Deep Mutational Scanning in Disease-related Genes with Saturation Mutagenesis-Reinforced Functional Assays (SMuRF).

Interpretation of disease-causing genetic variants remains a challenge in human genetics. Current costs and complexity of deep mutational scanning methods hamper crowd-sourcing approaches toward genome-wide resolution of variants in disease-related genes. Our framework, Saturation Mutagenesis-Reinforced Functional assays (SMuRF), addresses these issues by offering simple and cost-effective saturation mutagenesis, as well as streamlining functional assays to enhance the interpretation of unresolved variants. Applying SMuRF to neuromuscular disease genes FKRP and LARGE1, we generated functional scores for over 99.8% of all possible coding single nucleotide variants and resolved 310 clinically reported variants of uncertain significance with high confidence, enhancing clinical variant interpretation in dystroglycanopathies. SMuRF also demonstrates utility in predicting disease severity, resolving critical structural regions, and providing training datasets for the development of computational predictors. Our approach opens new directions for enabling variant-to-function insights for disease genes in a manner that is broadly useful for crowd-sourcing implementation across standard research laboratories.

Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases.

The progression of proteinuric kidney diseases is associated with podocyte loss, but the mechanisms underlying this process remain unclear. Podocytes reenter the cell cycle to repair double-stranded DNA breaks. However, unsuccessful repair can result in podocytes crossing the G1/S checkpoint and undergoing abortive cytokinesis. In this study, we identified Pfn1 as indispensable in maintaining glomerular integrity - its tissue-specific loss in mouse podocytes resulted in severe proteinuria and kidney failure. Our results suggest that this phenotype is due to podocyte mitotic catastrophe (MC), characterized histologically and ultrastructurally by abundant multinucleated cells, irregular nuclei, and mitotic spindles. Podocyte cell cycle reentry was identified using FUCCI2aR mice, and we observed altered expression of cell-cycle associated proteins, such as p21, p53, cyclin B1, and cyclin D1. Podocyte-specific translating ribosome affinity purification and RNA-Seq revealed the downregulation of ribosomal RNA-processing 8 (Rrp8). Overexpression of Rrp8 in Pfn1-KO podocytes partially rescued the phenotype in vitro. Clinical and ultrastructural tomographic analysis of patients with diverse proteinuric kidney diseases further validated the presence of MC podocytes and reduction in podocyte PFN1 expression within kidney tissues. These results suggest that profilin1 is essential in regulating the podocyte cell cycle and its disruption leads to MC and subsequent podocyte loss.

Death after High-Dose rAAV9 Gene Therapy in a Patient with Duchenne's Muscular Dystrophy.

We treated a 27-year-old patient with Duchenne's muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing dSaCas9 (i.e., "dead" Staphylococcus aureus Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR-transactivator therapy. The dose of rAAV used was 1×1014 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.).

Development of Assays to Measure GNE Gene Potency and Gene Replacement in Skeletal Muscle.

BACKGROUND: GNE myopathy (GNEM) is a severe muscle disease caused by mutations in the UDP-GlcNAc-2-epimerase/ManNAc-6-kinase (GNE) gene, which encodes a bifunctional enzyme required for sialic acid (Sia) biosynthesis. OBJECTIVE: To develop assays to demonstrate the potency of AAV gene therapy vectors in making Sia and to define the dose required for replacement of endogenous mouse Gne gene expression with human GNE in skeletal muscles. METHODS: A MyoD-inducible Gne-deficient cell line, Lec3MyoDI, and a GNE-deficient human muscle cell line, were made and tested to define the potency of various AAV vectors to increase binding of Sia-specific lectins, including MAA and SNA. qPCR and qRT-PCR methods were used to quantify AAV biodistribution and GNE gene expression after intravenous delivery of AAV vectors designed with different promoters in wild-type mice. RESULTS: Lec3 cells showed a strong deficit in MAA binding, while GNE-/-MB135 cells did not. Overexpressing GNE in Lec3 and Lec3MyoDI cells by AAV infection stimulated MAA binding in a dose-dependent manner. Use of a constitutive promoter, CMV, showed higher induction of MAA binding than use of muscle-specific promoters (MCK, MHCK7). rAAVrh74.CMV.GNE stimulated human GNE expression in muscles at levels equivalent to endogenous mouse Gne at a dose of 1×1013vg/kg, while AAVs with muscle-specific promoters required higher doses. AAV biodistribution in skeletal muscles trended higher when CMV was used as the promoter, and this correlated with increased sialylation of its viral capsid. CONCLUSIONS: Lec3 and Lec3MyoDI cells work well to assay the potency of AAV vectors in making Sia. Systemic delivery of rAAVrh74.CMV.GNE can deliver GNE gene replacement to skeletal muscles at doses that do not overwhelm non-muscle tissues, suggesting that AAV vectors that drive constitutive organ expression could be used to treat GNEM.

