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Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons., Competing Interests: Competing interest statement: L.C. holds a patent on gene therapy vectors for treating cardiomyopathy that was licensed to DiNAQOR, is a member of the Scientific Advisory Board, and has shares in DiNAQOR., (Copyright © 2021 the Author(s). Published by PNAS.)

Objective: The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INS C94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates., Methods: Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics., Results: MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples., Conclusions: The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension.

Background: Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model., Methods: α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples., Results: No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions., Conclusion: Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection., (© 2014 John Wiley & Sons A/S.)

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal musculature and myocardium. In DMD patients and in a corresponding pig model with a deletion of DMD exon 52 (DMDΔ52), expression of an internally shortened dystrophin can be achieved by skipping of DMD exon 51 to reframe the transcript. To predict the best possible outcome of this strategy, we generated DMDΔ51-52 pigs, additionally representing a model for Becker muscular dystrophy (BMD). DMDΔ51-52 skeletal muscle and myocardium samples stained positive for dystrophin and did not show the characteristic dystrophic alterations observed in DMDΔ52 pigs. Western blot analysis confirmed the presence of dystrophin in the skeletal muscle and myocardium of DMDΔ51-52 pigs and its absence in DMDΔ52 pigs. The proteome profile of skeletal muscle, which showed a large number of abundance alterations in DMDΔ52 vs. wild-type (WT) samples, was normalized in DMDΔ51-52 samples. Cardiac function at age 3.5 mo was significantly reduced in DMDΔ52 pigs (mean left ventricular ejection fraction 58.8% vs. 70.3% in WT) but completely rescued in DMDΔ51-52 pigs (72.3%), in line with normalization of the myocardial proteome profile. Our findings indicate that ubiquitous deletion of DMD exon 51 in DMDΔ52 pigs largely rescues the rapidly progressing, severe muscular dystrophy and the reduced cardiac function of this model. Long-term follow-up studies of DMDΔ51-52 pigs will show if they develop symptoms of the milder BMD. [ABSTRACT FROM AUTHOR]

Background: The mucociliary clearance system driven by beating cilia protects the airways from inhaled microbes and particles. Large particles are cleared by mucus bundles made in submucosal glands by parallel linear polymers of the MUC5B mucins. However, the structural organization and function of the mucus generated in surface goblet cells are poorly understood.Methods: The origin and characteristics of different mucus structures were studied on live tissue explants from newborn wild-type (WT), cystic fibrosis transmembrane conductance regulator (CFTR) deficient (CF) piglets and weaned pig airways using video microscopy, Airyscan imaging and electron microscopy. Bronchoscopy was performed in juvenile pigs in vivo.Results: We have identified a distinct mucus formation secreted from the surface goblet cells with a diameter less than two micrometer. This type of mucus was named mucus threads. With time mucus threads gathered into larger mucus assemblies, efficiently collecting particles. The previously observed Alcian blue stained mucus bundles were around 10 times thicker than the threads. Together the mucus bundles, mucus assemblies and mucus threads cleared the pig trachea from particles.Conclusions: These results demonstrate that normal airway mucus is more complex and has a more variable structural organization and function than was previously understood. These observations emphasize the importance of studying young objects to understand the function of a non-compromised lung. [ABSTRACT FROM AUTHOR]

Abstract: For clinical xenotransplantation, transplants must be free of porcine cytomegalovirus (PCMV). Piglets become infected primarily in the perinatal period by the mother sow. While individual donor animals can be protected from infection by isolation husbandry, success is not guaranteed and this strategy poses the risk of undetected infections and raises animal welfare questions. Here, we present the establishment of a completely PCMV‐negative pig herd for breeding donor animals for xenotransplantation. Eleven pregnant DanAvl Basic hybrid sows were purchased from a designated pathogen‐free (DPF), PCMV‐positive colony and transferred to a new pig facility at the Centre for Innovative Medical Models (CiMM) 4 weeks prior to farrowing. At the age of 24 hours, piglets were early‐weaned and transferred to a commercially available Rescue Deck system dedicated to motherless rearing of piglets. Sows were removed from the facility. The PCMV status of F1‐generation animals was determined at regular intervals over a period of 14 months by a sensitive real‐time PCR‐based detection method testing blood, nasal swabs and cultured peripheral blood mononuclear cells (PBMCs). F1 sows were used as recipients of genetically modified embryos to generate a xenotransplant donor herd. Offspring were tested for PCMV accordingly. All offspring have remained PCMV negative over the whole observation period of 14 months. A completely PCMV‐negative pig herd for xenotransplantation has thus been successfully established. [ABSTRACT FROM AUTHOR]

