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Prevention of muscular dystrophy by crispr/cpf1-mediated gene editing

A technology for muscular dystrophy and dystrophin, which is applied in the field of treatment of Duchenne muscular dystrophy, and can solve problems such as the inability to correct DMD mutations and restore the expression of dystrophin

Pending Publication Date: 2019-10-25
BOARD OF RGT THE UNIV OF TEXAS SYST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods cannot correct DMD mutations or permanently restore dystrophin expression

Method used

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  • Prevention of muscular dystrophy by crispr/cpf1-mediated gene editing
  • Prevention of muscular dystrophy by crispr/cpf1-mediated gene editing
  • Prevention of muscular dystrophy by crispr/cpf1-mediated gene editing

Examples

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Effect test

Embodiment 1

[0262] Example 1 - Materials and methods

[0263] The pLbCpf1-2A-GFP and pAsCpf1-2A-GFP plasmids were generated. From the (Addgene plasmid #69988) pY016 plasmid (Zetsche et al., 2015) (pcDNA3.1-hLbCpf1) kindly provided by Feng Zhang and the (Addgene plasmid #69982) pY010 plasmid (Zetsche et al., 2015 ) (pcDNA3.1-hAsCpf1) PCR amplified human codon-optimized LbCpf1 and AsCpf1, respectively. The Cpf1 cDNA and T2A-GFP DNA fragments were cloned into the backbone of the pSpCas9(BB)-2A-GFP(PX458) plasmid (Ran et al., 2015), which was kindly provided by Feng Zhang (Addgene plasmid #48138), and the plasmid was cloned with AgeI / EcoRI cut to remove SpCas9(BB)-2A-GFP. In-Fusion HD Cloning Kit (Takara Bio) was used. Cpf1 guide RNA (gRNA) targeting the human DMD or mouse Dmd locus was subcloned into newly generated pLbCpf1-2A-GFP and pAsCpf1-2A-GFP plasmids using BbsI digestion and T4 ligation. Detailed primer sequences can be found in Table C, genomic target sequences can be found in ...

Embodiment 2

[0284] Example 2 - Results

[0285] Correction of DMD iPSC-derived cardiomyocytes by Cpf1-mediated genome editing. Exon deletions preceding exon 51 of the human DMD gene disrupt the open reading frame (ORF) by juxtaposing exons out-of-frame and represent the most common type of human DMD mutation (Aartsma-Rus et al., 2009). In principle, skipping of exon 51 could restore the DMD ORF in 13% of DMD patients with exon deletion (Cirak et al., 2011). To test the potential of Cpf1 to correct this type of "hot spot" mutation, the inventors used DMD fibroblast-derived iPSCs (Riken HPS0164, abbreviated as Riken51), which have a deletion of exons 48 to 50, introducing exon A premature stop codon within 51 (Fig. 1A).

[0286] The splice acceptor region is generally T / C rich (Padgett, 2012), which generates an ideal PAM sequence for genome editing by the Cpf1 endonuclease (Fig. 1B). To rescue dystrophin expression in Riken51 iPSCs, the inventors used Cpf1 gRNA to target exon 51, introd...

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Abstract

Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. The disclosure reports CRISPR / Cpfl -mediated gene editing (Myo-editing) is effective at correcting the dystrophin gene mutation in the mdx mice, a model for DMD. Further, the disclosure reports optimization of germline editing of mdxmice by engineering the permanent skipping of mutant exon and extending exon skipping to also correct the disease by post-natal delivery of adeno- associated virus (AAV). AAV-mediated Myo-editing canefficiently rescue the reading frame of dystrophin in mdx mice in vivo. The disclosure reports means of Myo-editing-mediated exon skipping has been successfully advanced from somatic tissues in miceto human DMD patients- derived iPSCs (induced pluripotent stem cells). Custom Myo-editing was performed on iPSCs from patients with differing mutations and successfully restored dystrophin protein expression for all mutations in iPSCs-derived cardiomyocytes.

Description

[0001] priority claim [0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 426,853, filed November 28, 2016, and entitled "Prevention of Muscular Dystrophy by CRISPR / Cpf1-Mediated Gene Editing," the disclosure of which is hereby incorporated by reference in its entirety incorporated. [0003] Federal Funding Support Terms [0004] This invention was made with government support under DK-099653 and U54-HD 087351 awarded by the National Institutes of Health. The government has certain rights in this invention. [0005] sequence listing [0006] This application contains a Sequence Listing that has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 28, 2017, is named UTFD_3124WO.txt and is 189,059 bytes in size. technical field [0007] The present disclosure relates to the fields of molecular biology, medicine and genetics. More specifically, the p...

Claims

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Application Information

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IPC IPC(8): C12N15/10A61K48/00C07K14/47C12N15/113
CPCC07K14/4708C12N15/102C12N15/113C12N2320/33C12N2310/20C12N2750/14143A01K2227/105A01K2267/0306C12N9/22A61P21/00A61P21/04A61P25/02A61P43/00A61K9/0019A61K48/0075C12N5/0606C12N5/0696C12N7/00C12N15/11C12N15/86C12N2800/80C12N2830/50
Inventor 张宇龙城祖罗达·巴塞尔-达比埃里克·奥尔松
Owner BOARD OF RGT THE UNIV OF TEXAS SYST
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