The Muscular Dystrophy Association's neuroMuscular ObserVational Research Data Hub (MOVR): Design, Methods, and Initial Observations.

BACKGROUND: Neuromuscular disease (NMD) research is experiencing tremendous growth as a result of progress in diagnostics and therapeutics yet there continues to be a significant clinical data shortage for these rare diseases. To maximize the development and impact of new therapies, the Muscular Dystrophy Association (MDA) created the neuroMuscular ObserVational Research Data Hub (MOVR) as an observational research study that collects disease-specific measures from individuals living with NMDs in the United States. OBJECTIVE: This manuscript provides a description of MOVR, participants enrolled in MOVR, and longitudinal data availability. METHODS: MOVR collects longitudinal data from individuals diagnosed with ALS, BMD, DMD, FSHD, LGMD, Pompe disease, or SMA, and who are seen for care at a participating MDA Care Center. Data are entered from medical records into standardized electronic case report forms (eCRFs). These eCRFs capture participants' demographics, diagnostic journeys, clinical visits, and discontinuation from the study. RESULTS: From January 2019 to May 2022, MOVR collected data from 50 participating care centers and 1,957 participants. Data from 1,923 participants who participated in MDA's pilot registry were migrated into MOVR, creating a total of 3,880 participants in MOVR. Initial analysis of aggregated data demonstrated that 91% of eCRFs were complete. Forty-three percent of participants had 3 or more encounters and 50% of all encounters were 5 months or less from the previous encounter. DISCUSSION: As a centralized data hub for multiple NMDs, MOVR serves as a platform that can be used to inform disease understanding, guide clinical trial design, and accelerate drug development for NMDs.

Meeting Report: 2022 Muscular Dystrophy Association Summit on 'Safety and Challenges in Gene Transfer Therapy'.

Muscular Dystrophy Association (MDA) has invested over $125M in the development of gene therapy for neuromuscular disorders (NMD) over the past 20 years. As a lead initiator of progress in this important field of medicine and to help ensure continued progress towards therapies for patients, MDA organized a dedicated summit in January 2022 to address emerging challenges in safely delivering adeno-associated virus (AAV) mediated gene therapies with a focus on their application in NMD. In this meeting, chaired by Carsten Bönnemann (NINDS, NIH) and Barry Byrne (University of Florida), academic and industry experts and stakeholders convened to openly discuss adverse events linked to clinical trials, as well as other challenges emerging in preclinical studies associated with difficulties in the translation of AAV-mediated gene therapies.

