transfected with negative control siRNA (AD-1955 ± standard deviation (SD)).

[0769] Table 4a. Single-dose screening of HAO1 in primary mouse hepatocytes

[0770]

[0771]

[0772]

[0773] Table 4b. Additional HAO1 single-dose screens in primary mouse hepatocytes

[0774]

[0775]

[0776]

[0777] Example 4. Dose-response screening in primary monkey hepatocytes

[0778] The IC50 of modified and conjugated HAO1 siRNA duplexes was determined in primary monkey hepatocytes. HAO1 siRNA was transfected at a dose range of 10 nM to 36 fM final duplex concentration (8, 6-fold dilutions). The results of these analyses are shown in Table 5.

[0779] Table 5a. HAO1 dose response screen in primary mouse hepatocytes

[0780]

[0781]

[0782] Table 5b. Additional HAO1 dose response screens in primary mouse hepatocytes

[0783] Double Helix ID Species IC50 PCH(nM) AD-62984.2 H 0.017 AD-62994.2 H 0.029 AD-62989.2 H 0.175 AD-62974.2 H 0.288 AD-62975.2 H 0.399 AD-62933.2 Hs / Mm 0.019 AD-62944.2 Hs / Mm 0.027 AD-62935.2 Hs / Mm 0.137 AD-62965.2 Hs / Mm 0.155 AD-62941.2 Hs / Mm 0.245 AD-62940.2 Hs / Mm 0.927

[0784] Example 5. Dose-response screen in primary mouse hepatocytes

[0785] The IC50 of modified and conjugated HAO1 siRNA duplexes was determined in primary mouse hepatocytes. HAO1 siRNA was transfected at a dose range of 10 nM to 36 fM final duplex concentration (8, 6-fold dilutions). The results of these analyses are shown in Table 6.

[0786] Table 6a. HAO1 dose response screen in primary mouse hepatocytes

[0787] Double Helix ID Species IC50 PMH(nM) AD-62989 H 0.003 AD-62994 H 0.006 AD-62975 H 0.059 AD-62974 H 0.122 AD-62984 H 0.264 AD-62944 Hs / Mm 0.002 AD-62935 Hs / Mm 0.007 AD-62965 Hs / Mm 0.008 AD-62933 Hs / Mm 0.008 AD-62941 Hs / Mm 0.087 AD-62940 Hs / Mm 0.090

[0788] Table 6b. Additional HAO1 dose response screens in primary mouse hepatocytes

[0789]

[0790]

[0791] Table 7. Other HAO1 single-dose screens in primary cynomolgus monkey and mouse hepatocytes

[0792]

[0793]

[0794]

[0795] Table 8. Additional single-dose screens in primary cynomolgus monkey hepatocytes

[0796]

[0797]

[0798] Example 6. In vivo evaluation of GO-GalNac conjugates in C57B6 mice

[0799] GO-GalNAc conjugates were subcutaneously administered at 10, 5, 2.5 or 1.25 mg / kg in C57B6 mice and mRNA was evaluated using qPCR 72 hours after administration to block gene expression in the liver. The single dose ED50 of compound A (AD-62994) and compound B (AD-62933) was approximately 1.25 and 2.5 mg / kg, respectively. In a repeated dose study, the conjugate was subcutaneously administered weekly (QW) for 4 weeks and liver GO mRNA levels were evaluated 72 hours after the 4th dose. The repeated dose ED50 of both compounds was approximately 0.3 mg / kg. The results are shown in Figure 4.

[0800] Example 7. In vivo evaluation of GO knockdown in AGXT KO mice and the effect on oxalate content

[0801] Lipid nanoparticles (LNPs) containing GO siRNA (AD-40257) were administered intravenously at 1 mg / kg in AGXT KO mice (Salido et al. (2006) PNAS 103:18249). Urinary oxalate or glycolate levels were measured using ion chromatography / mass spectrometry on day 15. The results are shown in Figure 5. Data are expressed relative to pre-dose values ​​and normalized to creatinine (Cr) to control for urine dilution. N = 4 mice / group and error bars represent standard deviation.

