Constructs for expressing lysomal 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.

That which is claimed is:  
     
         1 . An isolated nucleic acid encoding a chimeric polypeptide comprising a secretory signal sequence operably linked to a lysosomal polypeptide.  
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein said secretory signal sequence is derived from a, secreted polypeptide.  
     
     
         3 . The isolated nucleic acid of  claim 2 , wherein said secretory signal sequence is derived from erythropoietin, Factor IX, prealbumin or α1-antitrypsin precursor polypeptide.  
     
     
         4 . The isolated nucleic acid of  claim 1 , wherein said secretory signal sequence comprises the amino acid sequence of SEQ ID NO:5.  
     
     
         5 . The isolated nucleic acid of  claim 1 , wherein said isolated nucleic acid is operatively linked to a transcriptional control element operable, in liver cells.  
     
     
         6 . The isolated nucleic acid of  claim 1 , wherein said lysosomal polypeptide is a lysosomal acid α-glucosidase (GAA) polypeptide.  
     
     
         7 . The isolated nucleic acid of  claim 6 , wherein said GAA polypeptide is a human GAA polypeptide.  
     
     
         8 . The isolated nucleic acid of  claim 6 , wherein said isolated nucleic acid further comprises a 3′ untranslated region.  
     
     
         9 . The isolated nucleic acid of  claim 8 , wherein: 
 (a) said 3′ untranslated region comprises a deletion therein; or    (b) wherein said 3′ untranslated region is less than 200 nucleotides in length and comprises a segment that is heterologous to said GAA coding region.    
     
     
         10 . The isolated nucleic acid of  claim 1 , wherein the isolated nucleic acid is 4 kilobases or less in length.  
     
     
         11 . A vector comprising the isolated nucleic acid of  claim 1 .  
     
     
         12 . The vector of  claim 11 , wherein said vector is an adeno-associated virus (AAV) vector.  
     
     
         13 . The vector of  claim 11 , wherein said lysosomal polypeptide is a GAA polypeptide.  
     
     
         14 . A pharmaceutical formulation comprising the isolated nucleic acid of  claim 1  in a pharmaceutically acceptable carrier.  
     
     
         15 . The pharmaceutical formulation of  claim 14 , wherein said pharmaceutical formulation comprises a vector comprising the isolated nucleic acid.  
     
     
         16 . The pharmaceutical formulation of  claim 15 , wherein said vector is an adeno-associated virus (AAV) vector.  
     
     
         17 . The pharmaceutical formulation of  claim 14 , wherein said lysosomal polypeptide is a GAA polypeptide.  
     
     
         18 . A cell comprising the isolated nucleic acid of  claim 1 .  
     
     
         19 . A chimeric polypeptide comprising a secretory signal sequence operably linked to a lysosomal polypeptide.  
     
     
         20 . The chimeric polypeptide of  claim 19 , wherein said lysosomal polypeptide is an acid α-glucosidase (GAA) polypeptide.  
     
     
         21 . A method of delivering a nucleic acid encoding a lysosomal polypeptide to a cell, comprising contacting a cell with an isolated nucleic acid according to  claim 1  under conditions sufficient for the isolated nucleic acid to be introduced into the cell, expressed to produce the chimeric polypeptide comprising the secretory signal sequence operably linked to the lysosomal polypeptide, and the lysosomal polypeptide secreted from the cell.  
     
     
         22 . The method of  claim 21 , wherein the cell is contacted with a vector comprising the isolated nucleic acid.  
     
     
         23 . The method of  claim 21 , wherein said lysosomal polypeptide is a lysosomal acid α-glucosidase (GAA) polypeptide.  
     
     
         24 . The method of  claim 21 , wherein the cell is a cultured cell.  
     
     
         25 . The method of  claim 21 , wherein the cell is a cell in vivo.  
     
     
         26 . A method of producing a lysosomal acid α-glucosidase (GAA) polypeptide in a cultured cell, comprising: 
 contacting a cultured cell with an isolated nucleic acid according to  claim 6  under conditions sufficient for the isolated nucleic acid to be introduced into the cultured cell, expressed to produce the chimeric polypeptide comprising the secretory signal sequence operably linked to the GAA polypeptide, and the GAA polypeptide secreted from the cultured cell, and  
 collecting the GAA polypeptide secreted into the cell culture medium.  
 
