E - Disease Models and Clinical Applications -> Musculo-skeletal Diseases
2: Split Intein-Mediated Protein trans-Splicing to Express Large Dystrophins
Type: General Session
Presentation Details
Session Title: Presidential Symposium and Presentation of Top Abstracts
Location: West Hall B
Start Time: 5/17/2023 14:45
End Time: 5/17/2023 15:00
Gene replacement therapies mediated by Adeno-Associated Viral (AAV) vectors represent a promising approach for treating genetic diseases. Despite tremendous progress in capsid design and identification of novel serotypes with higher tropism and transduction activity an important constraint on using AAVs is their modest packaging capacity (~4.7 kb). This size restriction can significantly limit their application for many genetic disorders involving large genes, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the 2.2 MB dystrophin gene that has an 11.2 kb coding region. Affected patients develop body-wide muscle wasting and die from cardio-respiratory failure in their late teens to early 30s. Our group has pioneered the development of miniaturized forms of dystrophin (including micro-dystrophins) that can fit within and be transported by a single AAV vector. Although these smaller dystrophins are surprisingly functional, many clones tested thus far are unstable or showed incomplete rescue of the dystrophic phenotype when tested in DMD animal models and in patients. These observations suggest that the expression of larger dystrophins with additional functional domains is necessary to fully protect from, or reverse, muscle pathophysiology. Here, we present SIMPLI-GT (Split Intein-Mediated Protein Ligation for Gene Therapy), a novel method that allows the expression of large and stable proteins with high specificity and efficiency. This approach exploits the intrinsic ability of split inteins to ligate seamlessly multiple polypeptide fragments into a functional protein via a protein trans-splicing mechanism. We identified several split intein pairs that can efficiently join two or three fragments and generate, respectively, a large midi-dystrophin or the entire full-length dystrophin. In a proof-of-concept study, we show that the delivery of two or three AAV vectors results in a strong expression of large and functional dystrophins with a significant improvement of muscle histology and force development. Moreover, using the potent myotropic AAVMYO capsid, we demonstrate that a low dose of 2e13 vg/kg (10-fold lower compared to what is used in clinical studies) is sufficient to express large dystrophins in striated muscles bodywide with an almost complete physiological rescue. Our data show a clear superiority of large dystrophins over miniaturized forms when tested in young or very old dystrophic mice. This novel strategy addresses emerging challenges of AAV-based gene replacement. If successful with DMD, it can be adapted to many other genetic disorders caused by loss-of-function mutations in large genes with a coding sequence beyond AAV packaging capacity.
Hichem Tasfaout1,2, Christine L. Halbert1,2, James M. Allen1,2, Tim S. McMillen3, Galina V. Flint3, Stephen D. Hauschka2,4, Michael Regnier2,3, Jeffrey S. Chamberlain1,2
1Department of Neurology, University of Washington School of Medicine, Seattle, WA,2Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Washington School of Medicine, Seattle, WA,3Department of Bioengineering, College of Engineering and School of Medicine, University of Washington, Seattle, WA,4Department of Biochemistry, University of Washington School of Medicine, Seattle, WA
H. Tasfaout: None.
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