Repeat Dosing of AAV2.5T to Ferret Lungs Elicits an Antibody Response That Diminishes Transduction in an Age-dependent Manner

Abstract Readministration of recombinant adeno-associated virus (rAAV) may be necessary to treat cystic fibrosis (CF) lung disease using gene therapy. However, little is known about rAAV-mediated immune responses in the lung. Here we demonstrate the suitability of the ferret for testing AAV2.5T-mediated CFTR delivery to the lung and characterization of neutralizing antibody (NAb) responses. AAV2.5T-SP183-hCFTRΔR efficiently transduced both human and ferret airway epithelial cultures, and complemented CFTR Cl– currents in CF airway cultures. Delivery of AAV2.5T-hCFTRΔR to neonatal and juvenile ferret lungs produced hCFTR mRNA at 200-300% greater levels than endogenous fCFTR. Single-dose (AAV2.5T-SP183-gLuc) or repeat-dosing (AAV2.5T-SP183-fCFTRΔR followed by AAV2.5T-SP183-gLuc) of AAV2.5T was performed in neonatal and juvenile ferrets. Repeat-dosing significantly reduced transgene expression (11-fold) and increased bronchioalveolar lavage fluid (BALF) NAbs only in juvenile but not neonatal ferrets, despite near equivalent plasma NAbs responses in both age groups. Notably, both age groups demonstrated a reduction in BALF anti-capsid binding IgG, IgM, and IgA antibodies following repeat-dosing. Unique to juvenile ferrets was a suppression of plasma anti-capsid binding IgM following the second vector administration. Thus, age-dependent immune system maturation and isotype switching may impact the development of high-affinity lung NAbs following repeat-dosing of AAV2.5T and may provide a path to blunt AAV neutralizing responses in the lung.