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Development of Antisense Therapies for Facioscapulohumeral and Duchenne Muscular Dystrophy

  • Author / Creator
    Lim, Kenji Rowel Q.
  • Facioscapulohumeral muscular dystrophy (FSHD) and Duchenne muscular dystrophy (DMD) are two of the most commonly inherited muscular disorders in the world. There is currently no cure for both of them. Antisense oligonucleotides (AOs) are short, synthetic, single-stranded nucleic acids that hybridize to target mRNAs via base-pairing. In doing so, AOs could inhibit gene expression or modulate splicing and serve as viable therapeutic options for genetic disorders. In this work, we aim to develop novel, effective AOs for treating FSHD and DMD.FSHD is an autosomal dominant disorder caused by mutations inducing aberrant double homeobox protein 4 (DUX4) gene expression in skeletal muscle. When present in differentiated muscle, DUX4 is a cytotoxic protein that dysregulates pathways involved in cell death and muscle development, among others. Previous groups have attempted to reduce DUX4 mRNA expression using steric-blocking AOs, but the efficacy of these therapeutics could be improved. Gapmers are a class of AOs that induce the degradation of their target mRNAs via the recruitment of RNase H, and may present a better alternative to DUX4 knockdown. Here, we designed and evaluated the efficacy of gapmers with the locked nucleic acid (LNA) and 2’-O-methoxyethyl (2’-MOE) chemistries towards reducing DUX4 expression. Using immortalized patient-derived muscle cells, we demonstrated that both gapmers could significantly knock down DUX4 mRNA expression to nearly undetectable levels. We observed restorative transcriptomic effects, and treatment improved muscle fiber size in vitro. Local treatment with these gapmers also significantly reduced DUX4 expression in an FSHD mouse model. DMD is an X-linked recessive disorder primarily caused by large out-of-frame mutations in the dystrophin gene (DMD). Dystrophin loss weakens muscle cell membranes and predisposes muscles to degeneration upon use. AOs can skip out-of-frame exons in the DMD transcript, restoring the reading frame as well as the production of truncated, partially functional dystrophin. This approach has met with much success, such that four exon skipping AOs have been approved by the U.S. Food and Drug Administration for DMD. However, efficacy could be improved, since most of these AOs only restored <2% dystrophin of healthy levels (versus the target 10%) in patients after 48-180 weeks of once-weekly treatment, and did not show convincing evidence of functional benefit. All these are also single-exon skipping AOs, and only treat <30% of all patients in total. Here, we first investigated the advantages of early exon skipping treatment using dystrophic dog neonates. As DMD is a progressive disorder, there is a strong rationale for early treatment, but its benefits are poorly understood. Early exon skipping was safe, and restored dystrophin to an average 2% of healthy levels in skeletal muscles after three systemic injections. Early treatment was most beneficial for respiratory muscles. Secondly, we developed an exons 45-55 skipping AO cocktail for DMD therapy. By targeting exons in a mutation hotspot of the DMD gene, exons 45-55 skipping could treat nearly half of all patients. We achieved exons 45-55 skipping and dystrophin restoration by targeting as few as 5 exons. Conjugating the novel cell-penetrating peptide DG9 to AOs in this cocktail led to dystrophin restoration upon local in vivo treatment. We also tested a DG9-conjugated AO for single-exon 51 skipping, and saw functional improvement upon systemic treatment of dystrophic mice.Overall, we identified DUX4-targeting gapmers as potential candidates for further pre-clinical testing towards FSHD therapy. We also showed proof-of-concept that DMD exons 45-55 can be skipped with a minimized AO cocktail, and identified a peptide that could be conjugated to exon skipping AOs to improve their in vivo efficacy. Together with our findings regarding early exon skipping treatment, our work not only produced candidates for further pre-clinical testing but also helps inform the development of future exon skipping AOs for DMD therapy.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.