     
     
         27 . The method of  claim 26 , wherein the cell is a mammalian cell.  
     
     
         28 . The method of  claim 27 , wherein the cell is a CHO cell, a 293 cell, a HT1080 cell, a HeLa cell or a C10 cell.  
     
     
         29 . The method of  claim 26 , wherein the cell is contacted with a vector comprising the isolated nucleic acid.  
     
     
         30 . A method of treating a deficiency of a lysosomal polypeptide in a subject, comprising administering to the subject a cell according to  claim 18  in a pharmaceutically acceptable carrier in a therapeutically effective amount.  
     
     
         31 . A method of treating a deficiency of a lysosomal polypeptide in a subject, comprising administering to the subject the pharmaceutical formulation of  claim 14  in a therapeutically effective amount.  
     
     
         32 . The method of  claim 31 , wherein the pharmaceutical formulation comprises a vector comprising the isolated nucleic acid.  
     
     
         33 . The method of  claim 31 , wherein the isolated nucleic acid encoding the chimeric polypeptide is delivered to the liver.  
     
     
         34 . A method of treating a deficiency of a lysosomal polypeptide in a subject, comprising administering to the subject the pharmaceutical formulation of  claim 17  in a therapeutically effective amount.  
     
     
         35 . The method of  claim 34 , wherein the pharmaceutical formulation comprises a vector comprising the isolated nucleic acid.  
     
     
         36 . The method of  claim 34 , wherein the isolated nucleic acid encoding the chimeric polypeptide is delivered to the liver.  
     
     
         37 . The method of  claim 36 , wherein the GAA polypeptide is secreted from the liver and there is uptake of the secreted GAA polypeptide by skeletal muscle tissue, cardiac muscle tissue, diaphragm muscle tissue or a combination thereof, wherein uptake of the secreted GAA polypeptide results in a reduction in lysosomal glycogen stores in the tissue(s).  
     
     
         38 . An isolated nucleic acid encoding a lysosomal acid α-glucosidase (GAA) polypeptide, said isolated nucleic acid comprising: 
 (a) a coding region encoding a GAA polypeptide, and  
 (b) a 3′ untranslated region, 
 (i) wherein said 3′ untranslated region comprises a GAA 3′ untranslated region comprising a deletion of at least 25 consecutive nucleotides, so that upon introduction into a cell, GAA polypeptide is produced at a higher level from said isolated nucleic acid as compared with GAA polypeptide production from an isolated nucleic acid comprising a full-length GAA 3′ untranslated region, or  
 (ii) wherein said 3′ untranslated region is less than 200 nucleotides in length and comprises a segment that is heterologous to said GAA coding region, so that upon introduction into a cell, GAA polypeptide is produced at a higher level from said isolated nucleic acid as compared with GAA polypeptide production from an isolated nucleic acid comprising a full-length GAA 3′ untranslated region.  
 
 
     
     
         39 . The isolated nucleic acid of  claim 38 , wherein said 3′ untranslated region comprises the deletion of subparagraph (i).  
     
     
         40 . The isolated nucleic acid of  claim 39 , wherein said deletion in said 3′ untranslated region comprises a deletion of at least 100 bases.  
     
     
         41 . The isolated nucleic acid of  claim 39 , wherein at least 50% of said 3′ untranslated region has been deleted.  
     
     
         42 . The isolated nucleic acid of  claim 39 , wherein said 3′ untranslated region is 200 nucleotides in length or less.  
     
     
         43 . The isolated nucleic acid of  claim 42 , wherein essentially all of said 3′ untranslated region has been deleted.  
     
     
         44 . The isolated nucleic acid of  claim 39 , wherein said 3′ untranslated region comprises a deleted form of the 3′ untranslated region at nucleotides 3301 to 3846 of SEQ ID NO:1.  
     
     
         45 . The isolated nucleic acid of  claim 44 , wherein said 3′ untranslated region comprises nucleotides 2878 to 3012 of SEQ ID NO:3.  
     
