Periodic Reporting for period 1 - CombGeneTher (Gene-Independent Combination Therapy for Rod-Cone Dystrophy)
Periodo di rendicontazione: 2023-09-01 al 2025-08-31
Sintesi del contesto e degli obiettivi generali del progetto
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.
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.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
During the reporting period, significant progress was made toward establishing and optimizing the tools required to evaluate RdCVF, RdCVFL, and optogenetic proteins (Jaws, GtACR1, GtACR2) in both mouse models and human iPSC-derived retinal organoids.
1. Vector Design and Production
- A comprehensive series of AAV2-7m8 constructs were designed and produced to drive expression of RdCVF, RdCVFL, and inhibitory opsins under photoreceptor-specific (PR1.7) and ubiquitous (CAG) promoters.
- Dual-promoter vectors (PR1.7-GFP-CAG-mCherry and CAG-mCherry-PR1.7-GFP) were developed to enable co-expression of RdCVF and RdCVFL—an innovative design rarely implemented in gene therapy.
- Although only single-promoter activity was initially observed, these experiments established the foundation for troubleshooting and optimization of dual-gene expression systems.
2. Optimization of Gene Expression in Retinal Organoids and Mice
- Jaws-GFP was successfully expressed in both human retinal organoids and C57Bl6/J mouse retinas, confirming effective AAV transduction and functionality of the PR1.7 promoter in photoreceptors.
- The stGtACR2-mCherry construct failed to express, likely due to the soma-targeting sequence; this led to the design of GtACR1 and GtACR2 alternatives, which are currently under evaluation. Preliminary data show GtACR1-GFP exhibits higher light responsiveness than Jaws-GFP in retinal organoids.
3. Methodological Advances and Collaboration
- In collaboration with Dr Kate Grieve’s team at the Institut de la Vision, a novel dynamic full-field optical coherence tomography (D-FF-OCT) method was established for non-invasive, real-time imaging of light-evoked responses in retinal organoids.
- This new imaging technique provides quantitative, longitudinal assessment of cellular activity without tissue dissociation and represents a major methodological innovation for functional analysis of optogenetic constructs.
4. Technical Breakthrough: AAV Titering Optimization
- High variability in AAV quantification (vg/mL) was identified as a major bottleneck. The laureate optimized the DNase digestion-based titering method, improving reproducibility across experiments.
- This work resulted in the project’s first publication, “Impact of DNase digestion on titer measurements of engineered adeno-associated virus serotypes,” which has been positively received by the AAV gene therapy community.
5. Ongoing and Future Work
- Dual-promoter cassette troubleshooting continues in retinal organoids to achieve co-expression of RdCVF/RdCVFL prior to in vivo validation in neonatal mice.
- Combined RdCVF + Jaws-GFP and RdCVF + GtACR1-GFP studies are underway, with D-FF-OCT-based functional analyses providing quantitative measures of light response and metabolic activity.
- Work is expanding to include retinal organoid lines modeling retinal degeneration, to test therapeutic efficacy in a disease-relevant system and reduce animal use in subsequent studies.
1. Vector Design and Production
- A comprehensive series of AAV2-7m8 constructs were designed and produced to drive expression of RdCVF, RdCVFL, and inhibitory opsins under photoreceptor-specific (PR1.7) and ubiquitous (CAG) promoters.
- Dual-promoter vectors (PR1.7-GFP-CAG-mCherry and CAG-mCherry-PR1.7-GFP) were developed to enable co-expression of RdCVF and RdCVFL—an innovative design rarely implemented in gene therapy.
- Although only single-promoter activity was initially observed, these experiments established the foundation for troubleshooting and optimization of dual-gene expression systems.
2. Optimization of Gene Expression in Retinal Organoids and Mice
- Jaws-GFP was successfully expressed in both human retinal organoids and C57Bl6/J mouse retinas, confirming effective AAV transduction and functionality of the PR1.7 promoter in photoreceptors.
- The stGtACR2-mCherry construct failed to express, likely due to the soma-targeting sequence; this led to the design of GtACR1 and GtACR2 alternatives, which are currently under evaluation. Preliminary data show GtACR1-GFP exhibits higher light responsiveness than Jaws-GFP in retinal organoids.
3. Methodological Advances and Collaboration
- In collaboration with Dr Kate Grieve’s team at the Institut de la Vision, a novel dynamic full-field optical coherence tomography (D-FF-OCT) method was established for non-invasive, real-time imaging of light-evoked responses in retinal organoids.