[0802] Example 8. In vivo evaluation of GO-GalNac conjugate in rat AGXT knockdown model

[0803] To generate a rat PH1 model, LNPs (AF-011-63102) containing AGXT siRNA (AD-63102) were administered intravenously to female Sprague Dawley rats at 1 mg / kg on days 1 and 7 to maintain knockdown of AGXT in the rat liver and 1% ethylene glycol was added to the drinking water to further stimulate oxalate production. Some rats were also given GO GalNAc-siRNA (AD-62994) conjugates or PBS controls on days 0 and 7. The results are shown in FIG6 . Fig. 6A Shown is the quantification of hepatic AGXT mRNA levels 72 hours after a single 1 mg / kg dose of AGXT siRNA in LNPs. Figure 6B Urinary oxalate levels were quantified in 24-hour urine collected from days -1 to 0, 3 to 4, 5 to 6, and 7 to 8. Data were normalized to creatinine to control for urine dilution. N = 3 for the AGXT group and N = 2 for the PBS control group. Figure 6C In these same rats (e.g. Figure 6B) continued weekly dosing until day 49, with AF-011-63102 and AD-62994 given on days 14 and 21 and 24-hour urine collected as shown. Ethylene glycol was maintained in the drinking water until day 28. Fig.6D In the last 4 doses (corresponding to Figure 6C The duration of HAO1 knockdown in rats is shown by mRNA levels one or four weeks after treatment with PBS (days 28 and 49 in ). Error bars always represent standard deviations.

[0804]

[0805]

[0806]

[0807] Example 9: In vivo evaluation of GO-GalNac conjugates

[0808] Female C57BL / 6 mice aged 6-8 weeks were given a single subcutaneous dose of the GO siRNA-GalNac conjugates in Table 7. Mice were sacrificed 72 hours later and the liver was analyzed for HAO mRNA by bDNA analysis. The results are shown in Fig.13 middle.

[0809] Table 7: GO(HAO) siRNA-GalNac conjugates

[0810]

[0811]

[0812]

[0813] Example 10: In vivo evaluation of GO-GalNAc conjugates in mice

[0814] Female C57 BL / 6 mice were given a single subcutaneous 3 mg / kg dose of various GO siRNA-GalNAc conjugates described herein or PBS control. Mice were sacrificed 72 hours later and HAO1 mRNA knockdown in the liver was assessed using qPCR. Results are shown in Fig.14 In the figure, the values ​​are expressed relative to the PBS control.

[0815] Example 11: Dose-Response Evaluation of GO-GalNAc Conjugates in Mice

[0816] Female C57 BL / 6 mice were given a single subcutaneous dose of 1 or 3 mg / kg of GO siRNA-GalNAc conjugate compound A (AD-62994), compound B (AD-62933), compound C (AD-65644), compound D (AD-65626), compound E (AD-65590), compound F (AD-65585) or one of the PBS controls. Mice were killed ten days later and qPCR was used to evaluate HAO1 mRNA knockdown in the liver. In a repeated dose study, compound C, D, F or PBS controls were subcutaneously administered weekly (QW) for 4 weeks and liver HAO1 mRNA content was evaluated on the 10th day after the last dose. The results of a single dose are shown in Fig.15 The results of the moderate and repeated dose studies are presented in Fig.16 These data demonstrate the improved potency of compounds AD-65644 and AD-65626 relative to AD-62933 and compounds AD-65590 and AD-65585 relative to AD-62994.

[0817] Example 12: Dose-Response Evaluation of Compound D in Mice

[0818] Female C57 BL / 6 mice were given a single subcutaneous dose of 0.1, 0.3, 1, 3 or 10 mg / kg of AD-65626 or PBS control. Mice were sacrificed ten days later and HAO1 mRNA knockdown in the liver was assessed using qPCR, and the results are expressed relative to the PBS control as Fig.17 These results demonstrate a greater than 3-fold improvement in potency compared to compound AD-62933.

