A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene.

Neuromuscular disorders are often caused by heterogeneous mutations in large, structurally complex genes. Targeting compensatory modifier genes could be beneficial to improve disease phenotypes. Here we report a mutation-independent strategy to upregulate the expression of a disease-modifying gene associated with congenital muscular dystrophy type 1A (MDC1A) using the CRISPR activation system in mice. MDC1A is caused by mutations in LAMA2 that lead to nonfunctional laminin-α2, which compromises the stability of muscle fibres and the myelination of peripheral nerves. Transgenic overexpression of Lama1, which encodes a structurally similar protein called laminin-α1, ameliorates muscle wasting and paralysis in mouse models of MDC1A, demonstrating its importance as a compensatory modifier of the disease1. However, postnatal upregulation of Lama1 is hampered by its large size, which exceeds the packaging capacity of vehicles that are clinically relevant for gene therapy. We modulate expression of Lama1 in the dy2j/dy2j mouse model of MDC1A using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators and single-guide RNAs that target the Lama1 promoter. When pre-symptomatic mice were treated, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis. However, for many disorders it is important to investigate the therapeutic window and reversibility of symptoms. In muscular dystrophies, it has been hypothesized that fibrotic changes in skeletal muscle are irreversible. However, we show that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic dy2j/dy2j mice with apparent hindlimb paralysis and muscle fibrosis. Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR-dCas9-mediated upregulation of Lama1, which may enable mutation-independent treatment for all patients with MDC1A. This approach has a broad applicability to a variety of disease-modifying genes and could serve as a therapeutic strategy for many inherited and acquired diseases.

Spell Checking Nature: Versatility of CRISPR/Cas9 for Developing Treatments for Inherited Disorders.

Clustered regularly interspaced short palindromic repeat (CRISPR) has arisen as a frontrunner for efficient genome engineering. However, the potentially broad therapeutic implications are largely unexplored. Here, to investigate the therapeutic potential of CRISPR/Cas9 in a diverse set of genetic disorders, we establish a pipeline that uses readily obtainable cells from affected individuals. We show that an adapted version of CRISPR/Cas9 increases the amount of utrophin, a known disease modifier in Duchenne muscular dystrophy (DMD). Furthermore, we demonstrate preferential elimination of the dominant-negative FGFR3 c.1138G>A allele in fibroblasts of an individual affected by achondroplasia. Using a previously undescribed approach involving single guide RNA, we successfully removed large genome rearrangement in primary cells of an individual with an X chromosome duplication including MECP2. Moreover, removal of a duplication of DMD exons 18-30 in myotubes of an individual affected by DMD produced full-length dystrophin. Our findings establish the far-reaching therapeutic utility of CRISPR/Cas9, which can be tailored to target numerous inherited disorders.

Generation and characterization of a novel mouse model of Becker Muscular Dystrophy with a deletion of exons 52 to 55.

Becker Muscular Dystrophy (BMD) is a rare X-linked recessive neuromuscular disorder frequently caused by in-frame deletions in the DMD gene that result in the production of a truncated, yet functional, dystrophin protein. The consequences of BMD-causing in-frame deletions on the organism are difficult to predict, especially in regard to long-term prognosis. Here, we employed CRISPR-Cas9 to generate a new Dmd del52-55 mouse model by deleting exons 52-55, resulting in a BMD-like in-frame deletion. To delineate the long-term effects of this deletion, we studied these mice over 52 weeks by performing histology and echocardiography analyses and assessing motor functions. Our results suggest that a truncated dystrophin is sufficient to maintain wildtype-like muscle and heart histology and functions in young mice. However, the truncated protein appears insufficient to maintain normal muscle homeostasis and protect against exercise-induced damage at 52 weeks. To further delineate the effects of this exon52-55 in-frame deletion, we performed RNA-Seq pre- and post-exercise and identified several differentially expressed pathways that reflect the abnormal muscle phenotype observed at 52 weeks in the BMD model.

Prevention of early-onset cardiomyopathy in Dmd exon 52-54 deletion mice by CRISPR-Cas9-mediated exon skipping.

