# US 11,999,971 B2 · Stem cell derived islet differentiation

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- Number: US 11,999,971 B2
- Document ID: US11999971B2
- Status: Granted
- Kind: B2
- Application number: 18/055,312
- Filed: 2022-11-14
- Published: 2023-03-09
- Granted: 2024-06-04
- Earliest claimed: 2018-08-10
- Assignees: Vertex Pharmaceuticals Incorporated
- Applicants: Vertex Pharmaceuticals Incorporated
- Inventors: Felicia J. Pagliuca; George Harb; Lillian Ye
- Web page: https://patentgrep.com/patent/US11999971B2

## CPC

- C12N 5/0676: {Pancreatic cells}
- C12N 5/0678: {Stem cells; Progenitor cells; Precursor cells}
- A61K 35/39: Pancreas; Islets of Langerhans (Langerhans cells of epidermis A61K35/36)
- A61P 5/50: for increasing or potentiating the activity of insulin
- C07K 14/4702: {Regulators; Modulating activity}
- A61K 2035/126: {Immunoprotecting barriers, e.g. jackets, diffusion chambers}
- C12N 2500/46: Amines, e.g. putrescine
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/117: Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
- C12N 2501/119: Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/19: Growth and differentiation factors [GDF]
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/45: from artificially induced pluripotent stem 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
- 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

Provided herein are methods of producing β cells and precursors thereof utilizing a Wnt signaling inhibitor or PKC activator, or both. Also provided herein are in vitro cultures comprising said cells, methods of treating a subject with a disease characterized by high blood sugar levels over a prolonged period of time by administering said cells, and devices for encapsulating said cells.

## Claims

### Claim 1

An in vitro composition comprising a population of cells derived from stem cells in vitro, wherein:
a) at least 30% of the cells in the population express NKX6.1 and ISL1;
b) at least 30% of the cells in the population express ISL1 but do not express NKX6.1;
c) the population comprises cells that express NKX6.1 but do not express ISL1, and less than 25% of the cells in the population express NKX6.1 but do not express ISL1;
d) less than 25% of the cells in the population express VMAT1; and
e) the population comprises at least one cell that is genetically modified.

### Claim 2 (depends on 1)

The composition of claim 1 wherein less than 15% of the cells in the population express NKX6.1 but do not express 1SL1.

### Claim 3 (depends on 1)

The composition of claim 1, wherein at least 20% of the cells in the population express ARX.

### Claim 4 (depends on 1)

The composition of claim 1, wherein at least 30% of the cells in the population express ARX.

### Claim 5 (depends on 1)

The composition of claim 1, wherein at least 10% of the cells in the population express glucagon.

### Claim 6 (depends on 1)

The composition of claim 1, wherein at least 15% of the cells in the population express glucagon.

### Claim 7 (depends on 1)

The composition of claim 1, wherein at least 20% of the cells in the population express glucagon.

### Claim 8 (depends on 7)

The composition of claim 7, wherein less than 20% of the cells in the population express VMAT1.

### Claim 9 (depends on 1)

The composition of claim 1, wherein at least 40% of the cells in the population express NKX6.1 and ISL1.

### Claim 10 (depends on 1)

The composition of claim 1 wherein at least 35% of the cells in the population express ISL1 but do not express NKX6.1.

### Claim 11 (depends on 1)

The composition of claim 1, wherein the population of cells are in one or more cell clusters.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the population comprises cells that express NKX6.1 and ISL1 and exhibit glucose stimulated insulin secretion (GSIS) in vitro.

### Claim 13 (depends on 1)

The composition population of claim 1, wherein the at least one cell is genetically engineered to reduce an immune response.

### Claim 14 (depends on 13)

The composition of claim 13, wherein the at least one cell contains a genetic disruption in the gene encoding for beta-2-microglobulin.

### Claim 15 (depends on 13)

The composition of claim 13, wherein the at least one cell contains a genetic disruption in the gene encoding for one or more of HLA-A, HLA-B, or HLA-C.

### Claim 16 (depends on 1)

The composition of claim 1, wherein the population is in a device suitable for implantation into a human subject.

### Claim 17 (depends on 1)

The composition of claim 1, wherein the levels of NKX6.1 and ISL1 are determined utilizing immunocytochemistry.

### Claim 18 (depends on 1)

The composition of claim 1, wherein the levels of NKX6.1 and ISL1 are determined utilizing flow cytometry.

### Claim 19 (depends on 1)

The composition of claim 1 wherein the population is in a composition further comprising one or more of: a transforming growth factor beta (TGF-beta) signaling pathway inhibitor, a bone morphogenesis protein (BMP) inhibitor, a Rho-associated kinase (ROCK) inhibitor, a histone methylation inhibitor, a thyroid hormone signaling pathway activator, or a broad kinase inhibitor.

### Claim 20 (depends on 1)

The composition of claim 1 wherein the population is in a composition further comprising one or more of: an Activin-like kinase 5 (ALK5) inhibitor, LDN193189, thiazovivin, staurosporine, GC-1, or 3-deazaneplanocin A (DZNEP).