JPWO2021188661A5 - - Google Patents

Description

Equivalents The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the embodiments described above are to be considered illustrative in all respects rather than limiting the present disclosure. The scope of the disclosure is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are hereby incorporated. intended.
The present application also includes the following embodiments.
[Aspect 1]
A double-stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand,
The molecule, wherein the antisense strand comprises a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13.
[Aspect 2]
dsRNA according to aspect 1, wherein the antisense strand comprises a sequence substantially complementary to the SNCA nucleic acid sequence set forth in any one of SEQ ID NOs: 14-28.
[Aspect 3]
dsRNA according to aspect 1, comprising complementarity to at least 10, 11, 12, or 13 contiguous nucleotides of said SNCA nucleic acid sequences of SEQ ID NOs: 1-13.
[Aspect 4]
dsRNA according to aspect 1 or 3, comprising no more than three mismatches with the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13.
[Aspect 5]
dsRNA according to aspect 1, comprising complete complementarity to said SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13.
[Aspect 6]
dsRNA according to any one of aspects 1 to 5, wherein the antisense strand comprises about 15 to 25 nucleotides in length.
[Aspect 7]
dsRNA according to any one of aspects 1 to 6, wherein the sense strand comprises about 15 to 25 nucleotides in length.
[Aspect 8]
dsRNA according to any one of aspects 1 to 7, wherein the antisense strand is 20 nucleotides long.
[Aspect 9]
dsRNA according to any one of aspects 1 to 7, wherein the antisense strand is 21 nucleotides long.
[Aspect 10]
dsRNA according to any one of aspects 1 to 7, wherein the antisense strand is 22 nucleotides long.
[Aspect 11]
dsRNA according to any one of aspects 1 to 10, wherein the sense strand is 15 nucleotides long.
[Aspect 12]
dsRNA according to any one of aspects 1 to 10, wherein the sense strand is 16 nucleotides long.
[Aspect 13]
dsRNA according to any one of aspects 1 to 10, wherein the sense strand is 18 nucleotides long.
[Aspect 14]
dsRNA according to any one of aspects 1 to 10, wherein the sense strand is 20 nucleotides long.
[Aspect 15]
15. The dsRNA according to any one of aspects 1 to 14, comprising a double-stranded region of 15 to 20 base pairs.
[Aspect 16]
dsRNA according to any one of aspects 1 to 15, comprising a double-stranded region of 15 base pairs.
[Aspect 17]
16. The dsRNA according to any one of aspects 1 to 15, comprising a double-stranded region of 16 base pairs.
[Aspect 18]
16. The dsRNA according to any one of aspects 1 to 15, comprising a double-stranded region of 18 base pairs.
[Aspect 19]
dsRNA according to any one of aspects 1 to 15, comprising a double-stranded region of 20 base pairs.
[Aspect 20]
20. The dsRNA according to any one of aspects 1 to 19, wherein the dsRNA comprises blunt ends.
[Aspect 21]
21. The dsRNA according to any one of aspects 1 to 20, wherein the dsRNA comprises at least one single-stranded nucleotide overhang.
[Aspect 22]
22. The dsRNA of aspect 21, wherein said dsRNA comprises a single-stranded nucleotide overhang of about 2 to 5 nucleotides.
[Aspect 23]
22. The dsRNA of aspect 21, wherein the dsRNA comprises a single-stranded nucleotide overhang of 2 nucleotides.
[Aspect 24]
22. The dsRNA of aspect 21, wherein the dsRNA comprises a single-stranded nucleotide overhang of 5 nucleotides.
[Aspect 25]
25. A dsRNA according to any one of aspects 1 to 24, wherein said dsRNA comprises naturally occurring nucleotides.
[Aspect 26]
25. The dsRNA according to any one of aspects 1 to 24, wherein said dsRNA comprises at least one modified nucleotide.
[Aspect 27]
The modified nucleotide is a 2'-O-methyl modified nucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2' - dsRNA according to aspect 26, comprising an alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising a nucleotide, or a mixture thereof.
[Aspect 28]
28. A dsRNA according to any one of aspects 1 to 27, wherein said dsRNA comprises at least one modified internucleotide linkage.
[Aspect 29]
29. The dsRNA of aspect 28, wherein the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage.
[Aspect 30]
dsRNA according to any one of aspects 1 to 29, comprising 4 to 16 phosphorothioate internucleotide linkages.
[Aspect 31]
dsRNA according to any one of aspects 1 to 29, comprising 8 to 13 phosphorothioate internucleotide linkages.
[Aspect 32]
dsRNA according to any one of aspects 1 to 28, wherein said dsRNA comprises at least one modified internucleotide linkage of formula I:
(In the formula,
B is a base pair part;
W is selected from the group consisting of O, OCH2, OCH, CH2, _and CH _;
X is selected from the group consisting of halo, hydroxy, and C _1-6 alkoxy;
Y is selected from the group consisting of O ⁻ , OH, OR, NH ⁻ , NH ₂ , S ⁻ , and SH;
Z is selected from the group consisting of O and _CH2 ;
R is a protecting group,
is any double bond).
[Aspect 33]
33. A dsRNA according to any one of aspects 1 to 32, wherein said dsRNA comprises at least 80% chemically modified nucleotides.
[Aspect 34]
34. The dsRNA according to any one of aspects 1 to 33, wherein the dsRNA is fully chemically modified.
[Aspect 35]
dsRNA according to any one of aspects 1 to 33, wherein said dsRNA comprises at least 70% 2'-O-methyl nucleotide modification.
[Aspect 36]
dsRNA according to any one of aspects 1 to 33, wherein the antisense strand comprises at least 70% 2'-O-methyl nucleotide modification.
