Dux4 Target Gene Expression in Mouse Muscle Transplanted with Muscle Cells from FSHD Patients

Facioscapulohumeral Muscular Dystrophy (FSHD) is one of the most prevalent forms of muscular dystrophy. However, because of the unique nature of the genetic abnormality underlying the disease, there is currently no widely available laboratory model. In order to gain insights into FSHD molecular pathology, we developed a xenograft model by transplanting myogenic cells from patients with FSHD (4qA contractions) as well as from their unaffected relatives into the tibialis anterior muscles of immunodeficient mice. Our findings show that muscle xenografts derived from FSHD myogenic cells express Dux4 target genes, recapitulating the expression of these disease biomarkers in muscle biopsies of FSHD patients. FSHD muscle xenografts provide an animal model for investigations of the molecular pathogenesis of FSHD muscles and for drug development

Outcome Measures in Facioscapulohumeral Muscular Dystrophy Clinical Trials

Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscular dystrophy with a variable age of onset, severity, and progression. While there is still no cure for this disease, progress towards FSHD therapies has accelerated since the underlying mechanism of epigenetic derepression of the double homeobox 4 (DUX4) gene leading to skeletal muscle toxicity was identified. This has facilitated the rapid development of novel therapies to target DUX4 expression and downstream dysregulation that cause muscle degeneration. These discoveries and pre-clinical translational studies have opened new avenues for therapies that await evaluation in clinical trials. As the field anticipates more FSHD trials, the need has grown for more reliable and quantifiable outcome measures of muscle function, both for early phase and phase II and III trials. Advanced tools that facilitate longitudinal clinical assessment will greatly improve the potential of trials to identify therapeutics that successfully ameliorate disease progression or permit muscle functional recovery. Here, we discuss current and emerging FSHD outcome measures and the challenges that investigators may experience in applying such measures to FSHD clinical trial design and implementation

Identification of the hyaluronic acid pathway as a therapeutic target for facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is linked to epigenetic derepression of the germline/embryonic transcription factor DUX4 in skeletal muscle. However, the etiology of muscle pathology is not fully understood, as DUX4 misexpression is not tightly correlated with disease severity. Using a DUX4-inducible cell model, we show that multiple DUX4-induced molecular pathologies that have been observed in patient-derived disease models are mediated by the signaling molecule hyaluronic acid (HA), which accumulates following DUX4 induction. These pathologies include formation of RNA granules, FUS aggregation, DNA damage, caspase activation, and cell death. We also observe previously unidentified pathologies including mislocalization of mitochondria and the DUX4- and HA-binding protein C1QBP. These pathologies are prevented by 4-methylumbelliferone, an inhibitor of HA biosynthesis. Critically, 4-methylumbelliferone does not disrupt DUX4-C1QBP binding and has only a limited effect on DUX4 transcriptional activity, establishing that HA signaling has a central function in pathology and is a target for FSHD therapeutics

Silencing DUX4 Expression in FSHD Cells by CRISPR

Facioscapulohumeral Muscular Dystrophy (FSHD) is an autosomal dominant neuromuscular disease affecting 1 in 20,000 to 1 in 15,000 individuals and is characterized by progressive weakness in the facial, scapular, humeral, truncal, and lower extremity muscles (Tawil and Van Der Maarel Muscle Nerve 2006). FSHD is associated with the contraction of the D4Z4 microsatellite repeat below a threshold number of repeats (Wijmenga et al., Nat. Genet, 1992), allowing the transcription of the DUX4 gene contained within the last repeat (Snider et al., PLoS Gen, 2010). The disease only develops when DUX4 is expressed from a chromosome with the permissive 4qA allele, which contains a polyadenylation signal (PAS) that stabilizes the DUX4 transcript (Lemmers et al., Science, 2010). We are using CRISPR technology to investigate the possibility that disruption of the PAS in cells derived from FSHD patients could prevent expression of the DUX4 protein and restore the cell to a less affected phenotype. We will then take advantage of the high reprogramming efficiency of FSHD cells and generate iPSC from FSHD muscle cells with the repressed DUX4 allele, and determine if they have a similar phenotype to iPS cells derived from non-affected individuals. Finally, we will use the highly-engraftable iPS cells in xenograft experiments to determine if the DUX4-silenced iPSCs repopulate injured muscle more efficiently than unaltered FSHD-derived iPSC, and evaluate their potential for use as therapeutics

