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Novel use of exon skipping technology to study structure-function relationship of dystrophin

Periodic Reporting for period 1 - DYS_FUNCTION (Novel use of exon skipping technology to study structure-function relationship of dystrophin)

Reporting period: 2016-01-01 to 2017-12-31

Patients with Duchenne or Becker muscular dystrophy either lack dystrophin or produce a non-functional version of dystrophin, leading to progressive skeletal muscle and heart disease. Heart disease is currently poorly managed with cardiac medications and has become a major cause of death in these patient populations. Very promising new treatment approaches (gene therapy and exon skipping) that lead to expression of a shorter but functional form of dystrophin are currently effective in skeletal muscles but sub-optimal for preventing heart failure. One primary obstacle to their optimization is the lack of information on specific regions of dystrophin that are important for cardiac function and need to be preserved by these new treatment strategies. Therefore, the primary objective of this fellowship was to establish whether a specific region of dystrophin (Hinge 3) that is often absent in shorter dystrophins produced by exon skipping and gene therapy approaches plays a role in heart disease. Our studies have identified a new region adjacent to Hinge 3 that is important for preventing heart disease. We have further determined why this region is important for heart function and have gained important insights into the mechanisms underlying heart disease in Duchenne and Becker muscular dystrophies. This new knowledge will enable the future development of optimized gene therapy and exon skipping strategies that can more effectively protect patients from heart failure. In addition, this knowledge can help identify patients that are at higher risk of early onset heart disease and of heart failure based on genetic testing.
The primary objectives of this study were 1) to experimentally test our prediction that short dystrophins with altered structure in the Hinge 3 region have impaired cardiac function, and 2) to demonstrate the use of the leading exon skipping chemistry (Pip Chemistry) as a tool to study structure-function relationship of dystrophin disease in vivo in both cardiac and skeletal muscles.

Collaborative efforts with the MDEX consortium and the laboratories of Profs. Muntoni (University College London), Wood (Oxford University) and Gait (Cambridge University) have led us to conclude that the Pip chemistry can successfully target the heart and improve cardiac function in animal models of Duchenne muscular dystrophy. However, it requires further optimization before it can be successfully used in healthy animals to study structure-function relationships as proposed in our fellowship application. As a result, the MDEX consortium has established new collaborations with industry and academia to further develop the Pip chemistry as well as explore new chemistries.

Efforts directed at determining the cardio-protective properties of the Hinge 3 region of dystrophin have yielded important new knowledge. I have identified a specific region of dystrophin close to Hinge 3 that mediates a new association with caveolae, specialized membrane structures that play essential functions in cardiac contraction and whose disruption causes cardiac disease in animals and humans. I have shown that micro-dystrophin constructs for gene therapy that lack this region cannot interact with caveolae. In collaboration with the laboratory of Dr. Duan (University of Missouri), we have demonstrated that addition of this region to micro-dystrophin restores the association with caveolae and leads to normalization of cardiac contraction and protection from cardiac disease.

This work has resulted in a recent publication in Human Molecular Genetics (Wasala et al., 2018, Hum Gene Ther., PMID: 29433343) and I am preparing a second manuscript for submission to Circulation Research (a top leading journal in cardiac research). I have also presented the data as an invited speaker at the MRC neuromuscular centre seminar series, the Developmental Neurosciences seminar series, and the Myology forum conference. The research has also been disseminated in community outreach activities. In particular, it has been highlighted on the MDUK website that is accessed by scientists and lay members of the community, including patients and their families. I have also participated in a community outreach special initiative to introduce young children and families to science and neuromuscular disorders.
The impact of the proposed project relies on the successful identification of a region of dystrophin near Hinge 3 that is important for cardio-protection. This information will be used to 1) optimize micro-dystrophin gene therapy constructs; 2) inform the design of exon skipping therapies; and 3) provide a more accurate cardiac prognosis to patients with mutations in dystrophin.

Socio-economic impact: This fellowship has created new jobs and will lead to the opening of additional future positions through successful exploitation of the data to obtain multi-year grants and additional fellowship support. To date the data generated through this Marie Curie fellowship has allowed me to secure a second fellowship from Duchenne Parent Project-Netherland to further support my career progression at UCL, as well as a multi-year grant from Muscular Dystrophy UK that has helped me establish my lab and build a research team. New research assistant positions have been created as part of the establishment of my laboratory in the UK. Public engagement with UCL students has resulted in the recruitment of a PhD student to my laboratory. The identification of a cardio-protective domain will lead to new research by my team into a new improved micro-dystrophin gene therapy design, with intellectual property potential down the line. My incorporation into the MDEX consortium has opened new avenues of research into cardiac targeting for exon skipping therapies. As a result new collaborations with academia and industry have been initiated for new product development efforts.

Societal implications: The new knowledge generated by this fellowship has important clinical and translational implications for patients with Duchenne and Becker muscular dystrophy and their families. In the short term, this will lead to optimization of gene therapy constructs and exon skipping strategies. Using the resources of the Dubowitz Neuromuscular Centre in genetics/diagnostics and of EU patient databases, we are planning a targeted comparative analysis of cardiac disease in patients with mutations that affect or spare this new cardio-protective region of dystrophin. This knowledge will be essential in predicting cardiac risk for individual patients based on their genetic mutation.
Hinge 3 region of dystrophin