# US 2020/0399609 A1 · Self replicating rna for inducing somatic differentiation of unmodified adult stem cells

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2020/0399609 A1
- Document ID: US20200399609A1
- Status: Published application
- Kind: A1
- Application number: 17/008,481
- Filed: 2020-08-31
- Published: 2020-12-24
- Earliest claimed: 2016-05-10
- Assignees: Ingeneron, Inc.
- Applicants: InGeneron, Inc.
- Inventors: Eckhard U. ALT; Tahereh KARIMI; Vladimir N. Potaman
- Web page: https://patentgrep.com/patent/US20200399609A1

## CPC

- C12N 5/0667: {Adipose-derived stem cells [ADSC]; Adipose stromal stem cells}
- C07K 14/4702: {Regulators; Modulating activity}
- C07K 14/4705: {stimulating, promoting or activating activity}
- C12N 5/0676: {Pancreatic cells}
- C12N 15/86: Viral vectors
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- C12N 2501/60: Transcription factors
- C12N 2501/65: MicroRNA
- C12N 2506/1307: from adult fibroblasts
- C12N 2506/1384: from adipose-derived stem cells [ADSC], from adipose stromal stem cells
- C12N 2510/00: Genetically modified cells
- C12N 2770/36143: viral genome or elements thereof as genetic vector
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

A self-replicating RNA for inducing somatic differentiation of unmodified adult stem cells is described. Methods of differentiating unmodified adult stem cells into functional beta-like cells are also described, as well as compositions, tissues and devices containing such cells. The method requires inducing sequential expression of PDX1 before NGN3, and NGN3 before MAFA in these stem cells to form reprogrammed beta-cells. Self-replicating RNAs are provided and introduced into the adult stem cells to induce the sequential expression. Methods of treating diabetes are also provided, comprising obtaining stem cells, preferably from a patient with diabetes, inducing sequential expression of PDX1>NGN3>MAFA, in said stem cells to form reprogrammed beta-cells, and introducing said reprogrammed beta-cells into a pancreas of said patient.

## Claims

### Claim 1

A self-replicating RNA (srRNA) for inducing adult somatic stem cells to differentiate into beta-cells, said srRNA comprising (a) a 5′ cap, (b) coding sequences of nonstructural proteins, (c) a promoter, (d) coding sequences of at least one of pancreatic and duodenal homeobox 1 (“Pdx1”), Neurogenin 3 (“Ngn3”), v-maf avian musculoponeurotic fibrosarcoma oncogene homology A (“MafA”) and optionally a coding sequence for a fluorescent protein, (e) an independent ribosome entry site (IRES), (f) optionally a selectable marker, and (g) 3′ poly A tail.

### Claim 2 (depends on 1)

The self-replicating RNA of claim 1, wherein the nonstructural proteins are nsP1, nsP2, nsP3 and nsP4 of Venezuelan equine encephalitis (VEE) virus.

### Claim 3 (depends on 2)

The self-replicating RNA of claim 2, wherein the promoter is a 26S subgenomic promoter.

### Claim 4 (depends on 1)

The self-replicating RNA of claim 1, further comprising coding sequence of Nkx6.1.

### Claim 5 (depends on 1)

A method of inducing adult somatic stem cells to differentiate into beta-cells, said method comprising:
a) inducing a sequential expression of PDX1 before NGN3, and NGN3 before MAFA in a population of adult unmodified stem cells by transducing a self-replicating RNA of claim 1 into said adult somatic stem cells in order to reprogram said stem cells, and
b) growing said reprogrammed stem cells until reprogrammed beta-cells form.

### Claim 6 (depends on 5)

The method of claim 5, wherein a first srRNA encoding PDX1, a second srRNA encoding NGN3 and a third srRNA encoding MAFA are sequentially transduced into the adult unmodified stem cells.

### Claim 7 (depends on 5)

The method of claim 5, further comprising inducing a subsequent expression of NKX6.1.

### Claim 8 (depends on 5)

The method of claim 5, wherein said stem cells are autologous stem cells.

### Claim 9 (depends on 5)

The method of claim 5, wherein said stem cells are autologous adipose derived stem cells.

### Claim 10 (depends on 5)

The method of claim 5, wherein said stem cells are adipose tissue derived stem cells.

### Claim 11 (depends on 5)

The method of claim 5, wherein a medium comprising B18R protein is used in step b).

### Claim 12 (depends on 5)

The method of claim 5, wherein PDX1, NGN3, and MAFA are each expressed for 1 to 6 days before initiating a next protein.

### Claim 13 (depends on 5)

The method of claim 5, wherein PDX1, NGN3, and MAFA are each expressed for about 3 days before initiating a next protein.

### Claim 14

A composition comprising a population of induced beta-cells differentiated from adult unmodified stem cells transduced with one or more self-replicating RNAs to allow sequential upregulation of genes encoding PDX1 before NGN3, and NGN3 before MAFA, thus forming reprogrammed beta-cells able to produce insulin in response to glucose.

### Claim 15 (depends on 14)

The composition of claim 14, said stem cells being adult stem cells.

### Claim 16 (depends on 14)

The composition of claim 14, said stem cells being adipose derived adult stem cells.

### Claim 17 (depends on 14)

The composition of claim 14, said stem cells being adipose derived adult stem cells from a patient with diabetes.

### Claim 18 (depends on 14)

A method of treating diabetes, comprising introducing said composition of claim 14 into a patient.

### Claim 19 (depends on 18)

The method of claim 18, comprising introducing said reprogrammed beta-cells into a pancreas of said patient.

### Claim 20

A method of inducing differentiation of mammalian adult unmodified stem cells into somatic cells, the method comprising:
a) transducing a self-replicating RNA (srRNA) into the adult unmodified stem cells, said srRNA comprising (i) a 5′ cap, (ii) coding sequences of nonstructural proteins, (iii) a promoter, (iv) coding sequences of transcription factors and optionally fluorescent proteins mCherry or GFP, (v) independent ribosome entry sites (IRES), (vi) optionally a puromycin-resistance gene (Puro), and (vii) 3′ poly A tail; and
b) growing said transduced stem cells until differentiated somatic cells form;
wherein said transcription factors induce differentiation of the adult unmodified stem cells into somatic cells.

### Claim 21 (depends on 20)

The method of claim 20, wherein the transcription factors are PDX1, NGN3 and MAFA, wherein the PDX1 is transduced before the NGN3 is transduced, and the NGN3 is transduced before the MAFA is transduced, and the somatic cells are beta cells.