JPWO2021041322A5

JPWO2021041322A5 -

Info

Publication number: JPWO2021041322A5
Application number: JP2022508573A
Authority: JP
Publication date: 2023-08-31

Claims

A method for producing a lentiviral packaging vector-containing mammalian cell comprising:
a. mammalian cells,
i. A packaging vector comprising an expression cassette,
1. a lentiviral regulator of expression of the virion protein (REV) gene under the control of a first promoter;
2. 3. a lentiviral envelope gene under the control of a second promoter; encoding a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter;
transfecting with a packaging vector, wherein the expression cassette is flanked on both the 5′ and 3′ ends by transposon-specific inverted terminal repeats (ITRs);
b. culturing the transfected mammalian cells;
c. isolating said lentiviral packaging vector containing mammalian cells.

A method of producing a lentiviral vector-producing mammalian cell comprising:
a. mammalian cells,
i. A packaging vector comprising an expression cassette,
1. a lentiviral regulator of virion protein (REV) gene expression under the control of a first promoter;
2. 3. a lentiviral envelope gene under the control of a second promoter; encoding a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter;
a packaging vector, wherein the expression cassette is flanked on both the 5′ and 3′ ends by transposon-specific inverted terminal repeats (ITRs); and
ii. a transfer vector,
1. comprising a nucleic acid sequence encoding a gene of interest under control of a fourth promoter;
transfecting with a transfer vector, wherein the nucleic acid sequence is flanked on both the 5′ and 3′ ends by transposon-specific inverted terminal repeats (ITRs);
b. culturing the transfected mammalian cells;
c. isolating said lentiviral vector-producing mammalian cells.

A mammalian cell for producing a lentiviral vector, comprising:
a. a chromosomally integrated nucleic acid molecule in said mammalian cell, said nucleic acid molecule comprising
i. a lentiviral regulator of virion protein (REV) gene expression under the control of a first promoter;
ii. a lentiviral envelope gene under the control of a second promoter;
iii. a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter;
A mammalian cell comprising a nucleic acid molecule, wherein said nucleic acid sequence is flanked at both the 5' and 3' ends by sequences resulting from recombination of transposon-specific inverted terminal repeats (ITRs).

4. The mammalian cell of claim 3 , further comprising a chromosomally integrated nucleic acid sequence encoding a gene of interest under the control of a fourth promoter.

A method of producing a lentiviral vector, comprising:
a. The mammalian cell of claim 3,
i. transfecting with a transfer vector containing a nucleic acid sequence encoding the gene of interest under the control of a fourth promoter;
b. inducing production of said expression cassette and said nucleic acid;
c. culturing the transfected mammalian cells;
d. recovering the lentiviral vector.

A method of producing a lentiviral vector, comprising:
a. inducing production of said chromosomally integrated nucleic acid sequence and said chromosomally integrated nucleic acid sequence encoding the mammalian gene of interest of claim 4 ;
b. culturing the mammalian cells;
c. recovering the lentiviral vector.

7. The method of claim 1, 2, 5 or 6, wherein said mammalian cell is a mammalian cell culture.

8. The method of claim 7, wherein said mammalian cell culture is a suspension culture.

9. The method of claim 1, 2, 7 or 8, wherein said mammalian cells are HEK293T cells.

The method of any one of claims 1, 2 and 7-9, wherein the GAG gene is the HIV GAG gene and the POL gene is the HIV POL gene.

The method of any one of claims 1, 2 and 7-10, wherein the lentiviral envelope gene is the vesicular stomatitis virus glycoprotein (VSV-G) gene.

The method of any one of claims 1, 2 and 7-11, wherein said first, second and third promoters are derepressible promoters.

13. The method of claim 12, wherein said expression cassette further encodes repressor elements of said first, second and third derepressible promoters.

Each of said derepressible promoters is a functional promoter and a tetracyclyl
and the repressor element comprises a tetracycline operator sequence (TetO).
14. The method of claim 13, which is a non-repressor protein.

The expression cassette contains a sequence encoding the tetracycline repressor protein
14. The method of claim 13, further comprising a following Kruppel-associated box sequence.

A method according to any one of claims 1, 2 and 7-15, wherein said transposon-specific ITR is a Lepidoptera transposon (PIGGYBAC®) ITR.

A method according to any one of claims 1, 2 and 7-16, wherein transfecting is in the presence of a transposase that recognizes said transposon-specific ITR.

18. The method of claim 17, wherein said transposase is a Lepidoptera (PIGGYBAC(R)) transposase mRNA or a Lepidoptera (PIGGYBAC(R)) transposing cDNA.

The method according to any one of claims 2, 5 and 6-18, wherein said gene of interest is a gene of therapeutic interest.