Novel Approach to treat Wet Age-Related Macular Degeneration (wAMD) and other Retinal Vascular Diseases

Abstract

Purpose : Develop an enhanced PhotoDynamic Therapy (ePDT) for wAMD. Deliver designer phototoxic molecules selectively to vascular endothelial cells (vECs) of pathological angiogenesis of retina/choroid. Use light specifically absorbed by the expressed phototoxic molecules to create a toxic dose of reactive oxygen species and targeted vEC cell death.

Methods : VLDLR-/- and C57BL/6(J) mice were sedated with avertin. OCT images were recorded (Bioptigen: UHR-SD-OCT). Intravenous fluorescein angiography (IVFA) images were recorded following a bolus injection of fluorescein sulfate via tail vein (Butler and Sullivan, 2015). Histological sections were from embedded frozen eyes. The pKillerRed and pSuperNova plasmids were transfected into HEK293S cells (Lipofectamine 2000). Cells were exposed to focused excitation light (Butler et al., 2007). SYTOX green assessed cell death. Liposomes were prepared by sonication method, DSPC (mol. 25%), cholesterol (mol. 50%), DOTAP (mol. 15%), DSPE-PEG/ DSPE-PEG-RGD peptide (mol. 10%). Varying PoP (Porphyrin phospholipid: PDT agent) liposomes were made by reducing the amount of DSPC with increasing PoP%. DPPG and DSPC liposomes were made by replacing DOTAP with the corresponding lipid. In binding/uptake studies U87 cells were incubated with liposomes, washed, and imaged prior to lysis and quantitating fluorescence (plate reader).

Results : Florid formation of pathologically leaky choroidal/retinal neovacularization occurred in the VLDLR-/- mouse starting at 3 weeks. Lead candidate phototoxic genes (PTG) pKillerRed^CYTO (X:585 nm/M:610 nm) and pSuperNova (X:579 nm/M:610nm) were non-toxic (room light) and exhibited high levels of fluorescence in cells relative to alternative PTG tested. Cellular arrays were efficiently killed through light-dependent phototoxicity. Selectively targeted delivery of RGD-decorated DSPC/DOTAP lipid vesicles to U87 cells expressing a_vb₃ integrin receptors in their cell membrane provides one vector for delivery of ePDT.

Conclusions : The VLDLR-/- mouse models leaky outer retinal pathological angiogenesis (e.g. wAMD) for testing ePDT efficacy/toxicity. Lead candidate light-dependent suicide gene products KillerRed^CYTO, SuperNova) are only toxic in cells (e.g. vECs) when exposed to excitation light (~580 nm). Positively charged DOTAP-RGD liposomes selective delivered payload to U87 cells expressing α_vβ₃ integrins.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.