Gene-Agnostic Metabolic Engineering in Inherited Retinal Degenerations

Abstract

Inherited retinal degenerations (IRDs) culminate in non-cell-autonomous cone loss following rod failure and destabilization of outer-retinal metabolism. This dissertation tests whether compartment-specific metabolic reprogramming in rods, cones, and the retinal pigment epithelium (RPE) can preserve cone structure and function independent of genotype in etiologically diverse mouse models, including phosphodiesterase 6B (6), rhodopsin (), and membrane frizzled-related protein () mutant lines. The studies herein establish that metabolism can be therapeutically redirected across these compartments, supporting a strategy that complements gene-specific augmentation while extending protection to most patients without access to tailored genetic therapies. By reframing retinal degeneration as a disorder of metabolic ecosystem collapse, this work lays the conceptual and experimental foundation for therapies that are both mutation-agnostic and scalable, with potential relevance to common degenerative conditions such as age-related macular degeneration. Aim 1 (rods – 1) used 6⌃(⁶²⁰/⁶²⁰) and ⌃(¹¹⁰/⁺) mice to modulate rod metabolism via conditional prolyl-hydroxylase disruption(PHD) [6-CreERT2] and dual-AAV CRISPR editing of 1. Outcomes combined electroretinography (ERG), optical coherence tomography (OCT), histology, cone flatmounts, lactate assays, and [U-¹³C]glucose tracing. PHD2 disruption induced a Warburg-like shift via enhanced ¹³C labeling of glycolytic intermediates with increased phospho-pyruvate dehydrogenase 1 (Ser293), without elevating bulk lactate, preserving cone morphology, and improving cone-mediated ERG across recessive and dominant models. One-year fluorescein angiography showed no neovascularization. These results demonstrate that rod-specific glycolytic reprogramming through PHD2 disruption preserves cones by stabilizing carbon flux and redox balance, establishing 1 as a dominant lever for mutation-agnostic metabolic rescue in murine rods. Aim 2 (cones – 1) targeted cones in 6⌃(¹/¹) and 6⌃(⁶²⁰/⁶²⁰) mice with 1 deletion [3-CreERT2] and evaluated photopic ERG, cone flatmounts, and whole-retina qPCR/immunoblotting of canonical nuclear factor erythroid 2-related factor 2 (NRF2) targets. 1 loss yielded consistent structural rescue, higher opsin-positive cone counts, and healthier segment morphology. While functional gains were modest, bulk assays showed minimal induction of canonical NRF2 targets. This suggests cone protection may arise through subtle redox stabilization or structural preservation rather than broad transcriptional reprogramming, refining the paradigm of NRF2-mediated rescue. Aim 3 (RPE – ) reprogrammed the RPE in ⌃(⁶/⁶) mice by deleting [65-CreERT2] and assessed longitudinal ERG/OCT, cone flatmounts, RPE whole-mount morphology, and [U-¹³C]palmitate tracing to -hydroxybutyrate (BHB). loss increased ¹³C-palmitate incorporation into BHB and qualitatively preserved RPE architecture, with a mid-course plateau in outer nuclear layer thinning, late-stage scotopic ERG improvements, and significant peripheral cone preservation. This indicates that mutation-agnostic RPE reprogramming can secondarily stabilize photoreceptors in the background of IRD. These experiments demonstrate that tuning rod glycolysis, cone redox balance, and RPE fattyacid -oxidation provide complementary, mutation-agnostic protection of cone vision, establishing retinal metabolism as a practical therapeutic axis alongside gene-specific repair. By showing that interventions in distinct retinal compartments converge on the shared goal of cone preservation, this work reframes IRDs not only as collections of genetic defects but as disorders of metabolic interdependence. This perspective expands the therapeutic landscape beyond mutation-specific augmentation, highlighting metabolism as a scalable axis applicable across the genetically heterogeneous spectrum of IRDs. Moreover, by identifying nodal regulators that can be targeted with genetic or pharmacologic tools, these findings create a translational bridge to common late-stage diseases such as age-related macular degeneration, where metabolic instability is a central driver of pathology. Together, this dissertation establishes metabolism-centered therapy as a unifying framework that can complement precision medicine and reshape how retinal degeneration is treated in both rare and common contexts