Deciphering the super relaxed state of human ß-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.

Mutations in ß-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human ß-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

Synchrotron X-ray absorption spectroscopy of melanosomes in vertebrates and cephalopods: implications for the affinity of Tullimonstrum.

Screening pigments are essential for vision in animals. Vertebrates use melanins bound in melanosomes as screening pigments, whereas cephalopods are assumed to use ommochromes. Preserved eye melanosomes in the controversial fossil Tullimonstrum (Mazon Creek, IL, USA) are partitioned by size and/or shape into distinct layers. These layers resemble tissue-specific melanosome populations considered unique to the vertebrate eye. Here, we show that extant cephalopod eyes also show tissue-specific size- and/or shape-specific partitioning of melanosomes; these differ from vertebrate melanosomes in the relative abundance of trace metals and in the binding environment of copper. Chemical signatures of melanosomes in the eyes of Tullimonstrum more closely resemble those of modern cephalopods than those of vertebrates, suggesting that an invertebrate affinity for Tullimonstrum is plausible. Melanosome chemistry may thus provide insights into the phylogenetic affinities of enigmatic fossils where melanosome size and/or shape are equivocal.

A novel porcine model of ataxia telangiectasia reproduces neurological features and motor deficits of human disease.

Ataxia telangiectasia (AT) is a progressive multisystem disorder caused by mutations in the AT-mutated (ATM) gene. AT is a neurodegenerative disease primarily characterized by cerebellar degeneration in children leading to motor impairment. The disease progresses with other clinical manifestations including oculocutaneous telangiectasia, immune disorders, increased susceptibly to cancer and respiratory infections. Although genetic investigations and physiological models have established the linkage of ATM with AT onset, the mechanisms linking ATM to neurodegeneration remain undetermined, hindering therapeutic development. Several murine models of AT have been successfully generated showing some of the clinical manifestations of the disease, however they do not fully recapitulate the hallmark neurological phenotype, thus highlighting the need for a more suitable animal model. We engineered a novel porcine model of AT to better phenocopy the disease and bridge the gap between human and current animal models. The initial characterization of AT pigs revealed early cerebellar lesions including loss of Purkinje cells (PCs) and altered cytoarchitecture suggesting a developmental etiology for AT and could advocate for early therapies for AT patients. In addition, similar to patients, AT pigs show growth retardation and develop motor deficit phenotypes. By using the porcine system to model human AT, we established the first animal model showing PC loss and motor features of the human disease. The novel AT pig provides new opportunities to unmask functions and roles of ATM in AT disease and in physiological conditions.

Genetically engineered livestock for biomedical models.

To commemorate Transgenic Animal Research Conference X, this review summarizes the recent progress in developing genetically engineered livestock species as biomedical models. The first of these conferences was held in 1997, which turned out to be a watershed year for the field, with two significant events occurring. One was the publication of the first transgenic livestock animal disease model, a pig with retinitis pigmentosa. Before that, the use of livestock species in biomedical research had been limited to wild-type animals or disease models that had been induced or were naturally occurring. The second event was the report of Dolly, a cloned sheep produced by somatic cell nuclear transfer. Cloning subsequently became an essential part of the process for most of the models developed in the last 18 years and is stilled used prominently today. This review is intended to highlight the biomedical modeling achievements that followed those key events, many of which were first reported at one of the previous nine Transgenic Animal Research Conferences. Also discussed are the practical challenges of utilizing livestock disease models now that the technical hurdles of model development have been largely overcome.

A Translational Model for Diet-related Atherosclerosis: Effect of Statins on Hypercholesterolemia and Atherosclerosis in a Minipig.

Models of atherosclerosis are used in preclinical studies but often fail to translate to humans. A model that better reflects human atherosclerosis is necessary. We recently engineered the ExeGen™ low-density lipoprotein receptor (LDLR) miniswine, in which the LDL receptor gene is modified to drive hypercholesterolemia and atherosclerosis, and showed diet-related exacerbation of these phenotypes. Five groups of animals, either wild type (+/+) or heterozygous (+/-), were fed either a normal or high-fat diet for 6 months. One group of heterozygous pigs fed a high-fat diet was also administered atorvastatin at 3 mg/kg/day. Clinical chemistry and anatomic pathology parameters were measured biweekly and at termination. The high-fat diet resulted in increased adiposity and interspersion of adipocytes within the salivary glands. The heterozygous pigs on the high-fat diet gained more weight and had significant increases in total cholesterol, high-density lipoprotein, and LDL compared to wild-type animals or heterozygous animals fed a normal diet. Atorvastatin attenuated these parameters, indicating the statin had a beneficial effect, even in a high-fat diet scenario. Atorvastatin treatment also reduced the intensity of Oil Red O staining in pigs on high-fat diet. Atorvastatin-related amelioration of several indices of cardiovascular pathophysiology in this model underscores its utility for drug discovery.

