Summary by Tasneem Al Mudarris

Protein stitching to create a full-length dystrophin: a go-to strategy for large genes?

Publication: Expression of full-length dystrophin reverses muscular dystrophy defects in young and old mdx4cv mice

Hichem Tasfaout, Timothy S. McMillen, Theodore R. Reyes, Christine L. Halbert, Rong Tian, Michael Regnier, and Jeffrey S. Chamberlain

J Clin Invest. 2025 Jun 10;135(15):e189075. doi: 10.1172/JCI189075. eCollection 2025 Aug 1.

https://pubmed.ncbi.nlm.nih.gov/40493400/

Summary

Duchenne muscular dystrophy (DMD) is common and severe form of muscular dystrophy caused by loss of expression/function mutations in the X-linked DMD gene. In affected boys with causative DMD variants, the disease is relentlessly progressive and uniformly fatal. Currently, treatment is centered around optimised supportive care and glucocorticoid therapy. There is a critical unmet need for the development and translation of new transformative therapeutics for DMD.

Genetic medicines, including ASO mediated exon skipping and AAV delivered micro dystrophin, have shown great promise for DMD in pre-clinical models and have been tested in clinical trials. However, none have shown dramatic benefit nor have any substantially impacted current clinical care and disease trajectory. One key limitation to existing gene therapy strategies has been that they do not promote production of, or provide via replacement, a full-length version of the Dystrophin transcript and protein.

Dystrophin is a large gene (approximately 12 kb) that far exceeds the carrying capacity of AAV vectors. In fact, micro dystrophin, which is the favored cargo of current gene therapy programmes, represents a version of DMD that is only 1/3 of its total size.  Among potential approaches for overcoming this size limitation while still taking advantage of AAVs ability to deliver to skeletal muscle and to provide persistent ‘single dose’ treatment, protein trans-splicing is perhaps the most attractive. First reported in Nature and now followed up in this featured manuscript in the Journal of Clinical Investigation, senior author Jeff Chamberlain, first author Hichem Tasfaout, and members of their research teams present the groundbreaking application of split inteins to achieve production of full-length dystrophin in a mouse model of Duchenne muscular dystrophy.

Tasfaout et at utilised an approach called protein trans-splicing to combine fragments of Dystrophin together to produce a full length, fully function version of the protein. The process they employed uses specific protein sequences, called inteins, that bind to each other, excise themselves, and ligate the remainder of the fragments (exteins) together.

Specifically, the authors used approximately equal amounts of three AAVMYO1 vectors containing contiguous fragments of intein-dystrophin to create full-length protein in mdx4cv mice. AAVMYO1 has high muscle tropism (as compared to AAV9), enabling a cumulative dose that is well within the predicted safe and therapeutic range. They used intein sequences that they previously demonstrated to have high efficiency of trans splicing. They tested this therapy in young and old mdx4cv mice, and the results were dramatic. In young mice, they detected high expression of full-length dystrophin in the Tibialis anterior (TA) muscle, diaphragm, and the heart three months post injection. Treated muscle fibres were essentially rescued, with significant amelioration of dystrophic pathology, as reflected by myofiber size, collagen content, and general histology. Additionally, muscle force was significantly improved. Importantly, in mice treated at a much older age (17 months), where significant advanced dystrophic changes and ‘clinical’ weakness were clearly demonstrable, they were able to detect important resolution of disease features, in both skeletal muscle (TA and diaphragm) and in the heart. In total, Tasfaout et al. concluded that gene replacement therapy with a full-length dystrophin protein, as achieved with protein trans-splicing delivered using multiple AAVs, can fully rescue young mdx mice, and can provide substantial benefit (including to key target tissues like diaphragm and heart) in even very old mice.

In summary, this work presents a promising technique to overcome the limiting packaging capacity of AAV, which may be helpful in considering gene therapy not only for DMD, but also for a whole group of disorders caused by mutations in large genes, including gene products that are not amenable to miniaturisation. Dr. Tasfaout told us: “I am truly excited about this work and the potential to deliver large and challenging proteins, such as dystrophin. The proof-of-concept study in a mouse model of Duchenne muscular dystrophy demonstrates the feasibility of this approach and shows significant improvements in muscle force, histology, and alleviation of heart defects. I hope this work will provide a new class of therapies that will benefit patients with muscular dystrophies.”

For this promising research to advance to the clinic for testing, there are still important areas that require exploration. Additional studies are needed to confirm that the by-products of trans-splicing (i.e. un-spliced fragments as well as the resulting intein sequences) are not toxic to the cell and do not promote a significant immune response. There are also questions related to efficiency (i.e. will sufficient trans-splicing occur in human muscle) and to the safety and tolerability of delivering three MYOAAVs. In all, though, given the strength of the data presented in this study, it will be exciting to see how research using this promising strategy of split inteins for gene therapy moves forward in the coming years.

Figure: Schematic of Protein Trans-splicing with Split inteins. Tasfaout et all used 3 MYOAAVs to deliver fragments of dystrophin fused to intein sequences. Matching intein sequences identify each other upon expression and then promote the fusion of the fragments they are linked with. The resulting paired inteins are removed, and the Dystrophin fragments (i.e. exteins) are stitched together to create a seamless full length Dystrophin protein.

About the author

Dr. Hichem Tasfaout is an Assistant Professor in the Neurology Department of the University of Washington (Seattle, WA). His research interests focus on developing novel gene therapy method that combines split inteins and myotropic vectors to deliver and express large proteins. Using this approach, multiple protein fragments are delivered specifically to striated muscles using potent myotropic adeno-associated viral (AAV) vectors. Upon their expression, these fragments are then joined into highly functional proteins.

About the reviewer

Tasneem Al Mudarris is an MSc student at the University of Toronto. She is part of the Dowling lab at the Research Institute of the Hospital for Sick Children (Toronto, Canada). Her research focuses on investigating a gene therapy for nebulin-based nemaline myopathy. She has been working on zebrafish and mouse models in the past two years to unravel some exciting nebulin science.