Spell Checking Nature: Versatility of CRISPR/Cas9 for Developing Treatments for Inherited Disorders

Wojtal, Daria; Kemaladewi, Dwi U.; Malam, Zeenat; Abdullah, Sarah; Wong, Tatianna Wai Ying; Hyatt, Elzbieta; Baghestani, Zahra; Pereira, Sérgio Luiz; Stavropoulos, James; Mouly, Vincent; Mamchaoui, Kamel; Muntoni, Francesco; Voït, Thomas; Gonorazky, Hernan; Dowling, James J.; Wilson, Michael D.; Mendoza-Londoño, Roberto; Ivakine, Evgueni A.; Cohn, Ronald D.

doi:10.1016/j.ajhg.2015.11.012

Cited by 97 publications

(80 citation statements)

References 70 publications

(85 reference statements)

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“…This expression was followed by a detectable and correctly localized dystrophin only in the #994 cell line ( Figures 3C and 3D), which carries a smaller duplication of 137 kb, and not in the #515 cell line, which carries a larger, 263-kb duplication, probably due to the limited transduction efficiency in this cell line. Our results are concordant with those reported by Wojtal et al 28 in terms of restoration of protein expression in cells bearing deletions of comparable size (139 and 137 kb). We extended the significance of those findings by reporting the correct localization of dystrophin at the sarcolemma in myotubes derived from #994 cells transduced with two out of three lentiviral vectors, despite the small number of myoblasts positive for DMD expression ( Figure 3D).…”

Section: Discussionsupporting

confidence: 83%

“…The attempt to treat duplications has been recently reported, in a multi-exonic duplication in the DMD gene. 28 Here,…”

Section: Discussionmentioning

confidence: 99%

“…This approach has been independently validated by the Cohn lab (University of Toronto) to remove a duplication of the exons 18-30 in the DMD gene. 28 To identify editing targets able to correct the DMD duplication of the exon 2 (dup2) in all patients bearing this type of mutation, we collected DNA from eight patients and characterized the extension of the copy number variation with the DMD-CGH array. 29 Data from ten more patients were collected from the literature [29][30][31][32] and aligned with the other duplications analyzed in order to identify a region shared among all the patients studied ( Figure 1A).…”

Section: Strategy For the Correction Of Duplications In Dmd And Grna mentioning

confidence: 99%

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Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system

Lattanzi

Duguez

Moiani

et al. 2017

Neuromuscular Disorders

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Exonic duplications account for 10%-15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one guide RNA (gRNA) directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications.

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Section: Discussionsupporting

confidence: 83%

“…The attempt to treat duplications has been recently reported, in a multi-exonic duplication in the DMD gene. 28 Here,…”

Section: Discussionmentioning

confidence: 99%

Section: Strategy For the Correction Of Duplications In Dmd And Grna mentioning

confidence: 99%

See 1 more Smart Citation

Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system

Lattanzi

Duguez

Moiani

et al. 2017

Neuromuscular Disorders

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“…According to the Monaco rule (5), DMD is mostly due to out-of-frame mutations in the DMD gene that result in a complete loss of the protein and a severe phenotype, while in-frame mutations of the DMD gene are mainly associated with BMD where modified dystrophin is produced resulting in reputed less severe phenotypes. Most BMD mutations are in-frame genomic deletions that lead to proteins lacking part of the central domain repeats (6,7) and constitute the pattern for therapeutic strategies aiming to transform DMD patients into BMD patients (8) either by exon skipping, by injection of micro-dystrophins (9)(10)(11)(12) or by CRSIPR/cas9 gene edition (13,14). Indeed, the central domain has been until now considered as a monotonous rod-shaped domain which could be internally truncated without dramatic functional effects (2,15).…”

Section: Introductionmentioning

confidence: 99%

Dystrophin's central domain forms a complex filament that becomes disorganized by in-frame deletions

