Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method

Iyombe-Engembe, Jean-Paul; Ouellet, Dominique L.; Barbeau, Xavier; Rousseau, Joël; Chapdelaine, Pierre; Lagüe, Patrick; Tremblay, Jacques P.

doi:10.1038/mtna.2015.58

Cited by 72 publications

(57 citation statements)

References 64 publications

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“…The relative expression was expressed as a ratio of the target gene to the control gene using the formula 2 -(∆∆Ct) , where ∆∆Ct=(Ct Target -Ct β-actin ) treatment -(Ct Target -Ct β-actin ) control . Relative expression was normalized and expressed as a ratio to the expression in the control group [24-26]. …”

Section: Methodsmentioning

confidence: 99%

Lysine Restriction Affects Feed Intake and Amino Acid Metabolism via Gut Microbiome in Piglets

Yin

Han

et al. 2017

Cell Physiol Biochem

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Background/Aims: Our previous reports suggested that dietary supplementation with lysine influenced intestinal absorption and metabolism of amino acids. In this study, we further investigated the effect of lysine restriction (30%) on feed intake and we also tested the hypothesis that gut microbiome contributed to the potential mechanism of lysine restriction-mediated feeding behavior. Here, we profiled gut microbial communities by sequencing 16S ribosomal ribonucleic acid (rRNA) genes from gut samples as well as growth performance, serum hormones, and intestinal lysine transport in a piglet model. Results: Piglets preferred to the lysine restricted diet when giving three diets and the feed intake was markedly higher in the lysine-restricted group than that in the control group. Altered hormones (leptin, CCK, and ghrelin) might contribute to the feeding behavior caused by lysine restriction. Meanwhile, lysine transporting ability (SLC7A1 and SLC7A2 expression, intestinal electrophysiological changes, and amino acid pool in mesenteric vein) was decreased in response to lysine restriction. Through deep sequencing of bacterial rRNA markers, we observed that bacterial diversity was enhanced in the lysine-restricted group (Shannon H, PD, and Chao1). At the phylum level, lysine restriction enhanced gut Actinobacteria, Saccharibacteria, and Synergistetes abundances. At the family level, Moraxellaceae, Halomonadaceae, Shewanellaceae, Corynebacteriaceae, Bacillaceae, Comamonadaceae, Microbacteriaceae, Caulobacteraceae, and Synergistaceae abundances were increased in response to lysine restriction. Predictive functional profiling of microbial communities by PICRUSt also confirmed that dietary lysine restriction affected gut microbiome, which might further mediate amino acid metabolism, membrane transport, and endocrine system. Conclusion: Our results indicated that lysine restriction inhibited intestinal lysine transport and promoted feed intake, which might be associated with gut microbiome.

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Section: Methodsmentioning

confidence: 99%

Lysine Restriction Affects Feed Intake and Amino Acid Metabolism via Gut Microbiome in Piglets

Yin

Han

et al. 2017

Cell Physiol Biochem

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“…It is therefore not enough to restore the reading frame only to produce an internally truncated dystrophin. It is for this reason that we have used the CRISPR-induced deletion method (CinDel) [105,106]. CinDel aims to generate DSBs with two sgRNA and the Cas9 nuclease in the exons preceding and following the patient deletion, to induce additional deletions of portions of the target exons and of intron sequences located between these two DSBs.…”

Section: Advances and Challenges Of Using The Crispr/cas9mentioning

confidence: 99%

The advances and challenges of Gene Therapy for Duchenne Muscular Dystrophy

Tremblay¹

2017

J Genet Med Gene Ther

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Since the discovery of the dystrophin gene (DMD gene) thirty years ago, several therapeutic approaches have been investigated to treat Duchenne muscular dystrophy (DMD). This includes cell therapy, exon jumping, exonic knockout, and the CinDel method. In this article, we present the challenges of developping a treatment for DMD and the advances of these various approaches. We included the new CRISPR-Cas9 system, which permits not only major progress in the development of new treatments based on genome editing but also the production of new animal models.

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“…The development of designer endonucleases facilitated the implementation of novel strategies correcting endogenous dystrophin gene in mouse models and in patient-derived cells (Iyombe-Engembe et al 2016; Nelson et al 2016; Ousterout et al 2013, 2015a, b). These encouraging results led to the efforts in correcting patient-specific iPSCs (Li et al 2015; Young et al 2016).…”

Section: Corrections Of Disease-associated Genetic Mutations In Patiementioning

confidence: 99%

Gene correction in patient-specific iPSCs for therapy development and disease modeling

Jang

2016

Hum Genet

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The discovery that mature cells can be reprogrammed to become pluripotent and the development of engineered endonucleases for enhancing genome editing are two of the most exciting and impactful technology advances in modern medicine and science. Human pluripotent stem cells have the potential to establish new model systems for studying human developmental biology and disease mechanisms. Gene correction in patient-specific iPSCs can also provide a novel source for autologous cell therapy. Although historically challenging, precise genome editing in human iPSCs is becoming more feasible with the development of new genome-editing tools, including ZFNs, TALENs, and CRISPR. iPSCs derived from patients of a variety of diseases have been edited to correct disease-associated mutations and to generate isogenic cell lines. After directed differentiation, many of the corrected iPSCs showed restored functionality and demonstrated their potential in cell replacement therapy. Genome-wide analyses of gene-corrected iPSCs have collectively demonstrated a high fidelity of the engineered endonucleases. Remaining challenges in clinical translation of these technologies include maintaining genome integrity of the iPSC clones and the differentiated cells. Given the rapid advances in genome-editing technologies, gene correction is no longer the bottleneck in developing iPSC-based gene and cell therapies; generating functional and transplantable cell types from iPSCs remains the biggest challenge needing to be addressed by the research field.

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Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method

Cited by 72 publications

References 64 publications

Lysine Restriction Affects Feed Intake and Amino Acid Metabolism via Gut Microbiome in Piglets

Lysine Restriction Affects Feed Intake and Amino Acid Metabolism via Gut Microbiome in Piglets

The advances and challenges of Gene Therapy for Duchenne Muscular Dystrophy

Gene correction in patient-specific iPSCs for therapy development and disease modeling

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