Epigenetic variability is a modifier of facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD), the most prevalent myopathy afflicting both children and adults, is strongly associated with epigenetic changes of the 4q35-localized macrosatellite D4Z4 repeat. Recent studies propose that FSHD pathology is caused by the misexpression and missplicing of the DUX4 (double homeobox 4) gene, encoded within the repeat array, resulting in production of a pathogenic protein, DUX4-FL. We have analyzed DUX4 mRNA and protein expression in a large collection of myogenic cells and muscle biopsies derived from muscles of FSHD1 affected subjects and their unaffected first-degree relatives. We confirmed that stable DUX4-fl mRNA and protein were expressed in myogenic cells and muscle tissues derived from FSHD affected subjects, including several genetically diagnosed adults yet to show clinical manifestations of the disease; however, there was great individual and familial variation in the levels of DUX4-FL. In addition, we found DUX4-fl mRNA and protein expression in muscle biopsies and myogenic cells from genetically unaffected relatives of the FSHD subjects, although at a significantly lower frequency. These results establish that DUX4-fl expression per se is not sufficient for FSHD muscle pathology. To investigate if subtle differences in the epigenetic status of the 4q35 region could account for the wide variation in DUX4-fl expression among FSHD1 subjects and potentially the spurious expression in certain unaffected controls, family cohorts of myogenic cells from FSHD1 subjects were tested for their sensitivity to small molecules that can alter the chromatin state. We find that myogenic cells from FSHD1 subjects are overall epigenetically poised to express DUX4 compared with unaffected subjects; however, FSHD1 subjects show individual differences in their capacity to express DUX4-fl in response to DNA demethylation and blocking histone deacetylation. Therefor, individual differences in the epigenetic status likely impacts progression of disease pathology, variability in age of onset, disease severity, and asymmetry of affected muscles

C1QBP Inhibits DUX4-Dependent Gene Activation and Can Be Targeted with 4MU

FSHD is linked to the misexpression of the DUX4 gene contained within the D4Z4 repeat array on chromosome 4. The gene encodes the DUX4 protein, a cytotoxic transcription factor that presumably causes the symptoms of the disease. However, individuals have been identified who express DUX4 in their muscle biopsies, but who remain asymptomatic, suggesting that there are other factors that modify FSHD penetrance or severity. We hypothesized that an FSHD-modifying factor would physically interact with DUX4, and we took a proteomic approach to identify DUX4-interacting proteins. We identified the multifunctional C1QBP protein as one such factor. C1QBP is known to regulate several processes that DUX4 affects, including gene expression, oxidative stress, apoptosis, and pre-mRNA splicing. We used siC1QBP knockdown assays to determine if C1QBP affects DUX4 activity. While C1QBP had little effect on DUX4 activity in myotubes, we found that it inhibits the kinetics of DUX4-target gene activation during myogenic differentiation. This identifies C1QBP as a regulator of DUX4 activity and a potential target for FSHD therapeutics. Importantly, C1QBP is regulated by binding to the signaling molecule hyaluronic acid (HA). Decreasing HA by treating cells with 4-methylumbelliferone (4MU), an inhibitor of HA synthesis, resulted in a sharp decline in DUX4 activity and also greatly reduced its cytotoxicity. We have found that DUX4-induced cytotoxicity is associated with severe mislocalizaton of C1QBP, which is prevented by 4MU. This defect is not a downstream result of DUX4-induced oxidative stress, as it could not be prevented by treating cells with an antioxidant, nor could it be recapitulated by exposing cells to oxidants. This identifies C1QBP as a target for the treatment of FSHD, and in particular indicates that 4MU, already an approved drug in Europe and currently under investigation for other indications, may be an effective C1QBP-targeting FSHD therapeutic compound

Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4 maintains the myogenic population

<p>Abstract</p> <p>Background</p> <p>A hallmark of muscular dystrophies is the replacement of muscle by connective tissue. Muscle biopsies from patients severely affected with facioscapulohumeral muscular dystrophy (FSHD) may contain few myogenic cells. Because the chromosomal contraction at 4q35 linked to FSHD is thought to cause a defect within myogenic cells, it is important to study this particular cell type, rather than the fibroblasts and adipocytes of the endomysial fibrosis, to understand the mechanism leading to myopathy.</p> <p>Results</p> <p>We present a protocol to establish clonal myogenic cell lines from even severely dystrophic muscle that has been replaced mostly by fat, using overexpression of CDK4 and the catalytic component of telomerase (human telomerase reverse transcriptase; hTERT), and a subsequent cloning step. hTERT is necessary to compensate for telomere loss during <it>in vitro </it>cultivation, while CDK4 prevents a telomere-independent growth arrest affecting CD56+ myogenic cells, but not their CD56- counterpart, <it>in vitro</it>.</p> <p>Conclusions</p> <p>These immortal cell lines are valuable tools to reproducibly study the effect of the FSHD mutation within myoblasts isolated from muscles that have been severely affected by the disease, without the confounding influence of variable amounts of contaminating connective-tissue cells.</p

Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics

Derepression of DUX4 in skeletal muscle has emerged as a likely cause of pathology in facioscapulohumeral muscular dystrophy (FSHD). Here we report on the use of antisense phosphorodiamidate morpholino oligonucleotides to suppress DUX4 expression and function in FSHD myotubes and xenografts. The most effective was phosphorodiamidate morpholino oligonucleotide FM10, which targets the polyadenylation signal of DUX4. FM10 had no significant cell toxicity, and RNA-seq analyses of FSHD and control myotubes revealed that FM10 down-regulated many transcriptional targets of DUX4, without overt off-target effects. Electroporation of FM10 into FSHD patient muscle xenografts in mice also down-regulated DUX4 and DUX4 targets. These findings demonstrate the potential of antisense phosphorodiamidate morpholino oligonucleotides as an FSHD therapeutic option

iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease modeling

Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for \u3e 12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics

BMQ

BMQ: Boston Medical Quarterly was published from 1950-1966 by the Boston University School of Medicine and the Massachusetts Memorial Hospitals