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Methods and compositions for the targeted modification of a genome

a genome and composition technology, applied in the field of genome targeted modification methods and compositions, can solve the problems of limited use of rats in human disease modeling

Inactive Publication Date: 2016-03-03
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a method for modifying a specific part of a genome in a eukaryotic cell using a targeted genetic modification approach. This involves introducing a large targeting vector (LTVEC) containing a first nucleic acid flanked by a 5′ homology arm and a 3′ homology arm, and a first expression construct comprising a first promoter and a Cas protein. Additionally, a second expression construct comprising a second promoter and a guide RNA is introduced. The LTVEC is at least 10 kb. The methods allow for the replacement of an endogenous nucleic acid sequence, the deletion of an endogenous nucleic acid sequence, or the insertion of a new nucleic acid sequence. The methods can be used in mammalian cells, such as fibroblasts, and can be used in pluripotent cells, such as human embryonic stem cells. The targeted genetic modification can be used for research and therapeutic purposes.

Problems solved by technology

While rats have been regarded as an important animal model system that can recapitulate the pathology of various human diseases, including, but not limited to, cardiovascular (e.g., hypertension), metabolic (e.g., obesity, diabetes), neurological (e.g., pain pathologies), and a variety of cancers, the use of rats in modeling human diseases has been limited as compared to mice, due in part to unavailability of germline-transmittable pluripotent rat cells, which can sustain their pluripotency following a series of genetic modifications in vitro, e.g., one or more serial electroporations, and due in part to lack of efficient targeting technologies that allow introduction or deletion of large genomic DNA sequences, or replacement of large endogenous genomic DNA sequences with exogenous nucleic acid sequences in pluripotent rat cells.

Method used

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  • Methods and compositions for the targeted modification of a genome
  • Methods and compositions for the targeted modification of a genome
  • Methods and compositions for the targeted modification of a genome

Examples

Experimental program
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example 1

Rat ES Cell Derivation and Characterization

[0676]1.1. Rat ES Cell Characterization

[0677]As shown in FIG. 1, rat ESCs grow as compact spherical colonies, which routinely detach and float in the dish (close-up, FIG. 8). Rat ESCs express pluripotency markers including Oct-4 (FIG. 2A) and Sox2 (FIG. 2B), and express high levels of alkaline phosphatase (FIG. 3). Karyotype for line DA.2B is 42X,Y (FIG. 4). Rat ESCs often become tetraploid; thus, lines were pre-screened by counting metaphase chromosome spreads; lines with mostly normal counts were then formally karyotyped.

[0678]ACI blastocysts were collected from super-ovulated females obtained commercially. DA blastocysts were cultured from frozen 8-cell embryos obtained commercially. Zona pellucidae were removed with Acid Tyrodes; and blastocysts were plated onto mitotically inactivated MEFs. Outgrowths were picked and expanded using standard methods. All blastocysts were plated, cultured and expanded using 2i media (Li et al. (2008) Ger...

example 2

Inactivation of Genomic Loci in Rats

[0837]2.1: Inactivation of Endogenous Genomic Loci Using an Endonuclease Agent

[0838]In order to introduce a mutant allele at an endogenous rat genomic locus, the rat ES cells described herein are electroporated with expression vectors (or mRNA) that express ZFNs 1 and 2 (or TALENs 1 and 2). These proteins bind their target sequences on opposite strands, separated by about 6 bp to about 40 bp. A double-stranded break is formed within the target locus, which the cell attempts to repair by Non-Homologous End-Joining (NHEJ). In many cases, NHEJ results in creation of a deletion, which often disrupts the function of the gene (most often by producing a frameshift mutation). In order to identify a positive clone comprising a mutant allele, the electroporated cells are plated at low density, because no drug selection is done. Colonies are picked and assayed at the target site to see if a mutation was produced (e.g., using a modification of allele (MOA) as...

example 3

Targeted Modification of Rat Genomic Loci

[0854]3.1: Rat ESC Targeting: The Rat Rosa26 Locus.

[0855]The rat Rosa26 locus lies between the Setd5 and Thumpd3 genes as in mouse, with the same spacing. The rat Rosa26 locus (FIG. 12, Panel B) differs from the mouse Rosa26 locus (FIG. 12, Panel A). The mouse Rosa26 transcripts consist of 2 or 3 exons. The rat locus contains a 2nd exon 1 (Ex1b) in addition to the homologous exon to mouse exon1 (Ex1a). No 3rd exon has been identified in rat. Targeting of a rat Rosa26 allele is depicted in FIG. 12C, where homology arms of 5 kb each were cloned by PCR using genomic DNA from DA rat ESC. The targeted allele contains a SA (splicing acceptor)-lacZ-hUb-neo cassette replacing a 117 bp deletion in the rat Rosa26 intron.

[0856]Targeting efficiency at the rat Rosa26 locus was determined (Table 17). Linearized vector was electroporated into DA or ACI rat ESCs, and transfected colonies were cultured in 2i media+G418, using standard techniques. Individual c...

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Abstract

Compositions and methods are provided for modifying a genomic locus of interest in a eukaryotic cell, a mammalian cell, a human cell or a non-human mammalian cell using a large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences as described herein. Further methods combine the use of the LTVEC with a CRISPR / Cas system. Compositions and methods for generating a genetically modified non-human animal comprising one or more targeted genetic modifications in their germline are also provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 14 / 515,503, filed Oct. 15, 2014, which claims the benefit of U.S. Provisional Patent Application No. 61 / 914,768, filed Dec. 11, 2013, U.S. Provisional Patent Application No. 62 / 017,416, filed Jun. 26, 2014, U.S. Provisional Patent Application No. 62 / 029,261, filed Jul. 25, 2014, U.S. Provisional Patent Application No. 62 / 052,906, filed Sep. 19, 2014, U.S. Provisional Patent Application No. 62 / 059,527, filed Oct. 3, 2014, and U.S. Provisional Patent Application No. 62 / 064,384, filed Oct. 15, 2014, each of which is herein incorporated by reference in its entirety for all purposes.REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB[0002]The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 470590SEQLIST.TXT, created on Oct. 30, 2015, and having a size of 27.6 kilobytes, a...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/90C12N15/10C12N15/85
CPCC12N15/907C12N15/85C12N15/1024C12N2015/8527C12N2810/00C12N9/22A01K67/0276A01K67/0278A01K2217/072A01K2217/075A01K2227/105A01K2267/0362A01K2267/0387C12N2800/40C12N2810/10C12N2999/007C12N15/8509A01K67/0275A01K2217/07C12N2810/40
Inventor FRENDEWEY, DAVIDAUERBACH, WOJTEKLAI, KA-MAN, VENUSMUJICA, ALEXANDER, O.LEE, JEFFREY, D.DROGUETT, GUSTAVOTRZASKA, SEANHUNT, CHARLEENGAGLIARDI, ANTHONYKUNO, JUNKOVALENZUELA, DAVID, M.YANCOPOULOS, GEORGE, D.
Owner REGENERON PHARM INC
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