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.

Genetically modified pigs offer a solution to the severe shortage of organs available for human transplantation. Inactivation of the α1,3‐galactosyltransferase gene, responsible for α1,3‐Gal epitope synthesis (1–2) was a major breakthrough and essentially solved the problem of hyperacute rejection. However, further modifications of donor pigs are required to enable long term xenograft survival. These include expression of complement regulatory, antithrombotic, endothelium protective and immuno‐regulatory transgenes. Current evidence indicates that complement regulator transgenes, such as CD46 and CD55, must be expressed at least fivefold more than their human endogenous equivalent to effectively protect grafted tissue (3). To explore means of achieving high expression, we produced a series of BAC‐ and PAC‐vector constructs containing different sized genomic complement regulatory genes, from 70 to 190 kb, under the control of endogenous or CAGGs (CMV enhancer chicken β actin) promoter sequences. Using these vectors we obtained high levels of CD46 and CD55 expression in vitro. To prepare multi‐transgene “packages” for introduction into animals, the most effective constructs were combined with one or two further xenoprotective transgenes, including A20 (4), HO‐1 (5), thrombomodulin (6) and CTLA4‐Ig (LEA 29Y). In cotransfection experiments single cell clones expressing up to five different xenogenes could be detected. Achieving desired levels of transgene expression in an animal is not only a function of the transgene construct, but also of the location at which it integrates. Position effect variation in expression of random transgenes is well‐documented. Transgene placement by gene targeting at a permissive locus offers a useful means of achieving reliable transgene expression. In mice the ROSA26 locus is widely used to support ubiquitous expression of inserted transgenes (7–8). Murine ROSA26 encodes no functional genes and extensive gene targeting of this locus has not led to deleterious effects on development or physiology (9). To enable a similar approach in pigs, we recently identified a strong candidate for the porcine ROSA26 locus. Gene targeted placement of a neomycin reporter gene construct under the control of the ROSA26 promoter showed expression in all examined porcine tissues of all three germ layers. This strongly suggests that porcine ROSA 26 may resemble the murine locus as a suitably permissive site for transgene expression. We are currently proceeding with gene targeting to place various xenoprotective transgene constructs at this locus. References:  1. Dai Y, Vaught TD, Boone J et al. Targeted disruption of the alpha1,3‐ galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002; 20: 251–255. 2. Lai L, Kolber‐Simonds D, Park K et al. Production of alpha‐1,3‐galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002; 295: 1089–1092. 3. Miyagawa S, Fukuta D, Kitano E et al. Effect of tandem forms of DAF(CD55) on complement‐mediated xenogeneic cell lysis. Xenotransplantation 20...

Abundant expression of a series of xenoprotective transgenes is vital for clinically useful xeno‐donor pigs. Breeding of such multi‐transgenic animals requires that xeno‐transgenes be placed at a single, or a small number of loci in the pig genome to avoid or minimise separation by genetic segregation. We are investigating methods that support transgene expression and also enable transgenes to be “stacked” at a single locus. These include the use of BAC based multi‐transgene vectors, and very recently serial transgene placement at a permissive locus by homologous recombination supported by TALENs [1]. BAC vector constructs containing genomic sequences for the complement activation inhibitors CD55, CD46 and CD59 were assessed to determine the minimum size without reduction of expression level. These constructs enable us to express all biologically active CD55 splice variants (membrane bound and soluble forms). Effective xenoprotection also requires ubiquitous expression. We are thus examining endogenous promoter sequences of various lengths as well as the widely used CAGGs (CMV enhancer/chicken β actin) promoter to direct complement inhibitory gene expression levels in all porcine tissues. To investigate means of coexpressing groups of transgenes that provide xeno‐protection at different levels, we developed a series of BAC vector constructs containing the CD55 genomic sequence plus two additional xeno‐transgene cDNAs, including A20 [2], HO1 [3], thrombomodulin [4] and CTLA4‐Ig (LEA29Y) [5]. Analysis of porcine adipose MSC stably transfected cell clones revealed expression of all inserted xeno‐transgenes most importantly high expression of complement inhibitory genes (Fig. 1). This demonstrates that xeno‐transgenes can be effectively combined in a single construct, thus minimising the number of transgene loci in the final donor pigs. The huge carrying capacity of BAC vectors enables the addition of further xeno‐transgenes in a single vector construct. We have further examined the system in vivo. Primary cells transfected with up to three BAC constructs were used for nuclear transfer, fetuses explanted and examined for expression with very promising results. References [1] Carlson D, Tan W, Lillico S et al. Efficient TALEN‐mediated gene knockout in livestock. Proc Nat Acad Sci U S A 2012; 109: 17382–17387 [2] Oropeza M, Petersen B, Carnwath JW et al. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation 2009; 16: 522–534. [3] Loboda A, Jazwa A, Grochot‐Przeczek A et al. Heme Oxygenase‐1 and the Vascular Bed: From Molecular Mechanisms to Therapeutic Opportunities. Antioxid Redox Signal 2008; 10: 1767–1812. [4] Roussel JC, Moran CJ, Salvaris EJ et al. Pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein C and TAFI. Am J Transplant 2008; 8: 1101–1112. [5] Larsen C, Pearson T, Adams A et al. Rational Development of LEA29Y (belatacept), a High‐Affinity Variant of CTLA4‐Ig with Potent Immunos...