NF1+/ex42del miniswine model the cellular disruptions and behavioral presentations of NF1-associated cognitive and motor impairment.

Cognitive or motor impairment is common among individuals with neurofibromatosis type 1 (NF1), an autosomal dominant tumor-predisposition disorder. As many as 70% of children with NF1 report difficulties with spatial/working memory, attention, executive function, and fine motor movements. In contrast to the utilization of various Nf1 mouse models, here we employ an NF1+/ex42del miniswine model to evaluate the mechanisms and characteristics of these presentations, taking advantage of a large animal species more like human anatomy and physiology. The prefrontal lobe, anterior cingulate, and hippocampus from NF1+/ex42del and wild-type miniswine were examined longitudinally, revealing abnormalities in mature oligodendrocytes and astrocytes, and microglial activation over time. Imbalances in GABA: Glutamate ratios and GAD67 expression were observed in the hippocampus and motor cortex, supporting the role of disruption in inhibitory neurotransmission in NF1 cognitive impairment and motor dysfunction. Moreover, NF1+/ex42del miniswine demonstrated slower and shorter steps, indicative of a balance-preserving response commonly observed in NF1 patients, and progressive memory and learning impairments. Collectively, our findings affirm the effectiveness of NF1+/ex42del miniswine as a valuable resource for assessing cognitive and motor impairments associated with NF1, investigating the involvement of specific neural circuits and glia in these processes, and evaluating potential therapeutic interventions.

A novel porcine model of CLN3 Batten disease recapitulates clinical phenotypes.

Mouse models of CLN3 Batten disease, a rare lysosomal storage disorder with no cure, have improved our understanding of CLN3 biology and therapeutics through their ease of use and a consistent display of cellular pathology. However, the translatability of murine models is limited by disparities in anatomy, body size, life span and inconsistent subtle behavior deficits that can be difficult to detect in CLN3 mutant mouse models, thereby limiting their use in preclinical studies. Here, we present a longitudinal characterization of a novel miniswine model of CLN3 disease that recapitulates the most common human pathogenic variant, an exon 7-8 deletion (CLN3Δex7/8). Progressive pathology and neuron loss is observed in various regions of the CLN3Δex7/8 miniswine brain and retina. Additionally, mutant miniswine present with retinal degeneration and motor abnormalities, similar to deficits seen in humans diagnosed with the disease. Taken together, the CLN3Δex7/8 miniswine model shows consistent and progressive Batten disease pathology, and behavioral impairment mirroring clinical presentation, demonstrating its value in studying the role of CLN3 and safety/efficacy of novel disease-modifying therapeutics.

Magnetic resonance brain volumetry biomarkers of CLN2 Batten disease identified with miniswine model.

Late-infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease (Batten disease) is a rare pediatric disease, with symptom development leading to clinical diagnosis. Early diagnosis and effective tracking of disease progression are required for treatment. We hypothesize that brain volumetry is valuable in identifying CLN2 disease at an early stage and tracking disease progression in a genetically modified miniswine model. CLN2R208X/R208X miniswine and wild type controls were evaluated at 12- and 17-months of age, correlating to early and late stages of disease progression. Magnetic resonance imaging (MRI) T1- and T2-weighted data were acquired. Total intercranial, gray matter, cerebrospinal fluid, white matter, caudate, putamen, and ventricle volumes were calculated and expressed as proportions of the intracranial volume. The brain regions were compared between timepoints and cohorts using Gardner-Altman plots, mean differences, and confidence intervals. At an early stage of disease, the total intracranial volume (- 9.06 cm3), gray matter (- 4.37% 95 CI - 7.41; - 1.83), caudate (- 0.16%, 95 CI - 0.24; - 0.08) and putamen (- 0.11% 95 CI - 0.23; - 0.02) were all notably smaller in CLN2R208X/R208X miniswines versus WT, while cerebrospinal fluid was larger (+ 3.42%, 95 CI 2.54; 6.18). As the disease progressed to a later stage, the difference between the gray matter (- 8.27%, 95 CI - 10.1; - 5.56) and cerebrospinal fluid (+ 6.88%, 95 CI 4.31; 8.51) continued to become more pronounced, while others remained stable. MRI brain volumetry in this miniswine model of CLN2 disease is sensitive to early disease detection and longitudinal change monitoring, providing a valuable tool for pre-clinical treatment development and evaluation.

A Novel Porcine Model of CLN2 Batten Disease that Recapitulates Patient Phenotypes.

