<scp>CRISPR</scp> applications for Duchenne muscular dystrophy: From animal models to potential therapies

Chey, Yu Chinn Joshua; Arudkumar, Jayshen; Aartsma‐Rus, Annemieke; Adikusuma, Fatwa; Thomas, Paul Q.

doi:10.1002/wsbm.1580

Cited by 16 publications

(12 citation statements)

References 171 publications

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“…Furthermore, derivatives of the hDMDTg line such as the del52 exon deletion disease model are more difficult to engineer and validate. While these double-copy humanised models can be used for the testing of ASOs [15], they are not ideal for evaluating strategies that are reliant on DNA double-stranded breaks such as CRISPR-Cas9 gene editing [16]. This is because large intervening deletions between the target sites of the two transgene copies can complicate the analysis of DNA repair outcomes.…”

Section: Discussionmentioning

confidence: 99%

Megabase-Scale Transgene De-Duplication to Generate a Functional Single-Copy Full-Length Human DMD Transgenic Mouse Model

Chey,

Corbett,

Arudkumar

et al. 2024

Preprint

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The development of sequence-specific precision treatments like CRISPR gene-editing therapies for Duchenne Muscular Dystrophy (DMD) requires sequence humanised animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length humanDMDgene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true copy number of theDMDgene. This duplication also complicates the analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanised DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation show that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murineDmdknockout. Our unique hDMDTgSc mouse model can potentially be used to further generation of DMD disease models, suited for the pre-clinical assessment of sequence-specific therapies.

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Section: Discussionmentioning

confidence: 99%

Megabase-Scale Transgene De-Duplication to Generate a Functional Single-Copy Full-Length Human DMD Transgenic Mouse Model

Chey,

Corbett,

Arudkumar

et al. 2024

Preprint

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“…Since the correction occurs at mRNA level, the patients need regular administration during their whole life. To overcome the inconvenience of regular administration, researchers have tried to rescue the fatal mutations with the CRISPR–Cas system [ 96 ]. The first attempt was performed by the Akitsu Hotta laboratory.…”

Section: Knockout Escaping For Gene Therapymentioning

confidence: 99%

Escaping from CRISPR–Cas-mediated knockout: the facts, mechanisms, and applications

Wang,

Zhai,

Zhang

et al. 2024

Cell Mol Biol Lett

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Clustered regularly interspaced short palindromic repeats and associated Cas protein (CRISPR–Cas), a powerful genome editing tool, has revolutionized gene function investigation and exhibits huge potential for clinical applications. CRISPR–Cas-mediated gene knockout has already become a routine method in research laboratories. However, in the last few years, accumulating evidences have demonstrated that genes knocked out by CRISPR–Cas may not be truly silenced. Functional residual proteins could be generated in such knockout organisms to compensate the putative loss of function, termed herein knockout escaping. In line with this, several CRISPR–Cas-mediated knockout screenings have discovered much less abnormal phenotypes than expected. How does knockout escaping happen and how often does it happen have not been systematically reviewed yet. Without knowing this, knockout results could easily be misinterpreted. In this review, we summarize these evidences and propose two main mechanisms allowing knockout escaping. To avoid the confusion caused by knockout escaping, several strategies are discussed as well as their advantages and disadvantages. On the other hand, knockout escaping also provides convenient tools for studying essential genes and treating monogenic disorders such as Duchenne muscular dystrophy, which are discussed in the end.

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“…These models offer a testing ground for sequencespecific therapies like CRISPR, which seek to reframe or skip DMD mutations to restore functional dystrophin expression. [41] Animal models are crucial for validating delivery systems inside living things. In addition, these models serve as a testing ground for novel therapeutics and a method for adverse events, such as toxicity and immunogenicity, to be detected.…”

Section: Preclinical Studiesmentioning

confidence: 99%

Gene Editing: The Regulatory Perspective

Niazi¹

2023

Preprint

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Gene or genome editing (GE) revises, removes, or replaces a mutated gene at the DNA level; it is a tool. Gene therapy (GT) offsets mutations by introducing a "normal" version of the gene into the body while the diseased gene remains in the genome; it is a medicine. So far, no GE product has been approved, as opposed to 22 GT products that cost up to millions of dollars per dose. The FDA has recently added a guideline specific to gene editing that should be understood to enable faster development of GE products; at the same time, the FDA also needs to bring more clarification and make several amendments to this guideline to make it more rational.

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CRISPR applications for Duchenne muscular dystrophy: From animal models to potential therapies

Cited by 16 publications

References 171 publications

Megabase-Scale Transgene De-Duplication to Generate a Functional Single-Copy Full-Length Human DMD Transgenic Mouse Model

Megabase-Scale Transgene De-Duplication to Generate a Functional Single-Copy Full-Length Human DMD Transgenic Mouse Model

Escaping from CRISPR–Cas-mediated knockout: the facts, mechanisms, and applications

Gene Editing: The Regulatory Perspective

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