Methods and constructs for production of lentiviral vector
AssigneeLonza Walkersville, Inc.
InventorsYoung Shin, Anandita Seth, Bingnan Gu
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Abstract
The present disclosure relates to methods for producing lentiviral vector-producing cells. Specifically the methods utilize two 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 producer cells, including suspension-based cells, and the production of high amounts of lentivirus. These methods allow for the production of cells that can be later induced to produce lentivirus, and can be tailored to include a specific gene of interest.
Claims14
A method of producing a lentiviral vector-producing mammalian cell, comprising:
- a. transfecting a mammalian cell with:
- i. a packaging vector including an expression cassette, encoding:
- 1. a lentiviral regulator of expression of virion proteins (REV) gene under control of a first promoter;
- 2. a lentiviral envelope gene under control of a second promoter;
- and
- 3. a lentiviral group specific antigen gag gene and a lentiviral polymerase pol gene both under control of a third promoter,
- wherein the expression cassette is flanked on both the 5′ and 3′ ends by transposon-specific inverted terminal repeats (ITR);
- and
- ii. a transfer vector, comprising:
- 1. a nucleic acid sequence encoding a gene of interest under control of a fourth promoter,
- wherein the nucleic acid sequence is flanked on both the 5′ and 3′ ends by transposon-specific inverted terminal repeats (ITR);
- b. culturing the transfected mammalian cell;
- and
- c. isolating the lentiviral vector-producing mammalian cell.
Dependencies are inferred from the claim text.