CombGeneTher · Gene-Independent Combination Therapy for Rod-Cone Dystrophy

Gene-Independent Combination Therapy for Rod-Cone Dystrophy

Inherited retinal dystrophies (IRDs) are genetic disorders causing progressive photoreceptor and retinal pigment epithelium (RPE) degeneration, often leading to blindness. Over 250 genes are implicated, including autosomal dominant, recessive, and X-linked mutations. Examples include retinitis pigmentosa (RP), involving >75 genes, and Leber’s Congenital Amaurosis type 2 (LCA2), caused by RPE65 deficiency. The first approved gene therapy, Luxturna, uses an adeno-associated virus (AAV) vector to deliver RPE65, demonstrating the potential of AAV-based treatments. Due to the large number of causative genes, developing mutation-specific therapies is impractical. Broadly applicable approaches targeting common disease mechanisms, such as neuroprotection, or addressing loss of light perception, such as optogenetic restoration, are therefore prioritized. AAVs are non-pathogenic DNA viruses with multiple serotypes and engineered variants exhibiting distinct retinal tropisms. For instance, AAV2.7m8 enables intravitreal transduction of photoreceptors and RPE, as well as infection and transduction of retinal organoids. In rod-cone dystrophies, rod loss leads to secondary cone degeneration. Rods secrete rod-derived cone viability factor (RdCVF), a product of the NXNL1 gene, which promotes cone glucose uptake via the BSG1–GLUT1 complex. Its isoform, RdCVFL, acts intracellularly as an antioxidant thioredoxin. Both factors have shown therapeutic benefits in rodent RP models, maintaining cone structure and function after AAV- or protein-based delivery. Combined expression of RdCVF and RdCVFL further enhances cone preservation, supporting their use as mutation-independent therapies. Nonetheless, once cones lose their outer segments, neuroprotection alone cannot restore vision. Optogenetic therapy offers a solution by expressing light-sensitive proteins in remaining retinal neurons. Microbial opsins such as halorhodopsin (NpHR) and Jaws have restored light responses and vision-guided behavior in rodent and primate models when targeted to cones using AAV2.7m8 and the cone-specific PR1.7 promoter. Jaws has also demonstrated functional expression in human photoreceptor cells, validating its translational potential. Next-generation opsins, GtACR1 and GtACR2, exhibit higher light sensitivity and lower activation thresholds than Jaws, although its use in the retina remained unexplored and requires safety validation due to its blue-shifted spectrum. This project proposed a combined AAV-mediated therapy co-expressing RdCVF and a hyperpolarizing opsin (e.g., Jaws or GtACR) in cones. The strategy aims to (1) sustain cone viability and function through RdCVF/RdCVFL’s trophic and antioxidant effects, and (2) restore light sensitivity via optogenetic activation of dormant cones, offering a unified, mutation-independent approach to treat rod-cone dystrophies. Objective 1: Establish functional expression of RdCVF, RdCVFL, Jaws, and stGtACR2 in rd10 mice and compare channel properties. Progress under this objective began with the design and production of AAV constructs to express the therapeutic and optogenetic genes. The laureate developed multiple plasmid cassettes, including dual-promoter systems to enable co-expression of RdCVF and RdCVFL—a configuration not well established in the literature. Initial constructs (AAV2-7m8-PR1.7-GFP-CAG-mCherry and AAV2-7m8-CAG-mCherry-PR1.7-GFP) showed partial expression in mouse retinas, confirming activity of one promoter but highlighting the technical challenge of achieving simultaneous dual-gene expression. Successful expression of Jaws-GFP was achieved in mouse retinas, while stGtACR2-mCherry failed to express, likely due to issues with its soma-targeting sequence. Alternative constructs (GtACR1-mCherry and GtACR2-mCherry) have since been developed and are under evaluation. Ethical approval for experiments in rd10 mice has been submitted to the French authorities, and in vivo testing will begin once expression optimization in organoids is complete. Objective 2: Evaluate cone function in animals treated with RdCVF, RdCVFL, and either Jaws or stGtACR2. This objective has been delayed pending optimization of AAV expression systems in Objectives 1 and 3. Preparations for animal studies are underway, and ethical authorization is being finalized. These experiments will proceed once robust co-expression of RdCVF and the selected opsin has been achieved. Objective 3: Express RdCVF, RdCVFL, and either Jaws or stGtACR2 in human iPSC retinal organoids and assess cone function. Significant progress has been achieved toward this objective. Human iPSC-derived retinal organoids were used to evaluate AAV infectivity and gene expression while adhering to the 3Rs principle (Replacement, Reduction, Refinement). The laureate successfully expressed Jaws-GFP in retinal organoids, while stGtACR2-mCherry showed no detectable expression. New constructs expressing GtACR1-mCherry and GtACR2-mCherry are currently being tested. A collaboration with Dr. Kate Grieve’s group (Institut de la Vision) led to the development of a novel imaging approach using dynamic full-field optical coherence tomography (D-FF-OCT) to quantify light-induced cellular responses in organoids. This non-invasive method enables longitudinal imaging of living tissue and has revealed wavelength-dependent metabolic responses in Jaws-GFP-expressing cones. Similar imaging is ongoing for GtACR1-mCherry and RdCVF constructs to assess functional rescue. Cross-Objective Technical Developments: During vector development, high variability in AAV titer measurements (vg/mL) was identified as a key bottleneck, affecting reproducibility of organoid transductions. The laureate systematically optimized the titering protocol, resulting in the project’s first publication, “Impact of DNase digestion on titer measurements of engineered adeno-associated virus serotypes.” To further enhance transduction efficiency and therapeutic protein levels, additional AAV capsids beyond AAV2-7m8 and AAV9-2YF are being explored. Overall Status: Objective 1: Partial progress; Jaws expression confirmed, GtACR1/GtACR2 testing ongoing. Objective 2: Delayed pending vector optimization and ethical approval. Objective 3: Strong progress; Jaws-GFP and RdCVF expression confirmed, functional imaging underway. Output: One peer-reviewed publication, establishment of new AAV expression systems, and development of an innovative D-FF-OCT assay for retinal organoids.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз