QUESTFORMD · Quantitative functional assessment of gene therapeutics for Muscular Dystrophy

Quantitative functional assessment of gene therapeutics for muscular dystrophy

Gene therapy holds promise for improving longevity and quality of life in many diseases but trial therapies in people have frequently been unsatisfactory. Duchenne muscular dystrophy is a progressive fatal disease amenable to gene therapy to replace the mutant protein, dystrophin. The QUESTFORMD project aims to develop time- and cost-effective initial screens for gene therapeutics, using the zebrafish. To develop efficient therapy for patients, it is essential to understand the dynamics of the dystrophin-dystroglycan complex assembly and turnover in vivo. Our first goal was to fulfil this unmet need. We adapted an existing technique (fluorescence recovery after photobleaching (FRAP)) and developed a new analysis method combining experimental data and mathematical modelling. A key issue for a gene therapy approach is that dystrophin gene length exceeds the limits of viral vectors. Interestingly, Becker dystrophy (BMD) patients have large dystrophin deletions that result in mild disease. Therefore, short dystrophins can function in people, although some work better than others. Understanding why this happens is essential for designing good therapies because a small amount of a persistent / effective dystrophin form might be more beneficial than a large amount of a less persistent / effective form. A second goal of the project was to test the hypothesis that distinct short dystrophins differ in their ability to replace natural dystrophin. We proposed to use our FRAP analysis method to compare their dynamics with that of full-length dystrophin. We also aimed to address the efficiency of a range of short dystrophins in rescuing the disease symptoms in mutant zebrafish. By collaborating with theoretical physicists, we now have a novel, validated and extensively tested method for quantifying dystrophin dynamics in vivo using FRAP, which is in the final stages of preparation for submission for publication. This includes an open software platform that will be made available for the scientific community. We have found that the membrane bound dystrophin can be in one of two pools: a very stable one with slow turnover, and a very mobile one with fast turnover. The biological significance of each is under study. Analysis of selected Becker dystrophins in under way. We focused on analysing the influence of two independent regions on the stability of the dystrophin protein: the neuronal nitric-oxide synthase (nNOS) binding site and the flexible hinge III. We have found a significant correlation between the absence of the flexible hinge III in some ((delta)45-55 and (delta)50-51) Becker dystrophin proteins and an increased accumulation at the membrane binding sites, suggesting an important conformational change affecting protein behaviour. The partial truncation of the nNOS binding site in (delta)45-47 does not affect the intracellular protein distribution, reinforcing the hypothesis that the absence of hinge III is responsible for the abnormal sub-membrane accumulation. The increased accumulation at sub-membrane binding sites does not translate into increased ability to rescue the zebrafish dmd mutant phenotype. On the contrary, (delta)45-55 shows decreased rescue of dystrophic fibres when compared with full-length dystrophin. How the binding dynamics at the cell membrane is affected is under investigation. The resources and knowledge developed through the QUESTFORMD project are an important step towards improving the planning of gene therapy clinical trials, specifically for Duchenne muscular dystrophy. Our team is part of the International Collaborative Effort for Duchenne Muscular Dystrophy (ICE for DMD) project that brings together researchers and clinicians working at different levels of translational research focused on ameliorating the condition of Duchenne patients and improving treatments.

Data: CORDIS, © European Union