Konrad Fischer

Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing

Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age.

Porcine familial adenomatous polyposis model enables systematic analysis of early events in adenoma progression

We compared gene expression in low and high-grade intraepithelial dysplastic polyps from pigs carrying an APC1311 truncating mutation orthologous to human APC1309, analysing whole samples and microdissected dysplastic epithelium. Gene set enrichment analysis revealed differential expression of gene sets similar to human normal mucosa versus T1 stage polyps. Transcriptome analysis of whole samples revealed many differentially-expressed genes reflecting immune infiltration. Analysis of microdissected dysplastic epithelium was markedly different and showed increased expression in high-grade intraepithelial neoplasia of several genes known to be involved in human CRC; and revealed possible new roles for GBP6 and PLXND1. The pig model thus facilitates analysis of CRC pathogenesis.

Porcine endogenous retroviruses: Quantification of the copy number in cell lines, pig breeds, and organs

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.

Assembling multiple xenoprotective transgenes in pigs

This review gives a brief overview of the genetic modifications necessary for grafted porcine tissues and organs to overcome rejection in human recipients. It then focuses on the problem of generating and breeding herds of donor pigs carrying modified endogenous genes and multiple xenoprotective transgenes. A xenodonor pig optimised for human clinical use could well require the addition of ten or more xenoprotective transgenes. It is impractical to produce the required combination of transgene by cross‐breeding animals bearing individual transgenes at unlinked genetic loci, because independent segregation means that huge numbers of pigs would be required to produce relatively few donor animals. A better approach is to colocate groups of transgenes at a single genomic locus. We outline current methods to assemble transgene arrays and consider their pros and cons. These include polycistronic expression systems, in vitro recombination of large DNA fragments in PAC and BAC vectors, transposon vectors, classical gene targeting by homologous recombination at permissive loci such as ROSA26, targeted transgene placement aided by gene editing systems such as CRISPR/Cas9, and transgene placement by site‐specific recombination such as Min‐tagging using the Bxb1recombinase.

Non-invasive assessment of porcine oocyte quality by supravital staining of cumulus-oocyte complexes with lissamine green B.

We evaluated the usefulness of lissamine green B (LB) staining of cumulus-oocyte complexes (COC) as a non-invasive method of predicting maturational and developmental competence of slaughterhouse-derived porcine oocytes cultured in vitro. Cumulus cells of freshly aspirated COCs were evaluated either morphologically on the basis of thickness of cumulus cell layers, or stained with LB, which penetrates only non-viable cells. The extent of cumulus cell staining was taken as an inverse indicator of membrane integrity. The two methods of COC grading were then examined as predictors of nuclear maturation and development after parthenogenetic activation. In both cases LB staining proved a more reliable indicator than morphological assessment (P < 0.05). The relationship between LB staining and cumulus cell apoptosis was also examined. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for DNA fragmentation revealed that oocytes within COCs graded as low quality by either LB staining or visual morphology showed significantly greater DNA fragmentation (P < 0.05) than higher grades, and that LB and visual grading were of similar predictive value. Expression of the stress response gene TP53 showed significantly higher expression in COCs graded as low quality by LB staining. However expression of the apoptosis-associated genes BAK and CASP3 was not significantly different between high or low grade COCs, suggesting that mRNA expression of BAK and CASP3 is not a reliable method of detecting apoptosis in porcine COCs. Evaluation of cumulus cell membrane integrity by lissamine green B staining thus provides a useful new tool to gain information about the maturational and developmental competence of porcine oocytes.

Multitransgenic Porcine Fibroblasts are Protected from Immunoglobulin Binding and Complement Deposition in a Xeno-Microfluidic Model

Background One of the main issues that hamper xenotransplantation to be clinically feasible is humoral rejection. Xenoreactive antibodies, both natural or elicited, contribute significantly to rejection of xenotransplants by activation of complement and interactions with a variety of effector cells. Sugars – namely Gal-&agr;1,3-Gal, Neu5Gc and &bgr;4GalNT2 – expressed on the surface of endothelial cells are thought to play an important role. In this study, we tested the potential xenoprotective role of human complement regulators – hCD46, hCD55, hCD59 – combined with A20 (NF-&kgr;B inhibitor and anti-apoptotic protein) and HO-1 (oxidative stress-response protein) using a microfluidic model where porcine kidney fibroblasts (PKF) grown on artificial 3D microvessels were perfused with normal human serum (NHS). Cell lines lacking the expression of Gal-&agr;1,3-Gal (GGTA1 KO), Neu5Gc (CMAH KO) and &bgr;4GalNT2 (&bgr;4GalNT2 KO) sugars were also tested in order to assess the effects on complement activation and immunoglobulin binding. Methods PKF WT and transgenic were seeded on artificial microvessels with diameters of 550 &mgr;m fabricated in PDMS. The cells were cultured overnight under static condition to allow them to reach confluence. A peristaltic pump was then connected and the cells were perfused for 2 hours in a close loop with 5mL of 1:10 diluted NHS. Control samples were perfused with medium without NHS. The channels were washed with PBS, fixed and stained to detect complement deposition (C3b/c and C4b/c) and immunoglobulin binding (IgG and IgM). Microfluidic chips were analyzed under laser scanning confocal microscope. Five pictures/microchannel were acquired and the fluorescence intensity was quantified and plotted in a graph. Results Data showed that the absence of sugars from the cell surface of transgenic fibroblasts resulted in a significant decrease of antibody binding and antibody dependent complement deposition. Furthermore, the addition of 5 transgenes (hCD46, hCD55, hCD59, A20 and HO-1) to the GGTA1 KO background significantly improved the protection from complement deposition as shown by the immunofluorescence pictures and by the quantification. Experiments with PKF with triple KO together with the expression of the 5 trangenes are currently ongoing. Conclusions This study shows that immunoglobulin binding and complement deposition on transgenic PKF are strongly reduced when xenoantigens (GGTA1, CMAH, &bgr;4GalNT2) are removed from the cell surface. The addition of the 5 human transgenes further enhances the protection of the graft from complement. Transgenic pigs carrying such genetic modification might represent suitable donors for future pig to baboon preclinical xenotransplantation experiments. Furthermore, the study reveals the potential of our in vitro microfluidic system in screening the effects of different transgenes prior to the production of the respective pigs for in vivo experiments.