Abstract
Purpose :
Vascular endothelial cells (VEC) are essential for retinal homeostasis and their dysfunction underlies pathogenesis in diabetic retinopathy (DR) and exudative age-related macular degeneration (AMD). Studies have shown that recombinant adeno-associated virus (rAAV) vectors can deliver the gene to neural cells of the retina, yet targeting VECs remains extremely challenging. In this study, we developed rAAV capsid mutant vectors with improved tropism to target retinal VEC through the incorporation of endothelial targeting peptides into the viral capsid.
Methods :
rAAV2/2, 2/2[QuadYF-TV], and rAAV2/9 serotype vectors (n=10, capsid mutants per serotype) expressing GFP were generated by inserting heptameric peptides (7AA) designed to increase endothelial targeting at positions 588 (2/2 and 2/2[QuadYF-TV] or 589 (2/9) of the virus protein (VP1-3). The packaging and transduction efficiency of the VEC targeting vectors was assessed on HEK293T cells to confirm infectivity. Subsequently, each vector (MOI=75000) was administered to ex vivo primary bovine VECs. After 72 hours, cells were labeled with CD31, and transduction efficiency was quantified using flow cytometry. Following identification of a mutant (EC5) with improved endothelial tropism, AAVs vectors were administered intra-ocularly and intravenously in C57Bl/6j mice under isoflurane anesthesia. After 4-months, cSLO imaging was performed to assess tropism before the eyes and organs (heart, liver, lungs, and brain) were collected for post-mortem histology.
Results :
All capsid mutant rAAV vectors generated were found to transduce HEK293T cells leading to expression of GFP, indicating insertions did not adversely affect packaging. The EC5 mutant demonstrated increased transduction of CD31+ primary bovine VECs in all serotypes, with a fold change of 1.7, 2.7- and 3.6-times higher transduction efficiency when incorporated in the rAAV2/9, 2/2, and 2/2[QuadYF-TV] capsids, respectively. cSLO imaging demonstrated visible expression of GFP in the retinal vasculature following intravenous administration for EC5 mutant vectors versus unmodified controls.
Conclusions :
The generation of rAAV vectors targeting retinal VECs could improve gene therapy treatment for DR and AMD. Our findings suggest that incorporating endothelial targeting peptide into rAAV capsid could improve tropism and allow delivery of therapeutic transgenes efficiently to the retinal VECs.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.