Bio-production of lentiviral vectors

Abstract

The present disclosure provides a method of generating a stable producer cell line. The generation of stable producer cell lines, such as those provided in accordance with the present invention, increases the reproducibility and ease of creating high titer lentiviral stocks while easing biosafety concerns and the variation in expressed envelope proteins defines the tropism of the generated virus. The present disclosure also provides for a novel lentiviral transfer vector plasmid.

1 . A recombinant plasmid comprising a nucleotide sequence having at least 80% identity to that of SEQ ID NO: 1, wherein the recombinant plasmid comprises a multiple cloning site comprising BstBI, MluI, NotI, and ClaI restriction endonuclease sites. 
     
     
         2 . The recombinant plasmid of  claim 1 , wherein the nucleotide sequence has at least 85% identity to that of SEQ ID NO: 1. 
     
     
         3 . The recombinant plasmid of  claim 1 , wherein the recombinant plasmid comprises between about 6500 nucleotides and about 6700 nucleotides. 
     
     
         4 . The recombinant plasmid of  claim 1 , wherein the recombinant plasmid further comprises a nucleotide sequence encoding a packaging signal; a nucleotide sequence encoding a central polypurine tract; a nucleotide sequence encoding a Rev response element; and a nucleotide sequence encoding a self-inactivating long terminal repeat. 
     
     
         5 . A recombinant plasmid comprising a vector cassette having a nucleotide sequence having at least 80% identity to that of SEQ ID NO: 2. 
     
     
         6 . The recombinant plasmid of  claim 5 , wherein the nucleotide sequence of the vector cassette has at least 85% identity to that of SEQ ID NO: 2. 
     
     
         7 . The recombinant plasmid of  claim 5 , wherein the vector cassette is flanked by at least two restriction endonuclease sites, the at least two restriction endonuclease sites independently selected from the group consisting of sfiI and Bsu36I. 
     
     
         8 . The recombinant plasmid of  claim 5 , wherein the recombinant plasmid comprises between about 6500 nucleotides and about 6750 nucleotides. 
     
     
         9 . The recombinant plasmid of  claim 5 , wherein the recombinant plasmid comprises between about 6600 nucleotides and about 6700 nucleotides. 
     
     
         10 . The recombinant plasmid of  claim 5 , wherein the vector cassette comprises a multiple cloning site including BstBI, MluI, NotI, and ClaI restriction endonuclease sites. 
     
     
         11 . The recombinant plasmid of  claim 5 , wherein the recombinant plasmid further comprises a tetracycline repressible promoter upstream of the vector cassette. 
     
     
         12 . The recombinant plasmid of  claim 5 , wherein the recombinant plasmid comprises a nucleotide sequence having at least 85% identity to that of SEQ ID NO: 1. 
     
     
         13 . The recombinant plasmid of  claim 5 , wherein the recombinant plasmid further comprises a nucleotide sequence encoding a packaging signal; a nucleotide sequence encoding a central polypurine tract; a nucleotide sequence encoding a Rev response element; and a nucleotide sequence encoding a self-inactivating long terminal repeat. 
     
     
         14 . A concatemeric array comprising: (i) first DNA fragments obtained from the recombinant plasmid of  claim 1 ; and (ii) second DNA fragments obtained from a bleomycin antibiotic resistance cassette; wherein a ratio of the first DNA fragments to the second DNA fragments ranges from about 25:1 to about 1:25, and wherein each of the first DNA fragments comprise a nucleotide sequence having at least 80% identity to SEQ ID NO: 1. 
     
     
         15 . The concatemeric array of  claim 14 , wherein the each of the first DNA fragments comprise a nucleotide sequence having at least 85% identity to SEQ ID NO: 1. 
     
     
         16 . A stable producer cell line, the stable producer cell line generated by: transfecting a packaging cell line selected from the group consisting of GPR packaging cell line cells, GPRG packaging cell line cells, GPRT packaging cell line cells, GPRGT packaging cell line cells, and GPRT-G packing cell line cells with the concatemeric array of  claim 14 . 
     
     
         17 . A lentiviral vector derived from the recombinant plasmid of  claim 1 . 
     
     
         18 . The lentiviral vector of  claim 17 , wherein the lentiviral vector comprises at least one additional nucleotide sequence. 
     
     
         19 . A recombinant plasmid comprising a nucleotide sequence having at least 85% identity to that of SEQ ID NO: 1. 
     
     
         20 . The recombinant plasmid of  claim 19 , wherein the recombinant plasmid further comprises a nucleotide sequence encoding a packaging signal; a nucleotide sequence encoding a central polypurine tract; a nucleotide sequence encoding a Rev response element; and a nucleotide sequence encoding a self-inactivating long terminal repeat.