Duchenne muscular dystrophy (DMD) is a disease with a life-threatening trajectory resulting from mutations in the dystrophin gene, leading to degeneration of skeletal muscle and fibrosis of cardiac muscle. The overwhelming majority of mutations are multiexonic deletions. We previously established a dystrophic mouse model with deletion of exons 52-54 in Dmd that develops an early-onset cardiac phenotype similar to DMD patients. Here we employed CRISPR-Cas9 delivered intravenously by adeno-associated virus (AAV) vectors to restore functional dystrophin expression via excision or skipping of exon 55. Exon skipping with a solitary guide significantly improved editing outcomes and dystrophin recovery over dual guide excision. Some improvements to genomic and transcript editing levels were observed when the guide dose was enhanced, but dystrophin restoration did not improve considerably. Editing and dystrophin recovery were restricted primarily to cardiac tissue. Remarkably, our exon skipping approach completely prevented onset of the cardiac phenotype in treated mice up to 12 weeks. Thus, our results demonstrate that intravenous delivery of a single-cut CRISPR-Cas9-mediated exon skipping therapy can prevent heart dysfunction in DMD in vivo.

Targeted genome editing in vivo corrects a Dmd duplication restoring wild-type dystrophin expression.

Tandem duplication mutations are increasingly found to be the direct cause of many rare heritable diseases, accounting for up to 10% of cases. Unfortunately, animal models recapitulating such mutations are scarce, limiting our ability to study them and develop genome editing therapies. Here, we describe the generation of a novel duplication mouse model, harboring a multi-exonic tandem duplication in the Dmd gene which recapitulates a human mutation. Duplication correction of this mouse was achieved by implementing a single-guide RNA (sgRNA) CRISPR/Cas9 approach. This strategy precisely removed a duplication mutation in vivo, restored full-length dystrophin expression, and was accompanied by improvements in both histopathological and clinical phenotypes. We conclude that CRISPR/Cas9 represents a powerful tool to accurately model and treat tandem duplication mutations. Our findings will open new avenues of research for exploring the study and therapeutics of duplication disorders.

A novel mouse model of Duchenne muscular dystrophy carrying a multi-exonic Dmd deletion exhibits progressive muscular dystrophy and early-onset cardiomyopathy.

Duchenne muscular dystrophy (DMD) is a life-threatening neuromuscular disease caused by the lack of dystrophin, resulting in progressive muscle wasting and locomotor dysfunctions. By adulthood, almost all patients also develop cardiomyopathy, which is the primary cause of death in DMD. Although there has been extensive effort in creating animal models to study treatment strategies for DMD, most fail to recapitulate the complete skeletal and cardiac disease manifestations that are presented in affected patients. Here, we generated a mouse model mirroring a patient deletion mutation of exons 52-54 (Dmd Δ52-54). The Dmd Δ52-54 mutation led to the absence of dystrophin, resulting in progressive muscle deterioration with weakened muscle strength. Moreover, Dmd Δ52-54 mice present with early-onset hypertrophic cardiomyopathy, which is absent in current pre-clinical dystrophin-deficient mouse models. Therefore, Dmd Δ52-54 presents itself as an excellent pre-clinical model to evaluate the impact on skeletal and cardiac muscles for both mutation-dependent and -independent approaches.

Improved electrospray ionization efficiency compensates for diminished chromatographic resolution and enables proteomics analysis of tyrosine signaling in embryonic stem cells.

Characterization of signaling pathways in embryonic stem cells is a prerequisite for future application of these cells to treat human disease and other disorders. Identification of tyrosine signaling cascades is of particular interest but is complicated by the relatively low levels of tyrosine phosphorylation in embryonic stem cells. These hurdles correlate with the primary limitations of mass spectrometry-based proteomics; namely, poor detection limit and dynamic range. To overcome these obstacles, we fabricated miniaturized LC-electrospray assemblies that provided approximately 15-fold improvement in LC-MS performance. Significantly, our characterization data demonstrate that electrospray ionization efficiency compensates for diminished chromatographic performance at effluent flow rates below Van Deemter minima. Use of these assemblies facilitated quantitative proteomics-based analysis of tyrosine signaling cascades in embryonic stem cells. Our results suggest that a renewed focus on miniaturized LC coupled to ultralow flow electrospray will provide a viable path for proteomic analysis of primary cells and rare post-translational modifications.

Patient-Derived Xenografts for Prognostication and Personalized Treatment for Head and Neck Squamous Cell Carcinoma.

Overall survival remains very poor for patients diagnosed as having head and neck squamous cell carcinoma (HNSCC). Identification of additional biomarkers and novel therapeutic strategies are important for improving patient outcomes. Patient-derived xenografts (PDXs), generated by implanting fresh tumor tissue directly from patients into immunodeficient mice, recapitulate many of the features of their corresponding clinical cancers, including histopathological and molecular profiles. Using a large collection of PDX models of HNSCC, we demonstrate that rapid engraftment into immunocompromised mice is highly prognostic and show that genomic deregulation of the G1/S checkpoint pathway correlates with engraftment. Furthermore, CCND1 and CDKN2A genomic alterations are predictive of response to the CDK4and CDK6 inhibitor abemaciclib. Overall, our study supports the pursuit of CDK4 and CDK6 inhibitors as a therapeutic strategy for a substantial proportion of HNSCC patients and demonstrates the potential of using PDX models to identify targeted therapies that will benefit patients who have the poorest outcomes.

