Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

López, Sara Mata; Balog-Alvarez, Cynthia; Vitha, Stanislav; Bettis, Amanda K.; Canessa, Emily H.; Kornegay, Joe N.; Nghiem, Peter P.

doi:10.1371/journal.pone.0228072

Cited by 29 publications

(22 citation statements)

References 45 publications

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“…In most of these cases, a “permanent” exon skipping approach was selected where a shorter protein would be produced 27,28 , while in some others full length dystrophin was the result of the edition 37–39 . Several studies have shown efficacy in mice models 40–42 , an most recently in dogs, which is currently the most advanced example of its application to DMD 29 but there are also examples of the challenges associated to gene editing 32 . Before this can be a credible therapeutic option, several delivery and manufacturing problems will need to be overcome.…”

Section: Discussionmentioning

confidence: 99%

“…CRISPR/Cas9 has been successfully employed to correct mutations and/or restore the open reading frame recovering dystrophin expression both in vitro and in vivo 30,31 . However, some hurdles have been reported, such as difficulties transfecting myoblast or a recent study in the golden retriever muscular dystrophy dog (GRMD), where no dystrophin restoration at protein level was evident after gene editing using this technology 32 . More importantly, as well as these preclinical problems others such as possible off-target problems or immunogenicity linked with the use of Cas9 33 may delay their clinical application.…”

Section: Introductionmentioning

confidence: 99%

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Duchenne Muscular Dystrophy Cell Culture Models Created By CRISPR/Cas 9 Gene Editing And Their Application To Drug Screening

Soblechero-Martín

Albiasu-Arteta

Anton-Martinez

et al. 2020

Preprint

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CRISPR/Cas9-mediated gene editing may allow treating and studying rare genetic disorders by respectively, correcting disease mutations in patients, or introducing them in cell cultures. Both applications are highly dependent on Cas9 and sgRNA delivery efficiency. While gene editing methods are usually efficiently applied to cell lines such as HEK293 or hiPSCs, CRISPR/Cas9 editing in vivo or in cultured myoblasts prove to be much less efficient, limiting its use. After a careful optimisation of different steps of the editing protocol, we established a consistent approach to generate human immortalised myoblasts disease models through CRISPR/Cas9 editing. Using this protocol we successfully created a coding deletion of exon 52 of the DYSTROPHIN (DMD) gene in wild type immortalised myoblasts modelling Duchenne muscular dystrophy (DMD), and a microRNA binding sites deletion in the regulatory region of the UTROPHIN (UTRN) gene leading to utrophin upregulation in in Duchenne muscular dystrophy patient immortalised cultures. Sanger sequencing confirmed the presence of the corresponding genomic alterations and protein expression was characterised using myoblots. To show the utility of these cultures as platforms for assessing the efficiency of DMD treatments, we used them to evaluate the impact of exon skipping therapy and ezutromid treatment. Our editing protocol may be useful to others interested in genetically manipulating myoblasts and the resulting edited cultures for studying DMD disease mechanisms and assessing therapeutic approaches.SummaryWe report two novel immortalised myoblast culture models for studying Duchenne muscular dystrophy (DMD), generated through CRISPR/Cas9 gene editing: one recapitulates a common DYSTROPHIN (DMD) deletion and the other a regulatory mutation leading to UTROPHIN (UTRN) ectopic upregulation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Duchenne Muscular Dystrophy Cell Culture Models Created By CRISPR/Cas 9 Gene Editing And Their Application To Drug Screening

Soblechero-Martín

Albiasu-Arteta

Anton-Martinez

et al. 2020

Preprint

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“…54 The ssODNs used in this study included the correct DMD sequence at the intron 6 acceptor splice site. 54 With this HDR-mediated repair, examination of muscle biopsies showed that DMD mRNA expression was increased and dystrophin expression was restored to 6% to 16% of normal levels. 54 There are several limitations to HDR-based DMD therapy.…”

