Research Institution: Center for Genetic Medicine Research, Children’s National Health System, Washington DC, USA

Principle Investigator: ​Dr Yi­-Wen Chen

Type: International

Project title: “Targeting DUX4 using gene-silencing oligonucleotides in FSHD models”

Status: Active


Gene-silencing oligonucleotides work by preventing the production of the protein product of gene transcription. This is significant in FSHD because they can be used to stop the production of toxic proteins produced through the lack of inhibition in the D4Z4 region.

Gene silencing oligonucleotides have been studied previously in models of FSHD with some promising results. The oligonucleotides that Professor Chen will be using for this newly funded grant represent a significant advancement on these early oligonucleotides. In addition, Idera Pharmaceuticals have identified four potential oligonucleotides that effectively inhibit DUX4 mRNA transcripts in cells.

The aims of this grant are to further develop these potential therapeutics in human FSHD cells and a mouse model of FSHD.



Update June 2016

The goal of the proposed study is to test a new generation of gene silencing agents called gene-silencing oligonucleotides (GSOs) for their efficacy in suppressing the pathogenic DUX4 expression in FSHD myoblasts and an FSHD mouse model. Several innovative designs of the GSOs by Idera Pharmaceuticals made them promising therapeutic agents. In the past six months, we have tested five GSOs and showed that all five successfully suppressed the DUX4 expression in patients’ muscle cells in culture. Higher concentration of the GSOs suppressed DUX4 expression more. Several genes that are regulated by DUX4 were also suppressed by the GSOs, indicating that the GSO treatments successfully reduced the harmful DUX4 expression effects in the human FSHD muscle cells. We are currently studying how the treatments correct the physiological defects that have been reported in the cells. We thank the FSHD Global Research Foundation for supporting this work.

Update January 2017

The goal of the proposed study is to test a new gene silencing agents called gene-silencing oligonucleotides (GSOs) for their efficacy in suppressing the pathogenic DUX4 expression in FSHD myoblasts and an FSHD mouse model. In the past grant period, we have tested five GSOs and showed that the GSOs successfully suppressed the DUX4 expression and genes that are regulated by DUX4. Higher concentration of the GSOs suppressed DUX4 expression more. We then studied whether the treatments could correct the cellular defects that had been reported previously. The defects include lower fusion index and higher number of atrophic myotubes in FSHD muscle cells, both suggest that the cells cannot form mature muscles well. In our studies, we showed that the GSO treatments completely or partially corrected the defects by increasing the fusion indexes and reducing the numbers of atrophic myotubes in cell culture. The results suggested that the treatments improved the ability of muscle growth. Currently, we are studying efficacy of the GSOs using a new mouse model of FSHD. We thank the FSHD Global Research Foundation for supporting this work.

Update July 2017

Gene-silencing oligonucleotides (GSOs) produced by Idera Pharmaceutical are designed to reduce the expression of the pathogenic DUX4 expression in FSHD. The goal of this study is to determine their efficacy using both human cell and mouse models of FSHD. In the first part of the study, we have tested five GSOs using FSHD muscle cells and showed that the GSOs reduced DUX4 levels in the cells. In addition, the FSHD cells behaved more similar to the healthy cells after the treatments. In this report period, we started animal studies to determine whether we can successfully deliver GSOs to muscles and whether the GSOs reduce expression of DUX4 and genes regulated by DUX4. To allow us visualize the GSOs, we added a florescent tag to the GSO compounds before we injected them into the mice. Our results showed that GSOs were able to enter muscle cells after intramuscular injection (direct injection into muscle), as well as after the subcutaneous injection (injection under the skin which allows systemic delivery). The results showed that the GSOs entered the muscles after either intramuscular or subcutaneous injection. Higher amount of GSOs was observed in muscles when the GSOs were delivered by intramuscular injection. We also showed that a gene regulated by DUX4 reduced its expression level after systemic delivery of GSOs for 12 days (6 subcutaneous injections). Our next step will be to increase the number of injections of GSOs to determine the efficacy of GSOs when delivered for longer period of time. We thank the FSHD Global Research Foundation for supporting this work.

Update February 2019

Major finding: GSO treatment improved muscle function and pathology in a FSHD mouse model

In the last grant period, our main effort was to characterize phenotypic improvements after the six-week treatment of GSO developed by the Idera Pharmaceutical. In addition, we further characterized the FLExDUX4 mice to determine phenotypic defects in this model. This is important because the phenotypic defects will be used to determine in vivo efficacy in our pre-clinical studies using the model. We examined pathological and muscle functional changes of the FLExDUX4 mice at 2 months, 4 months, 8 months and 1 year of age. Our data showed that while the average body and muscle weights of the FLExDUX4 mice were not significantly different from their wildtype littermates at a younger age (2m old), the body and muscle weights were lower in the older FLExDUX4 mice (4m, 8m and 1y). In addition, the muscle strength of the FLExDUX4 mice, measured by grip strength and in vivo physiological testing, was significantly weaker at all ages. Muscle degeneration and regeneration were observed occasionally in the muscle samples but not prominent. However, we observed significant fibrosis in the FLExDUX4 mice. In the old FLExDUX4 mice (1year old), we also observed large numbers of muscle lesions inside a specific type of myofibers (type IIb), suggesting that different types of muscle fibers have different susceptibilities to the aberrantly expressed DUX4. This is further supported by the fiber type data, which showed that different fiber types were affected differently. The slow muscle fibers (type I) were more affected than the other fiber types. Type IIa and IIx were more affected than the type IIb. In addition to fiber type differences, the fiber size distribution was different in the FLExDUX4 mice. We are quantifying fiber sizes of different fiber types currently.
In our latest six-week trial, one GSO from the original five GSOs was selected based on our in vitro assays and the short-term in vivo studies. Six male and six female FLExDUX4 mice received subcutaneous injections of the GSO for total 13 injections (two injections per week). Male and female FLExDUX4 mice that only received saline injections were used as controls. The mice received their first injection when they were 3 weeks old and the muscles were collected when they were 2 months old. Our data showed that there was no significant body weight difference between the treated and the untreated groups; however there was a trend that the treated group was slightly lighter than the saline treated (control) group. We conducted grip strength testing to determine whether the muscle weakness was improved by the treatment. We conducted the first testing before the first injection to determine the baseline. We then measured the muscle strength at two time points, 3 weeks and 6 weeks after the 1st injection. Our data showed that the grip strength of the FLExDUX4 mice improved significantly at both time points, and the treatment was able to completely restore the muscle strength after six weeks of treatment.

After the last injection, we collected muscles and other organs (heart, livers and kidneys) for histological and molecular studies. We have processed the muscle samples to determine the pathological improvement by the GSO treatment. Muscle sections were stained using antibodies against different types of myosin heavy chain to identify different fiber types and to evaluate their changes in size. Pico Sirius Red staining was used for visualizing fibrosis in the muscles. We also stained the sarcolemmal membrane using the antibody against dystrophin for fiber size measurements. We have completed the staining and image acquisition. We are currently quantifying the image data. Our preliminary data showed that the FLExDUX4 mice had a change of fiber size distribution, likely reflected the changes of fiber types. The 3GA treatment corrected the difference by shifting the distribution back to that similar to the wildtype littermates. We will continue the quantification and determine whether the treatment improved fibrosis. In addition, we have performed RNA-seq to evaluate how the treatment affected the transcriptome of the muscles. The data will be used to evaluate both intended molecular effects and potential off target effects.