EXPEDITE · EXPanding AAV gene therapy by EDITing

In vivo gene therapy based on a single administration of adeno-associated viral (AAV) vectors is emerging as an effective therapeutic option for both monogenic and complex diseases. Given the episomal nature of the AAV genome, its applications are limited to non-replicating tissues like retina or adult liver. Several AAV-based products that target these tissues are either approved or in advanced clinical development. Despite this, some limitations still remain, including: the potential for insertional mutagenesis associated with genome-wide AAV integration; the loss of transgene expression from replicating tissues like newborn liver; and the challenge to counteract toxic gain-of-function mutations, which cause dominant diseases for which canonical gene replacement is ineffective. EXPEDITE aims to integrate therapeutic DNA at desired genomic loci safely and effectively, thus overcoming the above limitations. EXPEDITE will go beyond the current state-of-the-art by implementing and comparing two parallel strategies: (i) novel Cas fusion proteins to recruit DNA repair machineries at induced double stand breaks (DSBs) to maximize on-target donor DNA integration; (ii) novel cleavage-free platforms for therapeutic DNA integration, based on transposases or bacterial single strand-DNA annealing proteins. The therapeutic relevance of these platforms will be tested in the retina and liver, two highly relevant tissues for gene therapy, using animal models of inherited retinal degenerations and lysosomal storage diseases, respectively, and, ultimately, non human primates. To reduce the risk of potential off-targets, EXPEDITE will also test non-viral vectors for transient delivery of the genome editing tools while delivering the donor DNA via AAV. The results from EXPEDITE will allow significant expansion of the patient population that can benefit from in vivo gene therapy and may represent a change of paradigm for gene therapy by replacing canonical gene addition approaches.