[0819] Example 13: Relationship between mRNA knockdown and serum glycolate levels in mice

[0820] Female C57 BL / 6 mice were given a single subcutaneous dose of 0.1, 0.3, 1, 3 or 10 mg / kg of AD-65585 or PBS control. Mice were sacrificed ten days later and HAO1 mRNA knockdown in the liver was assessed using qPCR, and the results were expressed relative to the PBS control. Glycolate content in serum samples from these same mice was quantified using ion chromatography coupled to mass spectrometry, as previously described (Knight et al., Anal. Biochem. 2012 Feb 1; 421(1): 121-124). The results of these experiments are shown in Fig.18 middle.

[0821] These results indicate that AD-65585 is as potent as AD-65626, with a single dose ED50 of approximately 0.3 mg / kg in WT mice. In addition, HAO1 mRNA silencing caused a dose-responsive increase in serum glycolate up to 4-fold (approximately 200 μM) at the highest two doses.

[0822] Example 14: Relationship between mRNA knockdown and serum glycolate in rats

[0823] Male Sprague-Dawley rats were given a single subcutaneous dose of 1, 3 or 10 mg / kg of AD-65626 or a PBS control. Fourteen days later, the rats were sacrificed and qPCR was used to assess HAO1 mRNA knockdown in the liver, with results expressed relative to the PBS control. Glycolate content in serum samples collected from these same rats before dosing and on day 14 was quantified using ion chromatography coupled to mass spectrometry, also as previously described (Knight et al., Anal. Biochem. 2012 Feb 1; 421(1): 121-124). The results of these experiments are shown in Fig.19 middle.

[0824] As observed in wild-type mice, these results indicate that HAO1 mRNA silencing in Sprague-Dawley rats caused a dose-responsive increase in serum glycolate up to 12-fold (approximately 140 μM) at the highest dose.

[0825] Example 15: Pharmacology studies with ALN-65585

[0826] HAO1 inhibition in hepatocytes

[0827] Primary cynomolgus monkey hepatocytes were transfected at 10 nM with RNAimax (Invitrogen) and serially diluted AD-65585 (ALN-65585, "ALN-GO1") or a non-targeted mRNA luciferase control (AD1955). Relative HAO1 mRNA levels were determined by normalization to GAPDH mRNA levels (as quantified by real-time RT-PCR). Data were plotted to calculate IC50 values ​​for 10 pM. Results are shown in Fig. 20 middle.

[0828] In vitro transfection of AD-65585 showed an ED50 of approximately 10 pM in primary cynomolgus monkey hepatocytes.

[0829] Single-dose pharmacology in mice

[0830] ALN-GO1 pharmacology was evaluated in mice by quantification of hepatic HAO1 mRNA and serum glycolate levels. Fig.21). A single SC dose of ALN-GO1 caused a dose-dependent inhibition of HAO1 mRNA, with a dose of 10 mg / kg causing ED90 silencing. The ED50 dose for GO1 silencing in mice was estimated to be 0.3 mg / kg. Serum glycolate levels increased in a dose-response manner at a maximum level approximately 4-fold above baseline levels. The results are presented in Fig.21 , illustrate liver HAO1 mRNA and serum glycolate levels in C57BL / 6 mice on day 10 after a single subcutaneous administration of ALN-65585. The bar graphs represent the mean of 3 or 4 animals and the error bars depict the standard deviation.

[0831] Single dose duration in mice

[0832] After a single SC administration, GO1 silencing was durable and reversible ( Fig. 22 ). A single SC administration of 3 mg / kg ALN-GO1 in mice cau...

