A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells

Abstract

Summary Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed for evaluation of dystrophin in disease relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.

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

“…Absence of nNOS binding to the deleted dystrophin could be related to the severity of this ∆45-47 in-frame deletion (16) and could account at least partly for the heterogeneity of BMD phenotypes encountered with different deletions starting from exon 45 and which may impair the nNOS binding to various degrees (41). Among these deletions, we are currently studying the deletion of exons 45 to 55 which could rescue 65% of DMD patients (13,42).…”

“…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).…”

Drusen in patient-derived hiPSC-RPE models of macular dystrophies

“…The correction of disease-causing mutations in hiPSCs is a powerful tool that provides access to isogenic controls that are necessary to clearly correlate an identified disease phenotype to a mutation. In fact, hiPSC technology in conjunction with CRISPR technology has previously been used to gain important insights into the underlying mechanism of several diseases (e.g., Huntington's disease, Duchenne muscular dystrophy) (18,19). Using hiPSCs as a platform to study human ocular diseases is particularly relevant to RPE-based disorders, as hiPSC-RPE in culture displays morphological, transcriptional, and functional characteristics similar to adult human RPE in vivo (20)(21)(22)(23)(24).…”

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

“…Therefore, an NHEJ-mediated gene knockout strategy is mainly used to evaluate the lossof-function effects and to remove the disease-causing sequences. In the case of Duchenne muscular dystrophy (DMD) patient-derived iPSCs, a 725-kb genomic region containing a premature stop codon in the disease-causing DMD gene was deleted by CRISPR/Cas9 to rescue the open reading frame and ensure partial protein function [101]. In addition, to treat HIV-infected patients, the CCR5 gene, which encodes a chemokine co-receptor required for HIV infection but not survival, of T cells was removed by ZFN, for which clinical trials are underway [102].…”