CYTOMEGALOVIRUS EARLY PROMOTER INDUCED EXPRESSION OF hCD59 IN PORCINE ORGANS PROVIDES PROTECTION AGAINST HYPERACUTE REJECTION1

Abstract

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.

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

“…Correspondingly, all fetuses showed a significantly decreased susceptibility to complement-mediated lysis comparable to the HR-derived GGTA1-KO cells described previously (23,29).…”

Analysis of pig-to-human porcine endogenous retrovirus transmission in a triple-species kidney xenotransplantation model

“…The unmodified pigs were obtained from Schweinezuchtverband Weser-Ems, Oldenburg, Germany. The transgenic pigs were produced by microinjection of a mini-gene construct that coded for the human complement-regulating protein CD59 [24] and were obtained from the FAL Mariensee, Germany.…”

The contribution of farm animals to human health

“…Survival rates reached 23 -135 days with porcine xenografts transgenic for one of the two complement regulators; survival rates were heavily affected by the strength of the immune-suppressive protocol (Table 6) [29 -31]. Similarly, transgenic expression of hCD59 was compatible with an extended survival of porcine hearts in a perfusion model or following transfer into primates [32,33]. These data show that HAR can be overcome in a clinically acceptable manner by expressing human complement regulators in transgenic pigs [22].…”