# US 2019/0316149 A1 · Genetic modification of rats

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- Number: US 2019/0316149 A1
- Document ID: US20190316149A1
- Status: Published application
- Kind: A1
- Application number: 16/401,539
- Filed: 2019-05-02
- Published: 2019-10-17
- Earliest claimed: 2013-02-20
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Jeffrey D. Lee; Wojtek Auerbach; David Heslin; David Frendewey; Ka-Man Venus Lai; David M. Valenzuela
- Web page: https://patentgrep.com/patent/US20190316149A1

## CPC

- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 67/02: Breeding vertebrates
- A01K 67/0271: Chimeric vertebrates, e.g. comprising exogenous cells
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- A61D 19/04: for embryo transplantation
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 15/8775: {Murine embryos}
- A01K 2207/12: Animals modified by administration of exogenous cells
- A01K 2217/15: Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
- A01K 2227/105: Murine
- C12N 2501/235: Leukemia inhibitory factor [LIF]
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/999: Small molecules not provided for elsewhere
- 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)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A61D: VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS

## Abstract

Compositions and methods are provided for making rat pluripotent and totipotent cells, including rat embryonic stem (ES) cells. Compositions and methods for improving efficiency or frequency of germline transmission of genetic modifications in rats are provided. Such methods and compositions comprise an in vitro culture comprising a feeder cell layer and a population of rat ES cells or a rat ES cell line, wherein the in vitro culture conditions maintain pluripotency of the ES cell and comprises a media having mouse leukemia inhibitory factor (LIF) or an active variant or fragment thereof. Various methods of establishing such rat ES cell lines are further provided. Methods of selecting genetically modified rat ES cells are also provided, along with various methods to generate a transgenic rat from the genetically modified rat ES cells provided herein. Various kits and articles of manufacture are further provided.

## Claims

### Claim at position 1

-128. (canceled)

### Claim 129

A method of making a genetically modified rat embryonic stem (ES) cell clone capable of generating a genetically modified rat comprising a targeted genetic modification and transmitting the targeted genetic modification through the germline, comprising:
(a) culturing a population of rat ES cells under conditions comprising a layer of feeder cells that are not modified to express leukemia inhibitory factor (LIF) and a medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor;
(b) introducing into the rat ES cells a nuclease agent that generates a single or double strand break at a targeted genomic locus and/or a targeting vector comprising upstream and downstream homology arms flanking an insert polynucleotide, wherein the homology arms correspond to genomic regions within the targeted genomic locus; and
(c) obtaining a rat ES cell clone comprising the targeted genetic modification, wherein the obtaining consists of identifying in a single cloning step a rat ES cell clone comprising the targeted genetic modification and capable of generating a genetically modified rat comprising the targeted genetic modification and transmitting the targeted genetic modification through the germline.

### Claim 130 (depends on 129)

The method of claim 129, wherein the LIF is a mouse LIF or has at least 91% sequence identity to SEQ ID NO: 1.

### Claim 131 (depends on 129)

The method of claim 129, wherein the concentration of LIF in the medium is between about 75 U/mL to about 125 U/mL.

### Claim 132 (depends on 131)

The method of claim 131, wherein the concentration of LIF in the medium is between about 90 U/mL to about 110 U/mL.

### Claim 133 (depends on 132)

The method of claim 132, wherein the concentration of LIF in the medium is about 100 U/mL.

### Claim 134 (depends on 133)

The method of claim 133, wherein the concentration of the MEK inhibitor is about 1 μM, and the concentration of the GSK3 inhibitor is about 3 μM.

### Claim 135 (depends on 129)

The method of claim 129, wherein the concentration of the MEK inhibitor is 0.8 μM to about 1.2 μM, and the concentration of the GSK3 inhibitor is about 2.5 μM to about 3 μM or 3 μM to about 3.5 μM.

### Claim 136 (depends on 135)

The method of claim 135, wherein the concentration of the MEK inhibitor is about 1 μM, and the concentration of the GSK3 inhibitor is about 3 μM.

### Claim 137 (depends on 129)

The method of claims 129, wherein the MEK inhibitor is PD0325901, and the GSK3 inhibitor is CHIR99021.

### Claim 138 (depends on 129)

The method of claim 129, wherein the feeder cell layer comprises a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs).

### Claim 138 (depends on 129)

The method of claim 129, wherein the medium further comprises DMEM/F12 basal medium, Neurobasal medium, N2 supplement, and B27 supplement.

### Claim 140 (depends on 139)

The method of claim 139, wherein the feeder cell layer comprises mitotically inactivated mouse embryonic fibroblasts, and the medium is a 2i medium comprising DMEM/F12 basal medium, Neurobasal medium, N2 supplement, B27 supplement, LIF, and a combination of inhibitors consisting of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021.

### Claim 141 (depends on 140)

The method of claim 140, wherein the concentration of the LIF is 100 U/mL, the concentration of the MEK inhibitor is about 1 μM, and the concentration of the GSK3 inhibitor is about 3 μM.

### Claim 142 (depends on 129)

The method of claim 129, wherein:
(I) the rat ES cells have a normal karyotype; and/or (II) the rat ES cells form spherical, free-floating colonies in culture.

### Claim 143 (depends on 129)

The method of claim 129, wherein the rat ES cells are derived from an ACI rat or a Dark Agouti (DA) rat.

