Chapter 4 —— 54 —— Besides DDR proteins, episomes of both non-viral and viral origins, including AAV vector genomes, are prone to a vast array of cellular restriction factors that dampen or silence transgene expression, e.g. via the acquisition of heterochromatin epigenetic marks [33,54]. In this context, additional realtime monitoring of AAV vector transduction kinetics in the presence and absence of AdVP.C9KARA was consistent with HDR-mediated AAV gene targeting occurring soon after site-specific DSB formation, which presumably results in heightened and earlier onset of transgene expression from integrated sequences over non-integrated episomes (Supplementary Figure S8). Such differential expression levels between chromosomally targeted and episomal AAV donor DNA have been in fact successfully exploited to enrich for gene knock-ins amongst transduced cell populations [15]. Directly editing specific endogenous genes for the purpose of tagging, modifying or knocking-out encoded gene product(s) is an alternative approach to inserting transgenes at safe harbor loci. A distinctive advantage of endogenous gene modification is maximizing appropriate physiologically regulated expression and subcellular positioning of the resulting gene product(s). Moreover, most genetic disorders are caused by numerous and diverse types of mutations scattered along gene bodies. Consequently, knocking-in recombinant coding sequences downstream of cis-acting regulatory elements offers an ‘universal’ strategy for rescuing disease phenotypes or properly tracing gene products. A broad range of mutations in the DMD gene, that normally codes for the long sarcolemma-stabilizing protein dystrophin (427 kDa), causes the lethal X-linked muscle-wasting disorder Duchenne muscular dystrophy (DMD; MIM no. 310200). Due to its prevalence (∼1:4700 boys) and severity, DMD is a primary target for genetic therapies despite the challenges posed by the vast expanse of the affected striated musculature and of the DMD gene itself (>2.3 Mb) [55]. Candidate in vivo and ex vivo DMD genetic therapies have defined sets of pros and cons [56–58]. For instance, although autologous transplantation of ex vivo corrected myogenic stem/progenitor cells currently presents notable
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