Evaluating the tropism of chimeric helper-dependent adenoviral vectors for delivery of large genes.

Abstract

Purpose : Despite success with retinal gene therapy using adeno-associated viruses (AAVs), many disease-causing genes, like ABCA4 or USH2A, are too large for packaging into a single AAV. One option for delivery of large retinal genes is helper-dependent adenoviruses (HDAds). However, we recently showed that HDAd5 primarily targets Müller cells, not photoreceptors and elicits an inflammatory response. The purpose of this project is to evaluate the tropism and photoreceptor transduction efficiency of two chimeric HDAds.

Methods : Chimeric HDAds carrying eGFP under the control of the cytomegalovirus promoter (CMVp) were purchased from Creative Biolabs: HDAd constructed with the adenovirus 5 (Ad5) backbone and either an Ad3 or Ad35 fiber [HDAd5/3-CMVp-eGFP (HDAd5/3) and HDAd5/35-CMVp-eGFP (HDAd5/35)]. For testing in vitro, hTERT-immortalized human induced pluripotent stem cell (iPSC)-derived retinal progenitor cells were transduced with 10 µL of HDAd5, HDAd5/3 or HDAd5/35 (1 x 10⁶ vg/µL per well) and assessed via fluorescent microscopy at 72-hours post-transduction. For in vivo analysis, we utilized Sprague Dawley and RNU^+/- rat lines and subretinally injected 10 µL of HDAd5/3 or HDAd5/35 viral particles (1 x 10⁶ vg/µL per eye) or sterile buffer and assessed injected eyes at 3- and 7-days post-injection via immunohistochemistry and confocal microscopy.

Results : EGFP-positive retinal progenitor cells were observed at 72-hours post-transduction in wells treated with HDAd5, HDAd5/3 or HDAd5/35. There were noticeably more eGFP-positive cells in HDAd5/3- and HDAd5/35-treated cultures compared to HDAd5-treated wells. Fundus photography demonstrated that both HDAd5/3 and HDAd5/35 produced a speckled eGFP expression pattern, suggestive of photoreceptor cell transduction at 3- and 7-days post-injection in each Sprague Dawley and RNU^+/- rats. Confocal microscopy showed prominent eGFP expression within the retinal pigmented epithelium and neural retina.

Conclusions : Compared to HDAd5, chimeric HDAd5/3 and HDAd5/35 produced more eGFP-positive cells when delivered to human iPSC-derived retinal progenitor cells. Subretinal injection of HDAd5/3 and HDAd5/35 also produced eGFP expression in the retinal pigmented epithelium and neural retina of Sprague Dawley and RNU^+/- rats. Future studies will further characterize the tropism and transduction efficiency of chimeric HDAd-mediated gene transfer in the neural retina.

This is a 2021 ARVO Annual Meeting abstract.