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.

最近，人们对高剂量重组腺相关病毒（rAAV）的安全性提出了担忧。因此，我们进行了一系列实验来测试病毒衣壳（rAAV9和rAAV-PHP.B）、剂量和给药途径（基质内、玻璃体内和静脉内），重点针对无虹膜症（一种目前无法治愈的先天性失明）。无虹膜基因治疗的成功可能取决于受损无虹膜角膜中功能性角膜缘干细胞 (LSC) 的存在以及 rAAV 是否​​可以转导它们。这两个问题都是未知的，因此我们的研究也解决了这些问题。我们首次报告玻璃体内或基质内注射 rAAV-PHP.B 病毒后出现共济失调和致死率。我们证明了病毒从眼睛逃逸并通过 rAAV9 和 rAAV-PHP.B 衣壳转导非眼组织。我们还表明，rAAV9 的基质内和玻璃体内递送可以分别转导功能性 LSC，以及无虹膜眼中所有四种表达 PAX6 的视网膜细胞类型。总体而言，不良事件的缺乏以及 LSC 和视网膜细胞的成功转导表明 rAAV9 是未来无虹膜基因治疗的衣壳选择。我们对眼内注射后 rAAV 致死率的发现将对其他开发基于 rAAV 的基因疗法的研究人员产生影响。