Abstract
Purpose :
Anti-VEGF therapies for neovascular age-related macular degeneration (nAMD) have significantly revolutionised outcomes for millions of people. However, anti-VEGF drug efficacy and vision deteriorates in a significant proportion of individuals due to subretinal fibrosis. We designed a gene therapy capable of preventing, or reversing, subretinal fibrosis and neutralising pathological VEGF concentrations responsible for neovascularisation.
Methods :
Bi-cistronic plasmids and rAAV2/2 vector constructs expressing both anti-VEGF and anti-fibrotic components were examined in HEK293T, ARPE-19 cells and on co-cultures of human fibroblast/HUVECs. The lead constructs were evaluated in rodent models.
Results :
The lead constructs express a novel anti-VEGF capture protein that has a reduced affinity for human IgG-Fc gamma receptors than aflibercept. The constructs reduce endogenous VEGF concentrations (ng/mL) in HEK293Ts (control Null plasmid = 663 ± 15, IKC116P = 275 ± 7***, aflibercept = 296 ± 4***; P<0.001 by ANOVA followed by Bonferroni modified t-tests, mean ± SEM of 3 replicates) and attenuates capillary formation in co-cultures with equivalent efficacy to aflibercept. The anti-fibrotic component attenuates transforming growth factor (TGF)-beta-induced epithelial-mesenchymal transition, a precursor to subretinal fibrotic scar formation, as shown by the reduction in ARPE-19 fibronectin (control Null plasmid = 2.64 ± 0.32, IKC116P = 1.96 ± 0.26; P<0.05, n=4) and release of matrix metalloprotease-2 (control Null plasmid = 1.46 ± 0.09, IKC116P = 0.28 ± 0.04; P<0.0001, n=4) by western blot. Intravitreal injection of the lead vectors completely prevented vascular leakage after laser-induced CNV in mice compared to the vehicle controls.
Conclusions :
The novel anti-VEGF component is equipotent to aflibercept in VEGF neutralisation. Moreover, the incorporation of an anti-fibrotic transgene reduced TGF-beta induced early scar formation in ARPE-19 cells. This bi-cistronic gene therapy has the potential to address subretinal fibrosis in patients with deteriorating vision currently treated with anti-VEGF therapies.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.