The K/BxN mouse model of inflammatory arthritis: theory and practice.

Mice expressing the KRN T cell receptor transgene and the MHC class II molecule A(g7) (K/BxN mice) develop severe inflammatory arthritis, and serum from these mice causes similar arthritis in a wide range of mouse strains, owing to pathogenic autoantibodies to glucose-6-phosphate isomerase (GPI). This model has been useful for the investigation of the development of autoimmunity (K/BxN transgenic mice) and particularly of the mechanisms by which anti-GPI autoantibodies induce joint-specific imflammation (serum transfer model). In this chaper, after a summary of findings from this model system, we describe detailed methods for the maintenance of a K/BxN colony, crossing of the relevant TCR and MHC genes to other strain backgrounds, evaluation of KRN transgenic T cells, measurement of anti-GPI antibodies, induction of arthritis by serum transfer, and clinical and histological evaluation of arthritis.

Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism.

Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases. Prevalence is higher in neuromuscular disorders (NMDs), owing to the unusually large size and multi-exonic nature of genes encoding muscle structural proteins. Therapeutic genome editing to correct disease-causing splice-site mutations has been accomplished only through the homology-directed repair pathway, which is extremely inefficient in postmitotic tissues such as skeletal muscle. Here we describe a strategy using nonhomologous end-joining (NHEJ) to correct a pathogenic splice-site mutation. As a proof of principle, we focus on congenital muscular dystrophy type 1A (MDC1A), which is characterized by severe muscle wasting and paralysis. Specifically, we correct a splice-site mutation that causes the exclusion of exon 2 from Lama2 mRNA and the truncation of Lama2 protein in the dy2J/dy2J mouse model of MDC1A. Through systemic delivery of adeno-associated virus (AAV) carrying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing components, we simultaneously excise an intronic region containing the mutation and create a functional donor splice site through NHEJ. This strategy leads to the inclusion of exon 2 in the Lama2 transcript and restoration of full-length Lama2 protein. Treated dy2J/dy2J mice display substantial improvement in muscle histopathology and function without signs of paralysis.

Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma.

Rare cancer stem cells (CSC) are proposed to be responsible for tumour propagation and re-initiation and are functionally defined by identifying tumour-initiating cells (TICs) using the xenotransplantation limiting dilution assay (LDA). While TICs in clear cell renal cell carcinoma (ccRCC) appeared rare in NOD/SCID/IL2Rγ(-/-) (NSG) mice, xenografts formed more efficiently from small tumour fragments, indicating the LDA underestimated ccRCC TIC frequency. Mechanistic interrogation of the LDA identified multiple steps that influence ccRCC TIC quantitation. For example, tissue disaggregation destroys most ccRCC cells, common assays significantly overestimate tumour cell viability, and microenvironmental supplementation with human extracellular factors or pharmacological inhibition of anoikis increase clonogenicity and tumourigenicity of ccRCC cell lines and primary tumour cells. Identification of these previously uncharacterized concerns that cumulatively lead to substantial underestimation of TICs in ccRCC provides a framework for development of more accurate TIC assays in the future, both for this disease and for other cancers.

Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.

Paradoxical dampening of anti-islet self-reactivity but promotion of diabetes by OX40 ligand.

Costimulatory signals received by diabetogenic T cells during priming by or upon secondary encounter with autoantigen are decisive in determining the outcome of autoimmune attack. The OX40-OX40 ligand (OX40L) costimulatory pathway is known to influence T cell responses, prompting us to examine its role in autoimmune diabetes. A null allele at OX40L completely prevented diabetes development in nonobese diabetic mice and strongly reduced its incidence in a TCR transgenic model (BDC2.5). However, somewhat paradoxically, the initial activation of T cells responsive to islet beta cell Ag was slightly faster and more efficient in the absence of OX40L, with an increased degree of cell proliferation and survival in the deficient hosts. Activated T cell migration into and retention within the islets was also slightly accelerated. When challenged in vitro, splenocytes from BDC2.5.OX40L(o/o) mice showed no altered reactivity to exogenously added peptide, no bias to the Th1 or Th2 phenotype, and no alteration in T cell survival. Thus, the OX40/OX40L axis has the paradoxical effect of dampening the early activation and migration of autoimmune T cells, but sustains the long-term progression to autoimmune destruction.