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Neurofibromatosis Type 1 (NF1) is a genetic disease caused by mutations in Neurofibromin 1 (NF1). NF1 patients present with a variety of clinical manifestations and are predisposed to cancer development. Many NF1 animal models have been developed, yet none display the spectrum of disease seen in patients and the translational impact of these models has been limited. We describe a minipig model that exhibits clinical hallmarks of NF1, including café au lait macules, neurofibromas, and optic pathway glioma. Spontaneous loss of heterozygosity is observed in this model, a phenomenon also described in NF1 patients. Oral administration of a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor suppresses Ras signaling. To our knowledge, this model provides an unprecedented opportunity to study the complex biology and natural history of NF1 and could prove indispensable for development of imaging methods, biomarkers, and evaluation of safety and efficacy of NF1-targeted therapies.

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Background & Aims The molecular motor, Myosin Vb (MYO5B), is well documented for its role in trafficking cargo to the apical membrane of epithelial cells. Despite its involvement in regulating apical proteins, the role of MYO5B in cell polarity is less clear. Inactivating mutations in MYO5B result in microvillus inclusion disease (MVID), a disorder characterized by loss of key apical transporters and the presence of intracellular inclusions in enterocytes. We previously identified that inclusions in Myo5b knockout (KO) mice form from invagination of the apical brush border via apical bulk endocytosis. Herein, we sought to elucidate the role of polarity complexes and tight junction proteins during the formation of inclusions. Methods Intestinal tissue from neonatal control and Myo5b KO littermates was analyzed by immunofluorescence to determine the localization of polarity complexes and tight junction proteins. Results Proteins that make up the apical polarity complexes—Crumbs3 and Pars complexes—were associated with inclusions in Myo5b KO mice. In addition, tight junction proteins were observed to be concentrated over inclusions that were present at the apical membrane of Myo5b-deficient enterocytes in vivo and in vitro. Our mouse findings are complemented by immunostaining in a large animal swine model of MVID genetically engineered to express a human MVID-associated mutation that shows an accumulation of Claudin-2 over forming inclusions. The findings from our swine model of MVID suggest that a similar mechanism of tight junction accumulation occurs in patients with MVID. Conclusions These data show that apical bulk endocytosis involves the altered localization of apical polarity proteins and tight junction proteins after loss of Myo5b.

BACKGROUND The MEK1/2 inhibitor selumetinib was recently approved for Neurofibromatosis type 1 (NF1)-associated plexiform neurofibromas, but outcomes could be improved and its pharmacodynamic evaluation in other relevant tissues is limited. The aim of this study was to assess selumetinib tissue pharmacokinetics and pharmacodynamics using a minipig model of NF1. METHODS Eight wild type (WT) and eight NF1  +/- (NF1) minipigs received a single oral dose of 7.3 mg/kg selumetinib. Peripheral blood mononuclear cells (PBMCs), cerebral cortex, optic nerve, sciatic nerve, and skin were collected for pharmacokinetic analysis and pharmacodynamic analysis of extracellular regulated kinase phosphorylation (p-ERK) inhibition and transcript biomarkers (DUSP6 & FOS). RESULTS Key selumetinib pharmacokinetic parameters aligned with those observed in human patients. Selumetinib concentrations were higher in CNS tissues from NF1 compared to WT animals. Inhibition of ERK phosphorylation was achieved in PBMCs (mean 60% reduction), skin (95%) and sciatic nerve (64%) from all minipigs, whereas inhibition of ERK phosphorylation in cerebral cortex was detected only in NF1 animals (71%). Basal p-ERK levels were significantly higher in NF1 minipig optic nerve compared to WT and were reduced to WT levels (60%) with selumetinib. Modulation of transcript biomarkers was observed in all tissues. CONCLUSIONS Selumetinib reduces MAPK signaling in tissues clinically relevant to NF1, effectively normalizing p-ERK to WT levels in optic nerve but resulting in abnormally low levels of p-ERK in the skin. These results suggest that selumetinib exerts activity in NF1-associated central nervous system tumors by normalizing Ras/MAPK signaling and may explain common MEK inhibitor-associated dermatologic toxicities.

