# US 10,865,387 B2 · Methods of obtaining pancreatic endocrine cells

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- Number: US 10,865,387 B2
- Document ID: US10865387B2
- Status: Granted
- Kind: B2
- Application number: 15/310,490
- Filed: 2015-05-14
- Published: 2017-03-16
- Granted: 2020-12-15
- Earliest claimed: 2014-05-14
- Assignees: The University Court of the University of Aberdeen Regent Walk
- Applicants: The University Court of the University of Aberdeen
- Inventors: Kevin Docherty; Hilary Margaret Docherty; Maria Joao Marques De Lima; Kenneth Ross Muir; John Joseph Casey
- Web page: https://patentgrep.com/patent/US10865387B2

## CPC

- C12N 5/0676: {Pancreatic cells}
- A61K 35/39: Pancreas; Islets of Langerhans (Langerhans cells of epidermis A61K35/36)
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- C12N 2500/22: Zinc; Zn chelators
- C12N 2501/06: Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
- C12N 2501/065: Modulators of histone acetylation
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/60: Transcription factors
- C12N 2501/72: Transferases [EC 2.]
- C12N 2501/73: Hydrolases (EC 3.)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/22: from pancreatic cells
- 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)
- 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

## Abstract

The present invention relates to methods of producing pancreatic endocrine cells and uses of the cells obtained using the methods. The method utilises inhibitors or combinations of factors to provide increased quantities of endocrine material, for example for transplantation purposes.

## Claims

### Claim 1

A method for ex-vivo reprogramming comprising:
a) providing pancreatic cells to be reprogrammed,
b) reprogramming the pancreatic cells, comprising:
(i) treating the pancreatic cells with PAX4, PDX1, MAFA and NGN3 transcription factors and
(ii) inhibiting ARX expression and/or function
c) thereby obtaining beta-like cells that express insulin mRNA.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the beta-like cells obtained in step (c) are capable of producing insulin protein in response to glucose stimulation.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the beta-like cells obtained in step (c) produce insulin protein at a level of at least 5% of that of adult human islets.

### Claim 4 (depends on 1)

The method according to claim 1, wherein ARX expression is inhibited.

### Claim 5 (depends on 4)

The method according to claim 4, wherein ARX expression is inhibited using RNA interference (RNAi).

### Claim 6 (depends on 4)

The method according to claim 4, wherein ARX expression is inhibited by siRNA.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the cells are cultured on laminin throughout the method, or
wherein during the reprogramming the cells are cultured in glucose, wherein the glucose concentration is between 0-5 mM, or
wherein the cells are cultured in adherent culture prior to reprogramming, optionally for about 2 days, or
a combination thereof.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the pancreatic cells provided in step (a) are human pancreatic cells, or
wherein the pancreatic cells provided in step (a) comprise exocrine cells, optionally from an exocrine enriched fraction of a pancreas, or
a combination thereof.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the
reprogramming further comprises the step of pre-treating the cells, before treatment with the one or more transcription factors, with an inhibitor of epithelial to mesenchymal transition, or
reprogramming further comprises the step of pre-treating the cells, before treatment with the one or more transcription factors, with a chromatin modifying agent, or
a combination thereof.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the inhibitor of epithelial to mesenchymal transition is a TGFbetal signaling pathway inhibitor, a Rho-associated protein kinase (Rock) signaling pathway inhibitor, or a combination thereof.

### Claim 11 (depends on 9)

The method according to claim 9, wherein the chromatin-modifying agent is a DNA-methyltransferase inhibitor, a histone deacetylase (HDAC) inhibitor, or a combination thereof.

### Claim 12 (depends on 1)

The method according to claim 1, further comprising culturing the cells in media comprising betacellulin, exendin-4, nicotinamide, or a combination thereof.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the reprogramming comprises treatment with zinc.