Haplotype editing with CRISPR/Cas9 as a therapeutic approach for dominant-negative missense mutations in .

Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplo-sufficient. We previously edited a dominant missense mutation with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different missense mutations cause disease.

Generation and characterization of an isogenic control line by correcting the BAG3 P209L mutation of a human induced pluripotent stem cell (hiPSC) line from a patient with myofibrillar myopathy-6.

BAG3 is a central component of the chaperone-assisted selective autophagy complex and thus important for proteostasis. This function is affected by a point mutation (p.P209L; c.

Advances and challenges in modeling inherited peripheral neuropathies using iPSCs.

Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date.

Functional analysis of a common BAG3 allele associated with protection from heart failure.

Multiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism remains elusive. In this study, we used induced pluripotent stem cells to test if the only coding variant in this allele block, BAG3, alters protein and cellular function in human cardiomyocytes. Quantitative protein interaction analysis identified changes in BAG3 protein partners specific to cardiomyocytes.

Allele-Specific Gene Editing Rescues Pathology in a Human Model of Charcot-Marie-Tooth Disease Type 2E.

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death.

Bioengineered optogenetic model of human neuromuscular junction.

Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users.

Rapid, precise quantification of large DNA excisions and inversions by ddPCR.

The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length.

Mapping cis-regulatory chromatin contacts in neural cells links neuropsychiatric disorder risk variants to target genes.

Mutations in gene regulatory elements have been associated with a wide range of complex neuropsychiatric disorders. However, due to their cell-type specificity and difficulties in characterizing their regulatory targets, the ability to identify causal genetic variants has remained limited. To address these constraints, we perform an integrative analysis of chromatin interactions, open chromatin regions and transcriptomes using promoter capture Hi-C, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing, respectively, in four functionally distinct neural cell types: induced pluripotent stem cell (iPSC)-induced excitatory neurons and lower motor neurons, iPSC-derived hippocampal dentate gyrus-like neurons and primary astrocytes.

Two patients with FOXF1 mutations with alveolar capillary dysplasia with misalignment of pulmonary veins and other malformations: Two different presentations and outcomes.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) describes a group of developmental disorders affecting the lungs with its pulmonary vasculature. Mutations in the FOXF1 gene have been reported in most cases, and extrapulmonary findings were described. We present two patients with ACDMPV and FOXF1 mutations that illustrate the variability in presentation and outcome of their disease.

Variable rescue of microtubule and physiological phenotypes in mdx muscle expressing different miniaturized dystrophins.

Delivery of miniaturized dystrophin genes via adeno-associated viral vectors is one leading approach in development to treat Duchenne muscular dystrophy. Here we directly compared the functionality of five mini- and micro-dystrophins via skeletal muscle-specific transgenic expression in dystrophin-deficient mdx mice. We evaluated their ability to rescue defects in the microtubule network, passive stiffness and contractility of skeletal muscle.

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism.

Miniaturized iPS-Cell-Derived Cardiac Muscles for Physiologically Relevant Drug Response Analyses.

Tissue engineering approaches have the potential to increase the physiologic relevance of human iPS-derived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires >1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening.

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs.

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes.

Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured over Different Spatial Scales.

Contractile motion is the simplest metric of cardiomyocyte health in vitro, but unbiased quantification is challenging. We describe a rapid automated method, requiring only standard video microscopy, to analyze the contractility of human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CM). New algorithms for generating and filtering motion vectors combined with a newly developed isogenic iPSC line harboring genetically encoded calcium indicator, GCaMP6f, allow simultaneous user-independent measurement and analysis of the coupling between calcium flux and contractility.

Isolation of single-base genome-edited human iPS cells without antibiotic selection.

Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.

Expression of the dystrophin isoform Dp116 preserves functional muscle mass and extends lifespan without preventing dystrophy in severely dystrophic mice.

Dp116 is a non-muscle isoform of dystrophin that assembles the dystrophin-glycoprotein complex (DGC), but lacks actin-binding domains. To examine the functional role of the DGC, we expressed the Dp116 transgene in mice lacking both dystrophin and utrophin (mdx:utrn(-/-)). Unexpectedly, expression of Dp116 prevented the most severe aspects of the mdx:utrn(-/-) phenotype.

The polyproline site in hinge 2 influences the functional capacity of truncated dystrophins.

Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild form of the disease, Becker muscular dystrophy. The hinge 3 region in the rod domain of dystrophin is particularly prone to deletion mutations. In-frame deletions of hinge 3 are predicted to lead to BMD, however the severity of disease can vary considerably.

Sarcolemmal nNOS anchoring reveals a qualitative difference between dystrophin and utrophin.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. In normal muscle, dystrophin helps maintain sarcolemmal stability. Dystrophin also recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma.

Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy.

Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered.

Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex.

Duchenne muscular dystrophy is a severe disorder caused by mutations in the dystrophin gene. Dystrophin is required for assembly of the dystrophin-glycoprotein complex and provides a mechanically strong link between the cytoskeleton and the extracellular matrix. Several proteins in the complex also participate in signaling cascades, but the relationship between these signaling and mechanical functions in the development of muscular dystrophy is unclear.

Discovery and SAR of a novel selective and orally bioavailable nonpeptide classical competitive inhibitor class of protein-tyrosine phosphatase 1B.

Reversible phosphorylation and dephosphorylation of key proteins on tyrosine residues are important parts of intracellular signaling triggered by hormones and other agents. Recent knock-out studies in mice have identified PTP1B as a potential target for the treatment of diabetes and obesity. As a consequence, a number of academic and industrial groups are aggressively pursuing the development of selective PTP1B inhibitors.

Protein engineering of protein kinase A catalytic subunits results in the acquisition of novel inhibitor sensitivity.

Analysis of the role of specific protein kinases in signal transduction networks has relied heavily on ATP analog inhibitors. Currently used agents, however, often do not distinguish between kinase family members. Genetic approaches can also be used to inactivate a specific kinase, but these techniques do not afford the rapid kinetics possible with pharmacological inhibitors.