Nonalcoholic steatohepatitis (NASH) is an advanced form of nonalcoholic fatty liver disease (NAFLD), resulting from the complex interplay of genetic and environmental risk factors, e.g. diet. The development of novel therapies has been hampered by the lack of a preclinical animal model with high physiologic fidelity to the human condition. A single mutation (I148M) in the patatin‐like phospholipase domain‐containing 3 (PNPLA3) gene is significantly associated with liver fat content and has been identified as the strongest genetic risk factor for NAFLD/NASH, cirrhosis, and hepatocellular carcinoma. We hypothesized that editing PNPLA3I148M mutation into a genome of Ossabaw, a miniature pig that already has a natural propensity to develop metabolic syndrome, will develop phenotypes that hallmark NAFLD/NASH, and improve preclinical modeling over existing rodent models. PNPLA3I148M/I148M founders were developed using TALEN‐stimulated homology directed repair in fibroblasts, followed by somatic cell nuclear transfer. Mutant pigs with age/gender‐matched wildtype controls, reared on standard (SD) and specialized western diet (WD), were clinically and histopathologically characterized. No significant differences in circulating triglyceride and liver enzyme levels was observed in our initial cohort on SD of a female mutant pig relative to the wildtype control. However, this mutant female exhibited increased ALP level and a more atherogenic lipoprotein profile with LDL particle concentration than the levels of the respective control. Histopathologically, the mutant female fed on the WD for 3 months exhibited augmented interlobular inflammation, ballooning, as well lipid and collagen buildup in the liver relative to the wildtype counterparts on the same WD or SD. Longitudinal studies in second larger cohort of barrows over six‐months period revealed significantly elevated liver enzymes (ALP, ALT, GGT), bile acids, and lipid profiles (cholesterol, triglyceride, triglyceride‐rich lipoprotein and vLDL) among the mutant versus wildtypes on western diet, substantiating liver pathology and the associated comorbidity of dyslipidemia. Liver histopathology from these barrows on WD over 6‐months did not reveal as significant difference as in the females of the initial cohort, implying gender differences and variances in western diet formulation in susceptibility to NAFLD/NASH that warrant future large‐scale studies. Levels of circulating extracellular vesicles (EVs) were significantly elevated in the sera of mutant pigs relative to wildtypes on the WD, validating this as an important biomarker in NAFLD/NASH pathology. Additionally, the mutant pigs on the WD had significantly greater coronary artery plaque burden than the control groups, in line with the predicted higher comorbidity level in mutants and reinforces the relevance to human clinical medicine. Support or Funding Information NIH (1R43OD023257‐01A1 to TM) and internal funds from Recombinetics Inc., Drs. Chalasani, Sturek, and Malhi.

Introduction: Most male pigs are surgically castrated to avoid puberty-derived boar taint and aggressiveness. However, this surgical intervention represents a welfare concern in swine production. Disrupting porcine KISS1 is hypothesized to delay or abolish puberty by inducing variable hypogonadotropism and thus preventing the need for castration.Methods: To test this hypothesis, we generated the first KISS1-edited large animal using CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides. The targeted region preceded the sequence encoding a conserved core motif of kisspeptin. Genome editors were intracytoplasmically injected into 684 swine zygotes and transferred to 19 hormonally synchronized surrogate sows. In nine litters, 49 American Yorkshire and 20 Duroc liveborn piglets were naturally farrowed.Methods: Thirty-five of these pigs bore KISS1-disruptive alleles ranging in frequency from 5% to 97% and did not phenotypically differ from their wild-type counterparts. In contrast, four KISS1-edited pigs (two boars and two gilts) with disruptive allele frequencies of 96% and 100% demonstrated full hypogonadotropism, infantile reproductive tracts, and failed to reach sexual maturity. Change in body weight during development was unaffected by editing KISS1. Founder pigs partially carrying KISS1-disruptive alleles were bred resulting in a total of 53 KISS1+/+, 60 KISS1+/−, and 34 KISS1−/− F1 liveborn piglets, confirming germline transmission.Discussion: Results demonstrate that a high proportion of KISS1 alleles in pigs must be disrupted before variation in gonadotropin secretion is observed, suggesting that even a small amount of kisspeptin ligand is sufficient to confer proper sexual development and puberty in pigs. Follow-on studies will evaluate fertility restoration in KISS1 KO breeding stock to fully realize the potential of KISS1 gene edits to eliminate the need for surgical castration.

Glioblastoma (GBM) is the most common and malignant primary brain tumor. Novel therapeutic development for GBM is desperately needed, as the standard of care universally fails to cure patients and the 5-year survival rate remains extremely low. GBM therapeutic development is hampered by the lack of relevant preclinical models for preclinical studies. To mitigate this problem, we have developed a genetic model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. We developed methods for introducing genome engineering tools to minipig brain in vivo by direct injection of gene delivery reagents to the lateral ventricle, altering major signaling pathways frequently changed in human GBM. Using this technique, we have delivered a combination of expression vectors for oncogenes and targeted nucleases to disrupt tumor suppressor genes commonly altered in human GBM. We have altered six major human GBM-associated signaling pathways and modeled molecular GBM subclasses. We have also engineered a secreted tumor reporter that can be used to monitor tumor size through a simple blood test. This somatic cell gene-modification platform we have developed in the minipig allows us to reproduce the genetic heterogeneity seen in GBM and understand the impact of the tumor microenvironment, immune system, and response to therapy. This minipig model of GBM Is being used to test the standard of care against novel therapies in preclinical studies, and biopsy, surgical, imaging, and radiation therapy techniques are being optimized in this mode to improve clinical trial success rates and patient outcomes. Funding for this study is provided by the National Institutes of Health though SBIR grant # 1R43CA235837-01A1.