This splicing interference avoids that exons disrupting the reading frame are incorporated into mature mRNA transcripts

This splicing interference avoids that exons disrupting the reading frame are incorporated into mature mRNA transcripts. We demonstrate that genome editing predicated on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing mutations in bulk populations of patient-derived muscle progenitor cells?(up to 37% of corrected?alleles. INTRODUCTION Duchenne muscular dystrophy (DMD) is a severe X-linked inherited disease caused by mutations that disrupt the reading frame of the dystrophin-encoding gene (1). The lack of functional dystrophin precludes the anchorage between cytoskeleton and sarcolemma structural components needed for the integrity of striated muscle tissue. This results in a cascade of events leading to progressive muscle degeneration and wasting followed by early death, typically between the third and fourth decade of life (2). The sheer size of (2.4 Mb) combined with its Cetrorelix Acetate mutational hotspots, regions linked to high rates of rearrangements and deletions, contribute to make DMD the most common muscular dystrophy in humans (1 in 3500 boys). Despite the identification in 1987 of the molecular basis responsible for MG149 DMD (1), to date there is no effective therapy available. Importantly, however, there is an increasing number of research lines based on molecular and cellular approaches aiming at tackling DMD (2,3). Among the broad array of mutations, the vast majority (>60%) comprises large intragenic deletions of one or more exons that disrupt the reading frame (4). In contrast, deletions within yielding in-frame transcripts often result in the synthesis of shorter dystrophin forms that underlie milder Becker muscular dystrophy (BMD) phenotypes (2,3). This observation provided a strong rationale for developing therapeutic strategies based on delivering recombinant microdystrophins and antisense oligonucleotides (AONs) for gene replacement and exon skipping, respectively (3). In the latter approaches, disrupted reading frames are restored at the RNA level by AON hybridization to specific splice site motifs in pre-mRNA templates with the consequent masking of these motifs from the splicing machinery. This splicing interference avoids that exons disrupting the reading frame are incorporated into mature mRNA transcripts. Therefore, similarly to microdystrophin delivery, the ultimate goal here is to convert DMD into milder BMD forms (2,3). transcript repair by exon skipping has entered clinical testing in the form of AONs targeting exon 51 (5,6). Despite initial indications of therapeutic benefit, the requirement for lifelong AON MG149 administrations and potential long-term AON toxicities, warrant the unabated pursuit of alternative or complementary DMD therapies. In addition, multi-exon skipping by AON multiplexing aiming at a wider mutant genotype coverage remains rather inefficient (7). Genome editing based on sequence-specific designer nucleases (also known as programmable nucleases) has recently been put forward as a potential therapeutic modality for restoring on a permanent basis the native reading frame in patient-own cells, including stem and progenitor cells with myogenic capacity (8C12). The value of designer nucleases arises from their ability to induce site-specific double-stranded DNA breaks (DSBs) that stimulate the two main cellular DNA repair pathways, i.e. non-homologous end-joining (NHEJ) and homologous recombination (HR). The former pathway involves the direct end-to-end ligation of DNA termini created by chromosomal DSBs, often resulting in the introduction of small insertions and deletions (indels) at the junction; the latter requires homologous donor DNA sequences to serve as templates for DNA synthesis-dependent DSB repair (13,14). Although extremely valuable to achieve precise endogenous gene repair and targeted addition of whole transgenes, current HR-based genome editing approaches are, to some extent, limited by the fact that DSBs are often repaired via NHEJ instead of HR (15). Moreover, the very large size of the gene coupled to the broad distribution and types of its mutations, complicates the delivery of donor DNA substrates harboring the complete coding sequence (11 kb) or mutation-correcting templates. Thus, the ligation of designer nuclease-induced chromosomal breaks by NHEJ provides for alternative, donor DNA-independent, approaches for repairing aberrant reading frames. Importantly, such direct repair of native defective alleles assures the physiological regulation of dystrophin synthesis by keeping expression under its endogenous promoter. Designer nuclease technologies are developing at a rapid MG149 pace and include zinc-finger nucleases (ZFNs), engineered meganucleases, transcription activator-like effector nucleases (TALENs) and, more recently, RNA-guided nucleases (RGNs) (13,14). Among these, TALENs seem to display a particularly favorable specificity profile (16), whereas the RGN platform outstands for its simplicity and versatility, especially in multiplexing contexts (17). TALENs are modular proteins consisting of a DNA-binding domain (DBD) bearing repetitive motifs fused via a linker to a catalytic domain, commonly derived from the endonuclease. These artificial proteins work in pairs with the binding of each monomer to its target site resulting in.