Chimeric peptides for antisense delivery

Abstract

This invention provides oligonucleotides, chimeric peptides, and peptide-oligonucleotide conjugates. It also provides methods for treating muscle diseases, viral infections, or bacterial infections in individuals of need, comprising administering to the individual the oligonucleotides, chimeric peptides, and peptide-oligonucleotide conjugates described herein.

Description

[Previous Technology] Antisense technology provides a way to modulate the expression of one or more specific gene products (including alternative splicing products), and this approach is uniquely applicable to many therapeutic, diagnostic, and research applications. The underlying principle of antisense technology is that an antisense compound (e.g., an oligonucleotide) hybridizing with a target nucleic acid modulates gene expression activity via any of many antisense mechanisms, such as transcription, splicing, or translation. The sequence specificity of antisense compounds makes them an attractive tool for therapies involving target validation and gene functionalization, as well as the selective modulation of disease-related gene expression. Despite significant progress in the field of antisense technology, oligonucleotides and peptide-oligonucleotide conjugates with modified antisense or antigene efficacy are still needed in this technology. [Summary of the Invention] This article provides chimeric peptide-oligonucleotide conjugates comprising oligonucleotides covalently bonded to a chimeric peptide (CP). This article also provides methods for treating diseases in individuals in need, comprising administering to the individual the chimeric peptide-oligonucleotide conjugates described herein. Therefore, in one embodiment, this document provides a chimeric peptide-oligonucleotide conjugate of formula I: [Figure] or a pharmaceutically acceptable salt thereof, wherein: A' is selected from -NHCH2C(O)NH2, -N(C1-6-alkyl)CH2C(O)NH2, [Figure], wherein R5 is -C(O)(O-alkyl)x-OH, wherein x is 3-10, and each alkyl group is independently C2-6-alkyl each time it appears, or R5 is selected from -C(O)C1-6-alkyl, triphenylmethyl, monomethoxytriphenylmethyl, -(C1-6-alkyl)-R6, -(C1-6-heteroalkyl)-R6, aryl-R6, heteroaryl-R6, -C(O)O-(C1-6-alkyl)-R6, -C(O)O-aryl-R6, -C(O)O-heteroaryl-R6, and [Figure]; R6 is selected from OH, SH, and NH2, or R6 is O, S, or NH, each of which is covalently linked to a solid support; each R1 is independently selected from OH and -N(R3)(R4), wherein each R3 and R4 is independently -C1-6-alkyl in each occurrence; each R2 is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemically protected group, wherein the nucleobase independently comprises a C3-6-heterocycle selected from pyridine, pyrimidine, triazine, purine, and denitro-purine in each occurrence; z is 8-40; and E' is selected from H, -C1-6-alkyl, -C(O)C1-6-alkyl, benzoyl, stearyl, triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, [Figure]; Where Q is -C(O)(CH2)6C(O)- or -C(O)(CH2)2S2(CH2)2C(O)-; R7 is -(CH2)2OC(O)N(R8)2, where R8 is -(CH2)6NHC(=NH)NH2; L is -C(O)(CH2)1-6-C1-6-heteroaryl-(CH2)1-6C(O), where L is covalently linked to the amino terminus of J via an amide bond; J is a cell-penetrating peptide with 2, 3, 4 or 5 covalently linked groups; G is selected from H, -C(O)C1-6-alkyl, benzoyl and stearyl, where G is covalently linked to the carboxyl terminus of J via an amide bond; and at least one of the following conditions is true: 1) A' is [Figure]; or 2) E' is [Figure]. In one embodiment, L is -C(O)(CH2)1-6-triazole-(CH2)1-6C(O). In some embodiments, at least one of the cell-penetrating peptides is an amphiphilic peptide, and at least one of the cell-penetrating peptides is an oligospermine peptide. In some embodiments, one of the cell-penetrating peptides is an amphiphilic peptide, and one of the cell-penetrating peptides is an oligospermine peptide. In one embodiment, J is two covalently linked cell-penetrating peptides, wherein one of the cell-penetrating peptides is an amphiphilic peptide and one of the cell-penetrating peptides is an oligospermine peptide. In one embodiment, the chimeric peptide-oligonucleotide conjugate of Formula I is the chimeric peptide-oligonucleotide conjugate of Formula Ia: [Figure] or a medically acceptable salt thereof, wherein J is as defined above. In some embodiments, at least one of the cell-penetrating peptides is an amphiphilic peptide, and at least one of the cell-penetrating peptides is an oligospermine peptide. In some embodiments, one of the cell-penetrating peptides is an amphiphilic peptide, and one of the cell-penetrating peptides is an oligospermine peptide. In some embodiments, J represents two covalently linked cell-penetrating peptides as defined above. In another embodiment, the chimeric peptide-oligonucleotide conjugate of Formula I is the chimeric peptide-oligonucleotide conjugate of Formula Ib: [Figure] or a pharmaceutically acceptable salt thereof, wherein J is as defined above. In some embodiments, at least one of the cell-penetrating peptides is an amphiphilic peptide, and at least one of the cell-penetrating peptides is an oligospermine peptide. In some embodiments, one of the cell-penetrating peptides is an amphiphilic peptide, and one of the cell-penetrating peptides is an oligospermine peptide. In some embodiments, the two covalently linked cell-penetrating peptides are as defined above. In another embodiment, this document provides a method for treating muscle diseases, viral infections, neuromuscular diseases, or bacterial infections in an individual in need, comprising administering to the individual the chimeric peptide-oligonucleotide conjugate of the present invention.

