Antisense conjugates for decreasing expression of DMPK
The disclosure provides novel conjugates comprising antisense oligonucleotides that hybridize to a DMPK transcript and a 3E10 antibody or binding fragment thereof. Also considered are these conjugates further comprising MBNL1 polypeptides. Methods of treating myotonic dystrophy using these conjugates and kits comprising these conjugates are also considered. Wherein the conjugates are suitable for delivery to muscle cells.
1. A conjugate comprising
a) an antisense oligonucleotide that hybridizes to a DMPK transcript wherein the oligonucleotide is capable of:
i) mediating RNase H-mediated degradation following hybridization to the DMPK transcript, and/or
ii) preventing a protein or nucleic acid from binding to the DMPK transcript; and
b) an antibody or antigen binding fragment: wherein the antibody or antigen binding fragment comprises a heavy chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13, or a humanized variant thereof, and a light chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14, or a humanized variant thereof; wherein the antibody or antigen binding fragment promotes delivery of the antisense oligonucleotide into muscle cells.
2. The conjugate of claim 1 , wherein the antibody or antigen binding fragment comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 13, or a humanized variant thereof, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 14, or a humanized variant thereof.
3. The conjugate of claim 1 , wherein the antisense oligonucleotide is a gapmer or a morpholino.
4. The conjugate of claim 1 , wherein the antisense oligonucleotide hybridizes to CUG repeats to at least a portion of the 3′UTR of a DMPK transcript.
5. The conjugate of claim 1 , wherein the antisense oligonucleotide hybridizes to any one of SEQ ID NOs: 24-27.
6. The conjugate of claim 1 , wherein the antisense oligonucleotide is a single stranded oligonucleotide.
7. The conjugate of claim 1 , wherein the antisense oligonucleotide comprises 14, 15, 16, 17, 18, 19, or 20 nucleotides.
8. The conjugate of claim 1 , wherein the antisense oligonucleotide comprises 14-30 nucleotides.
9. The conjugate of claim 1 , wherein the antisense oligonucleotide comprises a central portion of at least 7 nucleotides, which central portion of at least 7 nucleotides is capable of mediating RNase H-mediated degradation following hybridization to RNA.
10. The conjugate of claim 1 , wherein the antisense oligonucleotide sterically inhibits binding of MBNL1 to a nucleotide sequence.
11. The conjugate of claim 1 , wherein the antisense oligonucleotide prevents a protein or nucleic acid from binding to any one of SEQ ID NOs: 24-27.
12. The conjugate of claim 9 , wherein the central portion of at least 7 nucleotides is composed of unmodified DNA nucleotides, modified nucleotides, or a combination thereof.
13. The conjugate of claim 12 , wherein modified nucleotides are phosphorothioate nucleotides.
14. The conjugate of claim 1 , wherein the antisense oligonucleotide comprises two wing portions, each of which flank the central portion.
15. The conjugate of claim 14 , wherein each of the two wing portions comprises at least 3 nucleotides.
16. The conjugate of claim 14 , wherein all of the nucleotides of each of the two wings comprise modified nucleotides of the same class of modification selected from one of the following: locked nucleic acid (LNA) nucleotides, 2′-O-methoxyelthyl nucleotides, 2′-O-methyl nucleotides, a peptide nucleic acid, a hexitol nucleic acid, an N3′-P5′-phosphoroamidate, or conformationally restricted nucleotides (CRN).
17. The conjugate of claim 16 , wherein the antisense oligonucleotide that binds to the DMPK transcript comprises the primary nucleotide sequence set forth in any of SEQ ID NOs: 9-12 or 22-23.
18. The conjugate of claim 17 , wherein the two wing portions of the antisense oligonucleotide comprise LNA nucleotides or 2′-O-methoxyethyl nucleotides.
19. The conjugate of claim 17 , wherein the two wing portions of the antisense oligonucleotide comprise CRN nucleotides.
20. The conjugate of claim 1 , wherein the conjugate comprises an antigen binding fragment, and the antisense oligonucleotide is conjugated to the C-terminus of the antigen binding fragment.
