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Optimized strategy for exon skipping modifications using crispr/cas9 with triple guide sequences

A sequence, target sequence technology, applied in the composition of genome editing and in the field of correcting in vivo mutations using exon skipping method, can solve problems such as loss of dystrophin expression, muscle membrane fragility, muscle atrophy, etc.

Pending Publication Date: 2019-11-26
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

Mutations in the dystrophin gene result in loss of dystrophin expression, leading to muscle membrane fragility and progressive muscle atrophy

Method used

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  • Optimized strategy for exon skipping modifications using crispr/cas9 with triple guide sequences
  • Optimized strategy for exon skipping modifications using crispr/cas9 with triple guide sequences
  • Optimized strategy for exon skipping modifications using crispr/cas9 with triple guide sequences

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Experimental program
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Effect test

Embodiment 1

[0505] Example 1 - Materials and methods

[0506] Research Approval. All experimental procedures involving animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center.

[0507] CRISPR / Cas9-mediated exon 50 deletion in mice. Two single guide RNAs (sgRNAs) specific for the inner region surrounding the exon 50 sequence of the mouse Dmd locus were cloned into vector px330 using the following primers:

[0508]

[0509] For in vitro transcription of sgRNAs, add the T7 promoter sequence to the sgRNA template by PCR using the following primers:

[0510]

[0511] Gel-purified PCR products were used as templates for in vitro transcription using the MEGAshortscript T7 Kit (Life Technologies). sgRNA was purified by MEGAclear kit (Life Technologies) and eluted with nuclease-free water (Ambion). The concentration of guide RNA was measured by NanoDrop instrument (Thermo Scientific).

[0...

Embodiment 2

[0522] Example 2 - Results

[0523] Humanized model of DMD. The most common hotspot mutation region in DMD patients was the region between exons 45 to 51, and skipping of exon 51 was available for treatment in the largest group (13-14%) of patients. To study CRISPR / Cas9-mediated exon 51 skipping in vivo, the inventors generated a mouse model mimicking the human "hotspot" region by deleting exon 50 using the CRISPR / Cas9 system guided by 2 sgRNAs (Fig. 1A). The deletion of exon 50 was confirmed by DNA sequencing (Fig. 1B). Deletion of exon 50 puts the dystrophin gene out of frame, resulting in dystrophin deficiency in skeletal muscle and heart (Fig. 1C–E). Mice lacking exon 50 showed marked dystrophic muscle changes at 2 months of age (Fig. 1E). Serum analysis of delta exon 50 mice revealed significantly increased levels of creatine kinase (CK), indicative of muscle damage (Fig. 1F). In conclusion, dystrophin expression, muscle histology, and serum CK levels confirmed the d...

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Abstract

CRISPR / Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. Here, usingthree promoters to drive expression of the same DMD guide RNA, a more robust and safe form of genome editing was achieved in a humanized mouse model for DMD with a deletion in exon 50, and in a DeltaEx50-MD Dog.

Description

[0001] priority statement [0002] This application claims U.S. Provisional Application Serial No. 62 / 596,298 filed December 8, 2017, U.S. Provisional Application Serial No. 62 / 544,499 filed August 11, 2017, and U.S. Provisional Application Serial No. filed January 5, 2017 No. 62 / 442,606, the entire contents of which are hereby incorporated by reference in their entirety. [0003] Federal Funding Support Terms [0004] This invention was made with Government support under Grant No. 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, which has been filed electronically in ASCII format, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 5, 2018, is named UTFD_P3178WO.txt and is 1,316,974 bytes in size. technical field [0007] The present disclosure relates to the fields of molecular biology, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/864C12N9/22
CPCC07K14/4708C12N9/22C12N15/113C12N2320/33C12N2330/51C12N2310/20C12N2750/14143A61P21/00A61P21/04C12N5/0659C12N5/0696C12N15/102C12N15/86C12N15/907A61K48/00C12N2510/00C12N2800/90C12N5/0657C12N5/066C12N5/0661
Inventor 利奥内拉·阿莫阿西埃里克·奥尔松
Owner BOARD OF RGT THE UNIV OF TEXAS SYST
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