Purchase this article with an account.
Ramesh Periasamy, Dwani Patel, Shannon E Boye, Sanford L Boye, Daniel M Lipinski; Development of rAAV capsid mutant vectors with improved tropism for retinal vascular endothelial.. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1468.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinal vascular endothelial cells (VECs) are critical for maintaining retinal-homeostasis and play a key role in mediating vascular permeability, tone and contractility. Dysfunction of the vascular endothelium negatively affects the integrity of the blood-retinal barrier and underlies pathogenesis in vascular diseases affecting the eye, such as diabetic retinopathy (DR) and exudative age-related macular degeneration (AMD). As such, VECs represent a promising therapeutic target for the development of future gene therapy treatments, but remain particularly challenging to transduce using existing viral vectors. In this study, we investigate whether the incorporation of endothelial targeting peptides into the rAAV capsid increases VEC transduction efficiency in an ex vivo primary culture model.
rAAV2/2, 2/2[QuadYF-TV] and rAAV2/9 serotype vectors (n=10 capsid mutants per serotype) packaging a ubiquitously expressing GFP reporter construct were generated by inserting heptameric peptides (7AA) at position 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 first assessed on HEK293T cells using a picogreen dsDNA quantitation assay, fluorescence microscopy, and flow cytometry. After isolating and culturing primary bovine VECs, all vectors were subsequently applied at MOI=75,000. After 72 hours, cells were stained with CD31, and transduction efficiency quantified using flow cytometry.
All VEC targeting mutants packaged successfully and vectors at MOI=10,000 were found to be infectious in HEK293T cells, resulting in widespread GFP expression. Flow cytometry revealed that capsid mutant 5 demonstrated significantly increased normalized GFP expression in CD31+ primary bovine VECs in all serotypes, with 1.7-fold, 2.7-fold and 3.6-fold higher transduction efficiency in rAAV2/9, 2/2 and 2/2[QuadYF-TV], respectively.
Generating rAAV vectors capable of efficiently targeting retinal VECs is an essential first step towards the development of a successful gene therapy treatment for ocular diseases such as DR and AMD. Our initial findings indicate that incorporation of endothelial targeting peptides in the rAAV capsid is well tolerated and able to significantly alter vector tropism, leading to increased VEC transduction efficiency.
This is a 2021 ARVO Annual Meeting abstract.
This PDF is available to Subscribers Only