Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates

Abstract

Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber's congenital amaurosis and choroideremia. One general challenge for AAV is that pre-existing immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizing antibodies (NABs) in AAV transduction of tissues considered to be immune privileged, such as the eye, is unclear in large animals. Intravitreal AAV administration allows for broad retinal delivery, but is more susceptible to interactions with the immune system than subretinal administration. To assess the effects of systemic anti-AAV antibody levels on intravitreal gene delivery, we quantified the anti-AAV antibodies present in sera from non-human primates before and after intravitreal injections with various AAV capsids. Analysis showed that intravitreal administration resulted in an increase in anti-AAV antibodies regardless of the capsid serotype, transgene, or dosage of virus injected. For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies was the amount of virus delivered. In addition, post-injection antibody titers were highest against the serotype administered, but the antibodies were also cross-reactive against other AAV serotypes. Furthermore, neutralizing antibody levels in serum correlated with those in vitreal fluid, demonstrating both that this route of administration exposes AAV capsid epitopes to the adaptive immune system and that serum measurements are predictive of vitreous fluid NAB titers. Moreover, the presence of pre-existing neutralizing antibody titers in the serum of monkeys correlated strongly (R=0.76) with weak, decaying, or no transgene expression following intravitreal administration of AAV. Investigating anti-AAV antibody development will aid in understanding the interactions between gene therapy vectors and the immune system during ocular administration and can form a basis for future clinical studies applying intravitreal gene delivery.

AAV-mediated delivery of optogenetic constructs to the macaque brain triggers humoral immune responses

“…6) (Fig. 5B) is also 341 consistent with another report of cross-reactivity to AAV2 following injections of other serotypes 342 (Kotterman et al, 2015). A new contribution of our study is the finding that small vector doses 343 injected into the monkey CNS can produce high NAb titers.…”

Noninvasive gene delivery to foveal cones for vision restoration

“…We and others have shown transduction of macaque cones using AAV variants with ubiquitous promoters (16,(29)(30)(31)(32), but achieving cone transduction by vitreally administered AAV has only been possible at high doses, leading to inflammation (16,29). We reasoned that foveal cone targeting could be achieved if we use a strong cone-specific promoter at lower intravitreally injected AAV doses compatible with safety (29,33). To test if such "dose sparing" is possible, we injected 2 macaque eyes with AAV2-7m8-PR1.7-GFP and 2 other macaque eyes were injected with AAV2-7m8-GFP under the control of the cytomegalovirus (CMV) promoter at a dose of 1 × 10 11 viral genomes (vg) per eye (Table 1).…”

“…One shortcoming with the intravitreal injection route is the higher susceptibility of AAVs administered into this compartment to interactions with the immune system compared with subretinal administration (33). It has been shown that antibody neutralization poses a barrier to intravitreal AAV vector-mediated gene delivery in NHPs, and this will likely pose a challenge for human application.…”

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina

“…Ten NHP were chosen based on the absence of neutralizing antibody titers against AAV2 in their blood sera. 37 One primate was injected intravitreally with two vector doses (one eye with 1 Â 10 11 and the other with 5 Â 10 11 vg), with CatCh expressed under the SNCG in fusion with GFP to monitor gene expression in vivo (NHP1). As GFP can be immunogenic, 24 we used CatCh without a fluorescent tag for the remaining nine primates.…”