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 >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.

Muscular dystrophies are a group of inherited genetic diseases characterised by progressive muscle weakness. They lead to disability or even death, and no cure exists against these conditions. Advances in genome sequencing have identified many mutations that underly muscular dystrophies, opening the door to new therapies that could repair incorrect genes or rebuild damaged muscles. However, testing these ideas requires better ways to recreate human muscular dystrophy in the laboratory. One strategy for modelling muscular dystrophy involves coaxing skin or other cells from an individual into becoming 'induced pluripotent stem cells'; these can then mature to form almost any adult cell in the body, including muscles. However, this approach does not usually create myoblasts, the 'precursor' cells that specifically mature into muscle during development. This limits investigations into how disease-causing mutations impact muscle formation early on. As a response, Guo et al. developed a two-step protocol of muscle maturation followed by stem cell growth selection to isolate and grow 'induced myoblasts' from induced pluripotent stem cells taken from healthy volunteers and muscular dystrophy patients. These induced myoblasts can both make more of themselves and become muscle, allowing Guo et al. to model three different types of muscular dystrophy. These myoblasts also behave as stem cells when grafted inside adult mouse muscles: some formed human muscle tissue while others remained as precursor cells, which could then respond to muscle injury and start repair. The induced myoblasts developed by Guo et al. will enable scientists to investigate the impacts of different mutations on muscle tissue and to better test treatments. They could also be used as part of regenerative medicine therapies, to restore muscle cells in patients.

A non-viral genome editing platform for site-specific insertion of large transgenes.

BACKGROUND: The precise, functional and safe insertion of large DNA payloads into host genomes offers versatility in downstream genetic engineering-associated applications, spanning cell and gene therapies, therapeutic protein production, high-throughput cell-based drug screening and reporter cell lines amongst others. Employing viral- and non-viral-based genome engineering tools to achieve specific insertion of large DNA-despite being successful in E. coli and animal models-still pose challenges in the human system. In this study, we demonstrate the applicability of our lambda integrase-based genome insertion tool for human cell and gene therapy applications that require insertions of large functional genes, as exemplified by the integration of a functional copy of the F8 gene and a Double Homeobox Protein 4 (DUX4)-based reporter cassette for potential hemophilia A gene therapy and facioscapulohumeral muscular dystrophy (FSHD)-based high-throughput drug screening purposes, respectively. Thus, we present a non-viral genome insertion tool for safe and functional delivery of large seamless DNA cargo into the human genome that can enable novel designer cell-based therapies. METHODS: Previously, we have demonstrated the utility of our phage λ-integrase platform to generate seamless vectors and subsequently achieve functional integration of large-sized DNA payloads at defined loci in the human genome. To further explore this tool for therapeutic applications, we used pluripotent human embryonic stem cells (hESCs) to integrate large seamless vectors comprising a 'gene of interest'. Clonal cell populations were screened for the correct integration events and further characterized by southern blotting, gene expression and protein activity assays. In the case of our hemophilia A-related study, clones were differentiated to confirm that the targeted locus is active after differentiation and actively express and secrete Factor VIII. RESULTS: The two independent approaches demonstrated specific and functional insertions of a full-length blood clotting F8 expression cassette of ~ 10 kb and of a DUX4 reporter cassette of ~ 7 kb in hESCs. CONCLUSION: We present a versatile tool for site-specific human genome engineering with large transgenes for cell/gene therapies and other synthetic biology and biomedical applications.

A cross sectional study of two independent cohorts identifies serum biomarkers for facioscapulohumeral muscular dystrophy (FSHD).

Measuring the severity and progression of facioscapulohumeral muscular dystrophy (FSHD) is particularly challenging because muscle weakness progresses over long periods of time and can be sporadic. Biomarkers are essential for measuring disease burden and testing treatment strategies. We utilized the sensitive, specific, high-throughput SomaLogic proteomics platform of 1129 proteins to identify proteins with levels that correlate with FSHD severity in a cross-sectional study of two independent cohorts. We discovered biomarkers that correlate with clinical severity and disease burden measured by magnetic resonance imaging. Sixty-eight proteins in the Rochester cohort (n = 48) and 51 proteins in the Seattle cohort (n = 30) had significantly different levels in FSHD-affected individuals when compared with controls (p-value ≤ .005). A subset of these varied by at least 1.5 fold and four biomarkers were significantly elevated in both cohorts. Levels of creatine kinase MM and MB isoforms, carbonic anhydrase III, and troponin I type 2 reliably predicted the disease state and correlated with disease severity. Other novel biomarkers were also discovered that may reveal mechanisms of disease pathology. Assessing the levels of these biomarkers during clinical trials may add significance to other measures of quantifying disease progression or regression.

Endogenous DUX4 expression in FSHD myotubes is sufficient to cause cell death and disrupts RNA splicing and cell migration pathways.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by chromatin relaxation that results in aberrant expression of the transcription factor Double Homeobox 4 (DUX4). DUX4 protein is present in a small subset of FSHD muscle cells, making its detection and analysis of its effects historically difficult. Using a DUX4-activated reporter, we demonstrate the burst expression pattern of endogenous DUX4, its method of signal amplification in the unique shared cytoplasm of the myotube, and FSHD cell death that depends on its activation. Transcriptome analysis of DUX4-expressing cells revealed that DUX4 activation disrupts RNA metabolism including RNA splicing, surveillance and transport pathways. Cell signaling, polarity and migration pathways were also disrupted. Thus, DUX4 expression is sufficient for myocyte death, and these findings suggest mechanistic links between DUX4 expression and cell migration, supporting recent descriptions of phenotypic similarities between FSHD and an FSHD-like condition caused by FAT1 mutations.