Evaluation of LNA Gapmer efficacy in FSHD patients' muscle cells

• Author / Creator

  Guncay, Ashley K

• Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by early involvement of muscle weakness. FSHD is most commonly caused by a deletion of a subset of D4Z4 macrosatellite repeats at the locus located on chromosome 4q35. Within each D4Z4 repeat unit is a double homeobox sequence encoding the DUX4 protein. In FSHD patients, mis-expression of DUX4 results in the production of a pathogenic protein and causes a transcriptional deregulation cascade which triggers muscle atrophy, apoptosis, and oxidative stress. Currently, no curative treatment options for FSHD patients have been established, further illustrating the importance of determining the biological impact of DUX4 suppression via antisense oligonucleotides. Implication for antisense oligonucleotides in FSHD could help towards progressing the development of therapeutic approaches. Using in vitro methods, in immortalized control myoblasts and immortalized FSHD patient muscle cells, three culture media additives were investigated, fetal bovine serum (FBS), knockout serum replacement (KOSR) and dexamethasone, to determine their effects on DUX4 expression in culture. Various RT-conditions were also examined for sensitive detection of DUX4. In addition, suppression of DUX4 was examined via RNase H-mediated degradation using locked nucleic acids (LNA)-DNA chimeras, called gapmers, targeting DUX4. In this work, I have shown that SuperScript III reverse transcriptase (RT) and GoTaq® G2 green master mix is the most sensitive cDNA synthesis strategy to use for detection of DUX4. Supplementing culture medium with dexamethasone enabled better detection of DUX4 expression in immortalized healthy and FSHD myoblasts. Transfection with LNA gapmers 1, 1, 3, 4, 6, and 7, all which target exon 3 of the DUX4 mRNA, suppress the expression of DUX4 in immortalized FSHD patient muscle cells. Transfection with LNA gapmers 1 and 3 was also found to change the localization of MuRF1 in the nucleus of FSHD myotubes. In addition, I found that PITX1 expression in immortalized FSHD patient muscle cells is not suppressed by LNA gapmers targeting DUX4. These results establish a sensitive detection strategy and culture method for detection of DUX4, identify potential LNA gapmers sequences which prevent DUX4 expression and change the localization of MuRF1 in nucleus and indicate that PITX1 per se is not a direct target of DUX4. This thesis also outlines the therapeutic potential of antisense chemistries, specifically LNA gapmers, in FSHD targeting DUX4.

• Subjects / Keywords

  • FSHD
  • Antisense oligonucleotide
  • PITX1
  • locked nucleic acid
  • DUX4
  • LNA gapmer

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R32V2CS35

• 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.