Lysosomal targeting peptides and uses thereof
View Patent ↗The present invention provides further improved compositions and methods for efficient lysosomal targeting based on the GILT technology. Among other things, the present invention provides methods and compositions for targeting lysosomal enzymes to lysosomes using furin-resistant lysosomal targeting peptides. The present invention also provides methods and compositions for targeting lysosomal enzymes to lysosomes using a lysosomal targeting peptide that has reduced or diminished binding affinity for the insulin receptor.
1. A therapeutic fusion protein comprising an amino acid sequence at least 70% identical to wild type human α-N-acetylglucosaminidase (Naglu);
a lysosomal targeting moiety; and
wherein, once administered, the therapeutic fusion protein is targeted to lysosomes and is therapeutically active in vivo,
wherein the lysosomal targeting moiety is an IGF-II mutein comprising an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1), with a mutation within a region corresponding to amino acids 30-40 of SEQ ID NO:1 such that said mutation abolishes at least one furin protease cleavage site, wherein said mutation comprises a deletion of amino acids 30-40 of SEQ ID NO:1 or an amino acid substitution at a position corresponding to Arg37 of SEQ ID NO: 1, and
wherein the IGF-II mutein is (a) resistant to furin cleavage, (b) has diminished binding affinity for the insulin receptor relative to the affinity of naturally-occurring human IGF-II for the insulin receptor, and (c) binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.
2. The therapeutic fusion protein of claim 1 , wherein the Naglu comprises an amino acid sequence at least 80% identical to wild-type human Naglu.
3. The therapeutic fusion protein of claim 1 , wherein the fusion protein further comprises a linker between the Naglu and the lysosomal targeting moiety.
4. The therapeutic fusion protein of claim 3 , wherein the linker comprises the amino acid sequence GAP (SEQ ID NO:4).
5. The therapeutic fusion protein of claim 1 , wherein the lysosomal targeting moiety is fused directly or via a linker to the C-terminus of the Naglu.
6. The therapeutic fusion protein of claim 1 , wherein said mutation comprises a deletion of amino acids 30-40 of SEQ ID NO:1.
7. The therapeutic fusion protein of claim 1 , wherein said mutation comprises an amino acid substitution at a position corresponding to Arg37 of SEQ ID NO: 1.
8. The therapeutic fusion protein of claim 7 , wherein said amino acid substitution is an Ala substitution.
9. A method of treating Sanfilippo B disease (MPS IIIB) comprising a step of administering intrathecally to a subject in need of treatment the therapeutic fusion protein of claim 1 .
10. The method of claim 9 , wherein the Naglu comprises an amino acid sequence at least 80% identical to wild-type human Naglu.
11. The method of claim 9 , wherein the fusion protein further comprises a linker between the Naglu and the lysosomal targeting moiety.
12. The method of claim 11 , wherein the linker comprises the amino acid sequence GAP (SEQ ID NO:4).
13. The method of claim 9 , wherein the lysosomal targeting moiety is fused directly or via a linker to the C-terminus of the Naglu.
14. The method of claim 9 , wherein the lysosomal targeting moiety is fused directly or via a linker to the N-terminus of the Naglu.
15. The method of claim 9 , wherein the intrathecal administration results in reduced intensity, severity, or frequency, or delayed onset of at least one symptom or feature of MPS IIIB.
16. The method of claim 9 , wherein the intrathecal administration takes place at an interval selected from once every two weeks, once every month, and once every two months.
17. The method of claim 9 , wherein the intrathecal administration is used in conjunction with intravenous administration.
18. The method of claim 9 , wherein the intrathecal administration is not used in conjunction with intravenous administration.
19. The method of claim 9 , wherein said mutation comprises a deletion of amino acids 30-40 of SEQ ID NO:1.
20. The method of claim 9 , wherein said mutation comprises an amino acid substitution at a position corresponding to Arg 37 of SEQ ID NO: 1.
21. The method of claim 9 , wherein said amino acid substitution is an Ala substitution.