Methods and compositions for treatment of myotubular myopathy using chimeric polypeptides comprising myotubularin 1(MTM1) polypeptides
The present invention provides chimeric polypeptides comprising myotubularin 1 (MTMI) polypeptides and an internalizing moiety, wherein, the moiety can be an antibody, and is preferably monoclonal antibody 3E10, a functional variant or a fragment thereof. One aspect of the present invention provides compositions comprising these chimeric polypeptides together with a pharmaceutically acceptable carrier, and optionally, a further therapeutic agent. Another aspect of the present invention provides methods of treating Myotubular Myopathy comprising administering the polypeptides or compositions comprising the polypeptides to a subject in need.
1. A chimeric polypeptide comprising: (i) a myotubularin (MTM1) polypeptide, or a bioactive fragment thereof; wherein the MTM1 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and (ii) an internalizing moiety,
wherein the chimeric polypeptide has phosphoinositide phosphatase activity;
wherein said internalizing moiety is an antibody or antigen-binding fragment thereof,
wherein said antibody or antigen-binding fragment thereof is a monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antigen-binding fragment of any of the foregoing.
2. The chimeric polypeptide of claim 1 , wherein the MTM1 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.
3. The chimeric polypeptide of claim 2 , wherein the MTM1 polypeptide comprises a wildtype MTM1 polypeptide.
4. The chimeric polypeptide of claim 1 , wherein (i) comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.
5. The chimeric polypeptide of claim 1 , wherein (i) comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1.
6. The chimeric polypeptide of claim 1 , wherein the internalizing moiety promotes transport of said chimeric polypeptide into muscle cells.
7. The chimeric polypeptide of claim 1 or 3 , wherein said antibody or antigen-binding fragment thereof comprises a light chain variable domain that is a humanized variant of SEQ ID NO: 4, and wherein said antibody or antigen-binding fragment thereof further comprises a heavy chain variable domain that is a humanized variant of SEQ ID NO: 2.
8. The chimeric polypeptide of claim 7 , wherein the internalizing moiety is a Fab′.
9. The chimeric polypeptide of claim 7 , wherein the internalizing moiety is a full-length antibody.
10. The chimeric polypeptide of claim 1 , wherein the internalizing moiety is a Fab′.
11. The chimeric polypeptide of claim 1 , wherein the internalizing moiety is a F(ab′)2 fragment.
12. The chimeric polypeptide of claim 1 , wherein the internalizing moiety is a full-length antibody.
13. The chimeric polypeptide of claim 1 , comprising a linker joining the MTM1 polypeptide or bioactive fragment thereof to the internalizing moiety.
14. The chimeric polypeptide of claim 13 , wherein the internalizing moiety is conjugated to the N-terminal or C-terminal amino acid of the MTM1 polypeptide.
15. The chimeric polypeptide of claim 1 , wherein the MTM1 polypeptide or bioactive fragment thereof is chemically conjugated to the internalizing moiety.
16. The chimeric polypeptide of claim 1 , wherein the MTM1 polypeptide is expressed in bacterial cells.
17. A nucleic acid construct, comprising a nucleotide sequence that encodes a myotubularin (MTM1) polypeptide, or a bioactive fragment thereof, operably linked to a nucleotide sequence that encodes an internalizing moiety,
wherein the nucleic acid construct encodes a chimeric polypeptide having phosphoinositide phosphatase activity; wherein the MTM1 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.
18. The nucleic acid of claim 17 , wherein the MTM1 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.
19. The nucleic acid of claim 18 , wherein the MTM1 polypeptide comprises a wildtype MTM1 polypeptide.
20. The nucleic acid of claim 19 , wherein said antibody or antigen-binding fragment thereof comprises a light chain variable domain that is a humanized variant of SEQ ID NO: 4, and wherein said antibody or antigen-binding fragment thereof further comprises a heavy chain variable domain that is a humanized variant of SEQ ID NO: 2.
21. The nucleic acid of claim 17 , wherein said antibody or antigen-binding fragment thereof comprises a light chain variable domain that is a humanized variant of SEQ ID NO: 4, and wherein said antibody or antigen-binding fragment thereof further comprises a heavy chain variable domain that is a humanized variant of SEQ ID NO: 2.
22. A method of delivering a chimeric polypeptide into a muscle cell, comprising
contacting a muscle cell with a chimeric polypeptide, which chimeric polypeptide comprises (i) a myotubularin (MTM1) polypeptide, or a bioactive fragment thereof; wherein the MTM1 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and (ii) an internalizing moiety,
wherein the chimeric polypeptide has phosphoinositide phosphatase activity;
wherein said internalizing moiety is an antibody or antigen-binding fragment thereof,
wherein said antibody or antigen-binding fragment thereof is a monoclonal antibody 3E10, or a variant thereof that retains the cell penetrating activity of 3E10, or an antigen-binding fragment of any of the foregoing.
23. The method of claim 22 , wherein the MTM1 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.
24. The method of claim 23 , wherein the MTM1 polypeptide comprises a wildtype MTM1 polypeptide.
25. The method of claim 24 , wherein said antibody or antigen-binding fragment thereof comprises a light chain variable domain that is a humanized variant of SEQ ID NO: 4, and wherein said antibody or antigen-binding fragment thereof further comprises a heavy chain variable domain that is a humanized variant of SEQ ID NO: 2.
26. The method of claim 22 , wherein said antibody or antigen-binding fragment thereof comprises a light chain variable domain that is a humanized variant of SEQ ID NO: 4, and wherein said antibody or antigen-binding fragment thereof further comprises a heavy chain variable domain that is a humanized variant of SEQ ID NO: 2.
27. The method of claim 22 , wherein the cell is a skeletal muscle cell.