Yuan Zhou 1,2, Chen Zhang 1, Weidong Xiao 1, Roland W Herzog 1, Renzhi Han 1,✉
Posted: July 21, 2024
Abstract
Current gene therapy for Duchenne muscular dystrophy (DMD) utilizes adeno-associated virus (AAV) to deliver micro-dystrophin (µDys), which does not provide full protection for striated muscles as it lacks many important functional domains of full-length (FL) dystrophin. Here we develop a triple vector system to deliver FL-dystrophin into skeletal and cardiac muscles. We split FL-dystrophin into three fragments linked to two orthogonal pairs of split intein, allowing efficient assembly of FL-dystrophin. The three fragments packaged in myotropic AAV (MyoAAV4A) restore FL-dystrophin expression in both skeletal and cardiac muscles in male mdx4cv mice. Dystrophin-glycoprotein complex components are also restored at the sarcolemma of dystrophic muscles. MyoAAV4A-delivered FL-dystrophin significantly improves muscle histopathology, contractility, and overall strength comparable to µDys, but unlike µDys, it also restores defective cavin 4 localization and associated signaling in mdx4cv heart. Therefore, our data support the feasibility of a mutation-independent FL-dystrophin gene therapy for DMD, warranting further clinical development.
Subject terms: Genetic vectors, Neuromuscular disease
Current gene therapy for Duchenne muscular dystrophy utilizes adeno-associated virus to deliver a partially functional copy of truncated dystrophin. Here, Zhou et al. present a split intein strategy to systemically deliver full-length dystrophin.
NeoBiotechnologies’ products were used in this study:
Cell pellets and mouse tissue samples were lysed using a cold radioimmuno-precipitation analysis buffer (RIPA) supplemented with 1x protease inhibitor cocktail (Thermo Scientific, 78440). Protein concentrations were measured to ensure uniform loading (Bio-Rad DC protein assay kit, 5000111). Proteins were separated using 4–15% precast SDS-PAGE gel (Bio-Rad, 17000927) and transferred onto 0.45 μm nitrocellulose membranes (Bio-Rad, 1620115). After blocking with 5% non-fat dry milk, membranes were incubated with an anti-dystrophin antibody (anti-N-terminus: MANHINGE1B (10F9) or MANHINGE1C (5D12), 1:100, Developmental Studies Hybridoma Bank, Iowa City, IA, USA; anti-M-fragment: MANEX50 (6A9), 1:100, Developmental Studies Hybridoma Bank, Iowa City, IA, USA, or rabbit polyclonal DMD/8773 R, 1:200, NeoBiotechnologies, Union City, CA, USA; anti-C-terminus: ab15277, 1:1000, Abcam, Cambridge, UK), mouse monoclonal anti-p44/42 MAPK (Cell Signaling Technology, 4696, 1:1000), rabbit polyclonal anti-Phospho-p44/42 MAPK (Cell Signaling Technology, 9101, 1:500), or rabbit monoclonal anti-GAPDH antibody (Cell Signaling Technology, 2118 S, 1:2000). Subsequently, membranes were washed and incubated with Horseradish peroxide (HRP)-conjugated goat anti-mouse (7076S, 1:4000, Cell Signaling Technology) and goat anti-rabbit (7074S, 1:4000, Cell Signaling Technology) secondary antibodies. Chemiluminescent detection was employed using enhanced chemiluminescence (ECL) western blotting substrate (Pierce Biotechnology, Rockford, IL, USA), capturing the signal by ChemiDoc XRS+ system (Bio-Rad). Western blots were quantified using ImageJ 1.54f software.
Publication History:
Nat Commun. 2024 Jul 21;15:6141. doi: 10.1038/s41467-024-50569-6
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