- This new imaging technique provides quantitative, longitudinal assessment of cellular activity without tissue dissociation and represents a major methodological innovation for functional analysis of optogenetic constructs.
4. Technical Breakthrough: AAV Titering Optimization
- High variability in AAV quantification (vg/mL) was identified as a major bottleneck. The laureate optimized the DNase digestion-based titering method, improving reproducibility across experiments.
- This work resulted in the project’s first publication, “Impact of DNase digestion on titer measurements of engineered adeno-associated virus serotypes,” which has been positively received by the AAV gene therapy community.
5. Ongoing and Future Work
- Dual-promoter cassette troubleshooting continues in retinal organoids to achieve co-expression of RdCVF/RdCVFL prior to in vivo validation in neonatal mice.
- Combined RdCVF + Jaws-GFP and RdCVF + GtACR1-GFP studies are underway, with D-FF-OCT-based functional analyses providing quantitative measures of light response and metabolic activity.
- Work is expanding to include retinal organoid lines modeling retinal degeneration, to test therapeutic efficacy in a disease-relevant system and reduce animal use in subsequent studies.
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
Despite substantial progress in vector design, methodological development, and in vitro testing, several key objectives remain to be completed to fully achieve the project’s goals:
1. Optimization of Dual-Promoter AAV Constructs
- The dual-promoter system enabling co-expression of RdCVF and RdCVFL has not yet yielded simultaneous expression of both genes.
- Ongoing work focuses on testing alternative promoter orientations, spacer sequences, and bidirectional promoter configurations to ensure balanced and stable expression.
- Verification of expression in human retinal organoids is required before moving to in vivo studies in neonatal mice.
2. Validation of GtACR1 Construct
- While GtACR1-GFP shows promising responsiveness in retinal organoids, quantitative comparison with Jaws-GFP is still underway.
- Functional characterization of these optogenetic channels will be completed using D-FF-OCT imaging, multi-electrode array and patch clamp.
3. In Vivo Testing (Objective 2)
- Ethical approval for the use of rd10 mice is pending. Once obtained, combination therapy experiments involving RdCVF/RdCVFL with Jaws-GFP or GtACR1-GFP will begin.
- Long-term follow-up studies (up to 8 months post-injection) are required to assess cone survival, function, and safety of the therapeutic approach.
4. Functional Assessment in Degenerating Retinal Organoids
- The laureate plans to test the combination therapy (RdCVF + optogenetic channel) in patient-derived retinal organoids showing photoreceptor degeneration.
- These experiments will provide preclinical validation in a disease-relevant human model and support refinement of the in vivo design.
5. Quantitative Functional Analysis
- Further optimization of D-FF-OCT imaging parameters is needed to standardize quantification of metabolic and light-response data across constructs.
- Integration of D-FF-OCT data with complementary transcriptomic and protein expression analyses will provide a comprehensive understanding of therapeutic effects.
1. Optimization of Dual-Promoter AAV Constructs
- The dual-promoter system enabling co-expression of RdCVF and RdCVFL has not yet yielded simultaneous expression of both genes.
- Ongoing work focuses on testing alternative promoter orientations, spacer sequences, and bidirectional promoter configurations to ensure balanced and stable expression.
- Verification of expression in human retinal organoids is required before moving to in vivo studies in neonatal mice.
2. Validation of GtACR1 Construct
- While GtACR1-GFP shows promising responsiveness in retinal organoids, quantitative comparison with Jaws-GFP is still underway.
- Functional characterization of these optogenetic channels will be completed using D-FF-OCT imaging, multi-electrode array and patch clamp.
3. In Vivo Testing (Objective 2)
- Ethical approval for the use of rd10 mice is pending. Once obtained, combination therapy experiments involving RdCVF/RdCVFL with Jaws-GFP or GtACR1-GFP will begin.
- Long-term follow-up studies (up to 8 months post-injection) are required to assess cone survival, function, and safety of the therapeutic approach.
4. Functional Assessment in Degenerating Retinal Organoids
- The laureate plans to test the combination therapy (RdCVF + optogenetic channel) in patient-derived retinal organoids showing photoreceptor degeneration.
- These experiments will provide preclinical validation in a disease-relevant human model and support refinement of the in vivo design.
5. Quantitative Functional Analysis
- Further optimization of D-FF-OCT imaging parameters is needed to standardize quantification of metabolic and light-response data across constructs.
- Integration of D-FF-OCT data with complementary transcriptomic and protein expression analyses will provide a comprehensive understanding of therapeutic effects.