Adenoviral vectors (AdVs) constitute powerful gene delivery vehicles. However, so far, their potential for genome editing has not been extensively investigated. By tailoring AdVs as... Show more Adenoviral vectors (AdVs) constitute powerful gene delivery vehicles. However, so far, their potential for genome editing has not been extensively investigated. By tailoring AdVs as carriers of designer nucleases and donor DNA sequences, the research presented in this thesis expands the utility of the AdV platform to genome editing. In particular, in the first part of this thesis, AdVs are exploited for tackling two of the major bottlenecks of genome editing: (i) developing improved methods for delivering the sizable gene-editing tools, such as RNA-guided nuclease complexes, into target cells, and (ii) increasing the specificity and fidelity of the gene-editing procedures. In the second part of the thesis, the insights derived from these studies are further exploited for testing AdVs encoding nucleases as repairing agents of defective DMD alleles in muscle cell populations derived from patients with Duchenne muscular dystrophy (DMD). Finally, the application of AdVs as gene editing tools for repairing endogenous DMD alleles is discussed in the context of other viral vector-based DMD editing strategies. Taken together, the findings reported in this work are expected to aid in the designing and testing of new therapeutic interventions for tackling DMD and are anticipated to be applicable to other genetic disorders. Show less
Accurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA... Show moreAccurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA template into target cells. The exogenous template, called donor DNA, normally contains the foreign sequences flanked by DNA regions sharing sequence identity ("homologous") to those bracketing the target site. The strategies for mediating the formation of DSBs at the predefined genomic loci, have been undergoing intense investigation since the introduction of sequence-customizable zinc-finger nuclease (ZFN) technology. More recently, prokaryotic protein-based transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases (RGNs) derived from CRISPR-associated protein (Cas9) complexes have substantially broadened the availability and applicability of designer nuclease-mediated genome editing. A potential alternative research line to the use of designer nucleases, is to investigate whether specific DNA structures can, by themselves, serve as triggers of the DNA damage response and, in doing so, elicit targeted gene repair. Such an approach would simplify genome editing protocols, such as, by reducing the number of reagents needed to be introduced into target cells. In this thesis, the roles of these secondary structures as well as designer nucleases and donor-DNA templates, delivered via adenoviral vectors, is described. Show less
