Improving retinal vascular endothelial cell tropism through rational rAAV capsid design.
Ramesh PeriasamyDwani D PatelSanford L BoyeShannon E BoyeDaniel M LipinskiPublished in: PloS one (2023)
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 are effective at delivering new genetic material to neural and glial cells within the retina, but targeting VECs remains challenging. To overcome this limitation, herein we developed rAAV capsid mutant vectors with improved tropism towards retinal VEC. rAAV2/2, 2/2[QuadYF-TV], and rAAV2/9 serotype vectors (n = 9, 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 (VP 1-3). The packaging and transduction efficiency of the vectors were assessed in HEK293T and bovine VECs using Fluorescence microscopy and flow cytometry, leading to the identification of one mutant, termed EC5, that showed improved endothelial tropism when inserted into all three capsid serotypes. Intra-ocular and intravenous administration of EC5 mutants in C57Bl/6j mice demonstrated moderately improved transduction of the retinal vasculature, particularly surrounding the optic nerve head, and evidence of sinusoidal endothelial cell transduction in the liver. Most notably, intravenous administration of the rAAV2/2[QuadYF-TV] EC5 mutant led to a dramatic and unexpected increase in cardiac muscle transduction.
Keyphrases
- optic nerve
- endothelial cells
- optical coherence tomography
- diabetic retinopathy
- age related macular degeneration
- wild type
- gene therapy
- flow cytometry
- disease virus
- high glucose
- high dose
- dengue virus
- cancer therapy
- vascular endothelial growth factor
- single molecule
- high resolution
- induced apoptosis
- skeletal muscle
- left ventricular
- klebsiella pneumoniae
- gene expression
- oxidative stress
- spinal cord
- high fat diet induced
- dna methylation
- low dose
- escherichia coli
- cell cycle arrest
- drug delivery
- metabolic syndrome
- high throughput