Growth hormone receptor knockout: Relevance to xenotransplantation.

Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications, effective immunosuppressive therapy, and anti-inflammatory therapy to protect pig tissues from the primate immune and inflammatory responses and correct molecular incompatibilities. Further study is required regarding identification and investigation of physiological incompatibilities. Although the exact cause remains uncertain, we and others have observed relatively rapid growth of kidney xenografts after transplantation into nonhuman primates (NHPs). There has also been some evidence of growth, or at least ventricular hypertrophy, of the pig heart after orthotopic transplantation into NHPs. Rapid growth could be problematic, particularly with regard to the heart within the relatively restricted confines of the chest. It has been suggested that the problem of rapid growth of the pig organ after transplantation could be resolved by growth hormone receptor (GHR) gene knockout in the pig. The GHR, although most well-known for regulating growth, has many other biological functions, including regulating metabolism and controlling physiological processes. Genetically modified GHRKO pigs have recently become available. We provide data on their growth compared to comparable pigs that do not include GHRKO, and we have reviewed the literature regarding the effect of GHRKO, and its relevance to xenotransplantation.

Anti-gal antibodies in α1,3-galactosyltransferase gene-knockout pigs.

Serum anti-galactose-α1,3-galactose (Gal) IgM and IgG antibody levels were measured by ELISA in α1,3-galactosyltransferase gene-knockout (GTKO) pigs (78 estimations in 47 pigs). A low level of anti-Gal IgM was present soon after birth, and rose to a peak at 4-6 m, which was maintained thereafter even in the oldest pigs tested (at >2 yr). Anti-Gal IgG was also present at birth, peaked at 3 m, and after 6 m steadily decreased until almost undetectable at 20 m. No differences in this pattern were seen between pigs of different gender. Total IgM followed a similar pattern as anti-Gal IgM, but total IgG did not decrease after 6m. The data provide useful baseline data for future experimental studies in GTKO pigs, e.g., relating to the antibody response to WT pig allografts.

Comparison of hematologic, biochemical, and coagulation parameters in α1,3-galactosyltransferase gene-knockout pigs, wild-type pigs, and four primate species.

BACKGROUND: The increasing availability of genetically engineered pigs is steadily improving the results of pig organ and cell transplantation in non-human primates (NHPs). Current techniques offer knockout of pig genes and/or knockin of human genes. Knowledge of normal values of hematologic, biochemical, coagulation, and other parameters in healthy genetically engineered pigs and NHPs is important, particularly following pig organ transplantation in NHPs. Furthermore, information on parameters in various NHP species may prove important in selecting the optimal NHP model for specific studies. METHODS: We have collected hematologic, biochemical, and coagulation data on 71 α1,3-galactosyltransferase gene-knockout (GTKO) pigs, 18 GTKO pigs additionally transgenic for human CD46 (GTKO.hCD46), four GTKO.hCD46 pigs additionally transgenic for human CD55 (GTKO.hCD46.hCD55), and two GTKO.hCD46 pigs additionally transgenic for human thrombomodulin (GTKO.hCD46.hTBM). RESULTS: We report these data and compare them with similar data from wild-type pigs and the three major NHP species commonly used in biomedical research (baboons, cynomolgus, and rhesus monkeys) and humans, largely from previously published reports. CONCLUSIONS: Genetic modification of the pig (e.g., deletion of the Gal antigen and/or the addition of a human transgene) (i) does not result in abnormalities in hematologic, biochemical, or coagulation parameters that might impact animal welfare, (ii) seems not to alter metabolic function of vital organs, although this needs to be confirmed after their xenotransplantation, and (iii) possibly (though, by no means certainly) modifies the hematologic, biochemical, and coagulation parameters closer to human values. This study may provide a good reference for those working with genetically engineered pigs in xenotransplantation research and eventually in clinical xenotransplantation.

Allergic response to medical products in patients with alpha-gal syndrome.

