Reinhard Schwinzer

Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.

A point mutation in PTPRC is associated with the development of multiple sclerosis

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. It is widely accepted that a dysregulated immune response against brain resident antigens is central to its yet unknown pathogenesis. Although there is evidence that the development of MS has a genetic component, specific genetic factors are largely unknown. Here we investigated the role of a point mutation in the gene (PTPRC) encoding protein-tyrosine phosphatase, receptor-type C (also known as CD45) in the heterozygous state in the development of MS. The nucleotide transition in exon 4 of the gene locus interferes with mRNA splicing and results in altered expression of CD45 isoforms on immune cells. In three of four independent case-control studies, we demonstrated an association of the mutation with MS. We found the PTPRC mutation to be linked to and associated with the disease in three MS nuclear families. In one additional family, we found the same variant CD45 phenotype, with an as-yet-unknown origin, among the members affected with MS. Our findings suggest an association of the mutation in PTPRC with the development of MS in some families.

HLA-E/human beta2-microglobulin transgenic pigs: protection against xenogeneic human anti-pig natural killer cell cytotoxicity

Background. Natural killer (NK) cells participate in pig-to-primate xenograft rejection both by antibody-dependent and -independent mechanisms. A majority of human NK cells express the inhibitory receptor CD94/NKG2A, which binds specifically to human leukocyte antigen (HLA)-E, a trimeric complex consisting of the HLA-E heavy chain, &bgr;2-microglobulin (&bgr;2m), and a peptide derived from the leader sequence of some major histocompatibility complex class I molecules. Methods. To use this mechanism for protection of pig tissues against human NK cell-mediated cytotoxicity, we generated transgenic pigs by pronuclear microinjection of genomic fragments of HLA-E with an HLA-B7 signal sequence and of human &bgr;2-microglobulin (hu&bgr;2m) into zygotes. Results. Three transgenic founder pigs were generated. Northern blot analysis of RNA from peripheral blood mononuclear cells revealed the presence of the expected transcript sizes for both transgenes in two of the three founders. The founder with the highest expression and his offspring were characterized in detail. Fluorescence-activated cell sorting (FACS) and Western blot analyses demonstrated consistent expression of HLA-E and hu&bgr;2m in peripheral blood mononuclear cells. Immunohistochemistry revealed the presence of HLA-E and hu&bgr;2m on endothelial cells of many organs, including heart and kidney. In vitro studies showed that lymphoblasts and endothelial cells derived from HLA-E/hu&bgr;2m transgenic pigs are effectively protected against human NK cell-mediated cytotoxicity, depending on the level of CD94/NKG2A expression on the NK cells. Further, HLA-E/hu&bgr;2m expression on porcine endothelial cells inhibited the secretion of interferon (IFN)-&ggr; by co-cultured human NK cells. Conclusions. This novel approach against cell-mediated xenogeneic responses has important implications for the generation of multitransgenic pigs as organ donors for clinical xenotransplantation.

Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli.

Oropeza M, Petersen B, Carnwath JW, Lucas‐Hahn A, Lemme E, Hassel P, Herrmann D, Barg‐Kues B, Holler S, Queisser A‐L, Schwinzer R, Hinkel R, Kupatt C, Niemann H. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli.
Xenotransplantation 2009; 16: 522–534.

Pigs transgenic for human thrombomodulin have elevated production of activated protein C.

Petersen B, Ramackers W, Tiede A, Lucas‐Hahn A, Herrmann D, Barg‐Kues B, Schuettler W, Friedrich L, Schwinzer R, Winkler M, Niemann H. Pigs transgenic for human thrombomodulin have elevated production of activated protein C.
Xenotransplantation 2009; 16: 486–495.

Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double-transgenic pigs

The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single‐step transduction to introduce an autoregulative tetracycline‐responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue‐independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.—Kues, W.A., Schwinzer, R., Wirth, D., Verhoeyen, E., Lemme, E., Hermann, D., Barg‐Kues, B., Hauser, H., Wonigeit, K., and Niemann, H. Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double‐transgenic pigs. FASEBJ. 20, E357–E366 (2006)

Successful Outcome Of Acute Graft-versus-host Disease In A Liver Allograft Recipient By Withdrawal Of Immunosuppression

BACKGROUND Graft-versus-host disease (GVHD) after liver transplantation is uncommon, and the outcome is almost always fatal. Since 1987, about 30 cases have been described, and patient survival is mostly exceptional. METHODS A 29-year-old man underwent retransplantation due to chronic cholestatic syndrome, 5 years after his first liver transplantation. Indication for the first liver transplantation was acute liver failure caused by exsiccosis. After the second transplantation, the patient had an initially uneventful course, developing thrombocytopenia at day 21 followed by skin rash and septic complications. Diagnosis of acute GVHD was made by using serological techniques for HLA-A and HLA-DRB and subsequently by fluorogenic sequence-specific primed polymerase chain reaction. In addition, donor lymphocytes were marked by immunohistochemical methods via biopsies of the skin. Immunosuppressive therapy was withdrawn to allow the patients own immune system to eliminate donor cells. RESULTS By withdrawing the immunosuppressive therapy, clinical and morphological signs of GVHD vanished. The patient is doing well without recurrence 13 months after transplantation. CONCLUSION Withdrawal of immunosuppressive therapy is a promising approach in the treatment of acute GVHD to allow the patients immune system to reconstitute itself, reject offending lymphocytes, and avoid lethal septic complications.

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P <0.05) over nontransgenic controls (207.5±54.6 vs. 57.5±64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.

Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys

Petersen B, Ramackers W, Lucas‐Hahn A, Lemme E, Hassel P, Queißer A‐L, Herrmann D, Barg‐Kues B, Carnwath JW, Klose J, Tiede A, Friedrich L, Baars W, Schwinzer R, Winkler M, Niemann H. Transgenic expression of human heme oxygenase‐1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. Xenotransplantation 2011; 18: 355–368.

First inducible transgene expression in porcine large animal models.

The purpose of this study was to establish inducible transgene expression in pigs, a model organism with great promise for experimental physiology and translational medicine, using the binary tet‐on system. This expression system is activated by doxycycline (dox) via the tet‐controlled transactivator (TA). Binding of TA to the transactivator response element (TRE) results in transcription of downstream genes. First, we cloned transgenic founder pigs expressing TA under the control of the CMV enhancer/chicken β‐actin promoter (CAG). Then, cells from CAG‐TA transgenic founders were nucleofected with TRE‐controlled expression vectors for either porcine cytotoxic T‐lymphocyte associated antigen 4‐Fc domain of immunoglobulin G1 (CTLA‐4Ig) or soluble receptor activator of NF‐κB ligand (RANKL), and double‐transgenic offspring were cloned. Dox administration resulted in a dose‐dependent increase in expression of CTLA‐4Ig or RANKL, in nucleofected cells and in transgenic pigs, while in the absence of dox, the levels of both proteins were below the detection limit. Inducible transgene expression was reproduced in double‐transgenic offspring generated by cloning or breeding. Our strategy revealed the first two examples of inducible transgene expression in pigs. The CAG‐TA transgenic pigs generated in this study constitute an interesting basis for future pig models with inducible transgene expression.—Klymiuk, N., Böcker, W., Schönitzer, V., Bahr, A., Radic, T., Fröhlich, T., Wünsch, A., Keßler, B., Kurome, M., Schilling, E., Herbach, N., Wanke, R., Nagashima, H., Mutschler, W., Arnold, G. J., Schwinzer, R., Schieker, M., Wolf, E. First inducible transgene expression in porcine large animal models. FASEB J. 26, 1086‐1099 (2012). www.fasebj.org