Delalande

Molza

Morais

et al. 2018

Journal of Biological Chemistry

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Dystrophin, encoded by the DMD gene, is critical for maintaining plasma membrane integrity during muscle contraction events. Mutations in the DMD gene disrupting the reading frame prevent dystrophin production and result in the high severe Duchenne muscular dystrophy (DMD); in-frame internal deletions allow production of partly functional internally deleted dystrophin and result in the less severe Becker muscular dystrophy (BMD). Many known BMD deletions occur in dystrophin's central domain, generally considered to be a monotonous rod-shaped domain based on the knowledge of spectrin-family proteins. However, effects caused by these deletions, ranging from asymptomatic to severe BMD, argue against the central domain serving only as a featureless scaffold. We undertook structural studies combining small-angle X-ray scattering and molecular modeling in an effort to uncover the structure of the central domain as dystrophin has been refractory to characterization. We show that this domain appears to be a tortuous and complex filament that is profoundly disorganized by the most severe BMD deletion (loss of exon 45-47). Despite the preservation of large parts of the binding site for neuronal nitric oxide synthase (nNOS) in this deletion, computational approaches failed to recreate the association of dystrophin with nNOS. This observation is in agreement with a strong decrease of nNOS immunolocalization in muscle biopsies, a parameter related to the severity of BMD phenotypes. The structural description of the whole dystrophin central domain we present here is a first necessary step to improve the design of microdystrophin constructs in the goal of a successful gene therapy for DMD.

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“…Therefore, the in vivo safe delivery of this therapy should be evaluated using other extensive measures, such as wide-genome assessment, before such experiments can be developed into therapeutics in the clinic [41]. More interestingly, in Duchenne muscular dystrophy caused by exons duplication, snipping out the duplication of DMD exons 18-30 restored the full-length of dystrophin [44].…”

Section: Recent Advances and Applicationsmentioning

confidence: 99%

CRISPR/Cas9-Mediated Genome Nano-Surgery: An Update

Mokbel¹,

Mokbel²

2017

Biochem Mol biol J

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Recent advances in sequencing methods have prompted an upsurge in research into the modification of diseaseassociated genes or genes involved in drug resistance. In the early days of genome-editing, immunogenic and difficult to deliver tools with high off-target effects such as Zinc Finger Nucleases (ZFNs) and Transcription activatorlike effector nucleases (TALENs) proteins were used. This was followed by the discovery of the Clustered Regularly Interspaced Short Palindromic Repeat s, CRISPR/Cas9 system. This "self-non-self-discriminatory" natural defence mechanism in bacteria and archaea identifies and degrades extrachromosomal genomes or foreign genetic elements to prevent them from integrating into the prokaryotic genomes. Unlike traditional gene therapies that can merely insert a gene in a random pattern, the inexpensive and expedient CRISPR/ Cas9 system was harnessed by scientists to precisely cut, add or manipulate multiple DNA sequences at specific sites. This review will focus on the most recent studies in genome-editing by giving an overview of the past and modern methods in this field with an emphasis on the bacterial adaptive immune system called CRISPR/Cas9. This article will also analyze the applications of CRISPR/ Cas9 in rewriting the human genome in clinical and research settings and its potential future therapeutic applications. In addition, we shall consider the technical complexities which need to be overcome for the safe and effective delivery of this novel therapy and how the ethical concerns associated with this revolutionary genome engineering technique have constrained research in germ-line genome-editing. Furthermore, we shall highlight the role of scientific regulatory committees in evaluating and assessing safety issues such as potential complications before translating this proof-of-concept evidential approach to clinical reality. More research is required to explore the risk of hazardous genome edits and to fine-tune and improve this novel therapeutic system. Although the inexpensive and easy to use CRISPR/ Cas9 platform paves new ways for precise genome editing, clinical application is still at an early stage.

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Spell Checking Nature: Versatility of CRISPR/Cas9 for Developing Treatments for Inherited Disorders

Cited by 97 publications

References 70 publications

Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system

Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system

Dystrophin's central domain forms a complex filament that becomes disorganized by in-frame deletions

CRISPR/Cas9-Mediated Genome Nano-Surgery: An Update

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