Implementation Method

Claims

1. A chimeric peptide-oligonucleotide conjugate of formula I or a pharmaceutically acceptable salt thereof, wherein: A' is selected from -NHCH2C(O)NH2, -N(C1-6-alkyl)CH2C(O)NH2, where R5 is -C(O)(O-alkyl)x-OH, where x is 3-10, and each alkyl group is independently C2-6-alkyl each time it appears, or R5 is selected from -C(O)C1-6-alkyl, triphenylmethyl, monomethoxytriphenylmethyl, -(C1-6-alkyl)-R6, -(C1-6-heteroalkyl)-R6, -(C6-12-aryl)-R6, -(C1-9-heteroaryl)-R6, -C(O)O-(C1-6-alkyl)-R6, -C(O)O-(C6-12-aryl)-R6, -C(O)O-(C1-9-heteroaryl)-R6, and; R6 is selected from OH, SH, and NH2, or R6 is O, S, or NH, each covalently linked to a solid support; each R1 is independently selected from OH and -N(R3)(R4), wherein each R3 and R4 is independently -C1-6-alkyl in each occurrence; each R2 is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemically protected group, wherein the nucleobase independently contains a C3-6-heterocycle selected from pyridine, pyrimidine, triazine, purine, and denitro-purine in each occurrence; z is 8-40; and E' is selected from H, -C1-6-alkyl, -C(O)C1-6-alkyl, benzoyl, stearyl, triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, ; Where Q is -C(O)(CH2)6C(O)- or -C(O)(CH2)2S2(CH2)2C(O)-; R7 is -(CH2)2OC(O)N(R8)2, where R8 is -(CH2)6NHC(=NH)NH2; L is -C(O)(CH2)1-6-C1-6-heteroaryl-(CH2)1-6C(O)-, where L is covalently linked to the amino terminus of J via an amide bond; J is a cell-penetrating peptide with two covalently linked groups; G is selected from H, -C(O)C1-6-alkyl, benzoyl, and stearyl, where G is covalently linked to the carboxyl terminus of J via an amide bond; and at least one of the following conditions is true: 1) A' is; or 2) E' is; At least one of the cell-penetrating peptides is an amphiphilic peptide, and at least one of the cell-penetrating peptides is an oligospermine peptide; wherein the amphiphilic peptide comprises a hydrophobic peptide segment and a hydrophilic peptide segment; and wherein the oligospermine peptide comprises the sequence [(RYzR)x], wherein R is arginine, Y is independently selected from aminohexanoic acid (X) or β-alanine (B), z is 1, and x is 1, 2, 3, 4 or 5.

2. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein J is two covalently linked cell-penetrating peptides, wherein the two cell-penetrating peptides comprise an amphiphilic peptide and an oligospermine peptide, wherein the oligospermine peptide is the C-terminus of J and the amphiphilic peptide is the N-terminus of J.

3. The chimeric peptide-oligonucleotide conjugate of claim 1 or its pharmaceutically acceptable salt, wherein the oligospermine peptide comprises the sequence [(RXR)(RBR)]x or [(RBR)(RXR)]x, wherein R is arginine, X is aminohexanoic acid, B is β-alanine, and x is 1 or 2.

4. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the oligospermine peptide is [(RXR)(RBR)]2 (Bpep).

5. The chimeric peptide-oligonucleotide conjugate of claim 1 or a medically acceptable salt thereof, wherein the hydrophobic peptide fragment comprises a sequence of 2 to 10 amino acids independently selected from glycine, isoleucine, alanine, valine, leucine, phenylalanine or tryptophan.

6. The chimeric peptide-oligonucleotide conjugate of claim 1 or its pharmaceutically acceptable salt, wherein the two mediator peptides are pVEC (LLIILRRRIR KQAHAHSK), penetratin (RQIKIWFQNR RMKWKK), or melittin (GIGAVLKVLT TGLPALISWI KRKRQQ).

7. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the two mediator peptides are transmembrane peptides (RQIKIWFQNR RMKWKK).

8. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein J is a transmembrane peptide-Bpep (RQIKIWFQNR RMKWKKRXRR BRRXRRBR), pVEC-Bpep (LLIILRRRIR KQAHAHSKRX RRBRRXRR BR), or bee venom peptide-Bpep (GIGAVLKVLT TGLPALISWI KRKRQQRXRR BRRXRRBR).

9. The chimeric peptide-oligonucleotide conjugate of claim 1 or its pharmaceutically acceptable salt, wherein J is a transmembrane peptide-Bpep (RQIKIWFQNR RMKWKKRXRR BRRXRRBR).

10. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein E' is selected from H, -C1-6-alkyl, -C(O)C1-6-alkyl, benzoyl, stearyl, triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, and.

11. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein A' is selected from -N(C1-6-alkyl)CH2C(O)NH2.

12. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein E' is selected from H, -C(O)CH3, benzoyl, stearyl, triphenylmethyl, 4-methoxytriphenylmethyl, and.

13. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein A' is selected from -N(C1-6-alkyl)CH2C(O)NH2; and E' is [missing information].

14. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein A' is, and E' is selected from H, -C(O)CH3, triphenylmethyl, 4-methoxytriphenylmethyl, benzoyl, and stearyl.

15. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the chimeric peptide-oligonucleotide conjugate of formula I is selected from the following: wherein E' is selected from H, C1-6-alkyl, -C(O)CH3, benzoyl, and stearyl.

16. The chimeric peptide-oligonucleotide conjugate of claim 15 or a pharmaceutically acceptable salt thereof, wherein the chimeric peptide-oligonucleotide conjugate is of formula (Ia).

17. The chimeric peptide-oligonucleotide conjugate of claim 15 or a pharmaceutically acceptable salt thereof, wherein the chimeric peptide-oligonucleotide conjugate is of formula (Ib).

18. A chimeric peptide-oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, as claimed in any of claims 1 to 15, wherein each R1 is N(CH3)2.

19. A chimeric peptide-oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, as claimed in any of claims 1 to 15, wherein each R2 is a nucleobase, independently selected each time it appears from adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, and hypoxanthine.

