Lentiviral-based vector and its use in directed evolution of genomic regions, genes and polynucleotides

Abstract

Replication-defective lentiviral vectors are described. Using this vector, methods of directing evolution of a target polynucleotide of interest for obtaining variants of the target polynucleotide, methods to generate genetic variability by preparing a cell library, and methods to isolate and/or screen variants of a polynucleotide or variants of a protein able to impact the phenotype of a cell or to confer a desired phenotype to target cells and to identify theses polynucleotide variants or protein variants responsible for this phenotype are described.

1 . A replication-defective lentiviral vector deposited at the CNCM under accession number I-3991 on May 14, 2008. 
     
     
         2 . A cell library, preferably a mammalian cell library, comprising cells having integrated in their genome target polynucleotide or a nucleotide variant of a target polynucleotide, wherein said cell library is obtained following the transduction by a population of replication-defective lentiviral particles of a generation from F4 to F20 or at least the 8th generation which are obtained after implementing a method comprising:
 a) providing parental (P) replication-defective lentiviral particles comprising a genetic material encapsidated in said particles, wherein said genetic material comprises the regulatory elements necessary for the transfer, transcription and optionally expression of said genetic material into the genome of a cell host and further comprises at least one target polynucleotide;   b) transducing producer cells with the parental replication-defective lentiviral particles of step a) to obtain parental cells, optionally in the presence of a mutagen;   c) transcomplementing the parental producer cells of step b) with viral proteins necessary for the production of a first filial generation of replication-defective lentiviral particles (F1);   d) collecting the first filial generation of replication-defective lentiviral particles (F1); and   e) repeating, n consecutive times, with n being 1 or more, the at least three following steps:   e1) transducing fresh producer cells with the immediately previous filial generation of replication-defective lentiviral particles, optionally in the presence of a mutagen, thereby obtaining a Fn filial cell generation;   e2) transcomplementing the Fn producer cells of step e1) with viral proteins necessary for the production of a n+1 filial generation of replication-defective lentiviral particles (Fn+1); and   e3) collecting the n+1 filial generation of replication-defective lentiviral particles (Fn+1).   
     
     
         3 . A cell library, preferably a mammalian cell library, obtainable by a method comprising:
 a) providing parental (P) replication-defective lentiviral particles comprising a genetic material encapsidated in said particles, wherein said genetic material comprises the regulatory elements necessary for the transfer, transcription and optionally expression of said genetic material into the genome of a cell host and further comprises at least one target polynucleotide;   b) transducing producer cells with the parental replication-defective lentiviral particles of step a) to obtain parental cells, optionally in the presence of a mutagen;   c) transcomplementing the parental producer cells of step b) with viral proteins necessary for the production of a first filial generation of replication-defective lentiviral particles (F1);   d) collecting the first filial generation of replication-defective lentiviral particles (F1);   e) repeating, n consecutive times, with n being 1 or more, the at least three following steps:   e1) transducing fresh producer cells with the immediately previous filial generation of replication-defective lentiviral particles, optionally in the presence of a mutagen, thereby obtaining a Fn filial cell generation;   e2) transcomplementing the Fn producer cells of step e1) with viral proteins necessary for the production of a n+1 filial generation of replication-defective lentiviral particles (Fn+1); and   e3) collecting the n+1 filial generation of replication-defective lentiviral particles (Fn+1); and   f) transducing target cells with the filial Fn+1 generation of replication-defective lentiviral particles, in conditions enabling a cell to be transduced by one of said particles.   
     
     
         4 . The mammalian cell library of  claim 2 , which is the human cell library deposited at the CNCM under accession number CNCM 1-3992 on May 14, 2008. 
     
     
         5 . A nucleic acid comprising or consisting in an U3 region mutated in positions 24, 82, 108, 160, 164, 183, 198, 251, 291, 333, 421 and 447 with respect to the U3 region of a wild type LTR of HIV-1 virus, in particular an U3 region having the mutations 24A>C, 82A>G, 108G>A, 160CA, 164T>G, 183A>G, 198A>C, 251G>A, 291G>A, 333T>C, 421C>G and 447C>T with respect to the U3 region of a wild type LTR of HIV-1 virus, and more particularly an U3 region as defined in SEQ ID NO:6. 
     
     
         6 . A nucleic acid comprising or consisting in a LTR, in particular a HIV-1 LTR, wherein its U3 region is as defined in  claim 5 . 
     
     
         7 . A genetic material or a lentiviral vector comprising, as a LTR5′ and/or as a LTR3′, the nucleic acid of  claim 6 . 
     
     
         8 . A lentiviral particle comprising the genetic material of  claim 7 . 
     
     
         9 . A cell comprising inserted in its genome the genetic material of  claim 7 . 
     
     
         10 . A variant of a gene encoding deoxycytidine kinase, selected from the group consisting of:
 1) a polynucleotide, the sequence of which is derived from SEQ ID NO:1 by at least a G to A substitution at position 739 and a C to A substitution at position 745;   2) a polynucleotide, the sequence of which is derived from SEQ ID NO:1 by a G to A substitution at position 739 and a C to A substitution at position 745;   3) a polynucleotide, the sequence of which is derived from SEQ ID NO:1 by a G to A substitution at position 237, a G to A substitution at position 739 and a C to A substitution at position 745;   4) a polynucleotide, the sequence of which is derived from SEQ ID NO:1 by a G to A substitution at position 511, a G to A substitution at position 739 and a C to A substitution at position 745; and   5) a polynucleotide, the sequence of which is derived from SEQ ID NO:1 by a G to A substitution at position 521, a G to A substitution at position 739 and a C to A substitution at position 745.   
     
