Use of human serum resistant vector particles and cell lines for human gene therapy

Abstract

The present invention relates to the use of non-primate mammalian cell lines having substantially no endogenous retroviral sequences as producer and packaging lines for preparation of human serum-resistant retroviral vector particles with improved safety for use in gene therapy applications. In a preferred embodiment, the cell line used in the present invention is the α-galactosyl(αGal)-positive cell ferret brain cell line designated as Mpf or a cell line having those identifying characteristics of the Mpf cell line suitable for the practice of the invention.

I claim:  
     
         1 . A method for preparing a stable, retroviral packaging cell line for generation of human serum-resistant retroviral vector particles (RVP) which comprises 
 (a) introducing one or more packaging vectors into a non-primate mammalian cell line, wherein said cell line exhibits substantially no hybridization to a Moloney-MLV retrovirus probe under stringent washing conditions and is capable of producing human-serum-resistant RVP and wherein said vectors, either singly or collectively, express a cellular targeting protein and retroviral gag and pol genes in amounts sufficient to package said RVP; and    (b) recovering said packaging cell line.    
     
     
         2 . The method of  claim 1 , wherein said cell line is the Mpf cell line designated by ATCC accession number 1656-CRL.  
     
     
         3 . The method of  claim 1 , wherein said cell line is α-galactosyl positive.  
     
     
         4 . The method of  claim 1  or  2 , wherein said cellular targeting protein is an amphotropic retroviral env protein, a xenotropic retroviral env protein, a polytropic retroviral env protein, a JSRV env protein, vesicular stomatitis virus G protein or transferrin.  
     
     
         5 . A packaging cell line produced by the method of of  claim 1  or  2 .  
     
     
         6 . A method for preparing a stable, retroviral producer cells capable of producing human serum-resistant retroviral vector particles (RVP) which comprises 
 (a) introducing a retrovirus vector into the packaging cell line of  claim 1 , wherein said retrovirus vector is capable of being packaged into an RVP and comprises a heterologous gene capable of expression in a human; and    (b) recovering said producer cells.    
     
     
         7 . The method of  claim 6 , wherein said cells are Mpf cells designated by ATCC accession number 1656-CRL.  
     
     
         8 . The method of  claim 6 , wherein said cells are α-galactosyl positive.  
     
     
         9 . The method of  claim 6 , wherein said cellular targeting protein is an amphotropic retroviral env protein, a xenotropic retroviral env protein, a polytropic retroviral env protein, a JSRV env protein, vesicular stomatitis virus G protein or transferrin.  
     
     
         10 . Producer cells prepared by the method of  claim 6  or  7 .  
     
     
         11 . A method for preparing human serum-resistant retroviral vector particles (RVP) which comprises: 
 (a) introducing a retrovirus vector into the packaging cell line of  claim 1 , wherein said retrovirus vector is capable of being packaged into an RVP and comprises a heterologous gene capable of expression in a human;    (b) culturing said cell line for a time and under conditions sufficient to produce said RVP; and    (c) recovering said RVP.    
     
     
         12 . The method of  claim 11 , wherein said cell line is the Mpf cell line designated by ATCC accession number 1656-CRL.  
     
     
         13 . The method of  claim 11 , wherein said cell line is α-galactosyl positive.  
     
     
         14 . The method of  claim 11  or  12 , wherein said cellular targeting protein is an amphotropic retroviral env protein, a xenotropic retroviral env protein, a polytropic retroviral env protein, a JSRV env protein, vesicular stomatitis virus G protein or transferrin.  
     
     
         15 . The method of  claim 12  wherein said cell line produces RVP having a supernatant titer on mink cell line Mv-1-Lu of at least about 10 4  to about 10 8  colony forming units per milliliter.  
     
     
         16 . A method for preparing human serum-resistant retroviral vector particles (RVP) which comprises: 
 (a) culturing the producer cells of  claim 6  for a time and under conditions sufficient to produce said RVP; and    (c) recovering said RVP.    
     
     
         17 . The method of  claim 16 , wherein said cells are α-galactosyl positive.  
     
