Abstract
Purpose :
Retinal vascular endothelial cells (VEC) play a major role in retinal-homeostasis and their dysfunction underlies pathogenesis in vascular diseases affecting the eye, such as diabetic retinopathy (DR) and exudative age-related macular degeneration (AMD). While numerous pre-clinical trials have shown that recombinant adeno-associated virus (rAAV) vectors can successfully mediate gene delivery to cells of the neural retina, targeting VECs remains particularly challenging. In this study, we examine whether incorporation of endothelial targeting peptides into the rAAV capsid increases VEC transduction efficiency using an ex vivo primary culture model.
Methods :
VEC targeting capsid mutant rAAV2/2, 2/2[QuadYF-TV] and rAAV2/9 serotype vectors (n=10 per serotype) packaging a green fluorescent protein (GFP) reporter gene were created through introduction of a heptameric targeting peptide (7AA) at position 588 (2/2 and 2/2[QuadYF-TV] or 589 (2/9) of the virus protein (VP1-3). The infectivity and packaging efficiency of the newly generated VEC targeting vectors was first assessed on cultures of human embryonic kidney (HEK) 293T cells (N=3 wells per vector) using a picogreen dsDNA quantitation assay, fluorescence microscopy and flow cytometry. All infectious vectors were subsequently applied (MOI=100,000) to cultures of primary bovine VECs. After 72 hours, cells were fixed with 4% paraformaldehyde, stained with CD31 and transduction efficiency quantified using flowcytometry.
Results :
The majority of VEC targeting mutants packaged successfully, although at lower titres that unmodified rAAV2/2 and 2/9 serotypes, and were capable of infecting HEK293T cells. CD31 labeling confirmed that ~75% of cells isolated from bovine retinae using our protocol were VECs. Three rAAV2/2 and rAAV2/2[QuadYF-TV) mutants (5, 6 and 8) were observed to have improved expression in VECs; rAAV2/9 mutants are being screened currently.
Conclusions :
The development of rAAV vectors that efficiently target retinal VECs has the potential to substantially improve the treatment of ocular diseases, such as DR and AMD.Our preliminary findings indicate that incorporation of an endothelial targeting peptide into an exposed surface loop of the rAAV capsid may potentially improve the efficiency of gene delivery to VECs.
This is a 2020 ARVO Annual Meeting abstract.