Abstract
Purpose :
First-line anti-VEGF therapies for diabetic macular edema (DME) are efficacious in around half of DME patients and repeated administrations are associated with complications.
Anti-VEGF therapies fail to address inflammation and retinal cell loss associated with this disease and furthermore, hemolysis has been observed in primates treated with aflibercept through IgG-Fc gamma receptors. Therefore, we have designed a gene therapy to address the complex pathophysiology of DME that requires a single ocular injection.
Methods :
Multi-cistronic plasmids and rAAV2/2 vectors expressing a novel anti-VEGF component and Tie2 receptor agonist together with a retinoprotective PEDF polypeptide were evaluated in HEK293T, ARPE-19 cells and co-cultures of human fibroblast/HUVECs. HUVEC monolayers were grown on transwells to assess permeability (TEER). The lead vectors were evaluated using rodent models.
Results :
In a co-culture model of angiogenesis, lead constructs demonstrated significantly reduced capillary length and branch number versus controls equivalent to aflibercept (capillary length (mm); control = 13.7 ± 0.5, IKC113 = 3.8 ± 0.4***, aflibercept = 3.9 ± 0.3***; mean ± SEM of 12 replicates; ***P<0.001 by ANOVA with Bonferroni modified t-tests). A lead construct reduced angiopoietin-2 concentrations (ng/mL) in the co-culture medium (control = 1.29 ± 0.20, IKC113 = 0.09 ± 0.00***, aflibercept = 0.06 ± 0.00***; mean ± SEM of 3 replicates; ***P<0.001). Furthermore, a lead construct, acting through the Tie2 agonist component, attenuated TNF-alpha (TNFα)-induced reduction in TEER (Ohms/cm2) over 24h in HUVEC monolayers (control minus TNFα = 23.5 ± 0.3, control Null plasmid plus TNFα = 15.1 ± 0.6, IKC113 plasmid plus TNFα = 20.1 ± 0.4; mean ± SEM of 3-6 replicates; **P<0.001 versus Null plasmid with TNFα) demonstrating reduced cytokine-induced vascular permeability. In the laser CNV study a lead tri-cistonic rAAV demonstrated a 60% reduction in CNV leakage compared to the vehicle control.
Conclusions :
In addition to robust anti-VEGF activity, we have demonstrated that the multi-cistronic rAAV2/2 gene therapy has the ability to reduce vascular leakage and inflammation through Tie2 receptor activation. These novel gene therapies are set to become a valuable new treatment option for patients with DME.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.