CLN2 Batten disease is a lysosomal disorder in which pathogenic variants in CLN2 lead to reduced activity in the enzyme tripeptidyl peptidase 1. The disease typically manifests around 2 to 4 years of age with developmental delay, ataxia, seizures, inability to speak and walk, and fatality between 6 and 12 years of age. Multiple Cln2 mouse models exist to better understand the etiology of the disease; however, these models are unable to adequately recapitulate the disease due to differences in anatomy and physiology, limiting their utility for therapeutic testing. Here, we describe a new CLN2R208X/R208X porcine model of CLN2 disease. We present comprehensive characterization showing behavioral, pathological, and visual phenotypes that recapitulate those seen in CLN2 patients. CLN2R208X/R208X miniswine present with gait abnormalities at 6 months of age, ERG waveform declines at 6-9 months, vision loss at 11 months, cognitive declines at 12 months, seizures by 15 months, and early death at 18 months due to failure to thrive. CLN2R208X/R208X miniswine also showed classic storage material accumulation and glial activation in the brain at 6 months, and cortical atrophy at 12 months. Thus, the CLN2R208X/R208X miniswine model is a valuable resource for biomarker discovery and therapeutic development in CLN2 disease.

Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets.

Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases.

Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer.

Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (DeltaF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice.

Longitudinal phenotype development in a minipig model of neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a rare, autosomal dominant disease with variable clinical presentations. Large animal models are useful to help dissect molecular mechanisms, determine relevant biomarkers, and develop effective therapeutics. Here, we studied a NF1 minipig model (NF1+/ex42del) for the first 12 months of life to evaluate phenotype development, track disease progression, and provide a comparison to human subjects. Through systematic evaluation, we have shown that compared to littermate controls, the NF1 model develops phenotypic characteristics of human NF1: [1] café-au-lait macules, [2] axillary/inguinal freckling, [3] shortened stature, [4] tibial bone curvature, and [5] neurofibroma. At 4 months, full body computed tomography imaging detected significantly smaller long bones in NF1+/ex42del minipigs compared to controls, indicative of shorter stature. We found quantitative evidence of tibial bowing in a subpopulation of NF1 minipigs. By 8 months, an NF1+/ex42del boar developed a large diffuse shoulder neurofibroma, visualized on magnetic resonance imaging, which subsequently grew in size and depth as the animal aged up to 20 months. The NF1+/ex42del minipig model progressively demonstrates signature attributes that parallel clinical manifestations seen in humans and provides a viable tool for future translational NF1 research.

Development of a porcine model of cystic fibrosis.

Cystic Fibrosis (CF) is a common autosomal recessive disease that affects multiple organs. The lack of an animal model with manifestations like those typically found in humans has slowed understanding of its pathogenesis. Therefore, because of the similarities between human and swine anatomy, biochemistry, physiology, size, and genetics, we chose to develop a porcine model of CF. We used homologous recombination in primary cultures of porcine fibroblasts to disrupt the CFTR gene and then used those cells as nuclear donors for somatic cell nuclear transfer. After crossing heterozygous pigs, we produced CFTR-/- pigs. The newborn CFTR null piglets manifested meconium ileus, pancreatic destruction, early focal biliary cirrhosis, and gall bladder abnormalities that were very similar to those observed in humans with CF. At birth, there were no abnormalities in the airway epithelium or submucosal glands and no evidence of inflammation, consistent with findings in the newborn human. We hope that this porcine model will help elucidate the pathogenesis of CF and thereby lead to the development of new mechanism-based therapies.

Assessment of nociception and related quality-of-life measures in a porcine model of neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder resulting from germline mutations in the NF1 gene, which encodes neurofibromin. Patients experience a variety of symptoms, but pain in the context of NF1 remains largely underrecognized. Here, we characterize nociceptive signaling and pain behaviors in a miniswine harboring a disruptive NF1 mutation (exon 42 deletion). We present the first characterization of pain-related behaviors in a pig model of NF1, identifying unchanged agitation scores, lower tactile thresholds (allodynia), and decreased response latencies to thermal laser stimulation (hyperalgesia) in NF1 (females only) pigs. Male NF1 pigs with tumors showed reduced sleep quality and increased resting, 2 health-related quality-of-life symptoms found to be comorbid in people with NF1 pain. We explore these phenotypes in relationship to suppression of the increased activity of the N-type voltage-gated calcium (CaV2.2) channel by pharmacological antagonism of phosphorylation of a regulatory protein-the collapsin response mediator protein 2 (CRMP2), a known interactor of neurofibromin, and by targeting the interface between the α subunit of CaV2.2 and the accessory ß-subunits with small molecules. Our data support the use of NF1 pigs as a large animal model for studying NF1-associated pain and for understanding the pathophysiology of NF1. Our findings demonstrate the translational potential of 2 small molecules in reversing ion channel remodeling seen in NF1. Interfering with CaV2.2, a clinically validated target for pain management, might also be a promising therapeutic strategy for NF1-related pain management.

A porcine model of neurofibromatosis type 1 that mimics the human disease.

