Human miRNA miR-675 inhibits DUX4 expression and may be exploited as a potential treatment for Facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating myopathy caused by de-repression of the DUX4 gene in skeletal muscles. Effective therapies will likely involve DUX4 inhibition. RNA interference (RNAi) is one powerful approach to inhibit DUX4, and we previously described a RNAi gene therapy to achieve DUX4 silencing in FSHD cells and mice using engineered microRNAs. Here we report a strategy to direct RNAi against DUX4 using the natural microRNA miR-675, which is derived from the lncRNA H19. Human miR-675 inhibits DUX4 expression and associated outcomes in FSHD cell models. In addition, miR-675 delivery using gene therapy protects muscles from DUX4-associated death in mice. Finally, we show that three known miR-675-upregulating small molecules inhibit DUX4 and DUX4-activated FSHD biomarkers in FSHD patient-derived myotubes. To our knowledge, this is the first study demonstrating the use of small molecules to suppress a dominant disease gene using an RNAi mechanism.

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Life sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. The objective of the study was to explore new strategies for the treatment of FSHD. FSHD is caused by de-repression of the DUX4 gene, which is toxic to muscle. FSHD therapies are thus focused on inhibiting DUX4, which was our main goal in this paper. However, instead of using conventional approaches (i.e antisense oligos (ASO), siRNAs, shRNA or artificial miRNAs), here we decided to test a novel strategy to direct RNAi against DUX4. Specifically, we reasoned that we could use drugs to up-regulate endogenous human microRNAs that naturally direct RNAi against DUX4, and that this would offer a novel strategy to inhibit the gene with RNAi. To our knowledge, this has never been done before for any dominant genetic disease. In this study, we show that mir-675 inhibits DUX4 efficiently and reduces DUX4-associated phenotypes in human HEK293 cells and FSHD muscle cell lines. We also show that mir-675 functions within a gene therapy vector to inhibit DUX4-associated pathologies in vivo -in an AAV.DUX4 mouse model we previously developed and published (PMID: 21446026). In the small molecule treatment assay, we used three different FSHD cell lines. For the 15A FSHD cell line, we performed 6 independent experiments. For the 17A and 18A FSHD cell lines, we performed 3 independent experiments. For all in vitro experiments, no sample size calculation was performed. Sample size was determined based on our previously published studies (PMID: 21446026; PMID: 22508491; PMID: 29387734). Based on our aforementioned previously published studies, these sample sizes were found to be sufficient. For the in vivo study, sample size was chosen based on our previously published studies (PMID: 22508491; PMID: 29387734).
No data were excluded from the analyses.
For the in vitro study, we performed between N=3 to N=6 independent experiments depending on the assay. We performed 6 independent blinded western blots when testing mir-675 specific inhibition of DUX4 expression. All raw western blot images are included in the submission.
In the small molecule treatment assay, we used three different FSHD cell lines. For the 15A FSHD cell line, we performed 6 independent experiments. For the 17A and 18A FSHD cell lines, we performed 3 independent experiments. For the in vivo study, sample size was chosen based on our previously published studies (PMID: 22508491; PMID: 29387734). All attempts at replication were successful.
For the in vitro work, independent experiments were randomized by choosing different days, and for transfection of cells, by transfecting the different samples with random order every time. The blinded western blot experiments also helped with the randomization of the experiment. For the in vivo work, AAV vectors were injected in tibialis anterior muscles of randomly selected mice. Every mouse received a coinjection and the contralateral control were distributed evenly across randomly assigned wild-type mice.
We performed 6 independent blinded western blots when testing mir-675 specific inhibition of DUX4 expression. When we performed blinded western blots, we asked a lab member who is not involved in the study to blind the DUX4 and miRNA expression plasmids before we transfect them into HEK293 cells. We also performed the SDS-PAGE gel and immunoblotting under blinding conditions. At the end of every experiment, the same lab member unblinded the blots. For all other experiments, blinding was not required as all data was collected by objective measurements.
HEK293 cells were tested for mycoplasma contamination. No mycolplasma contamination was detected in HEK293 cell lines.