[Aspect 37]
37. The dsRNA according to aspect 36, wherein the antisense strand comprises 70% to 90% 2'-O-methyl nucleotide modification.
[Aspect 38]
dsRNA according to any one of aspects 1 to 33, wherein the sense strand comprises at least 65% 2'-O-methyl nucleotide modification.
[Aspect 39]
39. The dsRNA according to aspect 38, wherein the sense strand comprises 100% 2'-O-methyl nucleotide modification.
[Aspect 40]
40. The dsRNA according to any one of aspects 1 to 39, wherein the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.
[Aspect 41]
41. The dsRNA according to aspect 40, wherein the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5' end of the sense strand.
[Aspect 42]
41. The dsRNA according to aspect 40, wherein the nucleotide mismatches are present at positions 2, 6, and 12 from the 5' end of the sense strand.
[Aspect 43]
43. The dsRNA according to any one of aspects 1 to 42, wherein the antisense strand comprises a 5' phosphate, a 5'-alkyl phosphonate, a 5' alkylene phosphonate, or a 5' alkenyl phosphonate.
[Aspect 44]
44. The dsRNA according to aspect 43, wherein the antisense strand comprises a 5' vinyl phosphonate.
[Aspect 45]
dsRNA according to aspect 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end,
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides;
(3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides;
(7) The dsRNA, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.
[Aspect 46]
dsRNA according to aspect 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end,
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 70% 2'-O-methyl modification;
(3) the nucleotide at position 14 from the 5' end of the antisense strand is not a 2'-methoxy-ribonucleotide;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises at least 70% 2'-O-methyl modification;
(7) The dsRNA, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.
[Aspect 47]
dsRNA according to aspect 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end,
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 85% 2'-O-methyl modification;
(3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand contains 100% 2'-O-methyl modification;
(7) The dsRNA, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.
[Aspect 48]
dsRNA according to aspect 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end,
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand contains 100% 2'-O-methyl modification;
(7) The dsRNA, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.
[Aspect 49]
dsRNA according to aspect 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end,
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand contains 100% 2'-O-methyl modification;
(7) The dsRNA, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.
[Aspect 50]
dsRNA according to aspect 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end,
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises at least 65% 2'-O-methyl modification;
(7) the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides;
(8) The dsRNA, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.
[Aspect 51]
dsRNA according to aspect 1, comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end,
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises at least 75% 2'-O-methyl modification;
(7) the nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides;
(8) The dsRNA, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.
[Aspect 52]
52. The dsRNA according to any one of aspects 1 to 51, wherein a functional moiety is linked to the 5' and/or 3' end of the antisense strand.
[Aspect 53]
52. The dsRNA according to any one of aspects 1 to 51, wherein a functional moiety is linked to the 5' and/or 3' end of the sense strand.
[Aspect 54]
52. The dsRNA according to any one of aspects 1 to 51, wherein a functional moiety is linked to the 3' end of the sense strand.
[Aspect 55]
55. The dsRNA according to any one of aspects 52 to 54, wherein the functional part comprises a hydrophobic part.
[Aspect 56]
56. The dsRNA of embodiment 55, wherein the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.
[Aspect 57]
57. The dsRNA according to aspect 56, wherein the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).
[Aspect 58]
57. The dsRNA according to aspect 56, wherein the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA).
[Aspect 59]
57. The dsRNA according to aspect 56, wherein the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof.
[Aspect 60]
60. The dsRNA according to aspect 59, wherein the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.
[Aspect 61]
61. dsRNA according to any one of aspects 54 to 60, wherein said functional moiety is linked to said antisense strand and/or sense strand by a linker.
[Aspect 62]
62. The dsRNA according to aspect 61, wherein the linker comprises a divalent or trivalent linker.
[Aspect 63]
The divalent or trivalent linker is
63. The dsRNA according to aspect 62, wherein n is 1, 2, 3, 4, or 5.
[Aspect 64]
63. The dsRNA of embodiment 61 or 62, wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.
[Aspect 65]
64. The dsRNA according to aspect 62 or 63, wherein when the linker is a trivalent linker, the linker further connects a phosphodiester or phosphodiester derivative.
[Aspect 66]
dsRNA according to aspect 65, wherein the phosphodiester or phosphodiester derivative is selected from the group consisting of:
(wherein X is O, S or BH3) _.
[Aspect 67]
The nucleotides at positions 1 and 2 from the 3' end of the sense strand and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via a phosphorothioate bond. , dsRNA according to any one of aspects 1 to 66.
[Aspect 68]
A pharmaceutical composition for inhibiting synuclein (SNCA) gene expression in an organism, comprising a dsRNA according to any one of aspects 1 to 67 and a pharmaceutically acceptable carrier.
[Aspect 69]
69. The pharmaceutical composition of aspect 68, wherein said dsRNA inhibits said expression of said SNCA gene by at least 50%.
[Aspect 70]
79. The pharmaceutical composition of aspect 68, wherein said dsRNA inhibits said expression of said SNCA gene by at least 80%.
[Aspect 71]
A method for inhibiting SNCA gene expression in a cell, the method comprising:
(a) introducing the double-stranded ribonucleic acid (dsRNA) according to any one of aspects 1 to 67 into the cell;
(b) maintaining said cells produced in step (a) for a period sufficient to obtain degradation of said mRNA transcripts of said SNCA gene, thereby inhibiting expression of said SNCA gene in said cells; and the method.
[Aspect 72]
68. A method of treating or managing a neurodegenerative disease, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of a dsRNA according to any one of aspects 1 to 67.
[Aspect 73]
73. The method of aspect 72, wherein the dsRNA is administered to the brain of the patient.
[Aspect 74]
73. The method of embodiment 72, wherein the dsRNA is administered by intracerebroventricular (ICV) injection, intrastriatal (IS) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.