Flavones provide resistance to DUX4-induced toxicity via an mTor-independent mechanism.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common of the muscular dystrophies, affecting nearly 1 in 8000 individuals, and is a cause of profound disability. Genetically, FSHD is linked to the contraction and/or epigenetic de-repression of the D4Z4 repeat array on chromosome 4, thereby allowing expression of the DUX4 gene in skeletal muscle. If the DUX4 transcript incorporates a stabilizing polyadenylation site the myotoxic DUX4 protein will be synthesized, resulting in muscle wasting. The mechanism of toxicity remains unclear, as many DUX4-induced cytopathologies have been described, however cell death does primarily occur through caspase 3/7-dependent apoptosis. To date, most FSHD therapeutic development has focused on molecular methods targeting DUX4 expression or the DUX4 transcript, while therapies targeting processes downstream of DUX4 activity have received less attention. Several studies have demonstrated that inhibition of multiple signal transduction pathways can ameliorate DUX4-induced toxicity, and thus compounds targeting these pathways have the potential to be developed into FSHD therapeutics. To this end, we have screened a group of small molecules curated based on their reported activity in relevant pathways and/or structural relationships with known toxicity-modulating molecules. We have identified a panel of five compounds that function downstream of DUX4 activity to inhibit DUX4-induced toxicity. Unexpectedly, this effect was mediated through an mTor-independent mechanism that preserved expression of ULK1 and correlated with an increase in a marker of active cellular autophagy. This identifies these flavones as compounds of interest for therapeutic development, and potentially identifies the autophagy pathway as a target for therapeutics.

Nuclease-Deficient Clustered Regularly Interspaced Short Palindromic Repeat-Based Approaches for In Vitro and In Vivo Gene Activation.

Clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been adapted to achieve a wide range of genome modifications, including transcription regulation. The focus of this review is on the application of CRISPR-based platforms such as nuclease-deficient Cas9 and Cas12a, to achieve targeted gene activation. We review studies to date that have used CRISPR-based activation technology for the elucidation of biological mechanism and disease correction, as well as its application in genetic screens as a powerful tool for high-throughput genotype-phenotype mapping. In addition to our synthesis and critical analysis of published studies, we explore key considerations for the potential clinical translation of CRISPR-based activation technology.

Therapeutic Approaches in Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common types of muscular dystrophy, affecting roughly one in 8000 individuals. The complex underlying genetics and poor mechanistic understanding has caused a bottleneck in therapeutic development. Until the discovery of DUX4 and its causal role in FSHD, most trials were untargeted with limited results. Emerging approaches can learn from these early trials to increase their chance of success. Here, we explore the evolution of FSHD clinical trials from nonspecific anabolic or anti-inflammatory/oxidant strategies to cutting-edge molecular therapies targeting DUX4, and we discuss the importance of clinical outcome measures. With combined advances across multiple facets of FSHD research, the field is now poised to accelerate the process of therapeutic discovery and testing.

Cellular and animal models for facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common forms of muscular dystrophy and presents with weakness of the facial, scapular and humeral muscles, which frequently progresses to the lower limbs and truncal areas, causing profound disability. Myopathy results from epigenetic de-repression of the D4Z4 microsatellite repeat array on chromosome 4, which allows misexpression of the developmentally regulated DUX4 gene. DUX4 is toxic when misexpressed in skeletal muscle and disrupts several cellular pathways, including myogenic differentiation and fusion, which likely underpins pathology. DUX4 and the D4Z4 array are strongly conserved only in primates, making FSHD modeling in non-primate animals difficult. Additionally, its cytotoxicity and unusual mosaic expression pattern further complicate the generation of in vitro and in vivo models of FSHD. However, the pressing need to develop systems to test therapeutic approaches has led to the creation of multiple engineered FSHD models. Owing to the complex genetic, epigenetic and molecular factors underlying FSHD, it is difficult to engineer a system that accurately recapitulates every aspect of the human disease. Nevertheless, the past several years have seen the development of many new disease models, each with their own associated strengths that emphasize different aspects of the disease. Here, we review the wide range of FSHD models, including several in vitro cellular models, and an array of transgenic and xenograft in vivo models, with particular attention to newly developed systems and how they are being used to deepen our understanding of FSHD pathology and to test the efficacy of drug candidates.

Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy.

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

Transgenic zebrafish model of DUX4 misexpression reveals a developmental role in FSHD pathogenesis.

