# US 11,299,752 B2 · Bio-production of lentiviral vectors

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- Number: US 11,299,752 B2
- Document ID: US11299752B2
- Status: Granted
- Kind: B2
- Application number: 16/164,178
- Filed: 2018-10-18
- Published: 2019-03-21
- Granted: 2022-04-12
- Earliest claimed: 2015-05-13
- Assignees: CSL Behring Gene Therapy, Inc.
- Applicants: CSL Behring Gene Therapy, Inc.
- Inventors: Chi-Lin Lee; Jeffrey S Bartlett
- Web page: https://patentgrep.com/patent/US11299752B2

## CPC

- C12N 15/86: Viral vectors
- A61P 31/18: for HIV
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2740/16041: Use of virus, viral particle or viral elements as a vector
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N 2740/16052: relating to complementing cells and packaging systems for producing virus or viral particles
- C12N 2800/107: for mammalian
- C12N 2800/70: Vectors containing special elements for cloning, e.g. topoisomerase, adaptor sites
- C12N 2830/006: tet repressible
- C12N 2830/40: being an insulator
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present disclosure provides a method of generating a stable producer cell line. The generation of stable producer cell lines, such as those provided in accordance with the present invention, increases the reproducibility and ease of creating high titer lentiviral stocks while easing biosafety concerns and the variation in expressed envelope proteins defines the tropism of the generated virus. The present disclosure also provides for a novel lentiviral transfer vector plasmid.

## Claims

### Claim 1

A method of harvesting lentiviral vector supernatant comprising:
generating stable producer cell line cells, wherein the generated stable producer cell line cells are derived from one of GPRG, GPRGT, or GPRT-G packing cell line cells; inducing lentiviral vector production from the generated stable producer cell line cells; and repeatedly harvesting the lentiviral vectors from the induced generated stable producer cell line cells every about 40 to about 56 hours following an initial harvesting of the lentiviral vectors, wherein each repeated harvesting comprises adding fresh media to the induced generated stable producer cell line cells without introducing additional generated stable producer cell line cells, wherein the generated stable producer cell line cells are generated by (a) synthesizing a lentiviral vector by cloning one or more genes into a recombinant plasmid; (b) forming a concatemeric array from (i) a lentiviral transfer vector DNA excised from the synthesized lentiviral vector, and (ii) an expression cassette obtained from an antibiotic resistance cassette plasmid; (c) transfecting the one of the GPRG, GPRGT, or GPRT-G packaging cell line cells with the formed concatemeric array; and (d) isolating the generated stable producer cell line cells, and wherein the recombinant plasmid comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 1.

### Claim 2

A method of harvesting lentiviral vector supernatant comprising:
generating stable producer cell line cells, wherein the generated stable producer cell line cells are derived from one of GPRG, GPRGT, or GPRT-G packing cell line cells; inducing viral lentivector production from the generated stable producer cell line cells; and repeatedly harvesting the lentiviral vectors from the induced generated stable producer cell line cells every about 40 to about 56 hours following an initial harvesting of the lentiviral vectors, wherein each repeated harvesting comprises adding fresh media to the induced generated stable producer cell line cells without introducing additional generated stable producer cell line cells, wherein the stable producer cell line cells are generated by (a) synthesizing a lentiviral vector by cloning one or more genes into a recombinant plasmid; (b) forming a concatemeric array from (i) a lentiviral transfer vector DNA excised from the synthesized vector, and (ii) an expression cassette obtained from an antibiotic resistance cassette plasmid; (c) transfecting the one of the GPRG, GPRGT, or GPRT-G packaging cell line cells with the formed concatemeric array; and (d) isolating the generated stable producer cell line cells, and wherein the recombinant plasmid comprises a vector cassette having at least 80% sequence identity to the sequence of SEQ ID NO: 2.