Atypical Pathological Signaling as a Target to Regulate Retinal Vasculopathies - Over the next five years, more than 2-3 million patients will fail to respond to anti-VEGF therapy or display only partial therapeutic benefits. Furthermore, anti-VEGF itself can cause healthcare problems in patients. Therefore, there is a critical need to identify alternative anti-angiogenic and anti-inflammatory targets and selective therapeutics to supplement or replace current treatment strategies. Dysregulated vasculogenesis and vascular homeostasis are central drivers of retinal diseases, including retinopathy of prematurity (ROP) and diabetic retinopathy (DR). An understudied pathological atypical mitogen-activated protein kinase (MAPK) p38 pathway is one such alternative pathway. Our recent studies elucidated the molecular regulation of atypical inflammatory responses in vascular cells. Furthermore, preliminary studies demonstrate that a genetic mutation blocking atypical p38 protects the retina from vaso-obliteration and neovascular tufting in an oxygen-induced retinopathy (OIR) model. Despite strong evidence supporting the central role of p38 in many retinal diseases, prior promising broad-range p38 therapeutics have failed due to the inhibition of all physiological and pathological signaling. Conversely, atypical p38 utilizes an independent pathway activated only in response to disease signaling, representing an alternative therapeutic target. This project overcomes current limitations by developing models to investigate and selectively target atypical p38 signaling. Our central hypothesis is that atypical p38 activation plays a central role in propagating vascular inflammatory and angiogenic responses in retinal disease. We will explore two specific aims to test this hypothesis: Aim 1: Defining Atypical p38-Dependent Regulation of Retinal Proangiogenic, Proinflammatory Signaling, and Neovascularization. Using our OIR model, we will further define protection from pathological neovascular responses in an atypical p38 deficient C57BL/6 mouse. These data will be confirmed in a retinal explant model and 3D sprouting spheroids of human retinal cells, and an established cell-penetrating peptide inhibitor. Using cytokine arrays, RNAseq, and high-resolution microscopy, we will examine the role of atypical p38 in retinal neovascularization. Aim 2: Characterization of Intrabodies to Block Atypical Signaling in Retinal Vascular Cells. We have developed an array of inhibitory nanobodies (IBi) to selectively block atypical p38 signaling. We will characterize the capacity of adeno-associated virus (AAV1) dependent delivery of intrabodies to suppress atypical p38 signaling inflammation and neovascular responses in primary human retinal endothelial cells. Additionally assessing AAV1-IBi retinal expression and suppression of OIR. Upon completion of the proposed studies, we expect to have 1) evidence that Tab1-intrabody expression inflammation and angiogenesis in retinal endothelial cells, and 2) a model of atypical p38-enhanced retinal damage during OIR. Critically, AAV-delivered intrabodies are the first generation of atypical p38 inhibitors that can be developed as either a complementary or alternative anti-VEGF strategy to treat vascular retinopathies.
