Abstract
Purpose :
Adeno-associated virus serotype 2 (AAV2) is a viral vector that can be used to deliver therapeutic genes to the retina. AAV2 capsids can be altered in order to improve the efficiency of gene transfer, for instance, by mutating surface-exposed residues that mediate intracellular phosphorylation and degradation of viral particles. Whilst this strategy has demonstrated promise in terms of improving retinal transduction, the possibility of immune activation following the incorporation of phosphodegron mutations into AAV2 has not been explored.
Methods :
Adult mate C57BL/6J mice were injected intravitreally with either 2E8 viral particles (VP)/eye AAV2, AAV2 (Y444F), AAV2 (Y444F, K556E, S662V), 2E10 VP/eye AAV2 or PBS vehicle. After three weeks, mice were sacrificed, serum samples were extracted and eyes were enucleated for analysis. Serum neutralising antibody (NAb) titres were calculated using an in vitro neutralisation assay. Transduction efficiency was assessed via analysis of reporter expression (GFP), and immunohistochemical analysis was used to identify activation of adaptive and innate immune systems. An in vitro heparan binding assay was used to assess whether phosphodegron mutant AAV2 may bind to heparan sulphate proteoglycan (HSPG) with lower affinity than prototypical AAV2.
Results :
AAV2 (Y444F) and AAV2 (Y444F, K556E, S662V) exhibited higher transduction efficiency in the retina than wild type capsids. Both mutant capsids induced higher serum NAb titres and levels of CD4+ and CD8+ T-cell infiltration into the retina than wild type AAV2. We also observed elevated immunoreactivity of CD68 and Iba1, suggesting that microglia activation was induced by the mutant capsids. Similarly, we saw morphological changes and increased immunoreactivity in GFAP+ fibrils, suggesting that Muller glia were involved in the immune response to the mutant capsids. Finally, we showed that AAV2 (Y444F) and AAV2 (Y444F, K556E, S662V) exhibit slightly reduced binding affinity to heparan sulphate.
Conclusions :
Our results suggest that incorporation of phosphodegron mutations in AAV2 results in elevated immune activation vs. wild-type capsids. The underlying molecular basis of this requires further investigation, however, increased permeation of virions into the neural retina due to attenuated heparan binding in the inner limiting membrane may be involved.
This is a 2021 ARVO Annual Meeting abstract.