Abstract: Background: Porcine endogenous retroviruses (PERVs) may pose a risk of xenotransplantation using porcine cells, tissues, or organs. PERVs are integrated in the genome of all pigs, and some can infect certain human cells. The copy number of PERVs in different pig breeds has been determined by using different methods, with varying results. Methods: To determine the PERV copy number in pig cell lines and in animals, a new method, droplet digital polymerase chain reaction (ddPCR) was used. DNA was isolated from pig cell lines (PK15 and PTK75 cells), from Aachen, Göttingen, and Black minipigs, and from genetically modified and non‐modified German landrace pigs. Primers specific for the polymerase gene (pol) were used for the ddPCR. Results: The median copy number of integrated proviruses was found between 46 and 70 copies in three different PK15 cell lines, 49 copies in PTK75 cells, 64 copies in Göttingen minipigs, 69 copies in Aachen minipigs, 117 copies in Black minipigs, and 59 copies in genetically modified pigs generated for xenotransplantation. PERV copy numbers varied between different organs from a single pig, indicating proviral amplification. The study also revealed that different PK15 cell lines from different laboratories which had been used as virus source for infection experiments carry different PERV copies. Furthermore, different copy numbers of cellular reference genes (GAPDH, ACTB) were detected in different cell lines and pigs. Conclusion: The determination of the PERV copy number using ddPCR extended previous data showing differences between the pig breeds and between different organs of a single animal. The determination of PERV copy numbers can be used to select animals less likely to transmit PERVs during xenotransplantation. In addition, this method will be of special value when PERV proviruses are to be inactivated by CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

Background Lung xenografts remain susceptible to loss of vascular barrier function within hours in spite of significant incremental advances based on genetic engineering to remove the Gal 1,3-αGal antigen (Gal TKO) and express human membrane cofactor protein ( hCD46). Natural killer cells rapidly disappear from the blood during perfusion of Gal TKO. hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature. Here we asked whether porcine expression of the human NK cell inhibitory ligand HLA-E and β2 microglobulin inhibits Gal TKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models. Methods Lungs from pigs modified to express Gal TKO.h CD46 (n=37) and Gal TKO.h CD46. HLA-E (n=5) were harvested and perfused with human blood until failure or elective termination at 4 hours. Airway pressures and pulmonary artery hemodynamics were recorded in real time. Blood samples were also collected throughout the experiment for analysis. Porcine aortic endothelial cells ( PAECs) from each genotype were cultured in monolayers in microfluidic channels and used in fluorescent cytotoxicity assays using human NK cells. Results HLA-E expression on Gal TKO.h CD46 PAECs was associated with significantly decreased antibody-dependent and antibody-independent NK-mediated cytotoxicity under in vitro conditions simulating physiologic shear stress. Relative to Gal TKO. hCD46 pig lungs perfused with human blood on an ex vivo platform, additional expression of HLA-E increased median lung survival (>4 hours, vs 162 minutes, P=.012), and was associated with attenuated rise in pulmonary vascular resistance, and decreased platelet activation and histamine elaboration. As expected, HLA-E expression was not associated with a significant difference in NK cell adhesion to endothelial cells in vitro, or NK cell and neutrophil sequestration during organ perfusion. Conclusions We conclude human NK cell activation contributes significantly to Gal TKO.h CD46 pig endothelial injury and lung inflammation and show that expression of HLA-E is associated with physiologically meaningful protection of Gal TKO.h CD46 cells and organs exposed to human blood. [ABSTRACT FROM AUTHOR]

We have successfully established and characterized a genetically modified pig line with ubiquitous expression of LEA29Y, a human CTLA4-Ig derivate. LEA29Y binds human B7.1/CD80 and B7.2/CD86 with high affinity and is thus a potent inhibitor of T cell co-stimulation via this pathway. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via assisted breeding methods. The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level. They present with an immune system affected by transgene expression, but can be maintained until sexual maturity and propagated by assisted reproduction techniques. Based on previous experience with pancreatic islets expressing LEA29Y, tissues from CAG-LEA29Y transgenic pigs should be protected against rejection by human T cells. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting large animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action. [ABSTRACT FROM AUTHOR]

Background: Pathological cardiac hypertrophy is a result of afterload-increasing pathologies including untreated hypertension and aortic stenosis. It features progressive adverse cardiac remodeling, myocardial dysfunction, capillary rarefaction, and interstitial fibrosis often leading to heart failure.Objectives: This study aimed to establish a novel porcine model of pressure-overload-induced heart failure and to determine the effect of inhibition of microribonucleic acid 132 (miR-132) on heart failure development in this model.Methods: This study developed a novel porcine model of percutaneous aortic constriction by implantation of a percutaneous reduction stent in the thoracic aorta, inducing progressive remodeling at day 56 (d56) after pressure-overload induction. In this study, an antisense oligonucleotide specifically inhibiting miR-132 (antimiR-132), was regionally applied via intracoronary injection at d0 (percutaneous transverse aortic constriction induction) and d28.Results: At d56, antimiR-132 treatment diminished cardiomyocyte cross-sectional area (188.9 ± 2.8 vs. 258.4 ± 9.0 μm2 in untreated hypertrophic hearts) and improved global cardiac function (ejection fraction 48.9 ± 1.0% vs. 36.1 ± 1.7% in control hearts). Moreover, at d56 antimiR-132-treated hearts displayed less increase of interstitial fibrosis compared with sham-operated hearts (Δsham 1.8 ± 0.5%) than control hearts (Δsham 10.8 ± 0.6%). Of note, cardiac platelet and endothelial cell adhesion molecule 1+ capillary density was higher in the antimiR-132-treated hearts (647 ± 20 cells/mm2) compared with in the control group (485 ± 23 cells/mm2).Conclusions: The inhibition of miR-132 is a valid strategy in prevention of heart failure progression in hypertrophic heart disease and may be developed as a treatment for heart failure of nonischemic origin. [ABSTRACT FROM AUTHOR]