Multi‐transgenic pigs for xenotransplantation (2013)

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. D ai Y , V aught TD , B oone J et al. Targeted disruption of the alpha1,3‐ galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002; 20: 251–255. 2. L ai L , K olber‐ S imonds D , P ark K et al. Production of alpha‐1,3‐galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002; 295: 1089–1092. 3. M iyagawa S , F ukuta D , K itano E et al. Effect of tandem forms of DAF(CD55) on complement‐mediated xenogeneic cell lysis. Xenotransplantation 2006; 13: 433–439. 4. O ropeza M , P etersen B , C arnwath 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. 5. L oboda A , J azwa A , G rochot‐ P rzeczek A et al. Heme Oxygenase‐1 and the Vascular Bed: From Molecular Mechanisms to Therapeutic Opportunities. Antioxid Redox Signal 2008; 10: 1767–1812. 6. R oussel JC , M oran CJ , S alvaris 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. 7. S oriano P . Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 1999; 21: 70–71. 8. S rinivas S , W atanabe T , L in CS et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 2001; 1: 4. 9. Z ambrowicz BP , I mamoto A , F iering S et al. Disruption of overlapping transcripts in the ROSA βgeo 26 gene trap strain leads to widespread expression of β‐galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci USA 1997; 94: 3789–2794.