Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet

Differential Immune Response to Bioprosthetic Heart Valve Tissues in the α1,3Galactosyltransferase-Knockout Mouse Model

Structural valve deterioration (SVD) of bioprosthetic heart valves (BHVs) has great clinical and economic consequences. Notably, immunity against BHVs plays a major role in SVD, especially when implanted in young and middle-aged patients. However, the complex pathogenesis of SVD remains to be fully characterized, and analyses of commercial BHVs in standardized-preclinical settings are needed for further advancement. Here, we studied the immune response to commercial BHV tissue of bovine, porcine, and equine origin after subcutaneous implantation into adult a1,3-galactosyltransferase-knockout (Gal KO) mice. The levels of serum anti-galactose a1,3-galactose (Gal) and -non-Gal IgM and IgG antibodies were determined up to 2 months post-implantation. Based on histological analyses, all BHV tissues studied triggered distinct infiltrating cellular immune responses that related to tissue degeneration. Increased anti-Gal antibody levels were found in serum after ATS 3f and Freedom/Solo implantation but not for Crown or Hancock II grafts. Overall, there were no correlations between cellular-immunity scores and post-implantation antibodies, suggesting these are independent factors differentially affecting the outcome of distinct commercial BHVs. These findings provide further insights into the understanding of SVD immunopathogenesis and highlight the need to evaluate immune responses as a confounding factor

Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices

Transgenic Expression of Glucagon-Like Peptide-1 (GLP-1) and Activated Muscarinic Receptor (M3R) Significantly Improves Pig Islet Secretory Function.

Porcine islets show notoriously low insulin secretion levels in response to glucose stimulation. While this is somehow expected in the case of immature islets isolated from fetal and neonatal pigs, disappointingly low secretory responses are frequently reported in studies using in vitro-maturated fetal and neonatal islets and even fully differentiated adult islets. Herein we show that β-cell-specific expression of a modified glucagon-like peptide-1 (GLP-1) and of a constitutively activated type 3 muscarinic receptor (M3R) efficiently amplifies glucose-stimulated insulin secretion (GSIS). Both adult and neonatal isolated pig islets were treated with adenoviral expression vectors carrying sequences encoding for GLP-1 and/or M3R. GSIS from transduced and control islets was evaluated during static incubation and dynamic perifusion assays. While expression of GLP-1 did not affect basal or stimulated insulin secretion, activated M3R produced a twofold increase in both first and second phases of GSIS. Coexpression of GLP-1 and M3R caused an even greater increase in the secretory response, which was amplified fourfold compared to controls. In conclusion, our work highlights pig islet insulin secretion deficiencies and proposes concomitant activation of cAMP-dependent and cholinergic pathways as a solution to ameliorate GSIS from pig islets used for transplantation

The Applications of Genome Editing in Xenotransplantation

Higher living standards and better medical care are increasing the lifespan of people around the world. Aging populations, however, have an increased incidence of loss of function or failure of cell, tissue or organ. This has led to the development of new medical disciplines, such as organ transplantation and more recently regenerative medicine. Organ transplantation using human donors (allotransplantation) has made enormous progress thanks to the discovery of novel immunosuppressive drugs. However, the growing demand for organs far exceeds the number of organs potentially available from human donors. Xenotransplantation, namely transplantation between animal donors and man, offers the opportunity to use healthy and highly specialized cells, tissues or solid organs readily available for immediate transfer to patients requiring replacement therapy (Ekser et al., 2012). The therapeutic potential of xenotransplantation is wide, some already in clinical use like bioprosthetic heart valves, decellularized pig tissues (skin, ligaments, bone and cartilage), polyclonal antisera, and pancreatic islet, or in a pre-clinical phase like kidney, heart, liver, lung, cornea, and dopaminergic neurons. The pig is a very suitable species for xenotransplantation for reasons that are well documented in the literature, including physiological and anatomical features, and the availability of a high resolution map of the genome. Moreover, the use of pigs for clinical purposes raises little concern from the wider public, because they are already bred by the millions for meat production worldwide. At present, the use of bioprosthetic heart valves of animal origin is a well established xenotransplantation procedure in clinical practice; however, pig islet xenotransplantation has just entered clinical trials (http://www.lctglobal.com/products/ diabecell/about-type-1-diabetes), and life supporting solid organs transplanted into nonhuman primates still do not survive long enough to warrant implementation of clinical trials (Le Bas-Bernardet et al., 2011) although heterotopic heart transplantation in a primate model has resulted in the remarkable survival of almost three years (Mohiuddin et al., 2015). Therefore, several issues still need to be addressed from the safety point of view, and a number of immunological hurdles have been identified (Table 1) and are currently being addressed at multiple levels (Griesemer et al., 2014). It is expected that the development of novel immunosuppressive strategies for allotransplantation and xenotransplantation, the modification of the immunogenicity of the donor pig through genetic engineering and, possibly the induction of immune tolerance, a phenomenon occasionally observed in allotransplantation, will all contribute to bringing xenotransplantation closer to the clinic (Ekser et al., 2012)