[Aspect 75]
73. The method of aspect 72, wherein administering the dsRNA causes a decrease in SNCA gene mRNA in one or more of the hippocampus, striatum, cortex, cerebellum, thalamus, hypothalamus, and spinal cord.
[Aspect 76]
76. The method according to any one of aspects 71-75, wherein said dsRNA inhibits said expression of said SNCA gene by at least 50%.
[Aspect 77]
76. The method according to any one of aspects 71-75, wherein said dsRNA inhibits said expression of said SNCA gene by at least 80%.
[Aspect 78]
A vector comprising a regulatory sequence operably linked to a nucleotide sequence encoding a dsRNA molecule substantially complementary to the SNCA nucleic acid sequences of SEQ ID NOS: 1-13.
[Aspect 79]
79. The vector of embodiment 78, wherein said RNA molecule inhibits said expression of said SNCA gene by at least 30%.
[Aspect 80]
79. The vector of embodiment 78, wherein said RNA molecule inhibits said expression of said SNCA gene by at least 50%.
[Aspect 81]
79. The vector of embodiment 78, wherein said RNA molecule inhibits said expression of said SNCA gene by at least 80%.
[Aspect 82]
79. The vector of embodiment 78, wherein the dsRNA comprises a sense strand and an antisense strand, and the antisense strand comprises a sequence substantially complementary to the SNCA nucleic acid sequences of SEQ ID NOs: 1-13.
[Aspect 83]
A cell comprising a vector according to any one of aspects 78 to 82.
[Aspect 84]
A recombinant adeno-associated virus (rAAV) comprising a vector according to any one of aspects 78 to 82 and an AAV capsid.
[Aspect 85]
A branched RNA compound comprising two or more dsRNA molecules according to any one of aspects 1 to 67 covalently linked to each other.
[Aspect 86]
86. The branched RNA compound of embodiment 85, wherein the dsRNA molecules are covalently linked to each other by a linker, spacer, or branch point.
[Aspect 87]
two or more RNA molecules comprising a length of 15 to 35 nucleotides, and
comprising a sequence substantially complementary to SNCA mRNA;
A branched RNA compound, wherein said two RNA molecules are joined to each other by one or more moieties independently selected from linkers, spacers and branch points.
[Aspect 88]
88. The branched RNA compound of embodiment 87, comprising a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13.
[Aspect 89]
88. A branched RNA compound according to aspect 87, comprising a sequence substantially complementary to one or more of the SNCA nucleic acid sequences of any one of SEQ ID NOs: 14-28.
[Aspect 90]
90. A branched RNA compound according to any one of aspects 87 to 89, wherein the RNA molecule comprises one or both of ssRNA and dsRNA.
[Aspect 91]
91. A branched RNA compound according to any one of aspects 87-90, wherein said RNA molecule comprises an antisense oligonucleotide.
[Aspect 92]
92. A branched RNA compound according to any one of aspects 87 to 91, wherein each RNA molecule comprises 15 to 25 nucleotides in length.
[Aspect 93]
Each RNA molecule comprises a dsRNA comprising a sense strand and an antisense strand, each antisense strand independently comprising a sequence substantially complementary to any one of the SNCA nucleic acid sequences of SEQ ID NOs: 1-13. , the branched RNA compound according to any one of aspects 87 to 90.
[Aspect 94]
94. A branched RNA compound according to aspect 93, comprising complementarity to at least 10, 11, 12, or 13 contiguous nucleotides of the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13.
[Aspect 95]
94. A branched RNA compound according to aspect 93, wherein each RNA molecule comprises no more than three mismatches with the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13.
[Aspect 96]
94. The branched RNA compound of embodiment 93, comprising substantially to complete complementarity to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13.
[Aspect 97]
A branched RNA compound according to any one of aspects 93 to 96, wherein the antisense strand comprises a portion having the nucleic acid sequence of any one of SEQ ID NOs: 29 to 43.
[Aspect 98]
98. A branched RNA compound according to any one of aspects 93 to 97, wherein the antisense strand and/or the sense strand comprises about 15 to 25 nucleotides in length.
[Aspect 99]
99. A branched RNA compound according to any one of aspects 93 to 98, wherein the antisense strand is 20 nucleotides in length.
[Aspect 100]
99. A branched RNA compound according to any one of aspects 93 to 98, wherein the antisense strand is 21 nucleotides in length.
[Aspect 101]
99. A branched RNA compound according to any one of aspects 93 to 98, wherein the antisense strand is 22 nucleotides in length.
[Aspect 102]
A branched RNA compound according to any one of aspects 93 to 101, wherein the sense strand is 15 nucleotides in length.
[Aspect 103]
A branched RNA compound according to any one of aspects 93 to 101, wherein the sense strand is 16 nucleotides in length.
[Aspect 104]
A branched RNA compound according to any one of aspects 93 to 101, wherein the sense strand is 18 nucleotides in length.
[Aspect 105]
A branched RNA compound according to any one of aspects 93 to 101, wherein the sense strand is 20 nucleotides long.
[Aspect 106]
The branched RNA compound according to any one of aspects 90 to 105, wherein the dsRNA comprises a double-stranded region of 15 base pairs to 20 base pairs.
[Aspect 107]
107. The branched RNA compound according to any one of aspects 90 to 106, wherein the dsRNA comprises a 15 base pair double-stranded region.
[Aspect 108]
107. The branched RNA compound according to any one of aspects 90 to 106, wherein the dsRNA comprises a 16 base pair double-stranded region.
[Aspect 109]
107. The branched RNA compound according to any one of aspects 90 to 106, wherein the dsRNA comprises a double-stranded region of 18 base pairs.
