2025 Theses Doctoral

Mutation Agnostic Genome Editing Strategies for Treating Neuroretinal Degenerations

Caruso, Salvatore Marco

Inherited retinal disorders (IRDs) represent a collection of highly genetically heterogeneous neurodegenerative disorders including retinitis pigmentosa that ultimately result in blindness. While there has been substantial preclinical progress in precision medicines for more common forms of retinal degenerations, therapeutic development for rare mutations unfortunately remains handicapped by burdensome developmental costs. Currently, there is only one FDA approved treatment for retinitis pigmentosa, which addresses a highly narrow subset of patients with mutations specifically in the RPE65 gene. For the remaining >100,000 patients in the United States alone, there exists no treatment.

To develop commercially viable therapeutics with the potential for real world applications, we have focused our research efforts on two key fronts: 1) a metabolic reprogramming strategy aimed at treating the universal metabolic dysregulation that underlines disease progression and occurs regardless of genetic background and 2) an epidemiologically informed gene editing approach capable of addressing the most common forms of retinitis pigmentosa.

Naturally, the retina’s metabolic ecosystem relies on the highly glycolytic photoreceptors (PRs) to produce a carbon substrate that the supportive RPE can consume as an alternative carbon source to glucose. However, during retinal degeneration, these cells can become metabolically uncoupled, resulting in the RPE consuming glucose that is essential for PR survival and effectively starving the distal neuroretina.

To drive glycolysis in the PRs and provide more lactate to the RPE, thus restoring metabolic homeostasis, we have targeted hypoxia-inducible factors (HIFs)—well characterized regulators of cell metabolism that are abundantly expressed in the retina. By ablating their negatively regulating binding partner, Von Hippel-Lindau (VHL), specifically in the photoreceptors of disease-modeling mice, we demonstrate a HIF-dependent metabolic reprogramming that drives glycolysis, preserves cells, and improves functionality in a preclinical model of retinitis pigmentosa. Furthermore, we uncovered evidence that suggests a crosstalk mechanism from PRs to RPE where genetically unperturbed RPE cells experience altered metabolism that is essential for therapeutic effect.

While metabolic reprogramming has the potential to treat a wide range of diseases and thus overcome the financial barriers of rare disease drug development, these approaches do not address the underlying genetic defects causing degeneration and may not be curative. To this end, we have developed an AAV-based, therapeutic prime editing strategy generalized to treat multiple mutations in the most implicated IRD gene—rhodopsin (RHO). By capitalizing on a highly heterozygous SNP found within the 5’ untranslated region (UTR), we can selectively silence the mutant allele via a prime editing mediated deletion, leaving the wildtype allele intact to support basic biological function. To assess our optimized therapeutic in vivo, we report a humanized mouse model of retinitis pigmentosa possessing the targeted SNP in addition to the clinically relevant, heterozygous P347L dominant negative mutation that phenocopies clinical presentation of the disease.

Our findings demonstrate two unique and commercially viable therapeutic strategies that can address a wide base of retinitis pigmentosa patients for whom there is no current treatment, while also providing a framework for therapeutics capable of expanding to the neurodegenerations of the CNS and beyond.