RESUMEN
The efficacy of redosing the recombinant adeno-associated virus (rAAV) vector rAAV2.5T to ferret lung is limited by AAV neutralizing antibody (NAb) responses. While immunosuppression strategies have allowed for systemic rAAV repeat dosing, their utility for rAAV lung-directed gene therapy is largely unexplored. To this end, we evaluated two immunosuppression (IS) strategies to improve repeat dosing of rAAV2.5T to ferret lungs: (1) a combination of three IS drugs (Tri-IS) with broad coverage against cellular and humoral responses (methylprednisolone [MP], azathioprine, and cyclosporine) and (2) MP alone, which is typically used in systemic rAAV applications. Repeat dosing utilized AAV2.5T-SP183-fCFTRΔR (recombinant ferret CFTR transgene), followed 28 days later by AAV2.5T-SP183-gLuc (for quantification of transgene expression). Both the Tri-IS and MP strategies significantly improved transgene expression following repeat dosing and reduced AAV2.5T NAb responses in the bronchioalveolar lavage fluid (BALF) and plasma, while AAV2.5T binding antibody subtypes and cellular immune responses by ELISpot were largely unchanged by IS. One exception was the reduction in plasma AAV2.5T binding immunoglobulin G (IgG) in both IS groups. Only the Tri-IS strategy significantly suppressed splenocyte expression of IFNA (interferon α [IFN-α]) and IL4. Our studies suggest that IS strategies may be useful in clinical application of rAAV targeting lung genetic diseases such as cystic fibrosis.
RESUMEN
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 bronchoalveolar lavage fluid (BALF) NAbs only in juvenile, but not neonatal, ferrets, despite near-equivalent plasma NAb responses in both age groups. Notably, both age groups demonstrated a reduction in BALF anti-capsid binding immunoglobulin (Ig) G, IgM, and IgA antibodies after repeat dosing. Unique to juvenile ferrets was a suppression of plasma anti-capsid-binding IgM after the second vector administration. Thus, age-dependent immune system maturation and isotype switching may affect the development of high-affinity lung NAbs after repeat dosing of AAV2.5T and may provide a path to blunt AAV-neutralizing responses in the lung.
