Expression of large and highly functional dystrophins using split inteins and myotropic AAV vectors


Pre-Clinical Research

Poster Number: S28


Hichem Tasfaout, PharmD, PhD, University of Washington, Christine L. Halbert, PhD, University of Washington, James M. Allen, PhD, University of Washington, Tim S. McMillen, PhD, University of Washington, Galina V. Flint, PhD, University of Washington, Stephen D. Hauschka, PhD, University of Washington, Michael Regnier, PhD, University of Washington, Jeffrey S Chamberlain, MD, University of Washington School of Medicine

Adeno-associated viral (AAV) vector-based therapies are promising for treating many genetic disorders. Recent years have witnessed tremendous progress in capsid design and the identification of serotypes with enhanced tissue-specific tropism. However, AAV’s modest packaging capacity (~4.7 kb) remains an important constraint, limiting their broad application to many genetic disorders with large genes, such as Duchenne muscular dystrophy (DMD). With an 11.2 kb dystrophin coding region, it has not been possible to take full advantage of the promising potential of AAV-based gene replacement therapies for DMD. As an alternative, our group and others have developed a series of miniaturized forms, also known as micro-dystrophins, which can be delivered by a single AAV. Although these truncated dystrophins are functional, many clones are unstable or show incomplete rescue when tested in DMD animal models or patients. This suggests that the expression of larger dystrophins with additional functional domains is necessary to fully protect and improve strength in DMD muscles.
Here, we present SIMPLI-GT (Split Intein-Mediated Protein Ligation for Gene Therapy), a novel method allowing the expression of large and highly functional proteins. This approach exploits the protein trans-splicing mechanism mediated by split inteins to ligate precisely and seamlessly multiple polypeptide fragments into a functional protein. We identified several 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 delivering two or three AAV vectors results in a strong expression of large and functional dystrophins with significant improvements in muscle histology and force development. Moreover, using the potent myotropic AAVMYO, we demonstrate that a low dose of 2e13 vg/kg (10-fold lower compared to doses used in preclinical or clinical studies with conventional serotypes) is sufficient to express large dystrophins in striated muscles bodywide, leading to significant functional correction of the DMD phenotype. Our data show a clear superiority of large dystrophins over miniaturized forms when tested in young or old dystrophic mice.
This novel strategy addresses emerging challenges of AAV-based gene replacement. It can be adapted to other genetic disorders caused by loss-of-function mutations in large genes with a coding sequence beyond AAV capacity.