Gene therapy constructs and methods of use

Abstract

Provided herein are improved gene therapy vectors and methods of use, in some embodiments, comprising sequences for improved expression and cellular targeting of a therapeutic protein.

What is claimed is: 
     
         1 . A gene therapy vector comprising a nucleic acid construct encoding a polypeptide comprising:
 (a) a therapeutic protein;   (b) a peptide that binds to the cation-independent mannose 6-phosphate (M6P) receptor (CI-MPR) with high affinity; and   (c) a linker between the therapeutic protein and the peptide that binds CI-MPR.   
     
     
         2 . The gene therapy vector of  claim 1 , wherein the peptide is a variant IGF2 (vIGF2) peptide. 
     
     
         3 . The gene therapy vector of  claim 2 , wherein the vIGF2 peptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and having at least one substitution at one or more positions selected from the group consisting of positions 6, 26, 27, 43, 48, 49, 50, 54, 55, and 65 of SEQ ID NO: 1. 
     
     
         4 . The gene therapy vector of  claim 3 , wherein the at least one substitution is selected from the group consisting of E6R, F26S, Y27L, V43L, F48T, R495, S50I, A54R, L55R, and K65R of SEQ ID NO:1. 
     
     
         5 . The gene therapy vector of  claim 3 , wherein the vIGF2 peptide comprises at least two substitutions at two or more positions selected from the group consisting of positions 6, 26, 27, 43, 48, 49, 50, 54, 55, 65 of SEQ ID NO: 1. 
     
     
         6 . The gene therapy vector of  claim 5 , wherein the at least two substitutions are selected from the group consisting of E6R, F26S, Y27L, V43L, F48T, R495, S50I, A54R, L55R, K65R of SEQ ID NO: 1. 
     
     
         7 . The gene therapy vector of  claim 1 , wherein the vIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQ ID NO: 1. 
     
     
         8 . The gene therapy vector of  claim 1 , wherein the vIGF2 peptide has decreased affinity for insulin receptor and IGF1R as compared to native IGF2 peptide. 
     
     
         9 . The gene therapy vector of  claim 2 , wherein the vIGF2 peptide is capable of facilitating uptake of the therapeutic protein into a cell. 
     
     
         10 . The gene therapy vector of  claim 2 , wherein the vIGF2 peptide is capable of facilitating uptake of the therapeutic protein into a lysosome. 
     
     
         11 . The gene therapy vector of  claim 1 , wherein the therapeutic protein is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having the genetic disorder. 
     
     
         12 . The gene therapy vector of  claim 11 , wherein the genetic disorder is a lysosomal storage disorder. 
     
     
         13 . The gene therapy vector of  claim 11 , wherein the genetic disorder is selected from the group consisting of aspartylglucosaminuria, Batten disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteau-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, Schindler disease type II, adenosine deaminase severe combined immunodeficiency (ADA-SCID), chronic granulomatous disease (CGD), and neuronal ceroid lipofuscinosis. 
     
     
         14 . The gene therapy vector of  claim 11 , wherein the genetic disorder is Pompe disease. 
     
     
         15 . The gene therapy vector of  claim 11 , wherein the genetic disorder is a CLN1 disease. 
     
     
         16 . The gene therapy vector of  claim 1 , wherein the therapeutic protein comprises a soluble lysosomal enzyme or an enzymatically active fragment thereof. 
     
     
         17 . The gene therapy vector of  claim 1 , wherein the therapeutic protein comprises a lysosomal enzyme or an enzymatically active fragment thereof, wherein the lysosomal enzyme is selected from the group consisting of alpha-galactosidase A, β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, glycosaminoglycan alpha-L-iduronohydrolase, iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, palmitoyl protein thioesterases, cyclin dependent kinase like 5, and alpha-glucosidase. 
     
     
         18 . The gene therapy vector of  claim 17 , wherein the therapeutic protein is alpha-glucosidase or an enzymatically active fragment thereof. 
     
     
         19 . The gene therapy vector of  claim 17 , wherein the therapeutic protein is palmitoyl protein thioesterase-1 or an enzymatically active fragment thereof. 
     
     
         20 . The gene therapy vector of  claim 1 , wherein the nucleic acid construct further comprises a translation initiation sequence. 
     
     
         21 . The gene therapy vector of  claim 1 , wherein the nucleic acid construct further comprises a nucleic acid sequence encoding a signal peptide wherein the signal peptide is capable of increasing secretion of the therapeutic protein as compared to the therapeutic protein without the signal peptide. 
     
     
         22 . The gene therapy vector of  claim 21 , wherein the signal peptide is selected from a binding immunoglobulin protein (BiP) signal peptide and a  Gaussia  signal peptide. 
     
     
         23 . The gene therapy vector of  claim 1 , wherein the vIGF2 peptide comprises the sequence of SEQ ID NO:31. 
     
     
         24 . The gene therapy vector of  claim 1 , wherein the construct comprises SEQ ID NO:36. 
     
     
         25 . The gene therapy vector of  claim 1 , wherein the polypeptide comprises SEQ ID NO:23. 
     
     
         26 . The gene therapy vector of  claim 1 , wherein the construct comprises SEQ ID NO:38. 
     
     
         27 . The gene therapy vector of  claim 1 , wherein the vIGF2 at the N-terminus of the polypeptide. 
     
     
         28 . The gene therapy vector of  claim 1 , wherein the vIGF2 is at the C-terminus of the polypeptide. 
     
     
         29 . The gene therapy vector of  claim 1 , wherein the linker peptide comprises SEQ ID NO: 18-21 or SEQ ID NO: 33. 
     
     
         30 . The gene therapy vector of  claim 1 , wherein the gene therapy vector is a virus vector selected from the group consisting of an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a lentivirus vector, a pox virus vector, a vaccinia virus vector, an adenovirus vector, and a herpes virus vector.