Abstract
Purpose: :
Adeno-associated viral (AAV) vectors are commonly used for gene transfer. However, administration of AAV-vectors can lead to development of neutralizing antibodies against the vector capsid, thus decreasing the efficiency of therapeutic gene transfer and effective vector re-administration. In previous studies we have found that AAV vectors delivered into different ocular compartments elicited humoral immune responses differently. Intravitreal administration of AAV vectors generates humoral immune response against AAV capsid that blocks vector expression upon re-administration via the same route into the partner eye. In this study, we aimed to further characterize the nature of the humoral responses and to develop strategies to circumvent them.
Methods: :
Contralateral mouse eyes received two sequential intravitreal injections of AAV vectors expressing GFP. In one group, the first eye received 1ul of different dilutions of AAV2 vector (109, 108, 107, 106, 105 vg/eye respectively), while the second eyes were injected with full dose (109 vg/eye) two months later. A second group of animals again received sequential intravitreal injections but with different combinations of wildtype AAV2, mutant AAV2 and AAV8 containing tyr-to-phe(Y-F) capsid mutations, which showed enhanced transduction efficiency thus lowering the effective dose needed. All mice were analyzed two months after the second injection. Expression of GFP was analyzed by fluorescence imaging of retinal flatmount and sections. Antibodies against AAV2 capsid were analyzed by ELISA using serum samples collected prior to and at different time points after the injection.
Results: :
Intravitreal injection of higher AAV2 vector doses induced humoral immune responses against capsid, blocked transduction of the same vector in partner eyes and reduced efficiency of transduction of a different serotype in the second eye (AAV8). Lower doses at the first intravitreal injection did not affect transduction of the second intravitreal injection in partner eyes, and this correlated with dose-dependent serum antibody levels. Y-F mutant vectors showed enhanced transduction efficiency at much lower doses than wt AAV vectors.
Conclusions: :
Humoral immune responses elicited by intravitreal injection can be circumvented by (1) lowering AAV doses; (2) using capsid-modified AAV vectors with increased transduction efficiency at lower doses; and (3) combinations of different serotypes. These findings may have important implications for the design of AAV-mediated ocular gene transfer clinical trials, particularly if partner eyes require sequential treatment.
Keywords: gene transfer/gene therapy • immune tolerance/privilege • retina