Chapter 1 —— 11 —— therapeutically relevant cells, e.g., induced pluripotent stem cells (iPSCs) and T cells, leading to the manufacturing of potentially safer cell products for transplantation purposes. In Chapter 3, we further elaborate on the use of nickase variants in the form of base editors directed at therapeutic GE applications. In particular, we commented on the work conducted by Chai et al. (Mol. Ther. Nucleic Acids. 2023 32:522-535), investigating repair of defective DMD alleles causing Duchenne muscular dystrophy (DMD) via adeno-associated viral (AAV) vector delivery of trans-splicing base editors in vitro and in vivo settings. Instead of testing AAV/CRISPR-Cas9-based DMD reading frame restoration, associated with the potentially prevalent capture of Cas9-encoding AAV at on- and off-target nuclease sites, the authors investigated dual AAV delivery of trans-splicing adenine-base editor constructs yielding a Cas9 nickase fused to an adenine deaminase that, together with a cognate gRNA, mediates adenine (A) to guanine (G) substitutions. The resulting substitutions disrupt splice site motifs leading to exon skipping of DMD exon sequences bearing premature stop codons. On the top of overcoming the limited cargo capacity of AAV vectors (i.e., 4.7 kb), such dual AAV method provides for a DSB-free DMD reading frame correction option for treating DMD patients whose disease is caused by outof-frame deletions. Even though, conceptually, one expects installing desired therapeutic gene edits with the aid of different GE tools, it has become patently clear the challenge of achieving efficient and safe delivery of the necessary tools into target cells, especially those cells resistant to transfection or relevant to therapeutical applications. Moreover, one needs to contend with the fact with the increasing precision of the most advanced GE tools there is a concomitant trend towards larger size increase in the associated machineries, which further hampers their delivery efficiency. Taken these delivery and safety aspects together, we reasoned that combining distinct engineered viral vectors lacking all viral genes could serve as sources of GE tools upon robust transduction of target cells. In particular, based on the complementary characteristics of
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