Exon Skipping Therapy.

Exondys 51 is the first therapy for Duchenne muscular dystrophy (DMD) to have been granted accelerated approval by the FDA. Approval was granted based on using dystrophin expression as a surrogate marker. Exondys 51 targets DMD exon 51 for skipping to restore the reading frame for 13% of Duchenne patients.

Nanoparticles systemically biodistribute to regenerating skeletal muscle in DMD.

Skeletal muscle disease severity can often progress asymmetrically across muscle groups and heterogeneously within tissues. An example is Duchenne Muscular Dystrophy (DMD) in which lack of dystrophin results in devastating skeletal muscle wasting in some muscles whereas others are spared or undergo hypertrophy. An efficient, non-invasive approach to identify sites of asymmetry and degenerative lesions could enable better patient monitoring and therapeutic targeting of disease. In this study, we utilized a versatile intravenously injectable mesoporous silica nanoparticle (MSNP) based nanocarrier system to explore mechanisms of biodistribution in skeletal muscle of mdx mouse models of DMD including wildtype, dystrophic, and severely dystrophic mice. Moreover, MSNPs could be imaged in live mice and whole muscle tissues enabling investigation of how biodistribution is altered by different types of muscle pathology such as inflammation or fibrosis. We found MSNPs were tenfold more likely to aggregate within select mdx muscles relative to wild type, such as gastrocnemius and quadriceps. This was accompanied by decreased biodistribution in off-target organs. We found the greatest factor affecting preferential delivery was the regenerative state of the dystrophic skeletal muscle with the highest MSNP abundance coinciding with the regions showing the highest level of embryonic myosin staining and intramuscular macrophage uptake. To demonstrate, muscle regeneration regulated MSNP distribution, we experimentally induced regeneration using barium chloride which resulted in a threefold increase of intravenously injected MSNPs to sites of regeneration 7 days after injury. These discoveries provide the first evidence that nanoparticles have selective biodistribution to skeletal muscle in DMD to areas of active regeneration and that nanoparticles could enable diagnostic and selective drug delivery in DMD skeletal muscle.

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond.

This is a review describing advances in CRISPR/Cas-mediated therapies for neuromuscular disorders (NMDs). We explore both CRISPR-mediated editing and dead Cas approaches as potential therapeutic strategies for multiple NMDs. Last, therapeutic considerations, including delivery and off-target effects, are also discussed.

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells.

Targeted genome editing technology can correct the sickle cell disease mutation of the ß-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the ß-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.

Innate and adaptive AAV-mediated immune responses in a mouse model of Duchenne muscular dystrophy.

High systemic doses of adeno-associated viruses (AAVs) have been associated with immune-related serious adverse events (SAEs). Although AAV was well tolerated in preclinical models, SAEs were observed in clinical trials, indicating the need for improved preclinical models to understand AAV-induced immune responses. Here, we show that mice dual-dosed with AAV9 at 4-week intervals better recapitulate aspects of human immunity to AAV. In the model, anti-AAV9 immunoglobulin G (IgGs) increased in a linear fashion between the first and second AAV administrations. Complement activation was only observed in the presence of high levels of both AAV and anti-AAV IgG. Myeloid-derived pro-inflammatory cytokines were significantly induced in the same pattern as complement activation, suggesting that myeloid cell activation to AAV may rely on the presence of both AAV and anti-AAV IgG complexes. Single-cell RNA sequencing of peripheral blood mononuclear cells confirmed that activated monocytes were a primary source of pro-inflammatory cytokines and chemokines, which were significantly increased after a second AAV9 exposure. The same activated monocyte clusters expressed both Fcγ and complement receptors, suggesting that anti-AAV-mediated activation of myeloid cells through Fcγ receptors and/or complement receptors is one mechanism by which anti-AAV antigen complexes may prime antigen-presenting cells and amplify downstream immunity.

Release of plasmid DNA-encoding IL-10 from PLGA microparticles facilitates long-term reversal of neuropathic pain following a single intrathecal administration.

