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In situ gene editing

A genome and coding technology, applied in gene therapy, genetic engineering, plant genetic improvement, etc., can solve problems such as large transplant failure, infection risk, and limited applicability

Pending Publication Date: 2021-08-24
PRESIDENT & FELLOWS OF HARVARD COLLEGE 17 Q +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, solid tissue cannot necessarily be removed from the patient, limiting the applicability of these methods
Furthermore, removal of tissues such as hematopoietic cells poses a huge risk of graft failure and infection and requires expensive GMP facilities to handle ex vivo cells

Method used

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Experimental program
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Effect test

Embodiment 1

[0120] In vivo delivery of genome-modifying enzymes holds important promise for therapeutic applications and functional genetic screening. Of particular interest in this context is the delivery to endogenous tissue stem cells that provide a durable source of cell replacement in homeostasis and in response to regenerative cues. Here, a sensitive fluorescent reporter system activated by Cre recombinase was used to test the efficiency of genome modification following in vivo transduction of endogenous tissue stem cells by adeno-associated virus (AAV). We combined immunophenotyping with robust in vitro and in vivo stem cell functional assays to reveal efficient targeting of tissue-localized skeletal muscle satellite cells, mesenchymal progenitor cells, and hematopoietic stem cells using multiple AAV serotypes. The genome modification rate achieved by this method was as high as 65%, and the modified cells retained key functional properties. This study establishes a powerful new pl...

Embodiment 2

[0251] AAV carrying S. aureus Cas9 (saCas9) + gRNA targeting Dnmt3a or a control locus (Jak2) was injected into 2-month-old C57BL / 6 mice. After four months, liver and bone marrow were harvested from one mouse in each treatment group. Hematopoietic stem cells (HSC) and multipotent progenitor cells (MPP) were isolated from bone marrow by FACS. In addition, genomic DNA at the Dnmt3a locus was amplified and sequenced from each of these populations / organs (liver, bone marrow, HSC, and MPP).

[0252] result

[0253] Figure 11A Shown is the percentage of edited reads, ie, the percentage of sequenced reads containing indels in the amplified Dnmt3a sequence containing the gRNA-targeted site. Figure 11B Shown is the percentage of %WT reads, ie, the percentage of sequencing reads that map to the wild-type Dnmt3a sequence. Such as Figure 11A As shown, Dnmt3a-targeting gRNAs introduced about 30% of the Dnmt3a locus edits in the liver. Indels were also detected in ~0.5% of reads in...

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Abstract

Disclosed are methods of in situ genomic modification of cells (e.g., stem cells, tissue stem cells, muscle stem cells, Sca-1* mesenchymal progenitor cells in skeletal muscle, CD140a* dermal mesenchymal cells) using sequence-targeting nucleases delivered via a virus (e.g., an AAV).

Description

[0001] related application [0002] This application claims the benefit of U.S. Provisional Application No. 62 / 719,628, filed August 18, 2018. The entire teachings of the above applications are incorporated herein by reference. [0003] governmental support [0004] This invention was made with government support under Grant Numbers F32AG050395, R01 HL135287, DP1 AG048917, and R01 AR070825 awarded by the National Institutes of Health. The government has certain rights in this invention. Background technique [0005] Efficient functioning of body tissues and organs depends on the maintenance of appropriate cell numbers (homeostasis) and the replacement of damaged cells after injury (repair), both processes requiring regulation by tissue stem cells. Numerous studies spanning decades have attempted to define key molecular regulators of stem cell function. However, the speed at which researchers can interrogate and identify such mediators is constrained by technical limitation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N15/00C12N15/07C12N15/11
CPCA61K48/00C12N15/90C12N2750/14143C12N15/102C12N9/22C12N15/113C12N2310/20A61K9/0019C12N7/00C12N15/11C12N15/86C12N15/907C12N2750/14171C12N2800/80
Inventor 艾米·J·韦格斯吉尔·戈尔茨坦利欧·王亚-切赫·徐梅里姆·冈萨雷斯·塞莱罗
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE 17 Q
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