Characterizing an FSHD-like Mouse Model to Study a Novel Antisense Oligonucleotide Therapy
Open Access DepositedFacioscapulohumeral muscular dystrophy (FSHD) is caused by aberrant expression of the double homeobox 4 (DUX4) gene, which causes disruption in a number of vital cellular pathways and progressive muscle loss [1-13]. There is no cure for FSHD and drugs that underwent clinical trials had mixed success and only targeted the downstream effects of DUX4 [14-23]. Antisense oligonucleotide (AON) therapy would allow for direct targeting of DUX4 mRNA and possibly correct more than one FSHD phenotype [24]. A novel third generation antisense (3GA) links two identical, phosphorothioate DNA sequences with a glycerol linker [24, 25]. A major hurdle in developing AONs is that previous animal models have been limited in experimental use. The FLExDUX4 model expresses DUX4, but was not extensively studied past 23-weeks¬old [26, 27]. Since FSHD is a progressive muscle disorder that affects both adults and pediatric patients, it is important to track the FLExDUX4 mice for a longer period of time. Therefore, we hypothesize that the FLExDUX4 model would be suitable for testing AONs and that the novel 3GA would be efficacious in knocking down DUX4 and reduce FSHD phenotypes both in vivo and in vitro. To determine whether the FLExDUX4 model is suitable for AON therapy, we observed the FLExDUX4 model to determine if long-term exposure to DUX4 caused phenotypes that were not previously reported. In this study, we found that older FLExDUX4 mice present lower muscle weights together with muscle weakness. Transcriptomic analysis indicated that myofibers had altered expression changes in oxidative stress response pathways, muscle remodeling, and fibrosis pathways. Pathological analysis of muscle sections revealed TDP-43 aggregation and, like in FSHD, preferential decrease in type 2 fibers size. This allows us to conclude, in context of the project, that FLExDUX4 mice have measurable phenotypes which recapitulate human FSHD and can be used to determine if an antisense therapy targeting DUX4 is effective. To determine whether 3GA will reduce DUX4 mRNA in vivo, we treated FLExDUX4 mice and tested for levels of DUX4 mRNA, grip strength, and fibrosis levels. In this study, long-term 3GA treatment over 6.5 weeks did not reduce DUX4 mRNA levels, despite improving grip strength performance and reduced serum TGF-β1. Conversely, FLExDUX4 mice treated with 3GA had smaller heart weights, elevated liver enzymes, and increased transcription of immune response genes. The improvement in phenotypes without reduction of DUX4 mRNA levels suggested an alternate mechanism-of-action that delays degradation of DUX4 mRNA in the long-term. These findings suggest that 3GA treatment is able to improve the phenotypes, but appears to have toxicity which needs to be addressed when further developing treatment for FSHD. Due to the absence of downstream gene expression in the FLExDUX4 model, testing 3GA efficacy in a model that can validate DUX4 protein reduction was needed. We used human immortalized FSHD myoblasts to determine 3GA efficacity in reducing DUX4 mRNA and corrected FSHD phenotypes. Myoblasts treated with 3GA targeting DUX4 had reduced levels of DUX4 and DUX4 transcriptional targets compared to control 3GA. However, levels of DUX4 in 3GA-treated myoblasts were higher than untreated controls. Also, low concentrations of 3GA improved fusion index and atrophic tube frequency. These data suggested that two effects can be occurring: knock down of DUX4 and/or induction of DUX4 expression. We hypothesized that the 3GA maybe stressing the cells, but further experiments are needed to provide consistency and additional information. In conclusion, this dissertation showed that administration of 3GA in vivo and in vitro resulted in phenotypic improvements, but also exhibited signs of toxicity. Importantly, this study showed that constant expression of DUX4 at a low level in the FLExDUX4 mice recapitulated some FSHD phenotypes. Older FLExDUX4 mice do exhibit muscle pathology and muscle weakness. Moving forward, future studies in older FLExDUX4 mice and in female FLExDUX4 mice would greater our understanding of the model and help to further elucidate if muscle pathologies in the model are a usable marker of improvement for future therapies.
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Kelly_Murphy_Dissertation_09Aug2022_-_Kelly_Murphy.pdf | 2022-08-22 | Open Access |
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