Dual-acting gene therapy targeting HIF1A and HIF2A by RNA interference mitigates retinal degeneration in two mouse models of AMD

Abstract

BackgroundThe combination of reduced choroidal blood flow, increased Bruchs membrane (BM) thickness and drusen formation leads to reduced oxygenation of the outer retina in the aging eye and contributes to the pathology of age-related macular degeneration (AMD). This implies that the molecular response of photoreceptors to hypoxia, with chronic activation of hypoxia-inducible factors (HIFs) at its core, impacts disease development and progression.

MethodsWe used the shRNAmiR system to develop a dual-acting gene therapy based on a single AAV that reduces activity of HIF1 in photoreceptors and HIF2 in the retinal pigment epithelium (RPE). The virus was injected subretinally in two models of pseudo (Rod{Delta}Vhl) or true (RPE{Delta}Vegfa) hypoxia-related retinal degeneration and treated mice were followed for up to 61 weeks post-injection. Light microscopy, fluorescence funduscopy, and optical coherence tomography were used to quantify the therapeutic effect. In situ hybridization, real-time PCR, Western blotting, immunofluorescence, and flatmounts of the retina, RPE, and choroid were used to investigate the disease models and therapeutic effects of the treatment.

ResultsNo adverse effects were noted after subretinal injection of the AAV expressing shRNAs targeting Hif1a in photoreceptors and Hif2a in the RPE. The virus preserved ONL thickness and photoreceptor segment length in Rod{Delta}Vhl mouse retinas up to 22 weeks and in RPE{Delta}Vegfa mice up to 61 weeks after injection demonstrating a long-lasting rescue of the phenotype. The dual-acting virus showed significantly higher efficacy than single-acting viruses targeting solely Hif1a in photoreceptors or Hif2a in the RPE.

ConclusionThis study introduces a novel dual-acting AAV vector that effectively downregulates two different genes in two specific cell types, offering a promising therapeutic strategy for complex diseases such as AMD. By simultaneously targeting Hif1a in photoreceptors and Hif2a in the retinal pigment epithelium, this gene-agnostic therapy shows significant potential to protect retinal tissues from chronic hypoxic conditions. By targeting a common and conserved disease pathway in AMD, it is applicable to a wide range of patients.