# US 10,767,165 B2 · Reprogramming method for producing induced pluripotent stem cells (iPSC)

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- Number: US 10,767,165 B2
- Document ID: US10767165B2
- Status: Granted
- Kind: B2
- Application number: 15/547,212
- Filed: 2016-02-01
- Published: 2018-01-25
- Granted: 2020-09-08
- Earliest claimed: 2015-01-30
- Assignees: Centre National de la Recherche Scientifique (CNRS)
- Applicants: Centre National de la Recherche Scientifique (CNRS)
- Inventors: Pierre Roux
- Web page: https://patentgrep.com/patent/US10767165B2

## CPC

- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 9/12: Antihypertensives
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 35/00: Antineoplastic agents
- C12N 2501/40: Regulators of development
- C12N 2501/998: Proteins not provided for elsewhere
- C12N 2506/1307: from adult fibroblasts
- C12N 2510/00: Genetically modified cells
- 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 invention is in the field of stem cells. Particularly, the present invention relates to a reprogramming method for producing induced pluripotent stem cells (iPSC) by transducing somatic differentiated cells with a viral vector expressing Δ133p53β isoform, Δ133p53γ isoform or both Δ133p53β and Δ133p53γ isoforms; to induced pluripotent stem cells as produced by this method; to uses thereof in cell therapy and as an in vitro model for studying various diseases; and to a method for detecting iPSC.

## Claims

### Claim 1

An in vitro method for producing mammalian induced pluripotent stem cells (iPSCs), comprising:
a) transducing isolated mammalian somatic cells with:
i) at least one vector encoding a Δ133p53β isoform, Δ133p53γ isoform, or both Δ133p53β and Δ133p53γ isoforms, and
ii) vectors encoding reprogramming factors Oct4, Sox2, Klf4, and cMyc;
b) culturing the cells obtained in step a) in a medium supporting pluripotent cell expansion; and
c) isolating iPSCs from the culture in step b).

### Claim 2 (depends on 1)

The in vitro method according to claim 1, wherein the mammalian somatic differentiated cells comprise peripheral blood mononuclear cells (PBMC), fibroblasts or CD4+ lymphocytes.

### Claim 3 (depends on 1)

The method of claim 1, wherein the at least one vector encoding a Δ133p53β isoform, Δ133p53γ isoform, or both Δ133p53β and Δ133p53γ isoforms is a retroviral vector and comprises elements necessary to allow expression thereof selected from a promoter, an enhancer or a regulatory sequence.

### Claim 4 (depends on 1)

The in vitro method according to claim 1, wherein the medium of step b) is a basal medium comprising inorganic salts, amino acids, vitamins and glucose, β-mercaptoethanol, at least one antibiotic, and bFGF.

### Claim 5 (depends on 1)

The in vitro method according to claim 1, wherein the induced pluripotent stem cells (iPSC) are isolated by a selection based on stem cells surface markers, an embryoid formation or iPSC colony picking.

### Claim 6 (depends on 1)

The in vitro method according to claim 1, further comprising step a1) of culturing the mammalian somatic differentiated cells before the step a) of transducing said cells.

### Claim 7 (depends on 1)

The in vitro method according to claim 1, wherein the mammalian somatic differentiated cells are transduced with vector expressing Δ133p53β isoform.

### Claim 8 (depends on 1)

The in vitro method according to claim 1, wherein the mammalian somatic differentiated cells are fibroblasts.

### Claim 9 (depends on 1)

The method of claim 1, wherein the at least one vector encoding a Δ133p53β isoform, Δ133p53γ isoform, or both Δ133p53β and Δ133p53γ isoforms is a murine stem cell viral (MSCV) vector and comprises elements necessary to allow expression thereof selected from a promoter, an enhancer or a regulatory sequence.

### Claim 10 (depends on 1)

The in vitro method according to claim 1, said method further comprising step a1) of culturing the mammalian somatic differentiated cells before the step a) in which said cells are transduced with vector expressing Δ133p53β isoform.

### Claim 11 (depends on 10)

The in vitro method according to claim 10, wherein the mammalian somatic differentiated cells are fibroblasts.

### Claim 12 (depends on 11)

The in vitro method according to claim 11, wherein said fibroblasts are transduced with retroviral vector expressing Δ133p53β isoform.

### Claim 13 (depends on 1)

The in vitro method according to claim 1, wherein the induced pluripotent stem cells (iPSC) are isolated by iPSC colony picking.