     
         18 . The method of  claim 16 , wherein said cellular targeting protein is an amphotropic retroviral env protein, a xenotropic retroviral env protein, a polytropic retroviral env protein, a JSRV env protein, vesicular stomatitis virus G protein or transferrin.  
     
     
         19 . The method of  claim 16  wherein said cell line produces RVP having a supernatant titer on mink cell line Mv-1-Lu of at least about 10 4  to about 10 8  colony forming units per milliliter.  
     
     
         20 . Retroviral vector particles produced by the methods of any one of claims  11 ,  12 ,  16  or  41 .  
     
     
         21 . Retroviral vector particles prepared from the producer cells of  claim 10 .  
     
     
         22 . A method for transducing a cell with a retroviral vector in the presence of a body fluid which comprises administering the retroviral vector particles (RVP) of  claim 20  to said cell.  
     
     
         23 . The method of  claim 22 , wherein said RVP is administered to said cell ex vivo or in vivo.  
     
     
         24 . The method of  claim 23 , wherein said RVP is administered in vivo by aerosol, transmucosal, oral, intravenous, intraperitoneal, intramuscular, transdermal, intradermal, subdermal, transmucosal or intrathecal delivery.  
     
     
         25 . A method of gene therapy which comprises delivering a therapeutic molecule encoded on a retrovirus vector to a human cell via retroviral vector particles (RVP) of  claim 20 .  
     
     
         26 . The method of  claim 25  wherein said therapeutic molecule is a hormone, a growth factor, an enzyme, a lymphokine, a cytokine, a receptor, an angiogenic factor,or an anti-angiogenesis factor.  
     
     
         27 . The method of  claim 25 , wherein said RVP is administered to said cell ex vivo or in vivo.  
     
     
         28 . The method of  claim 27 , wherein said RVP is administered in vivo by aerosol, transmucosal, oral, intravenous, intraperitoneal, intramuscular, transdermal, intradermal, subdermal, transmucosal or intrathecal delivery.  
     
     
         29 . A method for transducing a cell with a retroviral vector in the presence of a body fluid which comprises administering the retroviral vector particles (RVP) of  claim 21  to said cell.  
     
     
         30 . The method of  claim 29 , wherein said RVP is administered to said cell ex vivo or in vivo.  
     
     
         31 . The method of  claim 30 , wherein said RVP is administered in vivo by aerosol, transmucosal, oral, intravenous, intraperitoneal, intramuscular, transdermal, intradermal, subdermal, transmucosal or intrathecal delivery.  
     
     
         32 . A method of gene therapy which comprises delivering a therapeutic molecule encoded on a retrovirus vector to a human cell via retroviral vector particles of  claim 21 .  
     
     
         33 . The method of  claim 32  wherein said therapeutic molecule is a hormone, a growth factor, an enzyme, a lymphokine, a cytokine, a receptor, an angiogenic factor,or an anti-angiogenesis factor.  
     
     
         34 . The method of  claim 32 , wherein said RVP is administered to said cell ex vivo or in vivo.  
     
     
         35 . The method of  claim 34 , wherein said RVP is administered in vivo by aerosol, transmucosal, oral, intravenous, intraperitoneal, intramuscular, transdermal, intradermal, subdermal, transmucosal or intrathecal delivery.  
     
     
         36 . A method for transferring a heterologous gene into a human cell which comprises contacting said human cell with the producer cells of  claim 10  under conditions such that said producer cells release RVP containing a retrovirus vector encoding said heterologous gene and thereby introducing said gene into said human cell.  
     
     
         37 . The method of  claim 36 , wherein said producer cells are implanted in a human.  
     
     
         38 . The method of  claim 37 , wherein said producer cells are implanted in a human brain.  
     
     
         39 . A pharmaceutical composition comprising the RVP of  claim 20  and a pharmaceutically acceptable carrier.  
     
     
         40 . A pharmaceutical composition comprising the RVP of  claim 21  and a pharmaceutically acceptable carrier.  
     
     
         41 . The method of  claim 16 , wherein said cells are Mpf cells designated by ATCC accession number 1656-CRL.