Lysosomal Targeting Peptides and Uses Thereof
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 method of treating Sanfilippo B disease (MPS IIIB) comprising administering to a subject in need of treatment a targeted therapeutic fusion protein comprising: a lysosomal enzyme which is α-N-Acetylglucosaminidase (Naglu); an IGF-II mutein comprising amino acids 8-67 of SEQ ID NO: 1 and an Ala substitution at position Arg37 of SEQ ID NO:1, wherein the IGF-II mutein (i) has diminished binding affinity for the insulin receptor relative to the affinity of naturally-occurring human IGF-II for the insulin receptor, (ii) is resistant to furin cleavage and (iii) binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner; and a spacer between the lysosomal enzyme and the IGF-II mutein, wherein the spacer comprises the amino acid sequence Gly-Ala-Pro.
2 . The method of claim 1 , wherein the IGF-II mutein is fused via the spacer to the C-terminus of the lysosomal enzyme.
3 . The method of claim 1 , wherein the IGF-II mutein is fused via the spacer to the N-terminus of the lysosomal enzyme.
4 . The method of claim 1 , wherein the IGF-II mutein consists of amino acids 8-67 of SEQ ID NO:1 having an Ala substitution at position Arg37 of SEQ ID NO:1.
5 . The method of claim 1 , comprising administering the targeted therapeutic fusion protein to the nervous system.
6 . The method of claim 5 , comprising administering the targeted therapeutic fusion protein intraventricularly and/or intrathecally.
7 . The method of claim 1 , comprising administering the targeted therapeutic fusion protein in an amount effective to reduce the severity or frequency, or delay the onset of, at least one symptom or feature of MPS IIIB.
8 . The method of claim 7 , comprising administering the targeted therapeutic fusion protein in an amount effective to decrease accumulated heparan sulfate in lysosomes.
9 . The method of claim 1 , comprising administering the targeted therapeutic fusion protein at an interval selected from daily, thrice weekly, twice weekly, weekly, biweekly, triweekly, monthly, and bimonthly.
10 . The method of claim 1 , comprising administering the targeted therapeutic fusion protein in a pharmaceutical composition comprising a water-soluble carrier.
11 . A method of ameliorating the symptoms of Sanfilippo B disease (MPS IIIB) comprising administering to a subject in need of treatment a targeted therapeutic fusion protein comprising: a lysosomal enzyme which is α-N-Acetylglucosaminidase (Naglu); an IGF-II mutein comprising amino acids 8-67 of SEQ ID NO: 1 and an Ala substitution at position Arg37 of SEQ ID NO:1, wherein the IGF-II mutein (i) has diminished binding affinity for the insulin receptor relative to the affinity of naturally-occurring human IGF-II for the insulin receptor, (ii) is resistant to furin cleavage and (iii) binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner; and a spacer between the lysosomal enzyme and the IGF-II mutein, wherein the spacer comprises the amino acid sequence Gly-Ala-Pro.
12 . The method of claim 11 , wherein the IGF-II mutein is fused via the spacer to the C-terminus of the lysosomal enzyme.
13 . The method of claim 11 , wherein the IGF-II mutein is fused via the spacer to the N-terminus of the lysosomal enzyme.
14 . The method of claim 11 , wherein the IGF-II mutein consists of amino acids 8-67 of SEQ ID NO:1 having an Ala substitution at position Arg37 of SEQ ID NO:1.
15 . A method of delivering α-N-Acetylglucosaminidase (Naglu) enzyme activity to a cell deficient in Naglu enzyme activity comprising contacting the cell with a fusion protein comprising: a lysosomal enzyme which is α-N-Acetylglucosaminidase (Naglu); an IGF-II mutein comprising amino acids 8-67 of SEQ ID NO: 1 and an Ala substitution at position Arg37 of SEQ ID NO:1, wherein the IGF-II mutein (i) has diminished binding affinity for the insulin receptor relative to the affinity of naturally-occurring human IGF-II for the insulin receptor, (ii) is resistant to furin cleavage and (iii) binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner; and a spacer between the lysosomal enzyme and the IGF-II mutein, wherein the spacer comprises the amino acid sequence Gly-Ala-Pro.
16 . The method of claim 15 , wherein the cell is a nerve cell.