Methods for treating progeroid laminopathies using oligonucleotide analogues targeting human LMNA

1. A method for treating Hutchinson-Gilford progeria syndrome in a subject in need thereof comprising administering to the subject an antisense oligonucleotide, wherein the oligonucleotide modulates aberrant splicing of a human LMNA pre-mRNA, the oligonucleotide being composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′-exocyclic carbon of an adjacent subunit, containing up to 40 nucleotide bases; and having a targeting sequence comprising any one of SEQ ID NOs: 3-8, 10-18, and 20-34.

2. The method of claim 1 , where the targeting sequence comprises any one of SEQ ID NOs: 3-7, 11, and 14-16.

3. The method of claim 1 , where the targeting sequence consists essentially of SEQ ID NO: 4.

4. The method of claim 1 , where the targeting sequence consists essentially of SEQ ID NO: 11.

5. The method of claim 1 , where the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO), a PMO comprising one or more piperazine-containing intersubunit linkages or a PMO comprising one or more 4-aminopiperidinyl-containing intersubunit linkages.

6. The method of claim 1 , where the oligonucleotide contains about 10%-50% intersubunit cationic linkages.

7. The method of claim 1 , where the morpholino subunits in the oligonucleotide are joined by phosphorus-containing linkages, in accordance with the following structure:

wherein Z is S or O,

X═NR 1 R 2 or OR 6 ,

Y═O or NR 7 ,

and each said linkage is selected from:

(a) uncharged linkage (a), wherein each of R 1 , R 2 , R 6 , and R 7 is independently selected from hydrogen and lower alkyl;

(b1) cationic linkage (b1), wherein X═NR 1 R 2 and Y═O, and NR 1 R 2 represents an optional substituted piperazino group, such that R 1 R 2 ═—CHRCHRN(R 3 )(R 4 )CHRCHR—, wherein

each R 4 is H, CH 3 or null, and

R 3 is selected from H, lower alkyl, C(═NH)NH 2 , Z-L-NHC(═NH)NH 2 , and

[C(O)CHR′NH] m H, wherein where Z is carbonyl (C(O)) or a direct bond, L is an optional linker up to 18 atoms in length having bonds selected from alkyl, alkoxy, and alkylamino, R′ is a side chain of a naturally occurring amino acid or a one- or two-carbon homolog thereof, and m is 1 to 6;

(b2) cationic linkage (b2), wherein X═NR 1 R 2 and Y═O, R 1 ═H or CH 3 , and R 2 =LNR 3 R 4 R 5 , wherein L, R 3 , and R 4 are defined as above, and R 5 is H, lower alkyl, or lower (alkoxy)alkyl; and

(b3) cationic linkage (b3), wherein Y═NR 7 and X═OR 6 , and R 7 =LNR 3 R 4 R 5 , wherein L, R 3 , and R 4 and R 5 are defined as above, and R 6 is H or lower alkyl; and at least one said linkage is selected from cationic linkages (b1), (b2), and (b3).

8. The method of claim 7 , where each of R 1 and R 2 , in linkages of type (a), is methyl.

9. The method of claim 7 , where at least one linkage is of type (b1), where each R is H, R 4 is H, CH 3 , or an electron pair, and R 3 is selected from H, CH 3 , C(═NH)NH 2 , and C(O)-L-NHC(═NH)NH 2 .

10. The method of claim 7 , where at least one linkage is of type (b1), where each R is H, R 4 is an electron pair, and R 3 is selected from C(═NH)NH 2 and C(O)-L-NHC(═NH)NH 2 .

11. The method of claim 7 , wherein at least one linkage is of type (b1), where each R is H, R 4 is an electron pair, and R 3 is selected from C(═NH)NH 2 and C(O)-L-NHC(═NH)NH 2 .

12. The method of claim 11 , where R 3 is C(O)-L-NHC(NH)NH 2 , and L is a hydrocarbon having the structure —(CH 2 ) n —, where n is 1 to 12.

13. The method of claim 7 , where at least one linkage is of type (b1), where each R is H, and each of R 3 and R 4 is independently H or CH 3 .

