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Background: Pig‐derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α‐Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. Methods: The N‐glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1‐KO and GGTA1/CMAH‐KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser‐induced fluorescence detection. Results: We identified biantennary and core‐fucosylated N‐glycans terminating with immunogenic α‐Gal‐ and α‐Gal‐/Neu5Gc‐epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH‐KO pigs, respectively. Levels of N‐glycans terminating with galactose bound in β(1‐4)‐linkage to N‐acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N‐glycans capped with Neu5Gc were increased in GGTA1‐KO pigs compared to WT, but were not detected in GGTA1/CMAH‐KO pigs. Similarly, the ganglioside Neu5Gc‐GM3 was found in WT and GGTA1‐KO but not in GGTA1/CMAH‐KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. Conclusion: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human‐like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic. [ABSTRACT FROM AUTHOR]

Background: The present study reports the development of a sensitive dot blot protocol for determining the level of preformed antibodies against porcine heart valve tissue derived from wild‐type (WT) and α‐Gal‐KO (GGTA1‐KO) pigs in human sera. Methods: The assay uses decellularized and solubilized heart valve tissue; antibody binding found in this dot blot assay could be correlated with antibody titers of preformed anti‐α‐Gal and anti‐Neu5Gc antibodies detected by a sensitive ELISA. Results: The ultimate protocol had an inter‐assay variance of 9.5% and an intra‐assay variance of 9.2%, showing that the test is reliable and highly reproducible. With the aid of this dot blot assay, we found significant variation with regard to antibody contents among twelve human sera. Binding of preformed antibodies to WT tissue was significantly higher than to GGTA1‐KO tissue. Conclusions: The dot blot assay described herein could be a valuable tool to measure preformed antibody levels in human sera against unknown epitopes on decellularized tissue prior to implantation. Ultimately, this prescreening may allow a matching of the porcine xenograft with the respective human recipients in demand and thus may become an important tool for graft long‐term survival similar to current allotransplantation settings. [ABSTRACT FROM AUTHOR]

Background Xenotransplantation is considered to be a promising solution to the growing demand for suitable donor organs for transplantation. Despite tremendous progress in the generation of pigs with multiple genetic modifications thought to be necessary to overcoming the severe rejection responses after pig-to-non-human primate xenotransplantation, the production of knockout pigs by somatic cell nuclear transfer ( SCNT) is still an inefficient process. Producing genetically modified pigs by intracytoplasmic microinjection of porcine zygotes is an alluring alternative. The porcine GGTA1 gene encodes for the α1,3-galactosyltransferase that synthesizes the Gal epitopes on porcine cells which constitute the major antigen in a xenotransplantation setting. GGTA1- KO pigs have successfully been produced by transfecting somatic cells with zinc-finger nucleases ( ZFNs), transcription activator-like effector nucleases ( TALENs), or CRISPR/Cas targeting GGTA1, followed by SCNT. Methods Here, we microinjected a CRISPR/Cas9 vector coding for a single-guide RNA (sg RNA) targeting exon 8 of the GGTA1 gene into the cytoplasm of 97 in vivo-derived porcine zygotes and transferred 86 of the microinjected embryos into three hormonally synchronized recipients. Fetuses and piglets were analyzed by flow cytometry for remaining Gal epitopes. DNA was sequenced to detect mutations at the GGTA1 locus. Results Two of the recipients remained pregnant as determined by ultrasound scanning on day 25 of gestation. One pregnancy was terminated on day 26, and six healthy fetuses were recovered. The second pregnancy was allowed to go to term and resulted in the birth of six healthy piglets. Flow cytometry analysis revealed the absence of Gal epitopes in four of six fetuses (66%), indicating a biallelic KO of GGTA1. Additionally, three of the six live-born piglets (50%) did not express Gal epitopes on their cell surface. Two fetuses and two piglets showed a mosaicism with a mixed population of Gal-free and Gal-expressing cells. Only a single piglet did not have any genomic modifications. Genomic sequencing revealed indel formation at the GGTA1 locus ranging from +17 bp to −20 bp. Conclusions These results demonstrate the efficacy of CRISPR/Cas to generate genetic modifications in pigs by simplified technology, such as intracytoplasmic microinjection into zygotes, which would significantly facilitate the production of genetically modified pigs suitable for xenotransplantation. Importantly, this simplified injection protocol avoids the penetration of the vulnerable pronuclear membrane, and is thus compatible with higher survival rates of microinjected embryos, which in turn facilitates production of genetically modified piglets. [ABSTRACT FROM AUTHOR]

An abstract of the article "Transmission of porcine endogenous retroviruses (PERVs): Animal models and inhibition by RNA interference," by Britta Dieckhoff, Alexander Karlas, Björn Petersen, Wilfred A. Kues, Reinhard Kurth, Heiner Niemann, and Joachim Denner is presented.