     
         11 . A variant of the deoxycytidine kinase encoded by a polynucleotide as defined in  claim 10 . 
     
     
         12 . An in vitro method of generating a cell library expressing genetic diversity variants of a target polynucleotide comprising:
 a) providing parental (P) replication-defective lentiviral particles comprising a genetic material encapsidated in said particles, wherein said genetic material comprises the regulatory elements necessary for the transfer, transcription and optionally expression of said genetic material into the genome of a cell host and further comprises at least one target polynucleotide;   b) transducing producer cells with the parental replication-defective lentiviral particles of step a) to obtain parental producer cells containing integrated in their genome the nucleotide sequence of the target polynucleotide, optionally in the presence of a mutagen;   c) transcomplementing the parental producer cells of step b) with viral proteins necessary for the production of a first filial generation of replication-defective lentiviral particles (F1);   d) collecting the first filial generation of replication-defective lentiviral particles (F1);   e) repeating, n consecutive times, with n being 1 or more, the at least three following steps:   e1) transducing fresh producer cells with the immediately previous filial generation of replication-defective lentiviral particles, optionally in the presence of a mutagen, thereby obtaining a Fn filial producer cell generation comprising cells containing integrated in their genome the nucleotide sequence of the target polynucleotide;   e2) transcomplementing the Fn producer cells of step e1) with viral proteins necessary for the production of a n+1 filial generation of replication-defective lentiviral particles (Fn+1); and   e3) collecting the n+1 filial generation of replication-defective lentiviral particles (Fn+1);   wherein transduction and integration of the nucleotide sequence of the target polynucleotide in the genome of the producer cells is independent from viral replication and results in clonal expansion of variants of the target polynucleotide; and   f) transducing target cells with the filial Fn+1 generation of replication-defective lentiviral particles, in conditions enabling a cell to be transduced by one of said particles;   thereby, obtaining a library of target cells comprising cells containing the target polynucleotide or nucleotide variant of the target polynucleotide.   
     
     
         13 . The in vitro method of  claim 12 , wherein said target cell is similar to or different from the producer cell, and is a mammalian cell, and in particular a human cell, such as Hela TK- cells, cells lines deleted for SLP-76 or reporter HL116 cells. 
     
     
         14 . An in vitro method of screening a variant of a target polynucleotide, associated with a cell phenotype, comprising:
 a) providing parental (P) replication-defective lentiviral particles comprising a genetic material encapsidated in said particles, wherein said genetic material comprises the regulatory elements necessary for the transfer, transcription and optionally expression of said genetic material into the genome of a cell host and further comprises at least one target polynucleotide;   b) transducing producer cells with the parental replication-defective lentiviral particles of step a) to obtain parental producer cells containing integrated in their genome the nucleotide sequence of the target polynucleotide, optionally in the presence of a mutagen;   c) transcomplementing the parental producer cells of step b) with viral proteins necessary for the production of a first filial generation of replication-defective lentiviral particles (F1);   d) collecting the first filial generation of replication-defective lentiviral particles (F1);   e) repeating, n consecutive times, with n being 1 or more, the at least three following steps:   e1) transducing fresh producer cells with the immediately previous filial generation of replication-defective lentiviral particles, optionally in the presence of a mutagen, thereby obtaining a Fn filial producer cell generation comprising cells containing integrated in their genome the nucleotide sequence of the target polynucleotide;   e2) transcomplementing the Fn producer cells of step e1) with viral proteins necessary for the production of a n+1 filial generation of replication-defective lentiviral particles (Fn+1); and   e3) collecting the n+1 filial generation of replication-defective lentiviral particles (Fn+1);   wherein transduction and integration of the nucleotide sequence of the target polynucleotide in the genome of the producer cells is independent from viral replication and results in clonal expansion of variants of the target polynucleotide;   f) transducing target cells having an original phenotype, with the filial Fn+1 generation of replication-defective lentiviral particles, in conditions enabling a cell to be transduced by one of said particles;   g) isolating the target cells having a final phenotype that is different from the original phenotype; and   h) identifying the variant of the target polynucleotide, which is integrated into the cell having said final phenotype, in particular by carrying out the sequencing of the integrated target polynucleotide variant.   
     
     
         15 . The in vitro method of  claim 14 , wherein step g) is chosen from the group consisting of:
 g1) isolating the target cells having a desired activity or expressing a desired molecule, whereas the original target cells respectively lack the desired activity or do not express the desired molecule;   g2) isolating the target cells not having a desired activity or not expressing a desired molecule, whereas the original target cells respectively lack the desired activity or do not express the desired molecule;   g3) isolating the target cells not having a desired activity or not expressing a desired molecule, whereas the original target cells respectively had the desired activity or express the desired molecule; and   g4) isolating the target cells which, once put in contact with secondary cells, are able to activate or inhibit the activity of secondary cells or the expression of molecules by these secondary cells.