Packaging cells comprising codon-optimized gagpol sequences and lacking lentiviral accessory proteins

Abstract

Novel packaging cell lines useful for generating viral accessory protein independent HIV-derived retroviral vector particles, methods of constructing such packaging cell lines and methods of using the viral accessory protein independent HIV-derived retroviral vector particles are disclosed.

1 . A packaging cell line for producing viral accessory protein independent HIV-derived retroviral vector particles, said method comprising: 
 a) a mammalian cell;    b) a first retroviral nucleic acid in the cell which comprises a codon optimized coding sequence for an HIV gagpol;    c) a second retroviral nucleic acid in the cell which comprises the coding sequence for a heterologous envelope protein; and    d) a third retroviral nucleic acid in the cell which encodes a heterologous therapeutic protein and which comprises HIV cis-acting sequences required for packaging, reverse transcription and integration;    wherein said packaging cell line produces HIV-derived retroviral vector particles.    
     
     
         2 . A packaging cell line comprising: 
 a) a mammalian cell;    b) a first retroviral nucleic acid in the cell which comprises a codon optimized coding sequence for an HIV gagpol; and    c) a second retroviral nucleic acid in the cell which comprises a nucleotide sequence of interest encoding a heterologous therapeutic protein, said nucleic acid further comprising HIV cis-acting sequences required for packaging, reverse transcription and integration;    wherein said packaging cell line produces HIV-derived retroviral vector particles.    
     
     
         3 . A method of producing a packaging cell line which produces HIV-derived retroviral vector particles, comprising co-transfecting mammalian host cells with: 
 a) a first plasmid comprising a codon-optimized nucleotide sequence which encodes HIV gagpol proteins;    b) a second plasmid comprising a nucleotide sequence which encodes a heterologous envelope protein; and    c) a third plasmid comprising a nucleotide sequence encoding a heterologous therapeutic protein and further comprising HIV cis-acting nucleotide sequences required for packaging, reverse transcription and integration;    thereby producing a packaging cell line which produces HIV-derived retroviral vector particles.    
     
     
         4 . A method of producing HIV-derived retroviral vector particles, said method comprising: 
 a) co-transfecting mammalian host cells with: 
 i) a first plasmid comprising a codon-optimized nucleotide sequence which encodes HIV gagpol proteins;  
 ii) a second plasmid comprising a nucleotide sequence which encodes a heterologous envelope protein; and  
 iii) a third plasmid comprising a nucleotide sequence encoding a heterologous therapeutic protein and further comprising HIV cis-acting sequences required for packaging, reverse transcription and integration;  
   thereby producing transfected cells;    b) maintaining the transfected cells under conditions suitable for production of virus particles; and    c) recovering the virus particles produced in step b).    
     
     
         5 . A packaging cell line comprising: 
 a) a mammalian cell;    b) a first retroviral nucleic acid in the cell, said nucleic acid comprising a codon optimized coding sequence for a lentivirus gagpol;    c) a second retroviral nucleic acid in the cell which comprises the coding sequence for a heterologous envelope protein; and    d) a third retroviral nucleic acid in the cell, said nucleic acid encoding a heterologous therapeutic protein and said nucleic acid comprising lentivirus cis-acting sequences required for packaging, reverse transcription and integration.    
     
     
         6 . A packaging cell line comprising: 
 a) a mammalian cell;    b) a first retroviral nucleic acid in the cell, said nucleic acid comprising a codon optimized coding sequence for lentivirus gagpol; and    c) a second retroviral nucleic acid in the cell, said nucleic acid encoding a heterologous therapeutic protein, and said nucleic acid comprising lentivirus cis-acting nucleotide sequences required for packaging, reverse transcription and integration.    
     
     
         7 . A method of producing a packaging cell line for producing lentivirus-derived retroviral vector particles, said method comprising co-transfecting mammalian host cells with: 
 a) a first plasmid comprising a codon optimized nucleotide sequence which encodes lentivirus gagpol proteins;    b) a second plasmid comprising a nucleotide sequence which encodes a heterologous envelope protein; and    c) a third plasmid comprising a nucleotide sequence encoding a heterologous therapeutic protein and further comprising lentivirus cis-acting nucleic acid sequences required for packaging, reverse transcription and integration;    thereby producing a packaging cell line which produces lentivirus-derived retroviral vector particles.    
     
