# US 12,286,639 B2 · LDLR negative packaging cell line for the production of VSV-G pseudotyped retroviral vector particles or virus particles thereof

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- Number: US 12,286,639 B2
- Document ID: US12286639B2
- Status: Granted
- Kind: B2
- Application number: 17/264,763
- Filed: 2019-08-28
- Published: 2021-09-30
- Granted: 2025-04-29
- Earliest claimed: 2018-08-30
- Assignees: Miltenyi Biotec GmbH
- Applicants: Miltenyi Biotec B.V. & Co. KG
- Inventors: Thomas Schaser; Johann-Christoph Dettmann; Martin Meyer; Ian Johnston
- Web page: https://patentgrep.com/patent/US12286639B2

## CPC

- C12N 15/86: Viral vectors
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 2740/15023: Virus like particles [VLP]
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2740/15052: relating to complementing cells and packaging systems for producing virus or viral particles
- 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 2810/6081: rhabdoviridae, e.g. VSV
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides the use of a packaging cell line for the production of VSV-G pseudotyped retroviral vector particles or virus like particles thereof, wherein said packaging cell line is negative for Low-Density Lipoprotein Receptor (LDLR), optionally said packaging cell line stably expresses VSV-G. A method for producing said VSV-G pseudotyped retroviral vector particles or virus like particles thereof is disclosed as well as said particles obtained by said method.

## Claims

### Claim 1

A method for producing vesicular stomatitis virus envelope protein (VSV-G) pseudotyped vectors that contain a Ψ-positive nucleic acid encoding one or more transgene products or virus-like particles thereof containing said transgene product(s),
the method comprising:
obtaining cells from a packaging cell line that has been stably adapted and/or selected to not express cell surface low density lipoprotein receptor (LDLR);
causing the cells to express VSV-G, gag/pol, and optionally rev; and
causing the cells to express said Ψ-positive nucleic acid or said transgene product(s), whereby the cells produce said pseudotyped vectors or virus-like particles; then
harvesting from the cells said pseudotyped vectors or virus-like particles.

### Claim 2 (depends on 1)

The method of claim 1, wherein the packaging cell line is a human cell line.

### Claim 3 (depends on 1)

The method of claim 1, wherein the packaging cell line has been genetically engineered to prevent expression of LDLR on the cell surface.

### Claim 4 (depends on 3)

The method of claim 3, wherein lentiviral mediated autotransduction by the packaging cell line is reduced by at least 50% as a result of being genetically engineered to prevent expression of LDLR on the cell surface.

### Claim 5 (depends on 3)

The method of claim 3, wherein the packaging cell line has been adapted by knocking out or mutating a gene encoding LDLR to prevent expression of LDLR on the surface of cells from said line.

### Claim 6 (depends on 1)

The method of claim 1, wherein the packaging cell line is selected from human embryonic kidney (HEK) cell line 293, HEK 293T, HEK EBNA, HEK 293F, HEK 293FT, and HEK 293-S.

### Claim 7 (depends on 1)

The method of claim 1, wherein the packaging cell line has been genetically engineered to stably express VSV-G.

### Claim 8 (depends on 7)

The method of claim 7, wherein the packaging cell line has been genetically engineered to stably express at least one additional gene selected from Gag/pol, rev, and said Ψ-positive nucleic acid that encodes said transgene product(s).

### Claim 9 (depends on 1)

The method of claim 1, further comprising formulating the harvested vectors or virus-like particles as a pharmaceutical composition in a pharmaceutically acceptable carrier.

### Claim 10 (depends on 1)

The method of claim 1, comprising co-transfecting the LDLR-negative packaging cells with a combination of vectors that include:
a Ψ-negative retroviral expression vector that encodes gag/pol,
a Ψ-negative retroviral expression vector that encodes VSV-G, and
said Ψ-positive nucleic acid.

### Claim 11 (depends on 1)

The method of claim 1, comprising co-transfecting the LDLR-negative packaging cells with a combination of vectors that include:
a Ψ-negative retroviral expression vector that encodes gag/pol,
a Ψ-negative retroviral expression vector that encodes rev,
a Ψ-negative retroviral expression vector that encodes VSV-G, and
said Ψ-positive nucleic acid.

### Claim 12 (depends on 1)

The method of claim 1, comprising co-transfecting the LDLR-negative packaging cells with a combination of vectors that include:
a Ψ-negative retroviral expression vector that encodes gag/pol and rev,
a Ψ-negative retroviral expression vector that encodes VSV-G, and
said Ψ-positive nucleic acid.

### Claim 13 (depends on 1)

The method of claim 1, comprising co-transfecting the LDLR-negative packaging cells with a combination of vectors that include:
a Ψ-negative retroviral expression vector that encodes gag/pol,
a Ψ-negative retroviral expression vector that encodes rev, and
said Ψ-positive nucleic acid.

### Claim 14 (depends on 1)

The method of claim 1, wherein the packaging cell line has been selected by sorting and recovering individual cells with relatively low levels of LDLR expression by flow cytometry, and establishing a cell line therefrom.

### Claim 15 (depends on 14)

The method of claim 14, wherein the packaging cell line has been cloned and selected for a low level of LDLR expression.

### Claim 16

A method for producing vesicular stomatitis virus G envelope protein (VSV-G) pseudotyped vectors that contain a Ψ-positive nucleic acid encoding one or more transgene products or virus-like particles thereof containing said transgene product(s),
the method comprising:
adapting and/or selecting a population of packaging cells to not express low density lipoprotein receptor (LDLR);
causing the cells to express VSV-G, gag/pol, and optionally rev; and
causing the cells to express said W-positive nucleic acid or said transgene product(s), whereby the cells produce said pseudotyped vectors or virus-like particles; then
harvesting from the cells said pseudotyped vectors or virus-like particles.

### Claim 17

A combination configured for producing vesicular stomatitis virus G envelope protein (VSV-G) pseudotyped vectors containing a nucleic acid that encodes one or more transgene products, the combination comprising:
cells from a packaging cell line that has been stably adapted and/or selected to not express cell surface low density lipoprotein receptor (LDLR); along with
retrovirus expression vector(s) that encode VSV-G, gag/pol, and optionally rev.

### Claim 18 (depends on 17)

The combination of claim 17, wherein the packaging cell line has been genetically engineered to prevent expression of LDLR on the surface of the cells.

### Claim 19 (depends on 17)

The combination of claim 17, wherein the packaging cell line has been genetically engineered to stably express VSV-G, and
wherein the combination comprises retrovirus expression vector(s) that encode gag/pol, and rev.

### Claim 20 (depends on 17)

The combination of claim 17, which further comprises an expression vector that contains the transgene.

### Claim 21 (depends on 19)

The combination of claim 19, wherein the retrovirus expression vector(s) that encode gag/pol, and rev are Ψ-negative.

### Claim 22 (depends on 20)

The combination of claim 20, wherein the expression vector that contains the transgene is Ψ-positive.

### Claim 23

A combination configured for producing vesicular stomatitis virus G envelope protein (VSV-G) pseudotyped virus-like particles containing one or more transgene products, the combination comprising:
cells from a packaging cell line that has been stably adapted and/or selected to not express cell surface low density lipoprotein receptor (LDLR); along with
retrovirus expression vector(s) that encode VSV-G, gag/pol, and optionally rev.

### Claim 24 (depends on 9)

A method of treating a subject in need thereof, comprising administering to the subject a pharmaceutical composition produced according to the method of claim 9.