PURPOSE: Interleukin-10 (IL-10) is an anti-inflammatory molecule that has achieved interest as a therapeutic for neuropathic pain. In this work, the potential of plasmid DNA-encoding IL-10 (pDNA-IL-10) slowly released from biodegradable microparticles to provide long-term pain relief in an animal model of neuropathic pain was investigated. METHODS: PLGA microparticles encapsulating pDNA-IL-10 were developed and assessed both in vitro and in vivo. RESULTS: In vitro, pDNA containing microparticles activated macrophages, enhanced the production of nitric oxide, and increased the production of IL-10 protein relative to levels achieved with unencapsulated pDNA-IL-10. In vivo, intrathecally administered microparticles embedded in meningeal tissue, induced phagocytic cell recruitment to the cerebrospinal fluid, and relieved neuropathic pain for greater than 74 days following a single intrathecal administration, a feat not achieved with unencapsulated pDNA. Therapeutic effects of microparticle-delivered pDNA-IL-10 were blocked in the presence of IL-10-neutralizing antibody, and elevated levels of plasmid-derived IL-10 were detected in tissues for a prolonged time period post-injection (>28 days), demonstrating that therapeutic effects are dependent on IL-10 protein production. CONCLUSIONS: These studies demonstrate that microparticle encapsulation significantly enhances the potency of intrathecally administered pDNA, which may be extended to treat other disorders that require intrathecal gene therapy.

Generation of PAX7 Reporter Cells to Investigate Skeletal Myogenesis from Human Pluripotent Stem Cells.

This protocol describes the use of CRISPR/Cas9-mediated homology-directed recombination to construct a PAX7-GFP reporter in human pluripotent stem cells (hPSCs). PAX7 is a key transcription factor and regulator of skeletal muscle stem/progenitor cells. We obtained heterozygous knockin reporter cells and validated their PAX7 expression using both artificial activation by the CRISPR/dCas9-VPR system and physiological activation during hPSC myogenic differentiation. These cells can serve as tools for better understanding of in vitro hPSC myogenesis and enriching myogenic cells for downstream analysis. For complete details on the use and execution of this protocol, please refer to Xi et al. (2017) and Xi et al. (2020).

A Human Skeletal Muscle Atlas Identifies the Trajectories of Stem and Progenitor Cells across Development and from Human Pluripotent Stem Cells.

The developmental trajectory of human skeletal myogenesis and the transition between progenitor and stem cell states are unclear. We used single-cell RNA sequencing to profile human skeletal muscle tissues from embryonic, fetal, and postnatal stages. In silico, we identified myogenic as well as other cell types and constructed a "roadmap" of human skeletal muscle ontogeny across development. In a similar fashion, we also profiled the heterogeneous cell cultures generated from multiple human pluripotent stem cell (hPSC) myogenic differentiation protocols and mapped hPSC-derived myogenic progenitors to an embryonic-to-fetal transition period. We found differentially enriched biological processes and discovered co-regulated gene networks and transcription factors present at distinct myogenic stages. This work serves as a resource for advancing our knowledge of human myogenesis. It also provides a tool for a better understanding of hPSC-derived myogenic progenitors for translational applications in skeletal muscle-based regenerative medicine.

ERBB3 and NGFR mark a distinct skeletal muscle progenitor cell in human development and hPSCs.

Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells remains unclear. We observed that hPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite cells. Using RNA sequencing, we found that the cell surface receptors ERBB3 and NGFR demarcate myogenic populations, including PAX7 progenitors in human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal muscle is immature, but inhibition of transforming growth factor-ß signalling during differentiation improved fusion efficiency, ultrastructural organization and the expression of adult myosins. This enrichment and maturation strategy restored dystrophin in hundreds of dystrophin-deficient myofibres after engraftment of CRISPR-Cas9-corrected Duchenne muscular dystrophy human induced pluripotent stem cell-SMPCs. The work provides an in-depth characterization of human myogenesis, and identifies candidates that improve the in vivo myogenic potential of hPSC-SMPCs to levels that are equal to directly isolated human fetal muscle cells.

"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies.

Creation of a Novel Humanized Dystrophic Mouse Model of Duchenne Muscular Dystrophy and Application of a CRISPR/Cas9 Gene Editing Therapy.

Duchenne muscular dystrophy is caused by mutations in DMD which disrupt the reading frame. Therapeutic strategies that restore DMD's reading frame, such as exon skipping and CRISPR/Cas9, need to be tested in the context of the human DMD sequence in vivo. We have developed a novel dystrophic mouse model by using CRISPR/Cas9 to delete exon 45 in the human DMD gene in hDMD mice, which places DMD out-of-frame. We have utilized this model to demonstrate that our clinically-relevant CRISPR/Cas9 platform, which targets deletion of human DMD exons 45-55, can be directly applied in vivo to restore dystrophin.

A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells.

Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725 kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed evaluation of dystrophin in disease-relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.