Chapter 3 —— 35 —— guided by their earlier in vitro experiments, the authors apply dual AAVs to deliver a split version of an ABE8e variant (i.e., ABE.TadA-8eV106W), selected for its reduced off-target nucleic acid deaminase activities [6], together with a gRNA targeting the exon 45 SA region (Figure 2). Figure 2. Dual AAV ABE trans-splicing system for dystrophin repair. Two AAV serotype-9 vectors expressing separated portions of an adenine base editor (ABE.TadA8eV106W) and a gRNA (gRNAEx45) lead to intein-mediated assembly of complete ABE:gRNA complexes. Base editing involving A·T-to-G·C transitions at the splice acceptor site of exon 45 establishes permanent exon 45 skipping in striated muscle cells. In dystrophin-defective muscle cells lacking exon 44, exon 45 skipping restores the reading of mature transcripts that code for a truncated Becker-like dystrophin with therapeutic potential for DMD patients. ITR, T-shaped hairpin-structured AAV serotype2 inverted terminal repeats (cis-acting elements needed for vector DNA replication and packaging in producer cells). CK8e and An, synthetic striated muscle-specific promoter and polyadenylation signal, respectively. The dystrophin diagram was generated via: http://edystrophin.genouest.org/index.php?page=home. To favor base editing in striated muscles over non-target organs of Dmd exon 44-deleted mice, the vector constructs were packaged in AAV serotype-9 capsids with the split ABE.TadA-8eV106W moieties being expressed through the tissue-specific CK8e promoter. Intramuscular dual AAV coadministrations of 1 x 1011 total vector genomes (VGs) per tibialis anterior
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