Optimized AAV rh.10 Vectors That Partially Evade Neutralizing Antibodies during Hepatic Gene Transfer

Abstract

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.

Post‐translational modifications in capsid proteins of recombinant adeno‐associated virus (AAV) 1‐rh10 serotypes

“…The presence of ubiquitination and phosphorylation on AAV capsids was expected as several studies [35,37,49] have highlighted their role during viral entry or the intracellular trafficking process. Indeed, mutagenesis of several in silico predicted phosphorylation sites at serine, threonine, or tyrosine residues or ubiquitination targets (lysine residues) have improved the transduction efficiency of multiple AAV serotypes [36,50]. Our study also identified for the first time, SUMOylation targets in AAV2, AAV5, AAV7, AAV9, and AAVrh10 due to the mass difference of +600.25 (SUMO-1) and +599.27 (SUMO-2/3).…”

“…While we have assessed for the native levels of ubiquitinated capsids in freshly packaged AAV2 vectors, previous studies have employed in vitro ubiquitination assays on AAV2 either directly [36] or after AAV isolation from infected cell lines by immunoprecipitation based methods and their subsequent incubation in the presence of excess amounts of a ubiquitin substrate and ligases [35]. To further confirm this, we utilized a previously reported AAVrh10-K333R mutant that showed comparable levels of ubiquitination to AAVrh10-WT vectors in an in vitro ubiquitination analysis with excess amount of ubiquitination enzymes [50]. These vectors when analyzed for native levels of ubiquitination, directly after packaging by a dot-blot analysis had a distinctly reduced ubiquitination profile from WT-vectors (Fig.…”

Mucopolysaccharidosis IVA: Diagnosis, Treatment, and Management

“…AAV therapy remains a work in progress, and the full potential of this therapy is limited by the immune response generated against the capsid, transgene product, and transduced cells [124,125]. Methods of overcoming the immune response include novel serotype generation [126], promoter design and liver targeting [127,128], exosome coupled AAV vectors [129,130], and several others currently being investigated.…”

Engineering the AAV capsid to evade immune responses

“…This variant also had higher transduction in vitro and in vivo and was 27-to 64-fold more resistant to NABs in mice passively immunized with human intravenous immunoglobulin (IVIG, pooled IgG from >1000 donors). [18] As another strategy to disrupt epitopes, capsid residues or regions from different serotypes can be combined to yield a chimera with mixed properties of its parents. The first such variant was AAV2.5, which introduced five amino acid residues from AAV1 into AAV2, and thereby combined the receptor attachment behavior of AAV2, the muscle tropism of AAV1, and modified antigenic epitopes to reduce antibody neutralization.…”