Development of novel approaches for gene editing therapies of cystic fibrosis - SUMMARY Chronic lung infection is the primary cause of death in patients with cystic fibrosis (CF). CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Currently, CFTR modulators are effective in treating most CF patients, but this pharmaceutical rescue is costly, necessitates a lifelong commitment and provides no benefit to those who produce insufficient or no CFTR. Gene therapy remains the best hope for a cure applicable to all CFTR genotypes, and it would be less expensive than current modulator therapy. For gene therapy to become feasible, current strategies for CFTR addition, which lead to unregulated CFTR expression, must be adapted to reproduce the cellular heterogeneity of CFTR expression in the lung. This is essential for bacterial clearance and innate immunity. CRISPR-based gene editing holds promise for correcting the defect in CFTR without altering its endogenous regulation, and permanent correction is achievable by targeting the airway progenitor stem cells. However, the cell-cycle dependent homology-directed repair (HDR) is inefficient for application to the terminally differentiated airway epithelial cells and quiescent basal stem cells of the lung airways. Here we propose to overcome the inefficiency of the current HDR approach with homology- independent targeted integration (HITI), which has shown promise for correcting genetic defects in post-mitotic cells in vivo. We hypothesize that a mutation-free CFTR gene can be rebuilt by using HITI to trap a pre-spliced CFTR mega exon (associated with splicing acceptor) in a desired intron. rAAV2.5T efficiently transduces multiple epithelial cell types (including basal stem cells) in polarized human airway epithelium (HAE) cultures, as well as in ferret lung airways. We will use rAAV2.5T to deliver the components necessary for editing by HITI and evaluate the effectiveness of the HITI approach in correcting the CF phenotype both in vitro, using polarized HAE cultures derived from CF patients, and in vivo, using a G551D CF ferret model. However, the clinical utility of rAAV2.5T is currently limited by the need of pharmaceutical augmentation, because its transduction in polarized airway epithelium requires the co-administration of doxorubicin (Dox) to overcome a significant post-entry block to nuclear entry. To address this limitation, we will apply the insights gained from our preliminary studies on rAAV2.5T transduction biology to develop a next-generation rAAV2.5T vector. Specifically, we will create random mutants of the N terminus of the large capsid protein VP1 (VP1N), which plays a crucial role in vector translocation from the trans-Golgi network (TGN) to the nucleus in productive transduction. By directed evolution of the libraries in polarized HAE cultures, we will screen for AAV2.5T variants that efficiently transduce airway basal cells in the absence of Dox. The lead mutants from the screening will be evaluated for efficiency of transduction in HAE cultures and ferret airways in the absence of Dox, the best-performing variant will be used in the HITI gene editing approach. We expect that a therapeutic genome editing approach is feasible for permanent correction of mutations in CFTR for CF gene therapy, using a Dox-independent rAAV2.5T vector.
