Lysosomal targeting peptides and uses thereof

Abstract

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 targeted therapeutic fusion protein comprising:
 a lysosomal enzyme; and   an IGF-II mutein having an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1), wherein the IGF-II mutein is resistant to furin cleavage and binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.   
     
     
         2 . A targeted therapeutic fusion protein comprising:
 a lysosomal enzyme; and   an IGF-II mutein having an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1), and having diminished binding affinity for the insulin receptor relative to the affinity of naturally-occurring human IGF-II for the insulin receptor;   wherein the IGF-II mutein binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.   
     
     
         3 . A targeted therapeutic fusion protein comprising:
 a lysosomal enzyme; and   an IGF-II mutein having an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1), and having diminished binding affinity for the insulin receptor relative to the affinity of naturally-occurring human IGF-II for the insulin receptor;   wherein the IGF-II mutein is resistant to furin cleavage and binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.   
     
     
         4 . The targeted therapeutic fusion protein of any one of  claims 1 - 3 , wherein the IGF-II mutein comprises a mutation within a region corresponding to amino acids 30-40 of SEQ ID NO:1. 
     
     
         5 . The targeted therapeutic fusion protein of any one of the preceding claims, wherein the IGF-II mutein comprises a mutation within a region corresponding to amino acids 34-40 of SEQ ID NO:1 such that the mutation abolishes at least one furin protease cleavage site 
     
     
         6 . The targeted therapeutic fusion protein of  claim 4  or  5 , wherein the mutation is an amino acid substitution, deletion and/or insertion. 
     
     
         7 . The targeted therapeutic fusion protein of  claim 6 , wherein the mutation is an amino acid substitution at a position corresponding to Arg37 or Arg40 of SEQ ID NO:1. 
     
     
         8 . The targeted therapeutic fusion protein of  claim 7 , wherein the amino acid substitution is a Lys or Ala substitution. 
     
     
         9 . The targeted therapeutic fusion protein of  claim 6 , wherein the mutation is a deletion or replacement of amino acid residues corresponding to positions selected from the group consisting of 31-40, 32-40, 33-40, 34-40, 30-39, 31-39, 32-39, 34-37, 32-39, 33-39, 34-39, 35-39, 36-39, 37-40, 34-40 of SEQ ID NO:1, and combinations thereof. 
     
     
         10 . The targeted therapeutic fusion protein of any one of the preceding claims, wherein the IGF-II mutein further comprises a deletion or a replacement of amino acids corresponding to positions 2-7 of SEQ ID NO:1. 
     
     
         11 . The targeted therapeutic fusion protein of any one of  claims 1 - 10 , wherein the IGF-II mutein further comprises a deletion or a replacement of amino acids corresponding to positions 1-7 of SEQ ID NO:1. 
     
     
         12 . The targeted therapeutic fusion protein of any one of the preceding claims, wherein the IGF-II mutein further comprises a deletion or a replacement of amino acids corresponding to positions 62-67 of SEQ ID NO:1. 
     
     
         13 . The targeted therapeutic fusion protein of any one of the preceding claims, wherein the IGF-II mutein further comprises an amino acid substitution at a position corresponding to Tyr27, Leu43, or Ser26 of SEQ ID NO:1. 
     
     
         14 . The targeted therapeutic fusion protein of  claim 13 , wherein the IGF-II mutein comprises at least an amino acid substitution selected from the group consisting of Tyr27Leu, Leu43 Val, Ser26Phe and combinations thereof. 
     
     
         15 . The targeted therapeutic fusion protein of any one of the preceding claims, wherein the IGF-II mutein comprises amino acids corresponding to positions 48-55 of SEQ ID NO:1. 
     
     
         16 . The targeted therapeutic fusion protein of any one of the preceding claims, wherein the IGF-II mutein comprises at least three amino acids selected from the group consisting of amino acids corresponding to positions 8, 48, 49, 50, 54, and 55 of SEQ ID NO:1. 
     
     
         17 . The targeted therapeutic fusion protein of any one of the preceding claims, wherein the IGF-II mutein comprises, at positions corresponding to positions 54 and 55 of SEQ ID NO:1, amino acids each of which is uncharged or negatively charged at pH 7.4. 
     
     
         18 . The targeted therapeutic fusion protein of any one of the preceding claims, wherein the IGF-II mutein has diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor. 
     
     
         19 . The targeted therapeutic fusion protein of any one of the preceding claims, wherein the lysosomal enzyme is human acid alpha-glucosidase (GAA), or a functional variant thereof. 
     
     
         20 . The targeted therapeutic fusion protein of  claim 18 , wherein the lysosomal enzyme comprises amino acids 70-952 of human GAA. 
     
     
         21 . The targeted therapeutic fusion protein of any one of the preceding claims, wherein the targeted therapeutic fusion protein further comprises a spacer between the lysosomal enzyme and the IGF-II mutein. 
     
     
         22 . The targeted therapeutic fusion protein of  claim 21 , wherein the spacer comprises an amino acid sequence Gly-Ala-Pro. 
     
     
         23 . A nucleic acid encoding the targeted therapeutic fusion protein of any one of the preceding claims. 
     
     
         24 . A cell containing the nucleic acid of  claim 23 . 
     
     
         25 . A pharmaceutical composition suitable for treating lysosomal storage disease comprising a targeted therapeutic fusion protein of any one of the preceding claims. 
     
     
         26 . A method of treating lysosomal storage disease comprising administering to a subject in need of treatment a targeted therapeutic fusion protein of any one of the preceding claims. 
     
     
         27 . The method of  claim 26 , wherein the lysosomal storage disease is Pompe Disease. 
     
     
         28 . The method of  claim 26 , wherein the lysosomal storage disease is Fabry Disease. 
     
     
         29 . The method of  claim 26 , wherein the lysosomal storage disease is Gaucher Disease. 
     
     
         30 . A method of producing a targeted therapeutic fusion protein comprising a step of:
 culturing mammalian cells in a cell culture medium, wherein
 the mammalian cells carry the nucleic acid of  claim 23 ; and 
   the culturing is performed under conditions that permit expression of the targeted therapeutic fusion protein.   
     
     
         31 . A method of producing a targeted therapeutic fusion protein comprising a step of:
 culturing furin-deficient cells in a cell culture medium, wherein   the furin-deficient cells carry a nucleic acid encoding a fusion protein comprising a lysosomal enzyme and an IGF-II mutein having an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1), wherein the IGF-II mutein binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner; and   wherein the culturing is performed under conditions that permit expression of the targeted therapeutic fusion protein.