BACKGROUND: Galactose-α-1,3-galactose (alpha-gal) is a carbohydrate that is ubiquitously expressed in all mammals except for primates and humans. Patients can become sensitized to this antigen and develop alpha-gal syndrome (AGS), or a red meat allergy. Symptoms range from generalized gastroenteritis and malaise to anaphylaxis, and in endemic areas, the prevalence can be as high as 20%. Although AGS patients commonly avoid alpha-gal by avoiding meat, patients have also developed symptoms due to animal-derived medical products and devices. With the rise in transcatheter aortic valve replacement, we investigate the immunogenicity of common cardiac materials and valves. OBJECTIVE: To assess the in vitro immunoglobulin E response toward common medical products, including cardiac patch materials and bioprosthetic valves in patients with AGS. METHODS: Immunoblot and immunohistochemistry techniques were applied to assess immunoglobulin E reactivity to various mammalian derived tissues and medical products for patients with AGS. RESULTS: AGS serum showed strong reactivity to all of the commercially available, nonhuman products tested, including various decellularized cardiac patch materials and bioprosthetic aortic valves. AGS serum did not react to tissues prepared using alpha-gal knockout pigs. CONCLUSIONS: Despite commercial decellularization processes, alpha-gal continues to be present in animal-derived medical products, including bioprosthetic valves. Serum from patients with AGS demonstrates a strong affinity for these products in vitro. This may have serious potential implications for sensitized patients undergoing cardiac surgery, including early valve failure and accelerated coronary artery disease.

Biomanufacturing of Axon-Based Tissue Engineered Nerve Grafts Using Porcine GalSafe Neurons.

Existing strategies for repair of major peripheral nerve injury (PNI) are inefficient at promoting axon regeneration and functional recovery and are generally ineffective for nerve lesions >5 cm. To address this need, we have previously developed tissue engineered nerve grafts (TENGs) through the process of axon stretch growth. TENGs consist of living, centimeter-scale, aligned axon tracts that accelerate axon regeneration at rates equivalent to the gold standard autograft in small and large animal models of PNI, by providing a newfound mechanism-of-action referred to as axon-facilitated axon regeneration (AFAR). To enable clinical-grade biomanufacturing of TENGs, a suitable cell source that is hypoimmunogenic, exhibits low batch-to-batch variability, and able to tolerate axon stretch growth must be utilized. To fulfill these requirements, a genetically engineered, FDA-approved, xenogeneic cell source, GalSafe® neurons, produced by Revivicor, Inc., have been selected to advance TENG biofabrication for eventual clinical use. To this end, sensory and motor neurons were harvested from genetically engineered GalSafe day 40 swine embryos, cultured in custom mechanobioreactors, and axon tracts were successfully stretch-grown to 5 cm within 25 days. Importantly, both sensory and motor GalSafe neurons were observed to tolerate established axon stretch growth regimes of ≥1 mm/day to produce continuous, healthy axon tracts spanning 1, 3, or 5 cm. Once stretch-grown, 1 cm GalSafe TENGs were transplanted into a 1 cm lesion in the sciatic nerve of athymic rats. Regeneration was assessed through histological measures at the terminal time point of 2 and 8 weeks. Neurons from GalSafe TENGs survived and elicited AFAR as observed when using wild-type TENGs. At 8 weeks postrepair, myelinated regenerated axons were observed in the nerve section distal to the injury site, confirming axon regeneration across the lesion. These experiments are the first to demonstrate successful harvest and axon stretch growth of GalSafe neurons for use as starting biomass for bioengineered nerve grafts as well as initial safety and efficacy in an established preclinical model-important steps for the advancement of clinical-grade TENGs for future regulatory testing and eventual clinical trials. Impact statement Biofabrication of tissue engineered medical products requires several steps, one of which is choosing a suitable starting biomass. To this end, we have shown that the clinical-grade, genetically engineered biomass-GalSafe® neurons-is a viable option for biomanufacturing of our tissue engineered nerve grafts (TENGs) to promote regeneration following major peripheral nerve injury. Importantly, this is a first step in clinical-grade TENG biofabrication, proving that GalSafe TENGs recapitulate the mechanism of axon-facilitated axon regeneration seen previously with research-grade TENGs.

Production and characterization of transgenic pigs expressing porcine CTLA4-Ig.

