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In vivo homology directed repair in heart, skeletal muscle, and muscle stem cells

A heart cell, muscle technology, applied in the field of in vivo homology-directed repair in heart, skeletal muscle and muscle stem cells, can solve the problems of untested multi-organ HDR feasibility and inefficiency

Pending Publication Date: 2021-03-16
PRESIDENT & FELLOWS OF HARVARD COLLEGE 17 Q
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Although NHEJ is active throughout the cell cycle and in nondividing cells, this error-prone pathway produces variable sequence outcomes due to highly unpredictable nucleotide insertions and deletions
[0004] In contrast, HDR provides more precise gene editing results, as well as the unique ability to introduce entirely new sequence elements, but is generally considered to be inefficient in post-mitotic organs and requires homologous DNA present on endogenous chromosomes or exogenous templates
While recent studies have investigated the use of CRISPR-induced HDR in cultured cells, fertilized eggs, and localized delivery to specific tissues, the feasibility of achieving multi-organ HDR in vivo in postnatal mammals has not been tested

Method used

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  • In vivo homology directed repair in heart, skeletal muscle, and muscle stem cells
  • In vivo homology directed repair in heart, skeletal muscle, and muscle stem cells
  • In vivo homology directed repair in heart, skeletal muscle, and muscle stem cells

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Embodiment

[0113] To sensitively detect in vivo gene editing events via CRISPR / Cas9, a strongly enhanced green fluorescent protein (GFP) signal using ubiquitous expression was developed 8 A fluorescent protein-based reporter system for transgenic mouse strains ( Figure 1A ). Based on published BFP variants 9-11 The blue fluorescent protein (BFP) sequence was designed to carry a minimum of 2 base substitutions (C197G and T199C) compared to the GFP sequence. This simple modification allows easy differentiation of two fluorescent proteins by fluorescence-activated cell sorting (FACS) ( Figure 5A-Figure 5D ). The same 2 base substitutions also created a Btgl site for restriction fragment length polymorphism (RFLP) analysis. A single guide RNA (sgRNA) targeting a substitution site in GFP was designed to be compatible with the Cas9 protein (SaCas9) from S. + / - ; Efficient disruption of GFP signaling was tested in tail tip fibroblasts (TTF) of mdx mice ( Figure 5B , Figure 5C ). Thi...

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Abstract

Disclosed are methods of genomic modification of skeletal and cardiac muscle using sequence-targeting nucleases and a donor sequence delivered via a virus.

Description

[0001] related application [0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 666,685, filed May 3, 2018, the contents of which are incorporated herein by reference in their entirety. Background technique [0003] Sequence-targeted nucleases such as CRISPR / Cas9 provide powerful tools to edit mammalian genomes through cellular mechanisms involved in DNA double-strand break (DSB) repair. Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are the main pathways used by cells to repair nuclease-generated DSBs and prevent genomic damage and cell death. Although NHEJ is active throughout the cell cycle and in non-dividing cells, this error-prone pathway produces variable sequence outcomes due to highly unpredictable nucleotide insertions and deletions. [0004] In contrast, HDR provides more precise gene editing results, as well as the unique ability to introduce entirely new sequence elements, but is generally considered to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N15/00C12N15/07
CPCA61K48/00C12N9/22C12N2750/14143C12N15/907C12N2510/00C12N2750/14142
Inventor 艾米·J·韦格斯朱克先
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE 17 Q
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