CRX gene therapy in mouse and human models of retinal dystrophies targeting dominant CRX-associated retinopathies – CuRAT

We developed a gene therapy approach to introduce a functional copy of the CRX gene into photoreceptors, the light-sensitive cells of the retina. To do this, we used a vector called AAV, commonly used in medicine to deliver genes into cells. Two experimental models were used to evaluate the therapy’s effectiveness:

O Study in mice with CRX-related retinal diseases

We injected the AAV-CRX vector directly under the retina in two types of disease-model mice. To specifically target photoreceptors (cones and rods), we used a promoter sequence from a gene that is normally active in these cells. Mice were treated between 1 and 2 months of age and monitored for up to a year. Treated mice were compared to those that received a control AAV without therapeutic effect. The effects of the therapy were evaluated using several tests: i) Retinal imaging (microscopy), ii) Vision tests (electroretinography, optomotor reflex, visual behavior), iii) Genetic and protein analyses to assess reactivation of genes required for vision.

o Study in retinal organoids made from patient cells

We generated retinal organoids (lab-grown mini-retinas) from stem cells derived from patients carrying CRX mutations. These organoids were treated with the AAV-CRX vector at different stages of development. The efficacy of the treatment was assessed through i) Fluorescence imaging to visualize treated cells, ii) Genetic and biochemical analyses to verify activation of vision-related genes, iii) Electron microscopy to observe the structure of the cells. All analyses were performed at multiple time points after the start of organoid differentiation.

The goal of all these methods is to determine whether our gene therapy product can restore the expression of essential vision genes and preserve photoreceptor survival, both in animal models and in patient-derived mini-retinas produced in the laboratory.

Mouse study: repaired photoreceptors and improved vision.

In mice with early-onset blindness (CrxRip/+ model), a single subretinal injection of our gene therapy restored the expression of key visual markers, such as rhodopsin and cone arrestin, and led to the reconstruction of functional photoreceptors. These effects were observed up to 10 months after treatment, indicating long-term benefits. Vision tests (electroretinograms and light-driven behavior assays) showed improved visual perception, comparable to that of healthy mice. At the molecular level, analysis of treated cells revealed reactivation of genes involved in vision, neuronal development, and cell protection. The next step will be to refine these results at single-cell resolution.

A second transgenic model, expressing a mutated form of human CRX (CRXR41W model), showed that even in the absence of visible degeneration, gene therapy could correct specific gene expression defects.

Human retinal organoid study: efficacy varies with mutation severity

We generated retinal organoids (lab-grown mini-retinas) from patient-derived cells carrying CRX mutations of varying severity. Organoids from patients with severe forms (LCA) showed a drastic reduction in the expression of visual genes, confirming a major developmental defect. In contrast, those derived from a patient with cone-rod dystrophy (CORD) and an asymptomatic individual displayed subtler differences, demonstrating the sensitivity of this model even to mild abnormalities.

We then applied gene therapy to these organoids:

In severe cases, CRX gene expression was restored, but the expression of other visual genes remained low. In organoids with a moderate phenotype (CORD), therapy reactivated many vision-related genes, including those involved in rod and cone function and light perception, reflecting CRX’s natural role in photoreceptor development.

This study shows that CRX-targeted gene therapy can restore visual function in mice and partially correct genetic defects in human patient-derived retinal organoids. Our gene therapy product offers hope for treating certain forms of inherited blindness, particularly when retinal damage is not yet too advanced.

The promising results obtained with the support of the French National Research Agency (ANR) for our gene therapy targeting rare retinal diseases linked to the CRX gene have attracted the attention of Professor Francesca Simonelli, a world-renowned expert based in Naples, Italy. She reached out to our team to initiate the preparation of a clinical trial for CRX patients. She is already leading a longitudinal study in these patients to thoroughly characterize the disease and its progression, an essential step for their future inclusion in a clinical trial. Building on our findings that support the relevance of such a trial, we now aim to produce the AAV-CRX viral vector under clinical-grade (GMP) conditions and to assess its safety—an essential step before launching human trials. The French biotech company Variant is also involved in advancing the development of this therapy.