[Aspect 110]
107. The branched RNA compound according to any one of aspects 90 to 106, wherein the dsRNA comprises a 20 base pair double-stranded region.
[Aspect 111]
A branched RNA compound according to any one of embodiments 90 to 110, wherein the dsRNA comprises blunt ends.
[Aspect 112]
A branched RNA compound according to any one of aspects 90 to 110, wherein the dsRNA comprises at least one single-stranded nucleotide overhang.
[Aspect 113]
A branched RNA compound according to any one of aspects 90 to 112, wherein the dsRNA comprises a single-stranded nucleotide overhang of 2 to 5 nucleotides.
[Aspect 114]
A branched RNA compound according to any one of aspects 90 to 113, wherein the dsRNA comprises naturally occurring nucleotides.
[Aspect 115]
A branched RNA compound according to any one of aspects 90 to 114, wherein said dsRNA comprises at least one modified nucleotide.
[Aspect 116]
The modified nucleotide is a 2'-O-methyl modified nucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2' - A branched RNA compound according to aspect 115, comprising a non-natural base comprising an alkyl-modified nucleotide, a morpholinonucleotide, a phosphoramidate, or a nucleotide.
[Aspect 117]
117. A branched RNA compound according to any one of aspects 90 to 116, wherein said dsRNA comprises at least one modified internucleotide linkage.
[Aspect 118]
118. The branched RNA compound of embodiment 117, wherein the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage.
[Aspect 119]
A branched RNA compound according to any one of aspects 90 to 118, comprising 4 to 16 phosphorothioate internucleotide linkages.
[Aspect 120]
A branched RNA compound according to any one of aspects 90 to 118, comprising 8 to 13 phosphorothioate internucleotide linkages.
[Aspect 121]
Branched RNA compound according to any one of aspects 90 to 117, wherein said dsRNA comprises at least one modified internucleotide linkage of formula (I):
(In the formula,
B is a base pair part;
W is selected from the group consisting of O, OCH2, OCH, CH2, _and CH _;
X is selected from the group consisting of halo, hydroxy, and C _1-6 alkoxy;
Y is selected from the group consisting of O ⁻ , OH, OR, NH ⁻ , NH ₂ , S ⁻ , and SH;
Z is selected from the group consisting of O and _CH2 ;
R is a protecting group,
is any double bond).
[Aspect 122]
122. A branched RNA compound according to any one of aspects 90 to 121, wherein said dsRNA comprises at least 80% chemically modified nucleotides.
[Aspect 123]
122. A branched RNA compound according to any one of aspects 90 to 121, wherein said dsRNA is fully chemically modified.
[Aspect 124]
122. A branched RNA compound according to any one of aspects 90 to 121, wherein said dsRNA comprises at least 70% 2'-O-methyl nucleotide modification.
[Aspect 125]
125. A branched RNA compound according to any one of aspects 90 to 124, wherein the antisense strand comprises at least 70% 2'-O-methyl nucleotide modification.
[Aspect 126]
126. The branched RNA compound of embodiment 125, wherein the antisense strand comprises 70% to 90% 2'-O-methyl nucleotide modification.
[Aspect 127]
125. A branched RNA compound according to any one of aspects 90 to 124, wherein the sense strand comprises at least 65% 2'-O-methyl nucleotide modification.
[Aspect 128]
128. The branched RNA compound of embodiment 127, wherein the sense strand comprises 100% 2'-O-methyl nucleotide modification.
[Aspect 129]
129. A branched RNA compound according to any one of aspects 93-128, wherein the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.
[Aspect 130]
130. The branched RNA compound of aspect 129, wherein the one or more nucleotide mismatches are at positions 2, 6, and 12 from the 5' end of the sense strand.
[Aspect 131]
130. The branched RNA compound of aspect 129, wherein the nucleotide mismatches are at positions 2, 6, and 12 from the 5' end of the sense strand.
[Aspect 132]
132. The branched RNA compound according to any one of aspects 93-131, wherein the antisense strand comprises a 5' phosphate, a 5'-alkyl phosphonate, a 5' alkylene phosphonate, a 5' alkenyl phosphonate, or a mixture thereof.
[Aspect 133]
121329. The branched RNA compound of embodiment 121329, wherein the antisense strand comprises a 5' vinyl phosphonate.
[Aspect 134]
The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where:
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides;
(3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides;
(7) The branched RNA compound according to aspect 90, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are bonded to each other via a phosphorothioate internucleotide linkage.
[Aspect 135]
The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where:
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 70% 2'-O-methyl modification;
(3) the nucleotide at position 14 from the 5' end of the antisense strand is not a 2'-methoxy-ribonucleotide;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3' end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises at least 70% 2'-O-methyl modification;
(7) The branched RNA compound according to aspect 90, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are bonded to each other via a phosphorothioate internucleotide linkage.
[Aspect 136]
The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where:
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 85% 2'-O-methyl modification;
(3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3' end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand contains 100% 2'-O-methyl modification;
(7) The branched RNA compound according to aspect 90, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are bonded to each other via a phosphorothioate internucleotide linkage.
[Aspect 137]
The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where:
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand contains 100% 2'-O-methyl modification;
(7) The branched RNA compound according to aspect 90, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are bonded to each other via a phosphorothioate internucleotide linkage.
[Aspect 138]
The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where:
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand contains 100% 2'-O-methyl modification;
(7) The branched RNA compound according to aspect 90, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are bonded to each other via a phosphorothioate internucleotide linkage.
[Aspect 139]
The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where:
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises at least 65% 2'-O-methyl modification;
(7) the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides;
(8) The branched RNA compound according to aspect 90, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are bonded to each other via a phosphorothioate internucleotide linkage.
[Aspect 140]
The dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, where:
(1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises at least 75% 2'-O-methyl modification;
(7) the nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides;
(8) The branched RNA compound according to aspect 90, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand are bonded to each other via a phosphorothioate internucleotide linkage.
[Aspect 141]
Branched RNA compound according to any one of aspects 93 to 140, wherein a functional moiety is linked to the 5' end and/or 3' end of the antisense strand.
[Aspect 142]
Branched RNA compound according to any one of aspects 93 to 140, wherein a functional moiety is linked to the 5' end and/or 3' end of the sense strand.
[Aspect 143]
141. A branched RNA compound according to any one of aspects 93 to 140, wherein a functional moiety is linked to the 3' end of the sense strand.
[Aspect 144]
The branched RNA compound according to any one of aspects 141 to 143, wherein the functional moiety comprises a hydrophobic moiety.
[Aspect 145]
145. The branched RNA compound of embodiment 144, wherein the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.
[Aspect 146]
146. The branched RNA compound of embodiment 145, wherein said steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).
[Aspect 147]
146. The branched RNA compound of aspect 145, wherein the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA).
[Aspect 148]
146. The branched RNA compound of aspect 145, wherein the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, derivatives thereof, and metabolites thereof.
[Aspect 149]
146. The branched RNA compound of embodiment 145, wherein the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.
[Aspect 150]
Branched RNA compound according to any one of aspects 141 to 149, wherein said functional moiety is linked to said antisense strand and/or sense strand by a linker.
[Aspect 151]
151. The branched RNA compound of embodiment 150, wherein the linker comprises a divalent or trivalent linker.
[Aspect 152]
The divalent or trivalent linker is
152. The branched RNA compound according to aspect 151 selected from the group consisting of (wherein n is 1, 2, 3, 4, or 5).
[Aspect 153]
153. The branched RNA compound of embodiment 150 or 152, wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.
[Aspect 154]
152. The branched RNA compound of aspect 151, wherein when the linker is a trivalent linker, the linker further connects a phosphodiester or phosphodiester derivative.
[Aspect 155]
The branched RNA compound according to aspect 151, wherein the phosphodiester or phosphodiester derivative is selected from the group consisting of:
(wherein X is O, S or BH3) _.
[Aspect 156]
The nucleotides at positions 1 and 2 from the 3' end of the sense strand and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via a phosphorothioate bond. , the branched RNA compound according to any one of aspects 93-155.
[Aspect 157]
Compound of formula (I):
During the ceremony,
L includes an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof, and formula (I) is optionally one or more further comprising a branch point B, and one or more spacers S, where:
B is independently at each occurrence a polyvalent organic species or a derivative thereof;
S independently at each occurrence includes an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof;
N is a double-stranded nucleic acid comprising a length of 15 to 35 bases, including a sense strand and an antisense strand;
the antisense strand comprises a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13;
The sense strand and antisense strand each independently include one or more chemical modifications;
n is 2, 3, 4, 5, 6, 7, or 8).
[Aspect 158]
The compound according to aspect 157, having a structure selected from formulas (I-1) to (I-9):
[Aspect 159]
A compound according to embodiment 157, wherein the antisense strand comprises a 5' terminal group R selected from the group consisting of:
[Aspect 160]
A compound according to aspect 157, having the structure of formula (II):
During the ceremony,
X is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
Y is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
- represents a phosphodiester internucleoside linkage;
= represents a phosphorothioate internucleoside linkage;
--- represents, independently at each occurrence, a base pair interaction or mismatch.
[Aspect 161]
A compound according to embodiment 157, having the structure of formula (IV):
(In the formula,
X is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
Y is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
- represents a phosphodiester internucleoside linkage;
= represents a phosphorothioate internucleoside linkage;
--- represents, independently at each occurrence, a base pair interaction or mismatch.
[Aspect 162]
A compound according to any one of embodiments 157-161, wherein L is structure L1:
[Aspect 163]
163. A compound according to embodiment 162, wherein R is R3 ^and n is 2.
[Aspect 164]
A compound according to any one of embodiments 157-161, wherein L is structure L2:
[Aspect 165]
165. A compound according to aspect 164, wherein R is R3 ^and n is 2.
[Aspect 166]
Delivery system for therapeutic nucleic acids having the structure of formula (VI):
(In the formula,
L includes an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof, and formula (VI) is optionally one or more further comprising a branch point B, and one or more spacers S, where:
B independently at each occurrence comprises a polyvalent organic species or a derivative thereof;
S independently at each occurrence includes an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof;
Each cNA is independently a carrier nucleic acid containing one or more chemical modifications;
each cNA independently comprises at least 15 contiguous nucleotides of a SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13;
n is 2, 3, 4, 5, 6, 7, or 8).
[Aspect 167]
Delivery system according to embodiment 166, having a structure selected from formulas (VI-1) to (VI-9):
.
[Aspect 168]
167. The delivery system of aspect 166, wherein each cNA independently comprises a chemically modified nucleotide.
[Aspect 169]
167. The delivery system of embodiment 166, further comprising n therapeutic nucleic acids (NA), each NA hybridizing to at least one cNA.
[Aspect 170]
170. The delivery system of aspect 169, wherein each NA independently comprises at least 16 contiguous nucleotides.
[Aspect 171]
171. The delivery system of aspect 170, wherein each NA independently comprises at least 16-20 contiguous nucleotides.
[Aspect 172]
170. The delivery system of aspect 169, wherein each NA comprises an unpaired overhang of at least 2 nucleotides.
[Aspect 173]
173. The delivery system of embodiment 172, wherein the nucleotides of the overhang are attached via a phosphorothioate linkage.
