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Gene therapy of inherited retinal diseases

Periodic Reporting for period 2 - EYEGET (Gene therapy of inherited retinal diseases)

Reporting period: 2018-07-01 to 2019-12-31

Inherited retinal degenerations (IRDs) with an overall prevalence of 1/2,000 worldwide are a major cause of blindness and affect over 200,000 people in the European Union. Gene therapy holds great promise for the treatment of IRDs, and proof-of-principle of its efficacy and safety in animal models and humans has been provided. Vectors based on the adeno-associated virus (AAV) are the most efficient for retinal gene therapy. Indeed Luxturna, the first ocular gene therapy drug that received market approval and that we contributed to develop, is based on AAV.
EYEGET (EYE GEne Therapy) exploits the versatile and safe AAV platform to overcome the remaining challenges in retinal gene therapy and make this strategy widely applicable to treat genetic blindness:
1. Many recessive IRDs require the transfer of large genes whose size exceeds AAV cargo capacity. Objective 1 of this proposal is to investigate the efficacy of co-injection of multiple AAVs each carrying one part of a large gene which reassemble in target cells.
2. Several common forms of dominant IRDs are due to gain-of-function mutations that result in production of toxic proteins. Objective 2 uses genome editing with CRISPR-Cas9 to either knock-down or correct these mutant alleles.
3. In several recessive and dominant IRDs accumulation of toxic products results in photoreceptor cell death. To address these disorders, we propose to use AAV-mediated gene transfer to induce clearance of these toxic products (Objective 3).
4. In vivo gene therapy can not be applied to late stage IRDs when the majority of photoreceptor cells are lost. In Objective 4 we propose to investigate a novel method to obtain in a dish photoreceptors that can be transplanted in the diseased retinas, with or without previous ex vivo AAV-mediated correction.
During the first 30 months of EYEGET we were able to significantly improve the ability of delivering large genes via AAV vectors to the retina. We have set-up a strategy that uses 3 AAV vectors at the same time which upon recombination in target cells can reconstitute therapeutic DNA which is about 3 times larger than what normally carried by a single AAV. In addition, as this system based on AAV DNA recombination is not efficient enough for some applications, we have designed a new system based on protein trans-splicing, in which two polypeptides flanked by split-inteins fuse together through the inteins which then excise seamlessly from the full length mature protein (a cut-and-paste at the protein level). We have shown that AAV-intein reconstitute large therapeutic proteins defective in IRDs like Stargardt disease or Leber congenital amaurosis 10 to levels that are therapeutic in mouse models of these diseases. We have also implemented AAV-based genome editing to specifically target common Rhodopsin mutations which cause dominant retinitis pigmentosa, and we are testing the efficacy of this strategy in mouse models. Similarly, we have generated AAV vectors delivering TFEB and Beclin-1-derived peptide which induce cellular clearance, and their therapeutic potential is being investigated in mice. Finally, we have developed 3D retinas in a dish which contain cells which resemble photoreceptors.
We believe the EYEGET project has contributed to overcoming one of the major challenges in AAV gene therapy which is AAV limited cargo capacity, by developing systems, like AAV-intein, which allow to express large proteins in the retina. The safety and efficacy of this platform will be further investigated until the end of the project.
We have also observed that genome editing can occur in photoreceptors of mice and pigs and we are currently characterizing whether this has therapeutic relevance in animal models. Similarly, we are testing the efficacy of AAV vectors that express key inducers of cellular clearance and autophagy to enhance degradation of toxic proteins in IRDs.
Finally, we have developed human 3D retina which contain photoreceptor-like cells that we are considering for cell replacement in diseased retinas as well as to model human IRDs in a dish.