Methods for treating progeroid laminopathies using oligonucleotide analogues targeting human LMNA

1. A method for treating HutchinsonGilford Progeria Syndrome (HGPS) 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 about 12-40 bases; and having a targeting sequence comprising any one of SEQ ID NOs: 3-8, 10-18, and 20-34,

wherein the antisense oligonucleotide is covalently attached to a cell-penetrating peptide and linker moiety of the formula G-CPP, wherein G is the linker moiety and is selected from glycine, cysteine, proline, 6-aminohexanoic acid (Ahx), β-alanine (B), and Ahx-B, and CPP is the cell-penetrating peptide and is selected from SEQ ID NOS: 39-54.

2. 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 ,

Pj is a purine or pyrimidine base-pairing moiety,

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′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 R7=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).

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

4. The method of claim 2 , 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 .

5. The method of claim 2 , 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 .

6. The method of claim 2 , 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 .

7. The method of claim 6 , 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.

8. The method of claim 2 , 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 .

9. The method of claim 1 , where the cell-penetrating peptide is attached at its C-terminus to the 5′ end of the oligonucleotide.

10. The method of claim 1 , where the cell-penetrating peptide is attached at its C-terminus to the 3′ end of the oligonucleotide.

11. A method for treating Hutchinson-Gilford Progeria Syndrome (HGPS) 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 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 and 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 the antisense oligonucleotide is covalently attached to a cell-penetrating peptide and linker moiety of the formula -G-CPP, wherein G is the linker moiety selected from glycine, cysteine, proline, 6-aminohexanoic acid (Ahx), β-alanine (B), and Ahx-B, and CPP is the cell-penetrating peptide and is selected from SEQ ID NOS: 39-54.

12. The method of claim 11 , where at least 5% of the intersubunit linkages are linkage (B).

13. The method of claim 11 , where 10% to 50% of the intersubunit linkages are linkage (B).

14. The method of claim 11 , where each linkage (B) has the same structure at each occurrence.

15. The method of claim 11 , where each Y and each W are O.

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

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

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

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

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

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

wherein:

R is a cell-penetrating peptide and linker moiety of the formula -G-CPP, wherein G is the linker moiety and is selected from glycine, cysteine, proline, 6-aminohexanoic acid (Ahx), (β- alanine (B), and Ahx-B, and CPP is the cell-penetrating peptide and is selected from SEQ ID NOS: 39-54, 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.

22. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 3 (CCGCTGGCAGATGCCTTGTCGGCAG) and X is 23.

23. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 4 (CTGAGCCGCTGGCAGATGCCTTGTC) and X is 23.

24. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 5 (GCTCCTGAGCCGCTGGCAGATGCCT) and X is 23.

25. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 6 (TGGGCTCCTGAGCCGCTGGCAGATG) and X is 23.

26. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 7 (CACCTGGGCTCCTGAGCCGCTGGCA) and X is 23.

27. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 8 (CCACCCACCTGGGCTCCTGAGCCGC) and X is 23.

28. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 10 (AGATGGGTCCACCCACCTGGGCTCC) and X is 23.

29. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 11 (GAGGAGATGGGTCCACCCACCTGGG) and X is 23.

30. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 12 (GCCAGAGGAGATGGGTCCACCCACC) and X is 23.

31. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 13 (AAGAGCCAGAGGAGATGGGTCCACC) and X is 23.

32. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 14 (CAGAAGAGCCAGAGGAGATGGGTCC) and X is 23.

33. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 15 (GAGGCAGAAGAGCCAGAGGAGATGG) and X is 23.

34. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 16 (ACTGGAGGCAGAAGAGCCAGAGGAG) and X is 23.

35. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 17 (ACGTGGTGGTGATGGAGCAGGTCAT) and X is 23.

36. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 18 (ACTCACGTGGTGGTGATGGAGCAGG) and X is 23.

37. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 20 (CGGCGGCTACCACTCACGTGGTGGT) and X is 23.

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

39. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 22 (CCTCAGCGGCGGCTACCACTCACGT) and X is 23.

40. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 23 (GGCCTCAGCGGCGGCTACCACTCAC) and X is 23.

41. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 24 (GCTCGGCCTCAGCGGCGGCTACCAC) and X is 23.

42. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 25 (CGAGTCTGGGACTGACCACTCAGGC) and X is 23.

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

44. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 27 (AGACAAGGCTCAGGCGGGACGGCGA) and X is 23.

45. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 28 (AGGGAGACAAGGCTCAGGCGGGACG) and X is 23.

46. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 29 (GGGAAGGGAGACAAGGCTCAGGCGG) and X is 23.

47. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 30 (GCCCTGGGAAGGGAGACAAGGCTCA) and X is 23.

48. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 31 (GTGGGAGCCCTGGGAAGGGAGACAA) and X is 23.

49. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 32 (CTGCTGCAGTGGGAGCCCTGGGAAG) and X is 23.

50. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 33 (AGCTGCTGCAGTGGGAGCCCTGGGA) and X is 23.

51. The method of claim 21 , wherein the targeting sequence is SEQ ID NO: 34 (CCCCCGAGCTGCTGCAGTGGGAGCC) and X is 23.