### Claim 144 (depends on 129)

The method of claim 129, wherein the rat ES cells are male (XY) rat ES cells.

### Claim 145 (depends on 129)

The method of claim 129, wherein the rat ES cells are female (XX) rat ES cells.

### Claim 146 (depends on 129)

The method of claim 129, wherein the rat ES cells have one or more of the following characteristics:
(a) the rat ES cells express at least one pluripotency marker selected from Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, and Utf1;
(b) the rat ES cells do not express one or more pluripotency markers selected from c-Myc, Ecat1, and Rexo1;
(c) the rat ES cells do not express one or more mesodermal markers selected from Brachyury and Bmpr2;
(d) the rat ES cells do not express one or more endodermal markers selected from Gata6, Sox17, and Sox7;
(e) the rat ES cells do not express one or more neural markers selected from Nestin and Pax6;
(f) the rat ES cells expresses one or more pluripotency markers selected from Oct-4, Sox2, and alkaline phosphatase; and
(g) the rat ES cells express of one or more rat ESC-specific genes selected from Adherens Junctions Associated Protein 1 (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1 (Igfbpl1), Interleukin 36 beta (Il1f4 Interleukin 28 receptor, alpha (I128ra), left-right determination factor 1 (Lefty1), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fanko1), Forkhead box E1 (thyroid transcription factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3 (Drosophila) (Sal13), SATB homeobox 1 (Satb 1), and miR-632.

### Claim 147 (depends on 129)

The method of claim 129, wherein the step (b) comprises one or more rounds of electroporation.

### Claim 148 (depends on 129)

The method of claim 129, wherein the targeted genetic modification comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof

### Claim 149 (depends on 148)

The method of claim 148, wherein the targeted genetic modification comprises an insertion of a heterologous polynucleotide into the genome of the rat ES cells.

### Claim 150 (depends on 129)

The method of claim 129, wherein the method comprises modifying the population of rat ES cells to comprise two or more targeted modifications, wherein the modified rat ES cells can transmit the two or more targeted genetic modifications through the germline.

### Claim 151 (depends on 129)

The method of claim 129, wherein the targeted genetic modification is generated via a homologous recombination event.

### Claim 152 (depends on 151)

The method of claim 151, wherein the targeted genetic modification is generated by employing the targeting vector.

### Claim 153 (depends on 129)

The method of claim 129, wherein the targeted genetic modification is generated using the nuclease agent.

### Claim 154 (depends on 153)

The method of claim 153, wherein the nuclease agent is a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, or a CRISPR/Cas system.

### Claim 155 (depends on 129)

The method of claim 129, wherein:
step (b) comprises introducing into the population of rat ES cells a heterologous polynucleotide comprising a selection marker, wherein the selection marker is operably linked to a promoter active in the rat ES cells, and wherein the selection marker is present in the targeting vector; and
(ii) step (c) comprises culturing in vitro the rat ES cells produced by step (b) in alternating first and second culture media, wherein the first culture medium comprises an effective amount of a selection agent for a first time period and the second culture medium does not comprise the selection agent, wherein the in vitro culture conditions are sufficient to maintain pluripotency, thereby selecting modified rat ES cells having the heterologous polynucleotide stably integrated into the genome.

### Claim 156 (depends on 155)

The method of claim 155, wherein the first and second culture media are alternated every 24 hours.

### Claim 157 (depends on 155)

The method of claim 155, wherein the selection marker imparts resistance to an antibiotic.

### Claim 158 (depends on 155)

The method of claim 155, wherein the selection marker comprises one or more of neomycin phosphotransferase (neon), hygromycin B phosphotransferase (hygr), puromycin-N-acetyltransferase (puror), blasticidin S deaminase (bse), xanthine/guanine phosphoribosyl transferase (gpt), and herpes simplex virus thymidine kinase (HSV-tk).

### Claim 159 (depends on 155)

The method of claim 155, wherein the selection marker has one or more of the following characteristics:
(a) the selection marker comprises a non-attenuated selection marker gene; and
(b) the selection marker has an increased activity compared to a wild type selection marker.

### Claim 160 (depends on 155)

The method of claim 155, wherein the modified rat ES cells comprise multiple copies of the selection marker stably incorporated into the genome.

### Claim 161 (depends on 129)

The method of claim 129, further comprising:
(d) introducing the rat ES cell clone from step (c) into a rat host embryo;
(e) gestating the rat host embryo comprising the rat ES cell clone in a surrogate mother, wherein the surrogate mother produces an F0 progeny genetically modified rat comprising the targeted genetic modification; and
breeding the F0 progeny genetically modified rat with another rat to produce an F1 progeny genetically modified rat comprising the targeted genetic modification, wherein the targeted genetic modification is transmitted through the germline.

### Claim 162 (depends on 161)

The method of claim 161, wherein step (f) comprises breeding a male F0 rat identified in step (e) with a wild type female rat to produce an F1 progeny that is heterozygous for the targeted genetic modification.

### Claim 163 (depends on 162)

The method of claim 162, further comprising:
(g) breeding a male rat of the F1 progeny with a female rat of the F1 progeny to obtain an F2 progeny that is homozygous for the targeted genetic modification.

### Claim 164 (depends on 162)

The method of claim 162, wherein at least 3%, at least 10%, or at least 60% of the F1 progeny are derived from the rat ES cell clone identified in step (c).