Periodic Reporting for period 4 - EYEGET (Gene therapy of inherited retinal diseases)
Reporting period: 2021-07-01 to 2022-12-31
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.
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.
Within 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 have demonstrated the efficacy of this strategy in mouse models. In addition we have set-up a novel mutation-independent genome editing strategy, i.e. than can be applied to any Rhodopsin mutation, that is based on homology-independent targeted integration of a donor DNA at the Rhodopsin locus. This approach, based on subretinal delivery of AAV, results in therapeutic efficacy in a mouse model of dominant retinitis pigmentosa. Finally, we have developed human 3D retinas in a dish which contain cells which resemble photoreceptors and we have successfully tested some of the gene therapy platforms developed within EYEGET in this human model.
EYEGET has contributed to overcome some of the major challenges in AAV gene therapy: the AAV limited cargo capacity, by developing systems, like AAV-intein, which allow to express large proteins in the retina; counteracting gain-of-function mutations causing dominant retinal diseases by both allele-specific and mutation-independent genome editing approaches; developing 3D human retinal organoids from both normal individual and patients cells which are precious human models in a dish to test gene therapy approaches. The novel gene therapy/genome editing platforms developed and tested within EYEGET have the potential to become therapeutic tools for diseases affecting the retina as well as other tissues that are targeted by AAV vectors.