Section: Therapeutic Approach: Exon Reframingmentioning

confidence: 99%

CRISPR technologies for the treatment of Duchenne muscular dystrophy

Choi

Koo

2021

Molecular Therapy

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The emerging clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing technologies have progressed remarkably in recent years, opening up the potential of precise genome editing as a therapeutic approach to treat various diseases. The CRISPR-CRISPR-associated (Cas) system is an attractive platform for the treatment of Duchenne muscular dystrophy (DMD), which is a neuromuscular disease caused by mutations in the DMD gene. CRISPR-Cas can be used to permanently repair the mutated DMD gene, leading to the expression of the encoded protein, dystrophin, in systems ranging from cells derived from DMD patients to animal models of DMD. However, the development of more efficient therapeutic approaches and delivery methods remains a great challenge for DMD. Here, we review various therapeutic strategies that use CRISPR-Cas to correct or bypass DMD mutations and discuss their therapeutic potential, as well as obstacles that lie ahead.Recently, therapeutic applications of genome editing have been explored for treating various genetic diseases. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated

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“…In research settings, CRISPR is most often used in reverse genetics applications to generate indel mutations in the genome in order to abrogate gene function and study resulting phenotypes, though CRISPR is also particularly powerful for forward genetic screens. Indeed, CRISPR gene knockouts have been used in canine reverse genetics to study cardiovascular disease (Feng et al, 2018), Duchenne's muscular dystrophy (Mata Lopez et al, 2020), and even to generate germline edits of the myostatin gene (Zou et al, 2015). NHEJ activity requires KU70/80 to stabilize the DNA ends, preventing end resection, and DNA-PKcs (Chang et al, 2017).…”

Section: Non-homologous End Joining (Nhej)mentioning

confidence: 99%

Gene Editing and Gene Therapy: Entering Uncharted Territory in Veterinary Oncology

Wierson¹,

Abel²,

Siegler³

et al. 2021

Preprint

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With rapid advances in gene editing and gene therapy technologies, the development of genetic, cell, or protein-based cures to disease are no longer the realm of science fiction but that of today’s practice. The impact of these technologies are rapidly bringing them to the veterinary market as both enhanced therapeutics and towards modeling their outcomes for translational application. Simply put, gene editing enables scientists to modify an organism’s DNA a priori through the use of site-specific DNA targeting tools like clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Gene therapy is a broader definition that encompasses the addition of exogenous genetic materials into specific cells to correct a genetic defect. More precisely, the U.S Food and Drug Administration (FDA) defines gene therapy as “a technique that modifies a person’s genes to treat or cure disease” by either (i) replacing a disease-causing gene with a healthy copy of the gene; (ii) inactivating a disease-causing gene that was not functioning properly; or (iii) introducing a new or modified gene into the body to help treat a disease. In some instances, this can be accomplished through direct transfer of DNA or RNA into target cells of interest or more broadly through gene editing. While gene therapy is possible through the simple addition of genetic information into cells of interest, gene editing allows the genome to be reprogrammed intentionally through the deletion of diseased alleles, reconstitution of wild type sequence, or targeted integration of exogenous DNA to impart new function. Cells can be removed from the body, altered, and reinfused, or edited in vivo. Indeed, manufacturing and production efficiencies in gene editing and gene therapy in the 21st century has brought the therapeutic potential of in vitro and in vivo reprogrammed cells, to the front lines of therapeutic intervention (Brooks et al., 2016). For example, CAR-T cell therapy is revolutionizing hematologic cancer care in humans and is being translated to canines by us and others, and gene therapy trials are ongoing for mitral valve disease in dogs.

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Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

Cited by 29 publications

References 45 publications

Duchenne Muscular Dystrophy Cell Culture Models Created By CRISPR/Cas 9 Gene Editing And Their Application To Drug Screening

Duchenne Muscular Dystrophy Cell Culture Models Created By CRISPR/Cas 9 Gene Editing And Their Application To Drug Screening

CRISPR technologies for the treatment of Duchenne muscular dystrophy

Gene Editing and Gene Therapy: Entering Uncharted Territory in Veterinary Oncology

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