21. The conjugate of claim 1 , wherein the conjugate comprises an antigen binding fragment, and the antisense oligonucleotide is conjugated to the N-terminus of the antigen binding fragment.
22. The conjugate of claim 1 , wherein the conjugate comprises an antibody, and the antisense oligonucleotide is conjugated to an Fc portion of the antibody.
23. The conjugate of claim 22 , wherein a free cysteine is added to the antibody or antigen binding fragment, and the antisense oligonucleotide is conjugated by site directed chemical conjugation via the free cysteine.
24. The conjugate of claim 1 , wherein the ratio of antisense oligonucleotide conjugated per antibody or antigen binding fragment is 1:1.
25. The conjugate of claim 1 , wherein the antisense oligonucleotide hybridizes to at least a portion of the 3′UTR region of the DMPK transcript.
26. The conjugate of claim 25 , wherein the antisense oligonucleotide hybridizes to a DMPK transcript and promotes degradation of the transcript.
27. The conjugate of claim 1 , wherein the conjugate further comprises a muscle blind polypeptide (MBNL1), or a functional fragment thereof comprising all four zinc finger motifs, interconnected to the antibody or antigen fragment thereof.
28. A conjugate comprising
a MBNL1 polypeptide or a functional fragment thereof comprising all four zinc finger motifs;
an antibody or antigen binding fragment, wherein the antibody or antigen binding fragment comprises a heavy chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13, or a humanized variant thereof, and a light chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14, or a humanized variant thereof; wherein the antibody or antigen binding fragment promotes delivery of the antisense oligonucleotide into muscle cells; and
an antisense oligonucleotide that hybridizes to a DMPK transcript; wherein the oligonucleotide is capable of:
i) mediating RNase H-mediated degradation following hybridization to the DMPK transcript, and/or
ii) preventing a protein or nucleic acid from binding to the DMPK transcript.
29. A composition comprising the conjugate of claim 1 formulated in a physiologically acceptable carrier.
30. The composition of claim 29 , wherein said composition is substantially pyrogen-free.
31. A method of treating myotonic dystrophy, comprising administering to a patient in need thereof a conjugate of claim 1 .
32. A method of promoting entry into muscle cells, comprising contacting cells or administering to a patient a conjugate of claim 1 .
33. A method of treating myotonic dystrophy, comprising administering to a patient in need thereof two therapeutics, either simultaneously or concurrently, and wherein the therapeutics comprise:
a conjugate of claim 1 ; and
a chimeric polypeptide comprising:
(a) an MBNL1 polypeptide or a functional fragment thereof comprising all four zinc finger motifs; and
(b) an antibody or antigen binding fragment; wherein the antibody or antigen binding fragment comprises a heavy chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13, or a humanized variant thereof, and a light chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14, or a humanized variant thereof; wherein the antibody or antigen binding fragment promotes delivery of the antisense oligonucleotide into muscle cells.
34. A kit comprising
a first container comprising the conjugate of claim 1 ; and
a second container comprising a chimeric polypeptide comprising:
(a) an MBNL1 polypeptide or a functional fragment thereof comprising all four zinc finger motifs; and
(b) an antibody or antigen binding fragment; wherein the antibody or antigen binding fragment comprises a heavy chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13, or a humanized variant thereof, and a light chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14, or a humanized variant thereof; wherein the antibody or antigen binding fragment promotes delivery of the antisense oligonucleotide into muscle cells.
35. The kit of claim 34 , comprising instructions for research or therapeutic use.
36. The kit of claim 34 , comprising suitable buffers to reconstitute a lyophilized agent in either the first or the second container.
37. The conjugate of claim 1 , wherein the antisense oligonucleotide is a gapmer, and wherein the antisense oligonucleotide comprises phosphorothioate nucleotides and locked nucleic acid (LNA) nucleotides.
38. The conjugate of claim 37 , wherein the antibody or antigen binding fragment comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 13, or a humanized variant thereof, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 14, or a humanized variant thereof.