[Aspect 174]
170. The delivery system of embodiment 169, wherein each NA is independently selected from the group consisting of DNA, siRNA, antagomiR, miRNA, gapmer, mixmer, and guide RNA.
[Aspect 175]
170. The delivery system of embodiment 169, wherein each NA is substantially complementary to a SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13.
[Aspect 176]
A pharmaceutical composition for inhibiting the expression of the SNCA gene in an organism, comprising a compound according to any one of aspects 85 to 165 or a system according to any one of aspects 166 to 175, and a pharmaceutically acceptable carrier. thing.
[Aspect 177]
177. The pharmaceutical composition of aspect 176, wherein said compound or said system inhibits said expression of said SNCA gene by at least 50%.
[Aspect 178]
177. The pharmaceutical composition of aspect 176, wherein said compound or said system inhibits said expression of said SNCA gene by at least 80%.
[Aspect 179]
A method for inhibiting SNCA gene expression in a cell, the method comprising:
(a) introducing a compound according to any one of aspects 85 to 165 or a system according to any one of aspects 166 to 175 into said cell;
(b) maintaining said cells produced in step (a) for a period sufficient to obtain degradation of said mRNA transcripts of said SNCA gene, thereby inhibiting expression of said SNCA gene in said cells; and the method.
[Aspect 180]
A method of treating or managing a neurodegenerative disease, comprising administering to a patient in need of such treatment or management a therapeutically effective amount of a compound according to any one of embodiments 85-165 or any one of embodiments 166-175. The method comprises administering the system according to.
[Aspect 181]
181. The method of aspect 180, wherein the dsRNA is administered to the brain of the patient.
[Aspect 182]
181. The method of aspect 180, wherein the dsRNA is administered by intracerebroventricular (ICV) injection, intrastriatal (IS) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.
[Aspect 183]
181. The method of aspect 180, wherein administering the dsRNA causes a decrease in SNCA gene mRNA in one or more of the hippocampus, striatum, cortex, cerebellum, thalamus, hypothalamus, and spinal cord.
[Aspect 184]
184. The method according to any one of aspects 179-183, wherein said dsRNA inhibits said expression of said SNCA gene by at least 50%.
[Aspect 185]
184. The method according to any one of aspects 179-183, wherein said dsRNA inhibits said expression of said SNCA gene by at least 80%.

Claims (17)

two or more RNA molecules comprising a length of 15 to 35 nucleotides and a sequence substantially complementary to alpha synuclein ( SNCA ) mRNA, said two RNA molecules independently selected from linkers, spacers and branch points; branched RNA compounds that are linked to each other by one or more moieties that are
The branched RNA compound may include a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOS: 1 to 13.
or
The branched RNA compound may include a sequence substantially complementary to one or more of the SNCA nucleic acid sequences of any one of SEQ ID NOs: 14-28.
The RNA molecule may include one or both of single-stranded (ss) RNA and double-stranded (ds) RNA.
The RNA molecules may include antisense oligonucleotides, and each RNA molecule may have a length of 15 to 25 nucleotides.
or
Each RNA molecule may include a dsRNA comprising a sense strand and an antisense strand, each antisense strand being independently substantially complementary to a SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13. The antisense strand may contain a portion having the nucleic acid sequence of any one of SEQ ID NOS: 29 to 43.
The branched RNA compound . A d sRNA molecule comprising a sense strand and an antisense strand, said antisense strand comprising a sequence substantially complementary to a SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13. A. 1) the antisense strand comprises a sequence substantially complementary to the SNCA nucleic acid sequence set forth in any one of SEQ ID NOs: 14-28 ;
2) said dsRNA comprises complementarity to at least 10, 11, 12, or 13 contiguous nucleotides of said SNCA nucleic acid sequence of SEQ ID NOS: 1-13; may contain no more than three mismatches with the SNCA nucleic acid sequence;
3) said dsRNA comprises three or fewer mismatches with said SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13; or
4) said dsRNA comprises perfect complementarity to said SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13;
B. 1) the antisense strand comprises about 15 to 25 nucleotides in length, and/or the sense strand comprises about 15 to 25 nucleotides in length;
2) the antisense strand is 20, 21 or 22 nucleotides long;
3) the sense strand is 15, 16, 18 or 20 nucleotides long;
4) the dsRNA comprises a double-stranded region of 15 to 20 base pairs; and/or
5) the dsRNA comprises a double-stranded region of 15, 16, 18 or 20 base pairs;
C. the dsRNA comprises blunt ends;
D. the dsRNA comprises at least one single-stranded nucleotide overhang;
1) the dsRNA may include a single-stranded nucleotide overhang of about 2 to 5 nucleotides;
2) the dsRNA may include a single-stranded nucleotide overhang of 2 nucleotides; or
3) the dsRNA may include a single-stranded nucleotide overhang of 5 nucleotides;
E. The dsRNA comprises a naturally occurring nucleotide or at least one modified nucleotide, the modified nucleotide being a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide. , locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases including nucleotides, or mixtures thereof;
F. 1) the dsRNA comprises at least one modified internucleotide linkage;
2) the dsRNA comprises a modified internucleotide linkage, the modified internucleotide linkage comprising a phosphorothioate internucleotide linkage;
3) said dsRNA comprises 4 to 16 phosphorothioate internucleotide linkages; or
4) said dsRNA comprises 8 to 13 phosphorothioate internucleotide linkages; or
5) the dsRNA has formula I:
(In the formula,
B is a base pair part;
W is selected from the group consisting of O, OCH2, OCH, CH2, _and CH _;
X is selected from the group consisting of halo, hydroxy, and C _1-6 alkoxy;
Y is selected from the group consisting of O ⁻ , OH, OR, NH ⁻ , NH ₂ , S ⁻ , and SH;
Z is selected from the group consisting of O and _CH2 ;
R is a protecting group,
is any double bond)
at least one modified internucleotide linkage of;
G. said dsRNA comprises at least 80% chemically modified nucleotides;
H. 1) the dsRNA is completely chemically modified;
2) said dsRNA comprises at least 70% 2'-O-methyl nucleotide modification;
3) the antisense strand comprises at least 70% 2'-O-methyl nucleotide modification; the antisense strand may comprise 70% to 90% 2'-O-methyl nucleotide modification; or
4) the sense strand may contain at least 65% 2'-O-methyl nucleotide modifications, and the sense strand may contain 100% 2'-O-methyl nucleotide modifications;
I. The sense strand includes one or more nucleotide mismatches between the antisense strand and the sense strand, and the one or more nucleotide mismatches are at positions 2, 6, and 12 from the 5' end of the sense strand. or the nucleotide mismatches may be present at positions 2, 6, and 12 from the 5' end of the sense strand;
and/or
J. The antisense strand includes a 5' phosphate, a 5'-alkylphosphonate, a 5' alkylenephosphonate, or a 5' alkenylphosphonate, and the antisense strand may include a 5' vinylphosphonate.
