(632) Evaluation of Gene Insertion Strategies for Restoration of CFTR Expression in Airway Epithelium

Background: CF can be caused by a diverse set of mutations distributed across the 250 thousand base pairs of the CFTR gene. Although a variety of editing tools are now available for correction of individual mutations, a strong justification can be made for a more universal gene insertion approach. Directly targeting either a partial or full-length CFTR cDNA into the endogenous CFTR locus has the potential advantage of correcting or compensating for all CFTR mutations downstream of the integration site. If this can be achieved while retaining the native CFTR chromatin structure and regulatory sequences, it also has the possibility of restoring appropriate cell-type specific expression [1]. We have previously demonstrated the ability to efficiently target integration of AAV-delivered partial CFTR cDNAs into CFTR introns 7 or 8 of CF airway basal cells with restoration of CFTR expression and function in the derived airway epithelium [2]. To correct for all or nearly all CFTR mutations, it likely would be necessary to target integration of the partial CFTR cDNA sequences into the most upstream regions of CFTR sequences. Our objective is to develop a single donor vector for efficient integration of CFTR exons 2-27 (CFTR2-27) into intron 1 – enabling appropriately regulated CFTR expression.

Methods: We have performed a comparative evaluation of targeted integration into a variety of positions distributed across the ~24 kb sequences of intron 1 with the goal of identifying target sites that can be efficiently targeted and transgene constructs that give rise to robust expression. This evaluation first utilized constructs that included both a fluorescent reporter and a chimeric CFTR-luciferase transgene. The approach enables us to assay efficiency of targeted integration as well as develop a comparative assessment of levels of transgene expression.

Results: This analysis has revealed clear differences in expression level as a function of integration site further modulated by the choice of poly adenylation (polyA) signals. For those integration sites and polyA signals exhibiting highest luciferase expression, we are now evaluating delivery and expression of the desired CFTR2-27 transgene.

Conclusions: Our studies suggest that choice of integration site, as well as critical regulatory elements such as the polyA signal, will affect the level of CFTR expression and thus the location to target should be carefully selected. Although this analysis is being performed in the context of AAV-mediated delivery and homologous recombination-mediated integration, we anticipate the examination will be highly informative irrespective of the vector used for delivery or precise method of integration.

Acknowledgements: This work was supported by the CFF [DAVIS19XX0 and DAVIS19XX1]

References: [1] Cristina Barillà, Suzuki S, Rab A, Sorscher EJ and Davis BR. Targeted Gene Insertion for Functional CFTR Restoration in Airway Epithelium. Front Genome Ed 2022; https://doi.org/10.3389/fgeed.2022.847645
[2] Suzuki S, Crane AM, Anirudhan V, Barillà C, Matthias N, Randell SH, Rab A, Sorscher EJ, Kerscher J, Yin S, Harris S, Mendel M, Kim K, Zhang L, Conway A, Davis BR. Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction. Mol Ther 2020;28(7):1684-1695. doi: 10.1016/j.ymthe.2020.04.021.