One size fits all: Transcriptional upregulation of a disease modifier gene as a mutation-independent approach in muscular dystrophy

ASHG 2018 Annual Meeting – research using mouse models

Guest post by Dwi Kemaladewi (ASHG talk: Wednesday, 6:15pm – 6:30pm in Room 6F)

Identification 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.

At the American Society of Human Genetics conference, I will be presenting our work on 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 α2, which compromises muscle fibers stability and axon myelination in peripheral nerves. Transgenic overexpression of Lama1, encoding a structurally similar protein Laminin α1, ameliorates muscle wasting and paralysis in the MDC1A mouse models, demonstrating its role as a protective disease modifier. 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 packaged these CRISPR/dCas9 components into AAV-9, which has high serotype in skeletal muscles and nerves, injected into dy2j/dy2j mouse model of MDC1A and assessed whether systemic upregulation of Lama1 would yield therapeutic benefits.

Indeed, 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. In this work, we also 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.

Collectively, 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.

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