Methods and constructs for transient production of lentiviral vector

Patent metadata

Field	Value
Publication number	US-12398404-B2
Application number	US-202017757041-A
Country	US
Kind code	B2
Filing date	Dec 17, 2020
Priority date	Dec 18, 2019
Publication date	Aug 26, 2025
Grant date	Aug 26, 2025

Abstract

The present disclosure relates to methods for producing lentiviral vectors using mammalian cells. Specifically, the methods utilize three plasmids, rather than four, to provide the required packaging elements and transfer vector to a cell, allowing for the production of a large number of lentiviral vectors in mammalian cells, including suspension-based cells. These methods allow for the production of lentiviral vectors that can be tailored to include a specific gene of interest.

First claim

What is claimed is:

1. A method of producing a lentiviral vector, comprising:
a. transfecting a mammalian cell with:
i. a first nucleic acid molecule encoding a lentiviral regulator of expression of virion proteins (REV) gene under control of a first promoter and an envelope glycoprotein gene under control of a second promoter;
ii. a second nucleic acid molecule encoding a gene of interest under control of a third promoter; and
iii. a third nucleic acid molecule encoding a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene both under control of a fourth promoter,

wherein the first, second, and third nucleic acid molecules are three separate nucleic acid molecules;
b. culturing the transfected mammalian cell; and
c. isolating the lentiviral vector.

2. The method of claim 1 , wherein the mammalian cell is a mammalian cell culture.

3. The method of claim 2 , wherein the mammalian cell culture is a suspension culture.

4. The method of claim 1 , wherein the mammalian cell is an HEK293T cell.

5. The method of claim 1 , wherein the envelope glycoprotein gene is a Vesicular Somatitis Virus Glycoprotein (VSV-G) gene.

6. The method of claim 5 , wherein the nucleic acids encoding VSV-G and REV are codon-optimized.

7. The method of claim 5 , wherein the VSV-G gene expression is driven by a Rous sarcoma virus (RSV) promoter.

8. The method of claim 1 , wherein the GAG gene is an HIV GAG gene and the POL gene is an HIV POL gene.

9. The method of claim 1 , wherein the REV gene expression is driven by a cytomegalovirus (CMV) promoter.

10. The method of claim 1 , wherein the gene of interest is a gene of therapeutic interest.

11. The method of claim 10 , wherein the gene of therapeutic interest encodes for a protein.

12. The method of claim 10 , wherein the gene of therapeutic interest encodes for an antibody.

13. The method of claim 10 , wherein the gene of therapeutic interest encodes for a chimeric antigen receptor (CAR).

14. A lentiviral vector produced by the method of claim 1 .

15. A method of treatment with a lentiviral vector, comprising:
a. administering the lentiviral vector of claim 14 to a mammalian subject.

16. The method of claim 15 , wherein the gene of interest encodes for a protein.

17. The method of claim 15 , wherein the gene of interest encodes for an antibody.

18. The method of claim 15 , wherein the gene of interest encodes for a CAR.

19. The lentiviral vector of claim 14 , wherein the gene of interest encodes for a protein.

20. The lentiviral vector of claim 14 , wherein the gene of interest encodes for a CAR.

Assignees

• Lonza Walkersville Inc

Inventors

• Shin Young
• Seth Anandita

Classifications

• C12N2740/15041

  Use of virus, viral particle or viral elements as a vector · CPC title

• C12N2510/02

  Cells for production · CPC title

• C12N5/0603

  Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies · CPC title

• A61K35/76

  Viruses; Subviral particles; Bacteriophages · CPC title

• C12N2740/16052

  relating to complementing cells and packaging systems for producing virus or viral particles · CPC title