Durable transgene expression and efficient re-administration after rAAV2.5T-mediated fCFTRAR gene delivery to adult ferret lungs

The dosing interval for effective recombinant adeno-associated virus (rAAV)-mediated gene therapy of cystic fibrosis lung disease remains unknown. Here, we assessed the durability of rAAV2.5T-fCFTRAR-mediated transgene expression and neutralizing antibody (NAb) responses in lungs of adult wildtype ferrets. Within the first 3 months following rAAV2.5TfCFTRAR delivery to the lung, CFTRDR transgene expression declined X5.6-fold and then remained stable to 5 months at X26% the level of endogenous CFTR. rAAV NAbs in the plasma and bronchoalveolar lavage fluid (BALF) peaked at 21 days, coinciding with peak ELISpot T cell responses to AAV capsid peptides, after which both responses declined and remained stable at 4-5 months post dosing. Administration of reporter vector rAAV2.5T-gLuc (gaussia luciferase) at 5 months following rAAV2.5T-fCFTRAR dosing gave rise to similar levels of gLuc expression in the BALF as observed in age-matched reporteronly controls, demonstrating that residual BALF NAbs were functionally insignificant. Notably, the second vector administration led to a 2.6-fold greater ELISpot T cell response and X2.3-fold decline in fCFTRDR mRNA and vector genomes derived from the initial rAAV2.5T-fCFTRAR administration, suggesting selective destruction of transduced cells from the first vector dose. These findings provide insights into humoral and cellular immune response to rAAV that may be useful for optimizing gene therapy to the cystic fibrosis lung.