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

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).

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.

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.

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.

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.