Chapter 3 —— 36 —— (TA) led to 29.5% ± 2.7% A·T-to-G·C edits with minimal indel formation (i.e., 0.2% ± 0.1%), as determined by deep sequencing at 3 weeks post injection. Systemic dual AAV co-administrations of 1.5 x 1014 VG kg-1 and 3 x 1014 VG kg-1 via the temporal facial veins of postnatal day 2 (P2) mice yielded, in TA muscles, 5.5% ± 1.2% and 8.1% ± 3.0% edits and, in hearts, 22.0% ± 2.2% and 26.2% ± 4.4% edits, respectively, with <0.1% indels detected at 8 weeks post injection. Critically, a general dose-dependent improvement of disease-associated molecular, cellular, and functional endpoints is reported. In this regard, up to 31% and 60% of wild-type dystrophin levels estimated in treated TA and heart muscles corresponded to over 76% and 95% of dystrophin-positive myofibers and cardiomyocytes, respectively. Partial dystrophin rescue translated, in turn, in noticeable reduction of myofiber central nucleation, diameter distribution, and fibrosis, all hallmarks of muscle degeneration. Finally, Dmd exon 44-deleted mice systemically treated at P2 with low and high doses of dual AAV ABE transsplicing particles registered 31% and 41% grip strength augmentation, respectively, when compared with their untreated counterparts. Follow-up studies will be instructive to determine the long-term effects of the local and systemic gene-editing procedures in the treated animals. Of notice, despite the aforementioned transductional and transcriptional targeting measures, significant base editing was detected in the liver (i.e., 11.1% ± 5.9%), which correlated with high VG copy numbers present specifically in this organ. These data confirm the importance of developing liver de-targeting protocols and strictly myotropic AAV capsids [10]. Indeed, as dose-dependent toxicity and immunological constrains have emerged during AAV clinical applications, optimization of tissue tropism and expression will be particularly important for dual AAV trans-splicing approaches due to the necessarily higher particle amounts required for maximizing co-expression and full-length protein assembly. Moreover, besides seeking the elimination of the observed editing at two of five top-ranked in silico-predicted candidate off-target sites [5], unbiased genome- and transcriptome-wide assessments of
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