NEW THERAPEUTIC APPROACHES AND THEIR READOUT: O.14A mutation-independent approach via transcriptional upregulation of a disease modifier gene rescues muscular dystrophy in vivo

Abstract

Targeting disease modifier genes is an attractive therapeutic strategy in cases where causative genes are large and 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 α2, which compromises muscle fibers stability and axon myelination in peripheral nerves. Transgenic overexpression of Lama1, encoding a structurally similar Laminin α1, ameliorates muscle wasting and paralysis in 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 genome packaging capacity of clinically relevant adeno-associated viral vectors (AAVs). In this study, we sought to upregulate Lama1 using CRISPR/dCas9-based system, comprised of catalytically inactive Cas9 (dCas9) fused to VP64 transcriptional activation domains and sgRNAs targeting the Lama1 promoter. We demonstrated robust upregulation of Lama1 in myoblasts, and following AAV9-mediated intramuscular delivery, in skeletal muscles of dy2j/dy2j mouse model of MDC1A. We then assessed whether systemic upregulation of Lama1 would yield therapeutic benefits in dy2j/dy2j mice. When the intervention started early in pre-symptomatic dy2j/dy2j mice, Lama1 upregulation prevented muscle fibrosis and hindlimb paralysis. An important question for future therapeutic approaches 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. 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.