Loss of the NF1 tumor suppressor gene causes the autosomal dominant condition, neurofibromatosis type 1 (NF1). Children and adults with NF1 suffer from pathologies including benign and malignant tumors to cognitive deficits, seizures, growth abnormalities, and peripheral neuropathies. NF1 encodes neurofibromin, a Ras-GTPase activating protein, and NF1 mutations result in hyperactivated Ras signaling in patients. Existing NF1 mutant mice mimic individual aspects of NF1, but none comprehensively models the disease. We describe a potentially novel Yucatan miniswine model bearing a heterozygotic mutation in NF1 (exon 42 deletion) orthologous to a mutation found in NF1 patients. NF1+/ex42del miniswine phenocopy the wide range of manifestations seen in NF1 patients, including café au lait spots, neurofibromas, axillary freckling, and neurological defects in learning and memory. Molecular analyses verified reduced neurofibromin expression in swine NF1+/ex42del fibroblasts, as well as hyperactivation of Ras, as measured by increased expression of its downstream effectors, phosphorylated ERK1/2, SIAH, and the checkpoint regulators p53 and p21. Consistent with altered pain signaling in NF1, dysregulation of calcium and sodium channels was observed in dorsal root ganglia expressing mutant NF1. Thus, these NF1+/ex42del miniswine recapitulate the disease and provide a unique, much-needed tool to advance the study and treatment of NF1.

Functional repair of a mutant chloride channel using a trans-splicing ribozyme.

RNA repair has been proposed as a novel gene-based therapeutic strategy. Modified Tetrahymena group I intron ribozymes have been used to mediate trans-splicing of therapeutically relevant RNA transcripts, but the efficiency of the ribozyme-mediated RNA repair process has not been determined precisely and subsequent restoration of protein function has been demonstrated only by indirect means. We engineered a ribozyme that targets the mRNA of a mutant canine skeletal muscle chloride channel (cClC-1) (mutation T268M in ClC-1 causing myotonia congenita) and replaces the mutant-containing 3' portion by trans-splicing the corresponding 4-kb wild-type sequence. Repair efficiency assessed by quantitative RT-PCR was 1.2% +/- 0.1% in a population of treated cells. However, when chloride channel function was examined in single cells, a wide range of electrophysiological activity was observed, with 18% of cells exhibiting significant functional restoration and some cells exhibiting complete rescue of the biophysical phenotype. These results indicate that RNA repair can restore wild-type protein activity and reveal considerable cell-to-cell variability in ribozyme-mediated trans-splicing reaction efficiency.

Engineering Large Animal Species to Model Human Diseases.

Animal models are an important resource for studying human diseases. Genetically engineered mice are the most commonly used species and have made significant contributions to our understanding of basic biology, disease mechanisms, and drug development. However, they often fail to recreate important aspects of human diseases and thus can have limited utility as translational research tools. Developing disease models in species more similar to humans may provide a better setting in which to study disease pathogenesis and test new treatments. This unit provides an overview of the history of genetically engineered large animals and the techniques that have made their development possible. Factors to consider when planning a large animal model, including choice of species, type of modification and methodology, characterization, production methods, and regulatory compliance, are also covered. © 2016 by John Wiley & Sons, Inc.

Genetically engineered SCN5A mutant pig hearts exhibit conduction defects and arrhythmias.

SCN5A encodes the α subunit of the major cardiac sodium channel Na(V)1.5. Mutations in SCN5A are associated with conduction disease and ventricular fibrillation (VF); however, the mechanisms that link loss of sodium channel function to arrhythmic instability remain unresolved. Here, we generated a large-animal model of a human cardiac sodium channelopathy in pigs, which have cardiac structure and function similar to humans, to better define the arrhythmic substrate. We introduced a nonsense mutation originally identified in a child with Brugada syndrome into the orthologous position (E558X) in the pig SCN5A gene. SCN5A(E558X/+) pigs exhibited conduction abnormalities in the absence of cardiac structural defects. Sudden cardiac death was not observed in young pigs; however, Langendorff-perfused SCN5A(E558X/+) hearts had an increased propensity for pacing-induced or spontaneous VF initiated by short-coupled ventricular premature beats. Optical mapping during VF showed that activity often began as an organized focal source or broad wavefront on the right ventricular (RV) free wall. Together, the results from this study demonstrate that the SCN5A(E558X/+) pig model accurately phenocopies many aspects of human cardiac sodium channelopathy, including conduction slowing and increased susceptibility to ventricular arrhythmias.

Porcine cancer models for translational oncology.

Large-animal cancer models are needed to advance the development of innovative and clinically applicable tumor diagnostic, therapeutic, and monitoring technologies. We developed a genetically modified porcine model of cancer based on a TP53 mutation, and established its utility for tracking tumorigenesis in vivo through non-invasive clinical imaging approaches.