     
         8 . A method of producing lentivirus-derived retroviral vector particles, said method comprising: 
 a) co-transfecting mammalian host cells with: 
 i) a first plasmid comprising a codon-optimized nucleotide sequence which encodes lentivirus gagpol proteins;  
 ii) a second plasmid comprising a nucleotide sequence which encodes a heterologous envelope protein; and  
 iii) a third plasmid comprising a nucleotide sequence encoding a heterologous therapeutic protein and further comprising lentivirus cis-acting sequences required for packaging, reverse transcription and integration;  
   thereby producing transfected cells;    b) maintaining the transfected cells under conditions suitable for production of virus particles; and    c) recovering the virus particles produced in step b).    
     
     
         9 . HIV-derived retroviral vector particles having no viral accessory proteins, said particles produced by a method comprising: 
 a) co-transfecting mammalian host cells with: 
 i) a first plasmid comprising a codon optimized coding sequence for HIV gagpol proteins but not comprising DNA sequences encoding HIV accessory proteins or constitutive transport elements;  
 ii) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and  
 iii) a third plasmid comprising a DNA sequence of interest and HIV cis-acting sequences required for packaging, reverse transcription and integration;  
   thereby producing transfected cells; and    b) maintaining the transfected cells under conditions suitable for production of virus particles.    
     
     
         10 . The HIV-derived retroviral particles of  claim 9  wherein the heterologous envelope protein is the G glycoprotein of vesicular stomatitis virus (VSV G).  
     
     
         11 . The HIV-derived retroviral particles of  claim 9  wherein the heterologous envelope protein is the amphotropic envelope of the Moloney leukemia virus.  
     
     
         12 . The HIV-derived retroviral particles of  claim 9  wherein the DNA sequence of interest encodes a heterologous therapeutic protein.  
     
     
         13 . Lentivirus-derived retroviral vector particles having no viral accessory proteins, said particles produced by the method comprising: 
 a) co-transfecting mammalian host cells with: 
 i) a first plasmid comprising a codon optimized DNA sequence which encodes lentivirus gagpol proteins but not comprising DNA sequences encoding lentivirus accessory proteins or constitutive transport elements;  
 ii) a second plasmid comprising a DNA sequence which encodes a heterologous envelope protein; and  
 iii) a third plasmid comprising a DNA sequence of interest and lentivirus cis-acting sequences required for packaging, reverse transcription and integration;  
   thereby producing transfected cells; and    b) maintaining the transfected cells under conditions suitable for production of virus particles.    
     
     
         14 . The retroviral vector particles of  claim 13  wherein the heterologous envelope protein is the G glycoprotein of vesicular stomatitis virus (VSV G).  
     
     
         15 . The retroviral vector particles of  claim 13  wherein the heterologous envelope protein is the amphotropic envelope of the Moloney leukemia virus.  
     
     
         16 . The retroviral vector particles of  claim 13  wherein the DNA sequence of interest encodes a heterologous therapeutic protein.  
     
     
         17 . Isolated DNA encoding a co don optimized HIV gagpol.  
     
     
         18 . Isolated DNA encoding a codon optimized HIV gag.  
     
     
         19 . Isolated DNA of  claim 18  comprising SEQ ID NO:4.  
     
     
         20 . Isolated DNA encoding a codon optimized HIV pol.  
     
     
         21 . Isolated DNA of  claim 20  comprising SEQ ID NO:10.  
     
     
         22 . A method of introducing a DNA sequence of interest into a mammal, said method comprising introducing into said mammal a viral accessory protein independent HIV-derived retroviral vector particle comprising the DNA sequence of interest.  
     
     
         23 . The method of  claim 22  wherein the mammal is a human.  
     
     
         24 . The method of  claim 22  wherein the DNA sequence of interest encodes a heterologous therapeutic protein.  
     
     
         25 . A method of introducing a DNA sequence of interest into a mammal, said method comprising: 
 a) introducing into cells a viral accessory protein independent HIV-derived retroviral vector particle comprising the DNA sequence of interest; and    b) introducing the cells obtained in step a) into the mammal, thereby introducing the DNA sequence of interest into the mammal.    
     
     
         26 . The method of  claim 25  wherein the mammal is a human.  
     
     
         27 . The method of  claim 25  wherein the DNA sequence of interest is a heterologous therapeutic protein.  
     
     
         28 . The method of  claim 25  wherein the cells of step a) are obtained from the mammal.