Effective combination of xenoprotective transgenes

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 Immunosuppressive Properties. Am J Transplant 2005; 5: 443–453. Fig. 1. Q‐RT‐PCR analysis of CD55‐CAGGs (left) and CD46‐CAGGs (right) vector constructs compared to the expression levels of human MSCs.