dsRNA according to claim 2 . the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3'end;
A. (1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides;
(3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; and
(7) the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage ;
B. (1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 70% 2'-O-methyl modification;
(3) the nucleotide at position 14 from the 5' end of the antisense strand is not a 2'-methoxy-ribonucleotide;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises at least 70% 2'-O-methyl modification; and
(7) the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage;
C. (1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 85% 2'-O-methyl modification;
(3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises 100% 2'-O-methyl modification; and
(7) the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage;
D. (1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises 100% 2'-O-methyl modification; and
(7) the nucleotides at positions 1 and 2 from the 5′ end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage;
E. (1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises 100% 2'-O-methyl modification; and
(7) the nucleotides at positions 1 and 2 from the 5′ end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage;
F. (1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises at least 65% 2'-O-methyl modification;
(7) the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides; and
(8) the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage; or
G. (1) the antisense strand includes a sequence substantially complementary to the SNCA nucleic acid sequence of any one of SEQ ID NOs: 1 to 13;
(2) the antisense strand comprises at least 75% 2'-O-methyl modification;
(3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides;
(4) the nucleotides from positions 1 to 2 to positions 1 to 7 from the 3′ end of the antisense strand are linked to each other via a phosphorothioate internucleotide linkage;
(5) a portion of the antisense strand is complementary to a portion of the sense strand;
(6) the sense strand comprises at least 75% 2'-O-methyl modification;
(7) the nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides; and
(8) the nucleotides at positions 1 and 2 from the 5′ end of the sense strand are bonded to each other via a phosphorothioate internucleotide linkage;
dsRNA according to claim 2 . 1) a functional moiety is linked to the 5' end and/or 3' end of the antisense strand ;
2) a functional moiety is linked to the 5' and/or 3' end of the sense strand; or
3) a functional moiety is linked to the 3' end of the sense strand;
4) In each of the above characteristics 1), 2) or 3), the functional part includes a hydrophobic part;
5) In feature 4), the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof;
6) In the feature 5),
a) said steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA);
b) said fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA), or
c) said vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof;
7) In feature 6), the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate;
8) The feature 3) is defined in any one of the features 4) to 7), the functional part is linked to the antisense strand and/or the sense strand by a linker, and the functional part is linked to the antisense strand and/or the sense strand by a linker, and The linker may include a divalent or trivalent linker, and the divalent or trivalent linker is
(where n is 1, 2, 3, 4, or 5)
May be selected from;
9) The feature 3) is defined in any one of the features 4) to 7), the functional part is linked to the antisense strand and/or the sense strand by a linker, and the functional part is linked to the antisense strand and/or the sense strand by a linker, and The linker may include a divalent or trivalent linker, and the linker may be an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or the like. including combinations of;
10) In the feature 8), the linker is a trivalent linker, the linker further connects a phosphodiester or a phosphodiester derivative, and the phosphodiester or phosphodiester derivative is
(wherein X is O, S or _BH3 )
may be selected from the group consisting of;
and/or
11) The nucleotides at positions 1 and 2 from the 3' end of the sense strand and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand are bonded to adjacent ribonucleotides via a phosphorothioate bond. be done,
dsRNA according to any one of claims 2 to 4 . A pharmaceutical composition for inhibiting the expression of the S NC A gene in an organism, comprising the dsRNA according to any one of claims 2 to 5 and a pharmaceutically acceptable carrier , the composition comprising: Said pharmaceutical composition, wherein said dsRNA may inhibit said expression of said SNCA gene by at least 50% or at least 80% . A method for inhibiting SNCA gene expression in a cell, the method comprising:
(a) introducing the d sRNA according to any one of claims 2 to 5 into the cell ; and
(b) maintaining said cells produced in step (a) for a sufficient period of time to obtain degradation of said mRNA transcripts of said SNCA gene, thereby inhibiting expression of said SNCA gene in said cells; and
The method comprises :
said dsRNA may inhibit said expression of said SNCA gene by at least 50%, or
the dsRNA may inhibit the expression of the SNCA gene by at least 80%;
Said method . A composition for the treatment or management of a neurodegenerative disease or disorder , comprising a dsRNA according to any one of claims 2 to 5 ,
1) the composition may be for administration to the brain of a patient in need of such treatment or management;
2) May be for administration by intracerebroventricular (ICV) injection, intrastriatal (IS) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof;
3) the dsRNA may cause a decrease in SNCA gene mRNA in one or more of the hippocampus, striatum, cortex, cerebellum, thalamus, hypothalamus, and spinal cord;
4) said dsRNA may inhibit said expression of said SNCA gene by at least 50%, or said dsRNA may inhibit said expression of said SNCA gene by at least 80%;
and/or
5) The neurodegenerative disease or disorder is Parkinson's disease, Parkinson's disease dementia, Lewy body dementia, multiple system atrophy, Alzheimer's disease, polyglutamine disorder, Huntington's disease, spinocerebellar ataxia type 12, spinal cord May be selected from the group consisting of trinucleotide repeat diseases such as cerebellar ataxia type 8, fragile X syndrome, fragile XE mental retardation, Friedreich's ataxia and myotonic dystrophy.
