Mol Ther. 2022 Jun 1;30(6):2176-2185.
doi: 10.1016/j.ymthe.2022.02.003. Epub 2022 Feb 8.
Comparison of dystrophin expression following gene editing and gene replacement in an aged preclinical DMD animal model
Niclas E Bengtsson, Julie M Crudele, Jordan M Klaiman, Christine L Halbert, Stephen D Hauschka, Jeffrey S Chamberlain
Dystrophin is one of the essential structural proteins that anchors the actin cytoskeleton to the sarcolemma membrane along with other proteins of the dystroglycan complex. The lack or truncated dystrophin protein results in either Duchenne or Becker muscular dystrophy, which results in devastating muscle wasting diseases that affect one in 3,500 to 5,000 male births worldwide. Significant progress has been made over the last few decades towards the development of therapies to treat those patients. There are several ongoing clinical trials that are evaluating single systemic injections of AAV-mediated micro-dystrophin (µDys) constructs (a mini version of dystrophin that is can be find in Becker patients which have a milder version of Duchenne disease). Although these approaches show very promising feasibility and efficacy in infants and young patients with a mild disease phenotype, clinical trials are exploring whether older patients with progressed pathophysiology will show similar outcomes as younger individuals.
In this work, Dr. Bengtsson and colleagues compared two promising approaches using canine X-linked muscular dystrophy (CXMD) model, a dog model, whose phenotype is more similar to human DMD patients. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. AAV.CK8e.µDys5 (3rd generation of micro-dystrophins) and AAV.CK8e.CRISPR/SaCas9 (genome editing) vectors containing two single U6.gRNAs targeting intron 5 and 8 of the canine dystrophin gene. Both vectors were delivered via intramuscular administration into 3- and 8-year-old CXMD, which were sacrificed 6 weeks post injection.
Overall, CXMD dogs treated with µDys5 vector revealed a higher number of dystrophin-positive fibers by immunofluorescent staining and western blot and less centrally nucleated fibers compared with CRISPR-treated dogs. This is consistent with previously published data, but 8-year-old CXMD dogs appeared to show less muscle transduction efficiency than 3-year-old animals regardless of treatment. Digital RT-PCR analyses of samples treated with CRISPR vector revealed the presence of two transcripts lacking exons 6-8 (1.13±0.24%) and exons 6-9 (2.93±0.31%) with the higher frequency of the last one. Both of these transcripts encode the internally but in frame dystrophin protein with a level around 1% detected by WB.
Niclas E. Bengtsson, PhD: Dr. Bengtsson is an Assistant Professor in the department of Neurology at the University of Washington. He received his PhD degree in molecular cell biology from the University of Florida in 2009. Dr. Bengtsson’s research is primarily focused on elucidating key disease mechanisms responsible for pathologies in muscular dystrophies [particularly in Duchenne muscular dystrophy (DMD)], and on the development of effective methods to correct them. His ongoing research efforts are focused on enhancing the applicability, safety, and efficacy of genome editing- and gene replacement strategies to facilitate advancement of these promising approaches towards clinical application.
Liubov Gushchina, PhD: Dr. Gushchina is a Sr. Research Scientist at the Center for Gene therapy, Nationwide Children’s Hospital. She received her PhD degree in molecular biology from the Institute of Protein Research of the Russian Academy of Science in 2009. Liubov Gushchina research interests focuses on viral and non-viral gene therapy development for DMD and LGMDs.
Published on 31 July 2022.
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This article is presented by the Publication Highlights Committee.