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

An in vitro method of generating genetic diversity in the nucleotide sequence 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 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;
• and
• e2) transcomplementing the Fn producer cells of step el) with viral proteins necessary for the production of a n+1 filial generation of replication-defective lentiviral particles (Fn+1);
• e3) collecting the n+1 filial generation of replication-defective lentiviral particles (Fn+1);
• thereby, obtaining a population of replication-defective lentiviral particles of the n+1 filial generation, wherein said replication-defective lentiviral particles contain the target polynucleotide or a nucleotide variant of the target polynucleotide.

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 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 el) 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;
• 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 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);
• 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.

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 el) 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.