Effects of Gene Correction by Exon Skipping in a Murine Model of Duchenne Muscular Dystrophy
Open AccessDystrophin deficiency is the genetic basis for Duchenne muscular dystrophy (DMD). Mutations that lead to out of frame transcripts and no protein production result in a severe and fatal phenotype, as DMD. Conversely, mutations that preserve the reading frame with the production of a truncated version of dystrophin lead to a milder phenotype, as Becker muscular dystrophy (BMD). Systemic delivery of antisense oligonucleotides (AOs) to DMD patients convert out of frame transcripts to in frame thereby inducing the production of dystrophin protein in muscle (exon-skipping). The use of AOs, such as morpholino oligomers, is a promising therapeutic approach tested in both animal models and DMD boys. However, restoration of dystrophin by exon skipping has been observed to be highly variable leading to contradictory functional outcomes in clinical trials. Understanding and characterizing the effects of gene correction by exon skipping at the molecular and physiological levels in pre-clinical models is needed to develop more effective protocols for therapy in human clinical trials. Here we hypothesize that the variability in exon skipping efficacy is multifactorial, likely due to dose concentration and frequency, target tissue, immune factors as well as reliability of therapy outcome measures. In this thesis project we aim to understand the contribution of the above factors in the efficacy of exon skipping.We initially investigated possible factors that lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model of DMD. We tested whether specific muscle groups or myofiber types showed better success than others and also correlated residual phosphorodiamidate morpholino oligomer (PMO) concentration in muscle with the amount of dystrophin protein elicited by acute dosing of morpholino. We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays. Here we show that after acute PMO treatment dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression. All three dystrophin detection methods were generally concordant for all muscles. While we found some evidence of muscle group enhancement and successful rescue, we show that other as yet undefined factors may underlie the observed variability in the success of exon skipping. Our results highlight the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.Next, we analyzed functional outcome of gene correction at the sarcolemma level in the mdx mice. In order to understand the effect of dystrophin rescue on membrane stability and function, we first established the molecular mechanisms responsible for poor myofiber repair in mdx muscle. Previous results from our laboratory had demonstrated that the lack of dystrophin causes reduced activity and dynamics of mitochondria in mdx mice. We showed that these deficits reduce the ability of the dystrophic sarcolemma to repair, and result in a compensatory increase in the expression of dysferlin and dysferlin-interacting sarcolemmal repair proteins. We expanded on these results to show that reduced activity and dynamics of mitochondria are amongst the earliest cellular deficits of dystrophic myofibers in two dystrophin-deficient mdx mouse models (mdx-23 and mdx-52). Genetic deletion of dysferlin in mdx mice worsen the sarcolemmal repair of dystrophic myofibers and enhanced the muscle pathology. This establishes mitochondria-mediated sarcolemmal repair as a distinct and complimentary mechanism to dysferlin-mediated sarcolemmal repair, one that is critical for DMD. Since dystrophin absence causes mitochondrial deficit, we investigated the effectiveness of dystrophin rescue by exon skipping to improve sarcolemmal repair of dystrophic muscle. We show that the acute rescue of dystrophin expression caused twice as many mdx myofibers to repair from sarcolemmal injury. The repair response was heterogeneous, a potential consequence of the variable dystrophin expression observed previously in our research. We also tested the effect of acute increase in mitochondrial metabolic activity. Overall, we demonstrate that the poor mitochondria-mediated sarcolemmal repair in dystrophic muscle contributes to myofiber death and exon skipping is a promising therapy to rescue function in the pre-clinical murine model of DMD. Next, we investigated the effect of chronic morpholino treatment and whether sustained dystrophin expression would improve the variable success of exon skipping therapy observed with our acute treatment. Here we show that repeated high doses of PMO increase the overall amount of dystrophin rescued in mdx mice, which was associated with muscle functional improvement, an important outcome in clinical trials of exon skipping. Yet, after chronic treatment dystrophin rescue remains variable, with dystrophin-positive myofibers spatially dispersed across muscle sections. Our results show that high doses of morpholino are necessary to improve muscle function; however, optimizing drug dose and uptake and/or dosing schedule is imperative to normalize the heterogeneous expression of dystrophin protein and therefore achieve consistent clinical improvement. In the context of DMD, exon skipping gives rise to a protein that presumably was never tolerized by the immune system before. The potential of the newly synthesized dystrophin to trigger an immune response in DMD patients is not well established. In this study, we evaluate whether sustained dystrophin expression after chronic morpholino treatment in mdx mice elicits a specific immune response to dystrophin. We previously showed that dystrophin rescue is achieved in all skeletal muscles and diaphragm. As a result, we observed significant reduction in macrophage infiltration in all PMO treated mice, as well as a decrease in expression of inflammatory and some immune cell markers. Antibodies directed against dystrophin were developed in 38% of the dystrophin-rescued animals, while no antibodies were found in the control mice. We show that reactive antibodies recognized full length and truncated mouse dystrophin, as well as dog and human dystrophin. Our results show the potential of chronic exon skipping therapy to trigger a humoral immune response to dystrophin in a murine model of DMD. At this time, it is not clear to what extent this humoral response affects the success of dystrophin rescue. This study highlights the importance of assessing patient’s immune status before and during exon skipping treatment to avoid immune responses against therapeutic dystrophin.Overall, this project demonstrates that although exon skipping is not a perfect therapy, it provides dystrophic muscles with enough dystrophin protein to achieve functional improvement in mice, as observed at both the molecular (sarcolemmal repair) and physiological (force increase) levels. Exon skipping therapy efficiently corrects the susceptibility to contraction induced injury of dystrophic muscle likely by enhancing mitochondrial-mediated repair; however the efficiency varies significantly between muscles. Factors that control exon skipping efficacy are clearly multifactorial. We demonstrate that treatment duration and dosing are major determinants of therapy efficacy, while muscle group and fiber type, as well as residual PMO concentration in muscle are not. In addition, we provide evidence that immune response to newly synthesized dystrophin may play a role in the success of this therapeutic modality.
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