Said composition . A vector comprising a regulatory sequence operably linked to a nucleotide sequence encoding a dsRNA molecule substantially complementary to the SNCA nucleic acid sequences of SEQ ID NOS: 1-13 ,
1) said RNA molecule may inhibit said expression of said SNCA gene by at least 30%, at least 50%, or at least 80%; or
2) the dsRNA may include a sense strand and an antisense strand, and the antisense strand may include a sequence substantially complementary to the SNCA nucleic acid sequences of SEQ ID NOS: 1 to 13;
Said vector . A cell comprising the vector according to claim 9 . A recombinant adeno-associated virus (rAAV) comprising the vector of claim 9 and an AAV capsid. A branched RNA compound comprising two or more dsRNA molecules according to any one of claims 2 to 5 covalently linked to each other , the dsRNA molecules being linked by linkers, spacers or branch points. The branched RNA compounds may be covalently linked to each other . Formula (I ):
( In the formula,
L includes an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof ; B, and one or more spacers S , where :
B is independently at each occurrence a polyvalent organic species or a derivative thereof;
S independently at each occurrence includes an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof;
N is a double-stranded nucleic acid having a length of 15 to 35 bases, including a sense strand and an antisense strand; contains a sequence complementary to;
each of the sense strand and antisense strand independently comprises one or more chemical modifications;
n is 2, 3, 4, 5, 6, 7, or 8)
A compound of
1) The compound has formulas (I-1) to (I-9):
It may have a structure selected from;
2) the antisense strand is
may include a 5' terminal group R selected from the group consisting of;
3) The compound has formula (II):
(In the formula,
X is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
Y is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
- represents a phosphodiester internucleoside linkage;
= represents a phosphorothioate internucleoside linkage;
--- represents a base pair interaction or mismatch, independently for each occurrence)
It may have the structure;
or
4) The compound has formula (IV):
(In the formula,
X is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
Y is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
- represents a phosphodiester internucleoside linkage;
= represents a phosphorothioate internucleoside linkage;
--- represents a base pair interaction or mismatch, independently for each occurrence)
It may have the structure;
5) L is structure L1:
may be, R may be R3 ^, and n may be 2;
or
L is structure L2:
, R may be R ³ , and n may be 2
Said compound . Formula (VI ):
(In the formula,
L includes an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof, and formula (VI) has one or more branch points. B, and one or more spacers S , where:
B independently at each occurrence comprises a polyvalent organic species or a derivative thereof;
S independently at each occurrence includes an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof;
Each cNA is independently a carrier nucleic acid containing one or more chemical modifications;
each cNA independently comprises at least 15 contiguous nucleotides of a SNCA nucleic acid sequence of any one of SEQ ID NOs: 1-13;
n is 2, 3, 4, 5, 6, 7, or 8)
A delivery system for therapeutic nucleic acids having the structure of
1) The delivery system has formulas (VI-1) to (VI-9):
It may have a structure selected from;
B. Each cNA may independently contain chemically modified nucleotides, or
C. The delivery system may further comprise n therapeutic nucleic acids (NAs), each NA hybridizing to at least one cNA;
1) each NA may independently include at least 16 contiguous nucleotides; each NA may independently include at least 16-20 contiguous nucleotides; or
2) each NA may include an unpaired overhang of at least 2 nucleotides, and the nucleotides of the overhang may be linked via a phosphorothioate linkage, or
3) each NA may be independently selected from the group consisting of DNA, siRNA, antagomiR, miRNA, gapmer, mixmer, and guide RNA; or
4) each NA may be substantially complementary to any one SNCA nucleic acid sequence of SEQ ID NOs: 1-13;
Said delivery system . A medicament for use in inhibiting the expression of the SNCA gene in an organism, comprising a compound according to any one of claims 1, 12 and 13 or a delivery system according to claim 14 , and a pharmaceutically acceptable carrier. Said pharmaceutical composition, wherein said compound or said system may inhibit said expression of said SNCA gene by at least 50% or at least 80%. A method for inhibiting SNCA gene expression in a cell, the method comprising:
(a) introducing a compound according to any one of claims 1, 12 and 13 or a delivery system according to claim 14 into the cell ; and
(b) maintaining said cells produced in step (a) for a sufficient period of time to obtain degradation of said mRNA transcripts of said SNCA gene, thereby inhibiting expression of said SNCA gene in said cells; and,
The method described above. 15. A composition for use in the treatment or management of a neurodegenerative disease or disorder comprising a compound according to any one of claims 1, 12 and 13 or a delivery system according to claim 14, the composition comprising : The disease or disorder is Parkinson's disease, Parkinson's disease dementia, Lewy body dementia, multiple system atrophy, Alzheimer's disease, polyglutamine disorder, Huntington's disease, spinocerebellar ataxia type 12, spinocerebellar ataxia 8, Fragile X syndrome, Fragile XE mental retardation, Friedreich's ataxia and myotonic dystrophy.