Abstract
Purpose :
Retinal Vascular endothelial cells (VEC) are a critical component of the blood-retinal barrier and their dysfunction underlies the pathogenesis of several blinding diseases, including diabetic retinopathy (DR) and age-related macular degeneration (AMD). Recombinant adeno-associated virus (rAAV) vectors have been shown to facilitate efficient transduction of neuronal/glia cells within the retina, but are relatively ineffective at targeting VECs. In this study, we evaluated whether incorporating endothelial targeting peptides into the rAAV capsid increases VEC tropism in the retinal vasculature.
Methods :
rAAV2/2, rAAV2/2[QuadYF-TV], and rAAV2/9 serotype vectors (n=10/serotype) expressing GFP were modified by inserting heptameric peptides (7AA) 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 each vector expressing a GFP reporter was evaluated on HEK293T cells using a pico green dsDNA quantitation assay and fluorescence microscopy. Transduction efficiency relative to unmodified controls was screened on primary bovine VECs after 72 hours and quantified using co-localization with CD31 via flow cytometry. Vectors with the highest tropism were injected intravitreally and intravenously in mice and after 4-months, tropism was assessed in the eyes using cSLO imaging and post-mortem histology.
Results :
7AA insertions in rAAV vectors did not adversely affect packaging, and GFP expression was validated on HEK292 cells. rAAV capsid mutant 5 (EC5) showed higher transduction in all serotypes in bovine VECs, with a fold change of 1.7- (rAAV2-EC5), 2.7- (rAAV2/2[QuadYF-TV]-EC5), and 3.6 (rAAV2/9-EC5). cSLO imaging demonstrated GFP expression in the retinal vasculature surrounding the optic nerve head following intravenous injection of both rAAV2/2[QuadYF-TV]-EC5 and rAAV2/9-EC5. No evidence of VEC transduction was observed following intravitreal injection for any serotype. EC5 mutant vectors showed de-targeting of hepatocytes (liver), limited transduction of alveolar endothelium (lung) and dramatic increase in cardiac muscle transduction (heart), particularly in rAAV2/2[QuadYF-TV]-EC5 injected mice.
Conclusions :
Incorporation of EC5 peptide into the capsids of rAAV2/2, rAAV2/2[QuadYF-TV] and rAAV2/9 vectors led to altered tropism, with an increased affinity for retinal VECs, lung endothelium and cardiac muscle, and detargeting of liver hepatocytes.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.