Facioscapulohumeral dystrophy type 1 (FSHD-1) is the most common autosomal dominant form of muscular dystrophy with a prevalence of ∼1 in 8000 individuals. It is considered a late-onset form of muscular dystrophy and leads to asymmetric muscle weakness in the facial, scapular, trunk and lower extremities. The prevalent hypothesis on disease pathogenesis is explained by misexpression of a germ line, primate-specific transcription factor DUX4-fl (double homeobox 4, full-length isoform) linked to the chromosome 4q35. In vitro and in vivo studies have demonstrated that very low levels of DUX4-fl expression are sufficient to induce an apoptotic and/or lethal phenotype, and therefore modeling of the disease has proved challenging. In this study, we expand upon our previously established injection model of DUX4 misexpression in zebrafish and describe a DUX4-inducible transgenic zebrafish model that better recapitulates the expression pattern and late onset phenotype characteristic of FSHD patients. We show that an induced burst of DUX4 expression during early development results in the onset of FSHD-like phenotypes in adulthood, even when DUX4 is no longer detectable. We also utilize our injection model to study long-term consequences of DUX4 expression in those that fail to show a developmental phenotype. Herein, we introduce a hypothesis that DUX4 expression during developmental stages is sufficient to induce FSHD-like phenotypes in later adulthood. Our findings point to a developmental role of DUX4 misexpression in the pathogenesis of FSHD and should be factored into the design of future therapies.

A limb-girdle muscular dystrophy 2I model of muscular dystrophy identifies corrective drug compounds for dystroglycanopathies.

Zebrafish are a powerful tool for studying muscle function owing to their high numbers of offspring, low maintenance costs, evolutionarily conserved muscle functions, and the ability to rapidly take up small molecular compounds during early larval stages. Fukutin-related protein (FKRP) is a putative protein glycosyltransferase that functions in the Golgi apparatus to modify sugar chain molecules of newly translated proteins. Patients with mutations in the FKRP gene can have a wide spectrum of clinical symptoms with varying muscle, eye, and brain pathologies depending on the location of the mutation in the FKRP protein. Patients with a common L276I FKRP mutation have mild adult-onset muscle degeneration known as limb-girdle muscular dystrophy 2I (LGMD2I), whereas patients with more C-terminal pathogenic mutations develop the severe Walker-Warburg syndrome (WWS)/muscle-eye-brain (MEB) disease. We generated fkrp-mutant zebrafish that phenocopy WWS/MEB pathologies including severe muscle breakdowns, head malformations, and early lethality. We have also generated a milder LGMD2I-model zebrafish via overexpression of a heat shock-inducible human FKRP (L276I) transgene that shows milder muscle pathology. Screening of an FDA-approved drug compound library in the LGMD2I zebrafish revealed a strong propensity towards steroids, antibacterials, and calcium regulators in ameliorating FKRP-dependent pathologies. Together, these studies demonstrate the utility of the zebrafish to both study human-specific FKRP mutations and perform compound library screenings for corrective drug compounds to treat muscular dystrophies.

Limited proteolysis as a tool to probe the tertiary conformation of dysferlin and structural consequences of patient missense variant L344P.

Dysferlin is a large transmembrane protein that plays a key role in cell membrane repair and underlies a recessive form of inherited muscular dystrophy. Dysferlinopathy is characterized by absence or marked reduction of dysferlin protein with 43% of reported pathogenic variants being missense variants that span the length of the dysferlin protein. The unique structure of dysferlin, with seven tandem C2 domains separated by linkers, suggests dysferlin may dynamically associate with phospholipid membranes in response to Ca2+ signaling. However, the overall conformation of the dysferlin protein is uncharacterized. To dissect the structural architecture of dysferlin, we have applied the method of limited proteolysis, which allows nonspecific digestion of unfolded peptides by trypsin. Using five antibodies spanning the dysferlin protein, we identified a highly reproducible jigsaw map of dysferlin fragments protected from digestion. Our data infer a modular architecture of four tertiary domains: 1) C2A, which is readily removed as a solo domain; 2) midregion C2B-C2C-Fer-DysF, commonly excised as an intact module, with subdigestion to different fragments suggesting several dynamic folding options; 3) C-terminal four-C2 domain module; and 4) calpain-cleaved mini-dysferlinC72, which is particularly resistant to proteolysis. Importantly, we reveal a patient missense variant, L344P, that largely escapes proteasomal surveillance and shows subtle but clear changes in tertiary conformation. Accompanying evidence from immunohistochemistry and flow cytometry using antibodies with conformationally sensitive epitopes supports proteolysis data. Collectively, we provide insight into the structural topology of dysferlin and show how a single missense mutation within dysferlin can exert local changes in tertiary conformation.

Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral Muscular Dystrophy is a common form of muscular dystrophy that presents clinically with progressive weakness of the facial, scapular, and humeral muscles, with later involvement of the trunk and lower extremities. While typically inherited as autosomal dominant, facioscapulohumeral muscular dystrophy (FSHD) has a complex genetic and epigenetic etiology that has only recently been well described. The most prevalent form of the disease, FSHD1, is associated with the contraction of the D4Z4 microsatellite repeat array located on a permissive 4qA chromosome. D4Z4 contraction allows epigenetic derepression of the array, and possibly the surrounding 4q35 region, allowing misexpression of the toxic DUX4 transcription factor encoded within the terminal D4Z4 repeat in skeletal muscles. The less common form of the disease, FSHD2, results from haploinsufficiency of the SMCHD1 gene in individuals carrying a permissive 4qA allele, also leading to the derepression of DUX4, further supporting a central role for DUX4. How DUX4 misexpression contributes to FSHD muscle pathology is a major focus of current investigation. Misexpression of other genes at the 4q35 locus, including FRG1 and FAT1, and unlinked genes, such as SMCHD1, has also been implicated as disease modifiers, leading to several competing disease models. In this review, we describe recent advances in understanding the pathophysiology of FSHD, including the application of MRI as a research and diagnostic tool, the genetic and epigenetic disruptions associated with the disease, and the molecular basis of FSHD. We discuss how these advances are leading to the emergence of new approaches to enable development of FSHD therapeutics. © 2017 American Physiological Society. Compr Physiol 7:1229-1279, 2017.

SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndrome.

Arhinia, or absence of the nose, is a rare malformation of unknown etiology that is often accompanied by ocular and reproductive defects. Sequencing of 40 people with arhinia revealed that 84% of probands harbor a missense mutation localized to a constrained region of SMCHD1 encompassing the ATPase domain. SMCHD1 mutations cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) via a trans-acting loss-of-function epigenetic mechanism. We discovered shared mutations and comparable DNA hypomethylation patterning between these distinct disorders. CRISPR/Cas9-mediated alteration of smchd1 in zebrafish yielded arhinia-relevant phenotypes. Transcriptome and protein analyses in arhinia probands and controls showed no differences in SMCHD1 mRNA or protein abundance but revealed regulatory changes in genes and pathways associated with craniofacial patterning. Mutations in SMCHD1 thus contribute to distinct phenotypic spectra, from craniofacial malformation and reproductive disorders to muscular dystrophy, which we speculate to be consistent with oligogenic mechanisms resulting in pleiotropic outcomes.

Emerging preclinical animal models for FSHD.

Facioscapulohumeral dystrophy (FSHD) is a unique and complex genetic disease that is not entirely solved. Recent advances in the field have led to a consensus genetic premise for the disorder, enabling researchers to now pursue the design of preclinical models. In this review we explore all available FSHD models (DUX4-dependent and -independent) for their utility in therapeutic discovery and potential to yield novel disease insights. Owing to the complex nature of FSHD, there is currently no single model that accurately recapitulates the genetic and pathophysiological spectrum of the disorder. Existing models emphasize only specific aspects of the disease, highlighting the need for more collaborative research and novel paradigms to advance the translational research space of FSHD.

MicroRNA-486-dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy-associated symptoms.

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmdmdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmdmdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle-specific miR-486 overexpression in Dmdmdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.