Development of Novel Splice Modulation Therapies For Muscular Dystrophy

• Author / Creator

  Lee, Joshua

• The muscular dystrophies are a heterogeneous group of over 30 genetic diseases which are characterized by progressive weakening and deterioration of muscle tissue and which vary with respect to age of onset, pattern of muscle involvement, and severity. To date, no effective therapeutic options exist for either halting or reversing disease progression for any form of muscular dystrophy, although some emerging strategies are promising.
  Antisense-mediated exon skipping therapy uses synthetic molecules called antisense oligonucleotides to modulate splicing, allowing exons harboring or near genetic mutations to be removed and the open reading frame corrected. Antisense-mediated exon skipping has made significant progress as a therapeutic platform in recent years, especially in the case of Duchenne muscular dystrophy (DMD). Despite FDA approval of eteplirsen – the first-ever antisense drug clinically marketed for DMD – exon skipping therapy still faces significant hurdles, including limited applicability and unknown function of truncated proteins.
  Key to the success of an exon skipping strategy is the identification of appropriate exon targets – exons which are dispensable in terms of the stability and function of the resulting truncated proteins. In the case of DMD, in-frame exon skipping of DMD exons 45-55 represents a significant approach to treating DMD, as patients harboring DMD exons 45-55 deletion mutations are reported to have exceptionally mild to asymptomatic phenotypes. Additionally, a large proportion of patients harbor mutations within this “hotspot” region.
  The dysferlinopathies are another form of muscular dystrophy, caused by mutations in the dysferlin (DYSF) gene which render dysferlin protein unable to facilitate plasma membrane repair. Unlike DMD, there is no reported mutation hotspot in DYSF, and it is largely unknown which exons are potentially amenable to exon skipping and which are not.
  In this work, we sought to establish novel antisense-mediated exon skipping therapeutic approaches for two major forms of muscular dystrophy: Duchenne muscular dystrophy and dysferlinopathy. Here, we demonstrate that a cocktail of antisense oligonucleotides can effectively
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  skip DMD exons 45-55 in vitro in myotubes transdifferentiated from DMD patient fibroblast cells. We also report that removal of DYSF exons 26-27 or 28-29 does not impair plasma membrane resealing in dysferlinopathy patient fibroblasts, and a cocktail of antisense oligos is able to achieve multi-exon skipping of DYSF exons 28-29 and rescue plasma membrane resealing in patient cells. This is the first report of substantive DMD exons 45-55 skipping in DMD patient cells, as well as the first description of exons 26-27 and 28-29 as exon skipping targets in DYSF.
  Taken together, these findings help validate the feasibility of DMD exons 45-55 skipping in DMD patients and DYSF exons 26-27 and 28-29 skipping in dysferlinopathy patients as potential therapeutic platforms for future translation into clinical practice.
  This work also describes efforts undertaken by our lab to elucidate the underlying mechanisms behind the generation and expansion of dystrophin-positive revertant fibers (RFs) in mdx and mdx52 mouse models of DMD. We assessed the number of RFs in these mice and determined that mutation types and aging differently affect RF expansion in mdx and mdx52 mice. An understanding of how these rare dystrophin-positive fibers are generated and expand could be very beneficial to the development of future DMD therapies.

• Subjects / Keywords

  • Duchenne Muscular Dystrophy
  • Antisense Oligonucleotide
  • Exon Skipping
  • Muscular Dystrophy
  • Dysferlinopathy

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3HX1667R

• License

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