rAAV-PHP.B escapes the mouse eye and causes lethality whereas rAAV9 can transduce aniridic corneal limbal stem cells without lethality.

Recently safety concerns have been raised in connection with high doses of recombinant adeno-associated viruses (rAAV). Therefore, we undertook a series of experiments to test viral capsid (rAAV9 and rAAV-PHP.B), dose, and route of administration (intrastromal, intravitreal, and intravenous) focused on aniridia, a congenital blindness that currently has no cure. The success of gene therapy for aniridia may depend on the presence of functional limbal stem cells (LSCs) in the damaged aniridic corneas and whether rAAV can transduce them. Both these concerns were unknown, and thus were also addressed by our studies. For the first time, we report ataxia and lethality after intravitreal or intrastromal rAAV-PHP.B virus injections. We demonstrated virus escape from the eye and transduction of non-ocular tissues by rAAV9 and rAAV-PHP.B capsids. We have also shown that intrastromal and intravitreal delivery of rAAV9 can transduce functional LSCs, as well as all four PAX6-expressing retinal cell types in aniridic eye, respectively. Overall, lack of adverse events and successful transduction of LSCs and retinal cells makes it clear that rAAV9 is the capsid of choice for future aniridia gene therapy. Our finding of rAAV lethality after intraocular injections will be impactful for other researchers developing rAAV-based gene therapies.