Claims

1. A double-stranded RNAi agent for inhibiting the expression of hydroxyacid oxidase 1 (HAO1) ​​in a cell, The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand consists of the nucleotide sequence 5'-GACUUUCAUCCUGGAAAUAUA-3' of SEQ ID NO:589 and the antisense strand consists of the nucleotide sequence 5'-UAUAUUUCCAGGAUGAAAGUCCA-3' of SEQ ID NO:706; wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand independently comprise a nucleotide modification selected from the group consisting of: 2'-O-methyl modification and 2'-fluoro modification, and The 3'-end of the sense strand is bound to one or more N-acetylgalactosamine (GalNAc) ligands.

2. The double-stranded RNAi agent of claim 1, wherein the ligand is 3 . The double-stranded RNAi agent of claim 1 , wherein the ligand is linked to the 3′ end of the sense strand via a divalent or trivalent branched linker.

4. The double-stranded RNAi agent of claim 3, wherein the double-stranded RNAi agent is bound to the ligand, as shown in the following schematic diagram Wherein X is O or S.

5. The double-stranded RNAi agent of claim 1, wherein the agent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond.

6. The double-stranded RNAi agent of claim 5, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 3'-end of one strand. The double-stranded RNAi agent of claim 6 , wherein the one strand is the antisense strand. The double-stranded RNAi agent of claim 6 , wherein the one strand is the sense strand.

9. The double-stranded RNAi agent of claim 5, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 5'-end of one strand.

10. The double-stranded RNAi agent of claim 9, wherein the one strand is the antisense strand. The double-stranded RNAi agent of claim 9 , wherein the one strand is the sense strand.

12. The double-stranded RNAi agent of claim 5, wherein the phosphorothioate or methylphosphonate internucleotide bonds are located at the 5'-end and the 3'-end of one strand. The double-stranded RNAi agent of claim 12 , wherein the one strand is the antisense strand.

14. The double-stranded RNAi agent of claim 5, wherein the double-stranded RNAi agent comprises 6-8 phosphorothioate internucleotide bonds.

15. The double-stranded RNAi agent of claim 14, wherein the antisense strand comprises two phosphorothioate internucleotide bonds at the 5'-end and two phosphorothioate internucleotide bonds at the 3'-end, and the sense strand comprises at least two phosphorothioate internucleotide bonds at either the 5'-end or the 3'-end. 16 . The double-stranded RNAi agent of claim 1 , wherein the base pair at position 1 of the 5′-end of the antisense strand of the double-stranded region is an AU base pair.

17. A double-stranded RNAi agent capable of inhibiting the expression of hydroxyacid oxidase 1 (HAO1) ​​in a cell. The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand consists of the nucleotide sequence 5'-GACUUUCAUCCUGGAAAUAUA-3' of SEQ ID NO:589 and the antisense strand consists of the nucleotide sequence 5'-UAUAUUUCCAGGAUGAAAGUCCA-3' of SEQ ID NO:706; wherein all of the nucleotides of the sense strand comprise a modification selected from the group consisting of: 2'-O-methyl modification and 2'-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-end, wherein all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide bonds at the 5'-end and two phosphorothioate internucleotide bonds at the 3'-end, and The sense strand is bound to one or more N-acetylgalactosamine (GalNAc) ligands, and the one or more N-acetylgalactosamine (GalNAc) ligands are connected via a branched divalent or trivalent linker at the 3'-end.

18. The double-stranded RNAi agent of claim 1 or 17, wherein the sense strand consists of the nucleotide sequence 5'-gsascuuuCfaUfCfCfuggaaauaua-3' of SEQ ID NO: 213 and the antisense strand consists of the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO: 330, wherein a, g, c and u are 2'-O-methyl (2'-OMe) modified A, G, C and U nucleotides, respectively; Af, Gf, Cf and Uf are 2'-fluoro A, G, C and U modified nucleotides, respectively; and s is a phosphorothioate bond.

19. The double-stranded RNAi agent of claim 18, wherein the sense strand consists of the nucleotide sequence 5'-gsascuuuCfaUfCfCfuggaaauaua-3' of SEQ ID NO: 213 and the antisense strand consists of the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO: 330, wherein a, g, c and u are 2'-O-methyl (2'-OMe) modified A, G, C and U nucleotides, respectively; Af, Gf, Cf and Uf are 2'-fluoro A, G, C and U modified nucleotides, respectively; and s is a phosphorothioate bond, Wherein the ligand is linked to the 3' end of the sense strand, as shown in the following schematic diagram Where X is O.

