# US 11,993,781 B2 · Stable pseudotyped lentiviral particles and uses thereof

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- Number: US 11,993,781 B2
- Document ID: US11993781B2
- Status: Granted
- Kind: B2
- Application number: 16/095,098
- Filed: 2017-04-21
- Published: 2019-10-03
- Granted: 2024-05-28
- Earliest claimed: 2016-04-21
- Assignees: Institut National de la Sante et de la Recherche Medicale; Universite D'Evry Val D'Essonne
- Applicants: Genethon; Inserm (Institut National De La Santé Et De La Recherche MéDicale); Universite D'Evry Val D'Essonne
- Inventors: Anne Galy
- Web page: https://patentgrep.com/patent/US11993781B2

## CPC

- C12N 15/86: Viral vectors
- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- A61K 35/76: Viruses; Subviral particles; Bacteriophages
- A61K 40/10: characterised by the cell type used
- A61K 40/13: B-cells
- A61K 40/24: Antigen-presenting cells [APC]
- A61K 40/46: Viral antigens
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/02: Immunomodulators
- C07K 14/005: from viruses
- C12N 5/0635: {B lymphocytes}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2239/38: characterised by the dose, timing or administration schedule
- C12N 2740/10022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2740/16022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- 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: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention relates to a method for obtaining stable pseudotyped lentiviral particles including a heterologous gene of interest, comprising the following steps: a) transfecting at least one plasmid in appropriate cell lines, wherein said at least one plasmid comprises the gene of interest, the rev. gag and pol genes, and a sequence coding for an ERV syncytin, wherein the rev, gag and pol genes are retroviral genes; b) incubating the transfected cells obtained in a), so that they produce the stable pseudotyped lentiviral particles in the supernatant; and c) harvesting and concentrating the stable lentiviral particles obtained in b). The present invention also relates to a method to transduce immune cells using lentiviral vectors pseudotyped with an ERV syncytin glycoprotein. The method can be performed on non-stimulated blood cells or on cells stimulated briefly with IL7, and the cells can be expanded. The stable pseudotyped lentiviral particles obtained are particularly useful in gene therapy.

## Claims

### Claim 1

A method for obtaining stable lentiviral particles pseudotyped with an endogenous retroviral syncytin (ERV syncytin) and packaging a heterologous gene of interest, which have a high physical and/or infectious titer(s), comprising the following steps:
a) transfecting four plasmids in appropriate cell lines, wherein said four plasmids comprise a first plasmid comprising the heterologous gene of interest, a second plasmid comprising the lentiviral rev gene, a third plasmid comprising the gag and pol genes, and a fourth plasmid comprising a nucleotide sequence coding for an envelope glycoprotein, wherein the envelope glycoprotein consists of an ERV syncytin selected from the group consisting of HERV-W, HERV-FRD, murine syncytin-A and murine syncytin-B;
b) incubating the transfected cells obtained in a), so that they produce the stable lentiviral particles pseudotyped with an ERV syncytin, and packaging the heterologous gene of interest;
c) harvesting the stable lentiviral particles obtained in b) 24 hours after the transfection, before fusion of the transfected cells, and
d) concentrating the stable lentiviral particles harvested in c) to obtain a preparation of concentrated stable lentiviral particles having a high infectious titer higher than 1×106 TU/mL or a high physical titer higher than 1.5×10 5 ng p24/mL after freezing and thawing of the preparation.

### Claim 2 (depends on 1)

Particles obtained by the method of claim 1.

### Claim 3 (depends on 1)

Particles obtained by the method of claim 1 having a titer of infectious particles produced at the end of step d) higher than 1×106 TU/mL, or 2>106 TU/mL, and/or a physical titer of particles produced at the end of step d) higher than 1.5×105 ng p24/mL.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the concentration of step d) comprises centrifugating and/or purifying the harvested stable lentiviral particles obtained in c).

### Claim 5 (depends on 1)

The method according to claim 1, wherein the preparation of stable concentrated lentiviral particles produced in step d) have a high infectious titer higher than 2×106 TU/mL after freezing and thawing of the preparation.