Post‐translational modifications in viral capsids are known to fine‐tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post‐translational modifications during intra‐cellular maturation of its capsid proteins. Data on such post‐translational modifications in the capsid of recombinant adeno‐associated virus serotypes ( AAV 1‐rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post‐translational modifications in AAV 1‐rh10 capsid protein. Our analysis revealed a total of 52 post‐translational modifications in AAV 2‐ AAV rh10 capsids, including ubiquitination (17%), glycosylation (36%), phosphorylation (21%), SUMO ylation (13%) and acetylation (11%). While AAV 1 had no detectable post‐translational modification, at least four AAV serotypes had >7 post‐translational modifications in their capsid protein. About 82% of these post‐translational modifications are novel. A limited validation of AAV 2 capsids by MALDI ‐ TOF and western blot analysis demonstrated minimal glycosylation and ubiquitination of AAV 2 capsids. To further validate this, we disrupted a glycosylation site identified in AAV 2 capsid ( AAV 2‐N253Q), which severely compromised its packaging efficiency (~ 100‐fold vs. AAV 2 wild‐type vectors). In order to confirm other post‐translational modifications detected such as SUMO ylation, mutagenesis of a SUMO ylation site(K258Q) in AAV 2 was performed. This mutant vector demonstrated reduced levels of SUMO ‐1/2/3 proteins and negligible transduction, 2 weeks after ocular gene transfer. Our study underscores the heterogeneity of post‐translational modifications in AAV vectors. The data presented here, should facilitate further studies to understand the biological relevance of post‐translational modifications in AAV life cycle and the development of novel bioengineered AAV vectors for gene therapy applications. Enzymes Trypsin, EC 3.4.21.4

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Of the 12 common serotypes used for gene delivery applications, Adeno-associated virus (AAV)rh.10 serotype has shown sustained hepatic transduction and has the lowest seropositivity in humans. We have evaluated if further modifications to AAVrh.10 at its phosphodegron like regions or predicted immunogenic epitopes could improve its hepatic gene transfer and immune evasion potential. Mutant AAVrh.10 vectors were generated by site directed mutagenesis of the predicted targets. These mutant vectors were first tested for their transduction efficiency in HeLa and HEK293T cells. The optimal vector was further evaluated for their cellular uptake, entry, and intracellular trafficking by quantitative PCR and time-lapse confocal microscopy. To evaluate their potential during hepatic gene therapy, C57BL/6 mice were administered with wild-type or optimal mutant AAVrh.10 and the luciferase transgene expression was documented by serial bioluminescence imaging at 14, 30, 45, and 72 days post-gene transfer. Their hepatic transduction was further verified by a quantitative PCR analysis of AAV copy number in the liver tissue. The optimal AAVrh.10 vector was further evaluated for their immune escape potential, in animals pre-immunized with human intravenous immunoglobulin. Our results demonstrate that a modified AAVrh.10 S671A vector had enhanced cellular entry (3.6 fold), migrate rapidly to the perinuclear region (1 vs. >2 h for wild type vectors) in vitro, which further translates to modest increase in hepatic gene transfer efficiency in vivo. More importantly, the mutant AAVrh.10 vector was able to partially evade neutralizing antibodies (~27–64 fold) in pre-immunized animals. The development of an AAV vector system that can escape the circulating neutralizing antibodies in the host will substantially widen the scope of gene therapy applications in humans.