Our project has expanded through new collaborations with Dr. Diego Di Bernardo (TIGEM, Italy) and Prof. Roman Jerala (Chemical Institute, Slovenia). Together, we are developing and testing "smart genetic circuits" to finely tune the expression of the therapeutic gene within cells. This technology is especially well-suited to CRX, which is highly dose-sensitive: enough protein must be produced to promote photoreceptor differentiation, but not so much as to cause potential toxicity. Our AAV-CRX vector is thus an ideal candidate for testing these precision-control strategies.

Our RNA-Seq data and deep understanding of CRX’s function have led us to propose a new hypothesis: AAV-CRX therapy might also benefit retinal diseases not directly caused by CRX mutations. We have already demonstrated a neuroprotective effect in a mouse model of retinitis pigmentosa, and additional studies are underway to test its efficacy in other models.

Finally, our analysis of patient-derived human retinal organoids led to an unexpected observation: in a patient carrying a CRX mutation but showing no symptoms, we detected differences in expression between the healthy and mutant alleles. This suggests that a natural compensation mechanism may be at work. We are now investigating this finding further, as it could pave the way for new therapeutic strategies to delay or even prevent disease onset.

Thanks to strong basic research, international collaborations, and solid institutional support, CRX gene therapy is now approaching a major milestone: human clinical trials. At the same time, our work is opening up new therapeutic perspectives for other retinal diseases—and perhaps even for preventing symptoms in currently asymptomatic patients.

Inherited retinal dystrophies (IRDs) are genetically and clinically heterogeneous. Despite tremendous progress in IRD research over the past 10 years, these diseases still lead to legal blindness due to limited therapeutic options. The recent success of gene supplementation therapy in patients with Leber Congenital Amaurosis (LCA) caused by RPE65 mutations has highlighted the real interest in expanding this approach to other IRDs. Our previous work suggests that diseases due to dominant mutations in CRX, a transcription factor essential to photoreceptor development and maturation, are relevant candidates for gene supplementation. To date, over 50 mutations in the CRX gene are responsible for cone-rod dystrophies (CORD), LCA, and retinitis pigmentosa (RP).
Due to the clinical heterogeneity of patients carrying CRX mutations, our proposal aims to restore and/or maintain vision in two mouse models of retinopathies associated with CRX: an LCA model that we have already well characterized, and a novel CORD model. In this context, we will evaluate whether a gene replacement approach is able to compensate for the deleterious effects of CRX mutants for both clinical forms. Towards this aim, a CRX-expressing AAV vector will be administered, and its long-term efficacy evaluated, in both models. The recovery and preservation of retinal function will be tested by electroretinogram and optokinetic reflex recordings, and retinal morphology by optical coherence tomography. This efficacy study will be completed by histological and molecular analyses. Furthermore, in order to better understand disease pathophysiology and to validate a therapeutic approach in humans, we will complement these in vivo studies on iPSC-derived retinal organoids generated from three patients with CRX-associated LCA, CORD, or RP. By analyzing differential gene and protein expression, as well as the morphology of the organoids throughout retinal differentiation and maturation, we will establish a genotype-phenotype correlation and, hence, elucidate the basis for the differential clinical profiles associated with each mutation. Furthermore, we will obtain pertinent read-outs to assay the efficiency of an AAV-mediated gene replacement following the transduction of the patient organoids. In this way, we will determine whether all forms can benefit from our therapeutic approach, without negative effects linked with CRX overexpression.
At the end of this preclinical study, our expectation is to have demonstrated the efficacy of our gene therapy in mouse and human models with dominant CRX mutations. The potential clinical value of this project lies in the development of gene therapy to treat a broad spectrum of CRX-associated retinopathies and might serve as proof of concept for other dominant IRDs. This study may serve as a basis for further development of clinical trials.