14. The method of claim 1 , where the antisense oligonucleotide is covalently attached to a cell-penetrating peptide.

15. The method of claim 14 , where the cell-penetrating peptide is an arginine-rich peptide.

16. The method of claim 15 , where the arginine-rich peptide is attached at its C-terminus to the 5′ end of the oligonucleotide through a one- or two-amino acid linker.

17. The method of claim 15 , where the peptide is attached at its C-terminus to the 3′ end of the oligonucleotide through a one- or two-amino acid linker.

18. A method for treating Hutchinson-Gilford progeria syndrome in a subject in need thereof comprising administering to the subject an antisense oligonucleotide, or a pharmaceutical composition comprising the same, wherein the oligonucleotide modulates aberrant splicing of a human LMNA pre-mRNA, the oligonucleotide comprising a backbone, the backbone comprising a sequence of morpholino ring structures joined by intersubunit linkages, the intersubunit linkages joining a 3′-end of one morpholino ring structure to a 5′-end of an adjacent morpholino ring structure, wherein each morpholino ring structure is bound to a base-pairing moiety, such that the oligonucleotide can bind in a sequence-specific manner to a target nucleic acid, comprising a targeting sequence comprising any one of SEQ ID NOS: 3-8, 10-18, or 20-34, wherein the intersubunit linkages have the following general structure (I):

or a salt or isomer thereof, and wherein each of the intersubunit linkages (I) are independently linkage (A) or linkage (B):

wherein for linkage (A):

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —N(CH 3 ) 2 , —NR 1 R 2 , —OR 3 or;

Y is, at each occurrence, independently O or —NR 2 ,

R 1 is, at each occurrence, independently hydrogen or methyl;

R 2 is, at each occurrence, independently hydrogen or -LNR 4 R 5 R 7 ;

R 3 is, at each occurrence, independently hydrogen or C 1 -C 6 alkyl;

R 4 is, at each occurrence, independently hydrogen, methyl, —C(═NH)NH 2 , —Z-L-NHC(═NH)NH 2 or —[C(O)CHR′NH] m H, where Z is carbonyl (C(O)) or a direct bond, R′ is a side chain of a naturally occurring amino acid or a one- or two-carbon homolog thereof, and m is 1 to 6;

R 5 is, at each occurrence, independently hydrogen, methyl or an electron pair;

R 6 is, at each occurrence, independently hydrogen or methyl;

R 7 is, at each occurrence, independently hydrogen C 1 -C 6 alkyl or C 1 -C 6 alkoxyalkyl;

L is an optional linker up to 18 atoms in length comprising alkyl, alkoxy or alkylamino groups, or combinations thereof; and

wherein for linkage (B):

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —NR 8 R 9 or —OR 3 ; and

Y is, at each occurrence, independently O or —NR 10 ,

R 8 is, at each occurrence, independently hydrogen or C 2 -C 12 alkyl;

R 9 is, at each occurrence, independently hydrogen, C 1 -C 12 alkyl, C 1 -C 12 aralkyl or aryl;

R 10 is, at each occurrence, independently hydrogen, C 1 -C 12 alkyl or -LNR 4 R 5 R 7 ;

wherein R 8 and R 9 may join to form a 5-18 membered mono or bicyclic heterocycle or R 8 , R 9 or R 3 may join with R 10 to form a 5-7 membered heterocycle, and wherein when X is 4-piparazino, X has the following structure (III):

wherein:

R 11 is, at each occurrence, independently C 2 -C 12 alkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylcarbonyl, aryl, heteroaryl or heterocyclyl; and

R is, at each occurrence, independently an electron pair, hydrogen or C 1 -C 12 alkyl; and

R 12 is, at each occurrence, independently, hydrogen, C 1 -C 12 alkyl, C 1 -C 12 aminoalkyl, —NH 2 , —NR 13 R 14 , —NR 13 R 14 R 15 , C 1 -C 12 alkylcarbonyl, oxo, —CN, trifluoromethyl, amidyl, amidinyl, amidinylalkyl, amidinylalkylcarbonyl guanidinyl, guanidinylalkyl, guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl, heterocycle, —SR 13 or C 1 -C 12 alkoxy, wherein R 13 , R 14 and R 15 are, at each occurrence, independently C 1 -C 12 alkyl; and

wherein at least one of the intersubunit linkages is linkage (B).

