Hyatt, E. | Search bioRxiv

bioRxiv2023

Generation and characterization of a novel mouse model of Becker Muscular Dystrophy with a deletion of exons 52 to 55

Becker Muscular Dystrophy (BMD) is a rare X-linked recessive neuromuscular disorder caused by in-frame deletions in the DMD gene that result in the production of a truncated, yet functional, dystrophin protein. BMD is often considered a milder form of Duchenne Muscular Dystrophy, in which mutations typically result in the disruption of the reading frame and the malfunction or loss of dystrophin. The consequences of BMD-causing in-frame deletions on the organism are more difficult to predict, especially in regard to long-term prognosis. Here, we employed CRISPR-Cas9 technology to generate a new Dmd del52-55 mouse model by deleting exons 52-55, resulting in a typical BMD-like in-frame deletion. To delineate the long-term effects of this deletion, we studied these mice over 52 weeks. Our results suggest that a truncated dystrophin is sufficient to maintain wildtype-like muscle and heart functions in young mice. However, the truncated protein appears insufficient to maintain normal muscle homeostasis and protect against exercise-induced damage at 52 weeks. To further delineate the effects of the exons 52-55 in-frame deletion, we performed RNA-Seq pre- and post-exercise and identified several differentially expressed pathways that could explain the abnormal muscle phenotype observed at 52 weeks in the BMD model. Summary StatementWe generated and characterized the long-term effects of a Becker Muscular Dystrophy-like in-frame deletion of exon 52 to 55 in mice.

genetics↗

bioRxiv2018

A mutation-independent approach via transcriptional upregulation of a disease modifier gene rescues muscular dystrophy in vivo.

Introductory paragraphIdentification of protective and/or pathogenic genetic modifiers provides important insight into the heterogeneity of disease presentations in individuals affected by neuromuscular disorders (NMDs), despite having well-defined pathogenic variants. Targeting modifier genes to improve disease phenotypes could be especially beneficial in cases where the causative genes are large, structurally complex and the mutations are heterogeneous. Here, we report a mutation-independent strategy to upregulate expression of a compensatory disease-modifying gene in Congenital Muscular Dystrophy type 1A (MDC1A) using a CRISPR/dCas9-based transcriptional activation system. MDC1A is caused by nonfunctional Laminin 2, which compromises muscle fibers stability and axon myelination in peripheral nerves 1. Transgenic overexpression of Lama1, encoding a structurally similar protein Laminin 1, ameliorates muscle wasting and paralysis in the MDC1A mouse models, demonstrating its role as a protective disease modifier 2. Yet, upregulation of Lama1 as a postnatal gene therapy is hampered by its large size, which exceeds the current genome packaging capacity of clinically relevant delivery vehicles such as adeno-associated viral vectors (AAVs). In this study, we sought to upregulate Lama1 using CRISPR/dCas9-based transcriptional activation system, comprised of catalytically inactive S. aureus Cas9 (dCas9) fused to VP64 transactivation domains and sgRNAs targeting the Lama1 promoter. We first demonstrated robust upregulation of Lama1 in myoblasts, and following AAV9-mediated intramuscular delivery, in skeletal muscles of dy2j/dy2j mouse model of MDC1A.\n\nWe therefore assessed whether systemic upregulation of Lama1 would yield therapeutic benefits in dy2j/dy2j mice. When the intervention was started early in pre-symptomatic dy2j/dy2j mice, Lama1 upregulation prevented muscle fibrosis and hindlimb paralysis. An important question for future therapeutic approaches for a variety of disorders concerns the therapeutic window and phenotypic reversibility. This is particularly true for muscular dystrophies as it has long been hypothesized that fibrotic changes in skeletal muscle represent an irreversible disease state that would impair any therapeutic intervention at advanced stages of the disease. Here, we demonstrate that dystrophic features and disease progression were significantly improved and partially reversed when the treatment was initiated in symptomatic 3-week old dy2j/dy2j mice with already-apparent hind limb paralysis and significant muscle fibrosis.\n\nCollectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR/dCas9-mediated modulation of a disease modifier gene, which opens up an entirely new and mutation-independent treatment approach for all MDC1A patients. Moreover, this treatment strategy provides evidence that muscle fibrosis can be reversible, thus extending the therapeutic window for this disorder. Our data provide a proof-of-concept strategy that can be applied to a variety of disease modifier genes and a powerful therapeutic approach for various inherited and acquired diseases.

genetics↗