20. Use of a therapeutically effective amount of a double-stranded RNAi agent in the preparation of a medicament for treating a subject suffering from primary hyperoxaluria type 1 (PH1), the treatment comprising subcutaneously administering to the subject the therapeutically effective amount of the double-stranded RNAi agent, The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand consists of the nucleotide sequence 5'-GACUUUCAUCCUGGAAAUAUA-3' of SEQ ID NO:589 and the antisense strand consists of the nucleotide sequence 5'-UAUAUUUCCAGGAUGAAAGUCCA-3' of SEQ ID NO:706; wherein all of the nucleotides of the sense strand comprise a modification selected from the group consisting of: 2'-O-methyl modification and 2'-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-end, wherein all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide bonds at the 5'-end and two phosphorothioate internucleotide bonds at the 3'-end, and The sense strand is bound to one or more N-acetylgalactosamine (GalNAc) ligands, and the one or more GalNAc ligands are connected via a branched divalent or trivalent linker at the 3'-end.

21. The use of claim 20, wherein the subject is a human.

22. The use of claim 20, wherein the RNAi agent is administered in two or more doses.

23. A double-stranded RNAi agent that inhibits the expression of hydroxyacid oxidase 1 (HAO1) ​​in a cell. The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand consists of the nucleotide sequence 5'-gsascuuuCfaUfCfCfuggaaauaua-3' of SEQ ID NO:213 and the antisense strand consists of the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO:330, wherein a, g, c and u are 2'-O-methyl (2'-OMe) modified A, G, C and U nucleotides, respectively; Af, Gf, Cf and Uf are 2'-fluoro A, G, C and U modified nucleotides, respectively; and s is a phosphorothioate bond, Wherein a ligand is attached to the 3' end of the sense strand as shown in the following schematic diagram Where X is O.

24. An isolated cell containing the double-stranded RNAi agent of claim 23.

25. A pharmaceutical composition comprising the double-stranded RNAi agent according to claim 23.

26. The pharmaceutical composition of claim 25, wherein the double-stranded RNAi agent is in an unbuffered solution.

27. The pharmaceutical composition of claim 26, wherein the unbuffered solution is water.

28. The pharmaceutical composition of claim 26, wherein the unbuffered solution is normal saline.

29. The pharmaceutical composition of claim 25, wherein the double-stranded RNAi agent is in a buffered solution.

30. The pharmaceutical composition of claim 29, wherein the buffer solution comprises acetate, citrate, prolamin, carbonate or phosphate or any combination thereof.

31. The pharmaceutical composition of claim 29, wherein the buffer solution is phosphate buffered saline (PBS).

32. Use of a therapeutically effective amount of a double-stranded RNAi agent in the preparation of a medicament for treating a subject suffering from primary hyperoxaluria type 1 (PH1), the treatment comprising subcutaneously administering to the subject the therapeutically effective amount of the double-stranded RNAi agent, The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand consists of the nucleotide sequence 5'-gsascuuuCfaUfCfCfuggaaauaua-3' of SEQ ID NO:213 and the antisense strand consists of the nucleotide sequence 5'-usAfsuauUfuCfCfaggaUfgAfaagucscsa-3' of SEQ ID NO:330, wherein a, g, c and u are 2'-O-methyl (2'-OMe) modified A, G, C and U nucleotides, respectively; Af, Gf, Cf and Uf are 2'-fluoro A, G, C and U modified nucleotides, respectively; and s is a phosphorothioate bond, Wherein a ligand is attached to the 3' end of the sense strand as shown in the following schematic diagram Where X is O.

33. The use of claim 32, wherein the subject is a human.

34. The use of claim 32, wherein the RNAi agent is administered in two or more doses.

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