Constructs for expressing lysosomal polypeptides

Abstract

Provided are isolated nucleic acids for expressing lysosomal polypeptides such as lysosomal acid α-glucosidase (GAA) and vectors comprising the same. In one embodiment, the invention provides an isolated nucleic acid encoding a chimeric polypeptide comprising a secretory signal sequence operably linked to a lysosomal polypeptide. In another representative embodiment, an isolated nucleic acid is provided comprising a coding region encoding a GAA and a GAA 3′ untranslated region (UTR), wherein the GAA 3′ UTR comprises a deletion therein. In another representative embodiment, the invention provides an isolated nucleic acid comprising a coding region encoding a GAA and a 3′ UTR, wherein the 3′ UTR is less than about 200 nucleotides in length and comprises a segment that is heterologous to the GAA coding region. Also provided are methods of making and using delivery vectors encoding lysosomal polypeptides, for example, to produce the lysosomal polypeptide or to treat subjects afflicted with a deficiency in the lysosomal polypeptide.

1 . A vector comprising a nucleic acid encoding a chimeric polypeptide comprising a secretory signal sequence operably linked to a lysosomal polypeptide, wherein said secretory signal sequence replaces the native secretory signal sequence of the lysosomal polypeptide. 
     
     
         2 . The vector of  claim 1 , wherein said nucleic acid is operatively linked to a transcriptional control element operable in liver cells. 
     
     
         3 . The vector of  claim 1 , wherein the secretory signal sequence is derived from a secreted polypeptide. 
     
     
         4 . The vector of  claim 1 , wherein the secretory signal sequence is derived from SP38, erythropoietin albumin, albumin, or coagulation factor IX. 
     
     
         5 . The vector of  claim 1 , wherein the secretory signal sequence comprises the amino acid sequence set forth in any of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 9, or an amino acid sequence having at least 98% sequence identity to the sequence set forth in any of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 9. 
     
     
         6 . The vector of  claim 1 , wherein the vector is an adeno-associated virus (AAV). 
     
     
         7 . The vector of  claim 1 , wherein secretory signal sequence replaces the amino terminus of the native lysosomal polypeptide 
     
     
         8 . A pharmaceutical formulation comprising the vector of  claim 1  in a pharmaceutically acceptable carrier. 
     
     
         9 . An isolated cell comprising the vector of  claim 1 . 
     
     
         10 . An in-vitro method of delivering a nucleic acid encoding a lysosomal polypeptide to a cell, comprising contacting a cell in vitro with the vector according to  claim 1  under conditions sufficient for the vector to be introduced into the cell and for the nucleic acid to be expressed to produce the chimeric polypeptide comprising the secretory signal sequence operably linked to the lysosomal polypeptide wherein said secretory signal sequence replaces the native secretory signal sequence of the lysosomal polypeptide. 
     
     
         11 . The method of  claim 10 , wherein the cell is a cultured cell. 
     
     
         12 . The vector of  claim 10 , wherein the secretory signal sequence is derived from SP38, erythropoietin albumin, albumin, or coagulation factor IX. 
     
     
         13 . A method of producing a lysosomal polypeptide in a cultured cell, comprising: contacting a cultured cell with the vector according to  claim 1  under conditions sufficient for the vector to be introduced into the cultured cell and for the nucleic acid to be expressed to produce the chimeric polypeptide comprising the secretory signal operably linked to the polypeptide wherein said secretory signal sequence replaces the native secretory signal sequence of the lysosomal polypeptide and the polypeptide is secreted from the cultured, and collecting the polypeptide secreted onto the cell culture medium. 
     
     
         14 . The method of  claim 13 , wherein the cell is a mammalian cell. 
     
     
         15 . The method of  claim 13 , wherein the cell is a CHO cell, a 293 cell, a HT1080 cell, a HeLa cell or a C10 cell. 
     
     
         16 . The method of  claim 13 , wherein the secretory signal sequence is derived from SP38, erythropoietin albumin, albumin, or coagulation factor IX. 
     
     
         17 . An in vitro method of delivering a nucleic acid encoding a lysosomal polypeptide to a cell, comprising contacting a cell in vitro with the vector according to  claim 1  under conditions sufficient for the vector to be introduced into the cell and for the nucleic acid to be expressed to produce the chimeric polypeptide comprising the secretory signal sequence operably linked to the lysosomal polypeptide wherein said secretory signal sequence replaces the native secretory signal sequence of the lysosomal polypeptide. 
     
     
         18 . The method of  claim 17 , wherein the secretory signal sequence is derived from SP38, erythropoietin albumin, albumin, or coagulation factor IX. 
     
     
         19 . A method of treating a deficiency of a lysosomal polypeptide in a subject, comprising administering to the subject the pharmaceutical formulation of  claim 8  in a therapeutically effective amount. 
     
     
         20 . The method of  claim 19 , wherein the secretory signal sequence is derived from SP38, erythropoietin albumin, albumin, or coagulation factor IX.