19. The method of claim 18 , where at least 5% of the intersubunit linkages are linkage (B).

20. The method of claim 18 , where 10% to 50% of the intersubunit linkages are linkage (B).

21. The method of claim 18 , where each linkage (B) has the same structure at each occurrence.

22. The method of claim 18 , where each Y and each W is O.

23. The method of claim 18 , where the targeting sequence comprises any one of SEQ ID NOs: 3-7, 11, and 14-16.

24. The method of claim 18 , where the targeting sequence consists essentially of SEQ ID NO: 4.

25. The method of claim 18 , where the targeting sequence consists essentially of SEQ ID NO: 11.

26. A method for treating Hutchinson-Gilford progeria syndrome in a subject in need thereof, comprising administering to the subject a compound of formula:

or pharmaceutically acceptable salt thereof,

wherein:

R is H or —C(O)CH 3 , and

each Pj is a purine or pyrimidine base-pairing moiety, which taken together form a targeting sequence, wherein the targeting sequence is selected from:

a) SEQ ID NO: 3 (CCGCTGGCAGATGCCTTGTCGGCAG), wherein X is 23,

b) SEQ ID NO: 4 (CTGAGCCGCTGGCAGATGCCTTGTC), wherein X is 23,

c) SEQ ID NO: 5 (GCTCCTGAGCCGCTGGCAGATGCCT), wherein X is 23,

d) SEQ ID NO: 6 (TGGGCTCCTGAGCCGCTGGCAGATG), wherein X is 23,

e) SEQ ID NO: 7 (CACCTGGGCTCCTGAGCCGCTGGCA), wherein X is 23,

f) SEQ ID NO: 8 (CCACCCACCTGGGCTCCTGAGCCGC), wherein X is 23

g) SEQ ID NO: 10 (AGATGGGTCCACCCACCTGGGCTCC), wherein X is 23,

h) SEQ ID NO: 11 (GAGGAGATGGGTCCACCCACCTGGG), wherein X is 23,

i) SEQ ID NO: 12 (GCCAGAGGAGATGGGTCCACCCACC), wherein X is 23,

j) SEQ ID NO: 13 (AAGAGCCAGAGGAGATGGGTCCACC), wherein X is 23,

k) SEQ ID NO: 14 (CAGAAGAGCCAGAGGAGATGGGTCC), wherein X is 23,

l) SEQ ID NO: 15 (GAGGCAGAAGAGCCAGAGGAGATGG), wherein X is 23,

m) SEQ ID NO: 16 (ACTGGAGGCAGAAGAGCCAGAGGAG), wherein X is 23,

n) SEQ ID NO: 17 (ACGTGGTGGTGATGGAGCAGGTCAT), wherein X is 23,

o) SEQ ID NO: 18 (ACTCACGTGGTGGTGATGGAGCAGG), wherein X is 23,

p) SEQ ID NO: 20 (CGGCGGCTACCACTCACGTGGTGGT), wherein X is 23,

q) SEQ ID NO: 21 (CAGCGGCGGCTACCACTCACGTGGT), wherein X is 23,

r) SEQ ID NO: 22 (CCTCAGCGGCGGCTACCACTCACGT), wherein X is 23,

s) SEQ ID NO: 23 (GGCCTCAGCGGCGGCTACCACTCAC), wherein X is 23,

t) SEQ ID NO: 24 (GCTCGGCCTCAGCGGCGGCTACCAC), wherein X is 23,

u) SEQ ID NO: 25 (CGAGTCTGGGACTGACCACTCAGGC), wherein X is 23,

v) SEQ ID NO: 26 (AGGCTCAGGCGGGACGGCGAGTCTG), wherein X is 23,

w) SEQ ID NO: 27 (AGACAAGGCTCAGGCGGGACGGCGA), wherein X is 23,

x) SEQ ID NO: 28 (AGGGAGACAAGGCTCAGGCGGGACG), wherein X is 23,

y) SEQ ID NO: 29 (GGGAAGGGAGACAAGGCTCAGGCGG), wherein X is 23,

z) SEQ ID NO: 30 (GCCCTGGGAAGGGAGACAAGGCTCA), wherein X is 23,

aa) SEQ ID NO: 31 (GTGGGAGCCCTGGGAAGGGAGACAA), wherein X is 23,

bb) SEQ ID NO: 32 (CTGCTGCAGTGGGAGCCCTGGGAAG), wherein X is 23,

cc) SEQ ID NO: 33 (AGCTGCTGCAGTGGGAGCCCTGGGA), wherein X is 23, and

dd) SEQ ID NO: 34 (CCCCCGAGCTGCTGCAGTGGGAGCC), wherein X is 23.

27. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 3 (CCGCTGGCAGATGCCTTGTCGGCAG), X is 23, and R is H.

28. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 4 (CTGAGCCGCTGGCAGATGCCTTGTC), X is 23, and R is H.

29. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 5 (GCTCCTGAGCCGCTGGCAGATGCCT), X is 23, and R is H.

30. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 6 (TGGGCTCCTGAGCCGCTGGCAGATG), X is 23, and R is H.

31. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 7 (CACCTGGGCTCCTGAGCCGCTGGCA), X is 23, and R is H.

32. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 8 (CCACCCACCTGGGCTCCTGAGCCGC), X is 23, and R is H.

33. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 10 (AGATGGGTCCACCCACCTGGGCTCC), X is 23, and R is H.

34. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 11 (GAGGAGATGGGTCCACCCACCTGGG), X is 23, and R is H.

35. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 12 (GCCAGAGGAGATGGGTCCACCCACC), X is 23, and R is H.

36. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 13 (AAGAGCCAGAGGAGATGGGTCCACC), X is 23, and R is H.

37. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 14 (CAGAAGAGCCAGAGGAGATGGGTCC), X is 23, and R is H.

38. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 15 (GAGGCAGAAGAGCCAGAGGAGATGG), X is 23, and R is H.

39. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 16 (ACTGGAGGCAGAAGAGCCAGAGGAG), X is 23, and R is H.

40. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 17 (ACGTGGTGGTGATGGAGCAGGTCAT), X is 23, and R is H.

41. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 18 (ACTCACGTGGTGGTGATGGAGCAGG), X is 23, and R is H.

42. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 20 (CGGCGGCTACCACTCACGTGGTGGT), X is 23, and R is H.

43. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 21 (CAGCGGCGGCTACCACTCACGTGGT), X is 23, and R is H.

44. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 22 (CCTCAGCGGCGGCTACCACTCACGT), X is 23, and R is H.

45. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 23 (GGCCTCAGCGGCGGCTACCACTCAC), X is 23, and R is H.

46. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 24 (GCTCGGCCTCAGCGGCGGCTACCAC), X is 23, and R is H.

47. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 25 (CGAGTCTGGGACTGACCACTCAGGC), X is 23, and R is H.

48. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 26 (AGGCTCAGGCGGGACGGCGAGTCTG), X is 23, and R is H.

49. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 27 (AGACAAGGCTCAGGCGGGACGGCGA), X is 23, and R is H.

50. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 28 (AGGGAGACAAGGCTCAGGCGGGACG), X is 23, and R is H.

51. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 29 (GGGAAGGGAGACAAGGCTCAGGCGG), X is 23, and R is H.

52. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 30 (GCCCTGGGAAGGGAGACAAGGCTCA), X is 23, and R is H.

53. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 31 (GTGGGAGCCCTGGGAAGGGAGACAA), X is 23, and R is H.

54. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 32 (CTGCTGCAGTGGGAGCCCTGGGAAG), X is 23, and R is H.

55. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 33 (AGCTGCTGCAGTGGGAGCCCTGGGA), X is 23, and R is H.

56. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 34 (CCCCCGAGCTGCTGCAGTGGGAGCC), X is 23, and R is H.

57. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 3 (CCGCTGGCAGATGCCTTGTCGGCAG), X is 23, and R is —C(O)CH 3 .

58. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 4 (CTGAGCCGCTGGCAGATGCCTTGTC), X is 23, and R is —C(O)CH 3 .

59. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 5 (GCTCCTGAGCCGCTGGCAGATGCCT), X is 23, and R is —C(O)CH 3 .

60. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 6 (TGGGCTCCTGAGCCGCTGGCAGATG), X is 23, and R is —C(O)CH 3 .

61. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 7 (CACCTGGGCTCCTGAGCCGCTGGCA), X is 23, and R is —C(O)CH 3 .

62. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 8 (CCACCCACCTGGGCTCCTGAGCCGC), X is 23, and R is —C(O)CH 3 .

63. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 10 (AGATGGGTCCACCCACCTGGGCTCC), X is 23, and R is —C(O)CH 3 .

64. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 11 (GAGGAGATGGGTCCACCCACCTGGG), X is 23, and R is —C(O)CH 3 .

65. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 12 (GCCAGAGGAGATGGGTCCACCCACC), X is 23, and R is —C(O)CH 3 .

66. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 13 (AAGAGCCAGAGGAGATGGGTCCACC), X is 23, and R is —C(O)CH 3 .

67. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 14 (CAGAAGAGCCAGAGGAGATGGGTCC), X is 23, and R is —C(O)CH 3 .

68. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 15 (GAGGCAGAAGAGCCAGAGGAGATGG), X is 23, and R is —C(O)CH 3 .

69. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 16 (ACTGGAGGCAGAAGAGCCAGAGGAG), X is 23, and R is —C(O)CH 3 .

70. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 17 (ACGTGGTGGTGATGGAGCAGGTCAT), X is 23, and R is —C(O)CH 3 .

71. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 18 (ACTCACGTGGTGGTGATGGAGCAGG), X is 23, and R is —C(O)CH 3 .

72. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 20 (CGGCGGCTACCACTCACGTGGTGGT), X is 23, and R is —C(O)CH 3 .

73. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 21 (CAGCGGCGGCTACCACTCACGTGGT), X is 23, and R is —C(O)CH 3 .

74. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 22 (CCTCAGCGGCGGCTACCACTCACGT), X is 23, and R is —C(O)CH 3 .

75. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 23 (GGCCTCAGCGGCGGCTACCACTCAC), X is 23, and R is —C(O)CH 3 .

76. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 24 (GCTCGGCCTCAGCGGCGGCTACCAC), X is 23, and R is —C(O)CH 3 .

77. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 25 (CGAGTCTGGGACTGACCACTCAGGC), X is 23, and R is —C(O)CH 3 .

78. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 26 (AGGCTCAGGCGGGACGGCGAGTCTG), X is 23, and R is —C(O)CH 3 .

79. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 27 (AGACAAGGCTCAGGCGGGACGGCGA), X is 23, and R is —C(O)CH 3 .

80. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 28 (AGGGAGACAAGGCTCAGGCGGGACG), X is 23, and R is —C(O)CH 3 .

81. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 29 (GGGAAGGGAGACAAGGCTCAGGCGG), X is 23, and R is —C(O)CH 3 .

82. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 30 (GCCCTGGGAAGGGAGACAAGGCTCA), X is 23, and R is —C(O)CH 3 .

83. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 31 (GTGGGAGCCCTGGGAAGGGAGACAA), X is 23, and R is —C(O)CH 3 .

84. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 32 (CTGCTGCAGTGGGAGCCCTGGGAAG), X is 23, and R is —C(O)CH 3 .

85. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 33 (AGCTGCTGCAGTGGGAGCCCTGGGA), X is 23, and R is —C(O)CH 3 .

86. The method of claim 26 , wherein the targeting sequence is SEQ ID NO: 34 (CCCCCGAGCTGCTGCAGTGGGAGCC), X is 23, and R is —C(O)CH 3 .