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Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model

Gene Therapy volume 29, pages 520–535 (2022)Cite this article

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A Comment to this article was published on 22 April 2022

Abstract

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and β1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD.

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Fig. 1: Structure of the dystrophin associated protein complex and of the different micro-dystrophin constructs evaluated in this study.
Fig. 2: Comparison of DAPC proteins abundance relative to MD/dystrophin between co-IP from rat skeletal and cardiac muscles obtained from the different experimental groups.
Fig. 3: Vector biodistribution and expression in tissues of DMDmdx rats injected with rAAV2/9-MD1 and rAAV2/9-MD3 vectors.
Fig. 4: Histopathological analysis in muscles of DMDmdx rats injected with rAAV2/9-MD1 and rAAV2/9-MD3 vectors.
Fig. 5: Analysis of calcium homeostasis in skeletal muscles and isolated cardiomyocytes of DMDmdx rats injected with rAAV2/9-MD1 and rAAV2/9-MD3 vectors.
Fig. 6: Analysis of muscle force in DMDmdx rats injected with rAAV2/9-MD1 and rAAV2/9-MD3 vectors.
Fig. 7: Analysis of cardiac function in DMDmdx rats injected with rAAV2/9-MD1 and rAAV2/9-MD3 vectors.

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Data availability

The data that support the findings of this study are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank all the personnel of the Boisbonne Center for Gene Therapy (ONIRIS, INSERM, Nantes, France) and of the UTE IRS2 (University of Nantes, France) for the handling and care of the rats included in this study. We also thank the vector core of UMR 1089 (CPV, INSERM and University of Nantes) for the cloning of the MDs pAAV plasmids and the production of the rAAV vectors used in this study. We thank the Mass Spectrometry and Proteomics Facility (Ohio State University, Columbus, Ohio, USA), which performed the proteomics experiments. We thank E. Marrosu (UCL Great Ormond Street Institute of Child Health, London, United Kingdom) for providing MANEX1011B and MW8 antibodies for co-immunoprecipitation experiments. We thank G. Potier for his valuable help during the analyses of label free quantitation. We also thank T. Cronin for editing the English language of this manuscript. Finally, we thank the MDA Monoclonal Antibody Resource for providing the MANEX 1011 C antibody.

Funding

This project was supported by the MDA (Muscular Dystrophy Association, Research Grant ID #513878), the AFM-Téléthon (Association Française contre les Myopathies), the “Fondation d’entreprise pour la thérapie génique en Pays de la Loire”, INSERM, INRA, the University of Nantes and the University Hospital of Nantes. The Fusion Orbitrap instrument used for proteomics study was supported by NIH Award Number Grant S10 OD018056.

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Authors and Affiliations

  1. Translational Gene Therapy Laboratory, Nantes Université, CHU Nantes, INSERM UMR, 1089, Nantes, France

    Audrey Bourdon, Virginie François, Bodvael Fraysse, Tiphaine Girard, Cyrielle Lebreton, Anna Creismeas, Marine Allais, Basile Marie, Justine Guguin, Véronique Blouin, Corinne Huchet, Philippe Moullier, Oumeya Adjali & Caroline Le Guiner

  2. Mass Spectrometry and Proteomics Facility, Campus Chemical Instrument Center, The Ohio State University, Columbus, OH, USA

    Liwen Zhang

  3. Therassay platform, Capacités, Nantes Université, Nantes, France

    Aude Lafoux & Corinne Huchet

  4. L’Institut du Thorax, Nantes Université, CNRS, INSERM UMR, 1087, Nantes, France

    Gilles Toumaniantz & Agnès Hivonnait

  5. INRAE, Oniris, PAnTher, APEX, Nantes, France

    Thibaut Larcher & Mireille Ledevin

  6. Center for Research in Transplantation and Immunology, INSERM UMR, 1064, Nantes, France

    Séverine Remy & Ignacio Anegon

  7. Department of Biological Sciences, Centre of Biomedical Sciences, Royal Holloway, University of London, Egham, UK

    Alberto Malerba, Betty Kao, Anita Le Heron, George Dickson & Linda Popplewell

  8. Asklepios BioPharmaceutical, Inc, Research Triangle Park, NC, USA

    Philippe Moullier

  9. UCL Great Ormond Street Institute of Child Health, Developmental Neurosciences Program, London, UK

    Federica Montanaro

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  1. Audrey Bourdon
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Contributions

Study design and reviewing of the data: AB, VF, FM, CLG. Experimental investigation: AB, LZ, AL, BF, GT, TL, TG, ML, CL, AH, AC. Resources: VF, MA, BM, JG, VB, SR, IA, CH, AM, BK, ALH, LP and FM. Writing – original draft: AB, LZ, AL, BF and CLG. Writing – Review & Editing: all authors. Supervision: CLG and OA. Funding acquisition: CLG, PM, GD and OA.

Corresponding author

Correspondence to Caroline Le Guiner.

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Competing interests

PM, GD and CLG are co-authors of a patent for systemic treatment of dystrophic pathologies (EP3044319A1, dated 27 June 2014). GD is an inventor on a PCT for production of large-sized micro-dystrophins in an AAV-based vector configuration (PCT/EP2016/060350, dated 17 May 2015). The remaining authors declare no competing financial interests.

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Bourdon, A., François, V., Zhang, L. et al. Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model. Gene Ther 29, 520–535 (2022). https://doi.org/10.1038/s41434-022-00317-6

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  • DOI: https://doi.org/10.1038/s41434-022-00317-6

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