BACKGROUND: Inhibition of the T-cell-mediated immune response is a necessary component of preventing rejection following xenotransplantation with pig alpha1,3-galactosyltransferase gene-knockout (GTKO) organs. Cytotoxic T lymphocyte-associated antigen (CTLA4) is a co-stimulatory molecule that inhibits T-cell activity and may be useful in prolonging graft rejection. METHODS: An expression vector was built containing the extracellular coding region of porcine (p) CTLA4 fused to the hinge and CH2/CH3 regions of human IgG1 (pCTLA4-Ig). Pigs transgenic for pCTLA4-Ig, on either a GTKO or wild-type (WT) genetic background, were produced by nuclear transfer and characterized using Western blot analysis, immunofluorescence, ELISA, and necropsy. RESULTS: Fifteen pCTLA4-Ig-transgenic piglets resulted from five pregnancies produced by nuclear transfer. All transgenic pigs exhibited robust expression of the pCTLA4-Ig protein and most expressed the transgene in all organs analyzed, with significant levels in the blood as well. Despite initial good health, these pigs exhibited diminished humoral immunity, and were susceptible to infection, which could be managed for a limited time with antibiotics. CONCLUSIONS: Viable pigs exhibiting robust and ubiquitous expression of pCTLA4-Ig were produced on both a WT and GTKO background. Expression of pCTLA4-Ig resulted in acute susceptibility to opportunistic pathogens due at least in part to a significantly compromised humoral immune status. As this molecule is known to have immunosuppressive activity, high levels of pCTLA4-Ig expression in the blood, as well as defective development related to exposure to pCTLA4-Ig in utero, may contribute to this reduced immune status. Prophylactic treatment with antibiotics may promote survival of disease-free transgenic pigs to a size optimal for organ procurement for transplantation. Additional genetic modifications and/or tightly regulated expression of pCTLA4Ig may reduce the impact of this transgene on the humoral immune system.

Sperm morphology and preparation method affect bovine embryonic development.

This study was conducted to evaluate the effect of sperm separation methods of semen samples collected from bulls subjected to scrotal insulation on embryonic development after in vitro fertilization (IVF) and to determine whether IVF results would be affected by various heparin concentrations. Morphologically abnormal semen samples were obtained and cryopreserved from Holstein bulls following scrotal insulation for 48 hours. Standard protocols using the Percoll gradient (90%/45%) method and the swim-up method were used to separate spermatozoa fractions in experiment I. The pellet (A(p)) and the 45% layer (B(p)) were isolated from the Percoll separation, while for the swim-up separation, the supernatant (A(s)) and the interphase (B(s)) were isolated. The overall blastocyst rate for our laboratory control semen was 23.1 +/- 2.1% for Percoll separations (A(p) and B(p)) and 18.2 +/- 2.0% for swim-up (A(s) and B(s)) separations. This rate was higher (P <.01) than the rate observed for the semen from the bull that had the greatest response to scrotal insult 5 days prior to the insult, when it was 9.2 +/- 2.1% for the Percoll separation and 20.7 +/- 2.3% for the swim-up separation, while semen from 27 days after scrotal insulation (D +27) resulted in no blastocyst formation for the Percoll separation and a 4.2 +/- 2.1% rate for the swim-up separation. In experiment II, semen was sampled from the bulls that responded in the greatest and least degrees to scrotal insult 5 days before scrotal insulation (D -5) and on days 23 (D +23) and 34 (D +34) after scrotal insulation. These samples were exposed to IVF mediums with 3 different heparin concentrations (0.1, 1.0, and 10 microg/mL). There was a significant difference (P <.05) in developmental scores between the D -5 (1.08 +/- 0.08), D +23 (0.9 +/- 0.08), and D +34 (0.8 +/- 0.08) samples, but no differences were observed in blastocyst formation based on the number of cleaved embryos. Increasing the heparin concentration resulted in higher (P <.01) embryonic developmental scores. In conclusion, when semen samples with high percentages of abnormal spermatozoa are used for IVF, semen separation preparation methods affect results. Our results show that the separation methods used under these conditions were inadequate in their ability to provide potentially competent sperm for IVF. However, selecting appropriate sperm separation procedures could improve in the IVF embryonic development of semen from bulls used in artificial insemination. Also, an increase in the heparin concentration was able to partially overcome deficiencies, which suggests that morphologically abnormal spermatozoa undergo capacitation despite possible structural changes to the plasma membrane.