# US 2014/0193913 A1 · Methods and compositions for homologous recombination in human cells

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

- Number: US 2014/0193913 A1
- Document ID: US20140193913A1
- Status: Published application
- Kind: A1
- Application number: 13/968,852
- Filed: 2013-08-16
- Published: 2014-07-10
- Earliest claimed: 2009-05-29
- Assignees: The General Hospital Corporation
- Applicants: The General Hospital Corporation
- Inventors: Niels Geijsen; Christa Buecker
- Web page: https://patentgrep.com/patent/US20140193913A1

## CPC

- C12N 15/85: for animal cells
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 15/873: Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
- C12N 15/907: {in mammalian cells}
- C12N 2501/235: Leukemia inhibitory factor [LIF]
- C12N 2501/602: Sox-2
- C12N 2501/603: Oct-3/4
- C12N 2501/604: Klf-4
- C12N 2501/605: Nanog
- C12N 2501/606: c-Myc
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2510/00: Genetically modified cells
- C12N 2740/16043: 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)

## Abstract

The methods and compositions described herein are based, in part, on the discovery of a stem cell state in human cells that resembles the morphology observed in murine-derived stem cells. Induction of such a state in human stem cells permits an increase in the efficiency of homologous recombination. Thus, the methods and compositions described herein relate to cells and methods for increasing the efficiency of homologous recombination in human stem cells.

## Claims

### Claim 1

A method for generating a genetically-modified human stem cell, the method comprising:
(a) culturing a human stem cell with a GSK3β inhibitor,
(b) modifying the genome of the stem cell in step (a) by introducing a homologous recombination cassette and inducing homologous recombination,
wherein a genetically-modified human embryonic stem-like cell is generated.

### Claim 2 (depends on 1)

The method of claim 1, wherein the homologous recombination event of step (b) adds a nucleic acid sequence, deletes a nucleic acid sequence, removes an exon, removes an intron, introduces a point mutation, or corrects a mutation.

### Claim 3 (depends on 1)

The method of claim 1, wherein homologous recombination is induced using electroporation techniques.

### Claim 4 (depends on 1)

The method of claim 1, wherein the human stem cell is an iPS cell, an embryonic stem cell, a stem cell line, a pluripotent stem cell, a multipotent stem cell, or an adult stem cell.

### Claim 5 (depends on 1)

The method of claim 1, further comprising culturing the human stem cell with a MEK inhibitor.

### Claim 6 (depends on 5)

The method of claim 5, wherein the MEK inhibitor is PD98059.

### Claim 7

A composition comprising:
(a) a human stem cell comprising at least each of the reprogramming factors: Oct4, Sox2, Nanog, c-MYC and KLf4; and
(b) a homologous recombination cassette.

### Claim 8 (depends on 7)

The composition of claim 7, wherein the homologous recombination cassette targets a non-developmentally regulated gene or a non-embryonic gene.

### Claim 9 (depends on 7)

The composition of claim 7, wherein the cell in the composition has an enhanced efficiency of homologous recombination compared to a corresponding human stem cell.

### Claim 10 (depends on 9)

The composition of claim 9, wherein the efficiency of homologous recombination for the cell is enhanced by at least 50% compared to a corresponding human stem cell not comprising a reprogramming factor.

### Claim 11 (depends on 7)

The composition of claim 7, wherein the cell of the composition can survive expansion from a single cell and wherein the corresponding human stem cell not comprising a reprogramming factor does not survive expansion from a single cell.

### Claim 12 (depends on 7)

The composition of claim 7, wherein the cell of the composition survives passaging as a single cell by a non-collagenase digest, and wherein the corresponding human stem cell does not survive passaging as a single cell by a non-collagenase digest.

### Claim 13 (depends on 7)

The composition of claim 7, wherein the cell of the composition survives passaging as a single cell by trypsin digest, and wherein the corresponding human stem cell does not survive passaging as a single cell by trypsin digest.

### Claim 14 (depends on 7)

The composition of claim 7, wherein the cell of the composition has a faster growth rate compared to the growth rate of a corresponding human stem cell not comprising a reprogramming factor.

### Claim 15 (depends on 14)

The composition of claim 14, wherein the growth rate is assessed by measuring doubling time.

### Claim 16 (depends on 15)

The composition of claim 15, wherein the doubling time is at least 2 hours faster than the growth rate of a corresponding human stem cell not in an admixture with the GSK3β inhibitor.

### Claim 17 (depends on 7)

The composition of claim 7, further comprising LIF.

### Claim 18

A kit for enhancing the efficiency of homologous recombination in a human stem cell, the kit comprising:
(a) a GSK3b inhibitor,
(b) a homologous recombination cassette, and
(c) instructions and packaging thereof.

### Claim 19 (depends on 18)

The kit of claim 18, wherein the GSK3β inhibitor is selected from the group consisting of: CHIR99021, BIO, Kenpaullone, and SB216763.

### Claim 20 (depends on 18)

The kit of claim 18, further comprising a MEK inhibitor.

### Claim 21 (depends on 20)

The kit of claim 20, wherein the MEK inhibitor is PD98059.