Novel transgenic animal model of Amyotrophic Lateral Sclerosis

The present invention concerns a novel transgenic animal as model of Amyotrophic Lateral Sclerosis (ALS)

EFFICIENT EXPRESSION OF HUMAN ENDOTHELIAL PROTEIN C RECEPTOR AND HUMAN THROMBOMODULIN IN TRANSFECTED PIG PRIMARY hCD55(+)-GAL(-/-) FIBROBLASTS USING F2A EXPRESSION VECTOR

The genetic engineering of the pig genome for xenotransplantation studies requires the insertion of different transgenes to create multi-transgenic pigs. In order to simultaneously add more transgene in a single genetic insertion, we constructed a polycistronic vector using the F2A self-cleaving peptide. Moreover, this solution has the added advantages of preventing possible segregation during breeding of the animals and of guaranteeing an equimolar production of chosen transgenes. The scope of this work was the construction and validation of an ubiquitous F2A-bicistronic expression vector for human thrombomodulin (hTM) and human endothelial protein C receptor (hEPCR) genes in pig primary hCD55-GAL\u2013/\u2013 cells to establish transgenic fibroblasts colonies, to be used for somatic cell nuclear transfer (SCNT) to generate pigs for xenotransplantation research. The expression vector consisted of pCAGGS promoter (CMV-IE+chicken \u3b2 actin) followed by hEPCR-furinF2A-hTM coding sequence. The resulting expression cassette was inserted between 2 insulators obtained from the 5\u2032 MAR region of chicken lysozyme. Outside of this insulated structure, there is a loxable puromycin selection cassette. The resulting purified and linearized expression vector (pEFTM/Lgu I = 5 \u3bcg) was transfected into hCD55-GAL\u2013/\u2013 primary fibroblasts (1 7 106), using Nucleofector (Amaxa, Lonza, Cologne, Germany), in parallel for comparative purposes we cotransfected the 2 pCAGGS-monocistronic vectors for the same transgenes (hEPCR and hTM = 1:3, 5 \u3bcg). Transfected cells were selected with puromycin (1 \u3bcg mL\u20131) for 15 days. After 8 days of selection, resistant colonies were picked up and expanded into 24-well plates for cryopreservation and analyses. Bicistronic transfection produced 20 clones and cotransfection only 8 clones that were analysed by Western blot (WB) and by immunocytochemistry (ICC) using polyclonal antibody anti-EPCR (1:250, R&D) and monoclonal antibody ab6980-Abcam (1:5000, Abcam, Cambridge, UK) in WB; polyclonal antibody RCR252 (1:100, Sigma-Aldrich, St. Louis, MO, USA) and monoclonal antibody ab6980-Abcam (1:100, Abcam) for ICC. Seventeen bicistronic clones (85%) and 2 cotransfected monocistronic clones (25%) were positive for both transgenes using WB. After ICC analyses, only 11 bicistronic colonies (55%) and 1 cotransfected colony (12.5%) uniformly expressed the desired transgenes and were selected for SCNT. The pCAGGS promoter maintained its strong expression also using the hEPCR-FurinF2A-hTM coding sequence and this bicistronic solution permitted us to improve our results obtained with co-transfection. Availability of hEPCR+ hTM+ hCD55+-GAL\u2013/\u2013 colonies will allow us to obtain a new transgenic background for future xenotransplantation projects