     
         46 . The isolated nucleic acid of  claim 39 , wherein said isolated nucleic acid comprises SEQ ID NO:3.  
     
     
         47 . The isolated nucleic acid of  claim 38 , wherein said GAA polypeptide is a human GAA polypeptide.  
     
     
         48 . The isolated nucleic acid of  claim 38 , wherein said isolated nucleic acid is operatively linked to a transcriptional control element that is operable in liver cells.  
     
     
         49 . The isolated nucleic acid of  claim 38 , wherein said isolated nucleic acid is 4 kilobases or less in length.  
     
     
         50 . A vector comprising the isolated nucleic acid of  claim 38 .  
     
     
         51 . The vector of  claim 50 , wherein said vector is an adeno-associated virus (AAV) vector.  
     
     
         52 . A pharmaceutical formulation comprising the isolated nucleic acid of  claim 38  in a pharmaceutically acceptable carrier.  
     
     
         53 . The pharmaceutical formulation of  claim 52 , wherein said pharmaceutical formulation comprises a vector comprising the isolated nucleic acid.  
     
     
         54 . A cell comprising the isolated nucleic acid of  claim 38 .  
     
     
         55 . A method of delivering a nucleic acid encoding a lysosomal acid α-glucosidase (GAA) polypeptide to a cell, comprising contacting a cell with the isolated nucleic acid according to  claim 38  under conditions sufficient for the isolated nucleic acid encoding the GAA polypeptide to be introduced into the cell and expressed to produce the GAA polypeptide.  
     
     
         56 . The method of  claim 55 , wherein the cell is contacted with a vector comprising the isolated nucleic acid encoding the GAA polypeptide.  
     
     
         57 . The method of  claim 56 , wherein the vector is an adeno-associated virus (AAV) vector.  
     
     
         58 . The method of  claim 55 , wherein the cell is a cultured cell.  
     
     
         59 . A method of producing lysosomal acid α-glucosidase (GAA) polypeptide in a cultured cell, comprising: 
 contacting a cultured cell with an isolated nucleic acid according to  claim 38  under conditions sufficient for the isolated nucleic acid to be introduced into the cultured cell and expressed to produce the GAA polypeptide, and  
 collecting the GAA polypeptide.  
 
     
     
         60 . The method of  claim 59 , wherein the GAA polypeptide is secreted into the cell culture medium and collected therefrom.  
     
     
         61 . The method of  claim 59 , wherein the cell is a mammalian cell.  
     
     
         62 . The method of  claim 62 , wherein the cell is a CHO cell, a 293 cell, a HT1080 cell, a HeLa cell or a C10 cell.  
     
     
         63 . The method of  claim 59 , wherein the cell is contacted with a vector comprising the isolated nucleic acid.  
     
     
         64 . The method of  claim 63 , wherein the vector is an adeno-associated virus (AAV) vector.  
     
     
         65 . A method of treating lysosomal acid α-glucosidase (GAA) deficiency in a subject, comprising administering to the subject a cell according to  claim 54  in a pharmaceutically acceptable carrier in a therapeutically effective amount.  
     
     
         66 . A method of treating lysosomal acid α-glucosidase (GAA) deficiency in a subject, comprising administering to the subject the pharmaceutical formulation of  claim 52  in a therapeutically effective amount.  
     
     
         67 . The method of  claim 66 , wherein the subject is a human subject.  
     
     
         68 . The method of  claim 66 , wherein the isolated nucleic acid encoding GAA is delivered to the liver.  
     
     
         69 . The method of  claim 68 , wherein the GAA polypeptide is secreted from the liver into the systemic circulation.  
     
     
         70 . The method of  claim 69 , wherein there is uptake of the secreted GAA polypeptide by skeletal muscle tissue, cardiac muscle tissue, diaphragm muscle tissue or a combination thereof, wherein uptake of the secreted GAA polypeptide results in a reduction in lysosomal glycogen stores in the tissue(s).  
     
     
         71 . The method of  claim 66 , wherein the pharmaceutical formulation comprises a vector comprising-the isolated nucleic acid encoding the GAA polypeptide.  
     
     
         72 . The method of  claim 71 , wherein the vector is an adeno-associated virus (AAV) vector.