20. A chimeric peptide-oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, as claimed in any of claims 1 to 15, wherein L is -C(O)(CH2)1-6-triazole-(CH2)1-6C(O)-.

21. A chimeric peptide-oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, as claimed in any of claims 1 to 15, wherein L is...

22. A chimeric peptide-oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, as claimed in any of claims 1 to 15, wherein G is selected from H, C(O)CH3, benzoyl, and stearyl.

23. A chimeric peptide-oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, as claimed in any of claims 1 to 15, wherein G is H or -C(O)CH3.

24. A chimeric peptide-oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, as claimed in any of claims 1 to 15, wherein G is H.

25. A chimeric peptide-oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, as claimed in any of claims 1 to 15, wherein G is -C(O)CH3.

26. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the chimeric peptide-oligonucleotide conjugate shows an improvement in uptake of at least 20 times compared to an unconjugated oligonucleotide.

27. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the chimeric peptide-oligonucleotide conjugate shows at least a two-fold improvement in uptake compared to a non-chimeric peptide-oligonucleotide conjugate, wherein the non-chimeric peptide is Bpep ([(RXR)(RBR)]2).

28. The chimeric peptide-oligonucleotide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the chimeric peptide-oligonucleotide conjugate shows improved uptake compared to the corresponding non-chimeric peptide-oligonucleotide conjugate, wherein the non-chimeric peptide is a transmembrane peptide (RQIKIWFQNRRMKWKK).

29. A pharmaceutical composition comprising a chimeric peptide-oligonucleotide conjugate or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 28, and at least one pharmaceutically acceptable carrier.

30. A chimeric peptide-oligonucleotide conjugate of formula I or a pharmaceutically acceptable salt thereof, wherein: A' is selected from -NHCH2C(O)NH2, -N(C1-6-alkyl)CH2C(O)NH2, where R5 is -C(O)(O-alkyl)x-OH, where x is 3-10, and each alkyl group is independently C2-6-alkyl each time it appears, or R5 is selected from -C(O)C1-6-alkyl, triphenylmethyl, monomethoxytriphenylmethyl, -(C1-6-alkyl)-R6, -(C1-6-heteroalkyl)-R6, -(C6-12-aryl)-R6, -(C1-9-heteroaryl)-R6, -C(O)O-(C1-6-alkyl)-R6, -C(O)O-(C6-12-aryl)-R6, -C(O)O-(C1-9-heteroaryl)-R6, and; R6 is selected from OH, SH, and NH2, or R6 is O, S, or NH, each covalently linked to a solid support; each R1 is independently selected from OH and -N(R3)(R4), wherein each R3 and R4 is independently -C1-6-alkyl in each occurrence; each R2 is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemically protected group, wherein the nucleobase independently contains a C3-6-heterocycle selected from pyridine, pyrimidine, triazine, purine, and denitro-purine in each occurrence; z is 8-40; and E' is selected from H, -C1-6-alkyl, -C(O)C1-6-alkyl, benzoyl, stearyl, triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, ; Where Q is -C(O)(CH2)6C(O)- or -C(O)(CH2)2S2(CH2)2C(O)-; R7 is -(CH2)2OC(O)N(R8)2, where R8 is -(CH2)6NHC(=NH)NH2; L is -C(O)(CH2)1-6-C1-6-heteroaryl-(CH2)1-6C(O)-, where L is covalently linked to the amino end of J via an amide bond; J is Bpep-Bpep (RXRRBRRXRR BRRXRRBRRX RRBR) (SEQ ID NO: 4), where R is arginine, X is aminohexanoic acid, and B is β-alanine; G is selected from H, -C(O)C1-6-alkyl, benzoyl, and stearyl, wherein G is covalently linked to the carboxyl terminus of J by an amide bond; and wherein at least one of the following conditions is true: 1) A' is true; or 2) E' is true.

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