Therapeutics for the chorioretina

National Eye Institute

Investigators

Linked publications & trials

Abstract

We completed work demonstrating that deletion of Pnpla2 (PEDF-R) causes structural and functional defects in mouse photoreceptors. We also finalized a study on H105A peptide eye drops that promoted photoreceptor survival in murine and human models of retinal degeneration. Photoreceptor death underlies inherited blinding retinal diseases such as retinitis pigmentosa (RP). Because disease progression often outpaces therapeutic development, effective treatments are urgently needed. We evaluated the efficacy of PEDF-derived peptides, which restrict common cell death pathways associated with retinal disease. Chemically synthesized peptides (17-mer and H105A) with affinity for the PEDF receptor (PEDF-R) were delivered as eye drops to two RP mouse models, rd10 (phosphodiesterase 6b mutation) and RhoP23H/+ (rhodopsin P23H mutation), and AAV-H105A vectors were engineered for intravitreal delivery in RhoP23H/+ mice (collaboration with Dr. Marigo). Retinal organoids exposed to cigarette smoke extract (collaboration with N Vergara) were used to assess peptide effects in human tissue. Photoreceptor survival, morphology and function were evaluated. Both peptides promoted photoreceptor survival and improved retinal function in RP mouse models, while H105A prevented in human organoids. These results support PEDF peptide-based eye drops as a minimally invasive therapy for retinal degeneration. We continued evaluating the neurotrophic activity of modified H105A PEDF peptides in rd10 mouse photoreceptors. Peptides with various N- and C-terminal modifications, amino acid substitutions, or incorporation of non-alpha-amino acid linker were administered topically as eye drops. Photoreceptor cell death was assessed in vivo by phosphatidylserine (PS) externalization using PSVue™ and fundoscopy, and postmortem by BAX and BCL2 immunolabeling. Retinal morphology and function were evaluated by histology and electroretinography (ERG). Several PEDF peptides derived from H105A demonstrated translational potential for retinal degenerative diseases. Full-length PEDF[H105A] was also tested for protective effects against oxidative stress-induced retinal degeneration in a rabbit model (collaboration with P Campochiaro). This study provided a platform for surgical and drug intervention studies. To assess PSVue as a diagnostic tool for RP, PSVue eye drops were administered to rd10, Rpe65-/-, and RhoP23H+/- mice at ages P15 to P90 to monitor photoreceptor cell death over time. Fundoscopy of a phosphatidylserine-binding conjugate of Bis (zinc (II)-dipicolylamine) (PSVue®) was used to detect phosphatidylserine externalization in vivo, and retinal morphology and outer nuclear layer thickness was assessed histologically. Visual function was evaluated by ERG, establishing the natural history of photoreceptor cell death and demonstrating correlation between PSVue imaging, histology and ERG findings. We investigated the role of Pnpla2, a lipid-regulating gene, in RPE senescence and aging relevant to AMD. RPE flat mounts and retinal cryosections from Pnpla2-/- and Pnpla2+/+ mice were analyzed at three months of age. Senescence-associated-β-galactosidase activity and multinucleation were quantified, while immunofluorescence assessed tight junctions and markers of senescence and AMD (ZO-1, P-γ-H2AX, ApoE, HMGB1). Fundus imaging and ERG assessed retinal structure and function. Pnpla2-/- RPE exhibited elevated senescence markers, multinucleation, HMGB1 cytoplasmic translocation, disrupted tight junctions, increased P-γ-H2AX-positive RPE cells, and ApoE accumulation in Bruch’s membrane and subretinal regions. ERG c-wave attenuation progressed with age and white spots appeared by seven months in Pnpla2+/- mice. These findings indicate that Pnpla2 deficiency accelerates RPE aging and induces AMD-like features, highlighting its importance in maintaining retinal health. We studied epithelial–mesenchymal transition (EMT) in RPE using iPSC-derived RPE cells from AMD patients and a control individual. RPE differentiation was confirmed by morphology, RPE marker expression, and tight junction formation. EMT was induced, and changes were assessed by immunocytochemistry and functional assays, including wound healing and transwell migration. Treatment with the GSK3 inhibitor CHIR99021 maintained epithelial morphology, restored tight junctions, reduced EMT markers, and reversed pro-inflammatory factor-induced changes. RNA-seq analysis revealed gene expression differences between untreated and treated cells, demonstrating that GSK3 inhibition both suppressed and reversed EMT in RPE, and identifying CHIR99021 as a promising candidate for preventing RPE dysfunction in AMD. We investigated potential interventions for AMD using hiPSC-derived RPE cells from AMD patients and a control line. RPE differentiation and long-term culture were performed on standard or Matrigel-coated plates. Lipid accumulation was assessed by Bodipy staining and complement factor H (CFH) expression was evaluated by immunostaining. Dome-like structures, indicative of pigment epithelial detachment, were monitored throughout the culture period to track AMD-like pathology. This approach provided a platform to evaluate the effects of Matrigel on structural and molecular features associated with AMD in hiPSC-RPE cells.