Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non‐immunosuppressed baboons

Abstract

Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement‐mediated rejection, as shown here using non‐immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high‐level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement‐mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti‐Galα(1,3)Gal epitope (anti‐GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre‐sensitized with antibody were highly resistant to human complement‐mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non‐immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at ∼24‐h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median > 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti‐GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced ∼8‐fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.

Immunological Challenges and Therapies in Xenotransplantation

“…Several efforts have been made to eliminate the detrimental effect of the complement cascade's activation on the xenograft, including the use of specific soluble complement inhibitors, such as soluble complement receptor type 1 (Lam et al 2005), or the cobra venom factor (Kobayashi et al 1997), and the production of animals transgenic for human complement regulatory proteins (CRP), such as hCD55, hCD46, or hCD59 (Fodor et al 1994;Cozzi and White 1995;McCurry et al 1995;Cowan et al 2000;Loveland et al 2004;Menoret et al 2004). This approach represents an elegant strategy for down-regulating local complement activation on the xenograft while preserving systemic complement activity as a first line of defense.…”

Multi‐transgenic pigs for xenotransplantation

“…One pig line has however been reported as expressing abundant and ubiquitous expression of CD46, from a minigene, and this prevented hyperacute kidney rejection in non-immunosuppressed baboons for several days even without GGTA1 inactivation (Loveland et al, 2004). …”

“…The substituted CD46 cDNA sequence was used for the construction of a CD46 minigene according to Loveland et al, (2004).…”

“…As the CD46 expression levels of the mg2 minigene were not quantified by Loveland et al, (2004), a direct comparison of expression levels is not possible.…”

“…The transplantation of CD46 transgenic porcine kidneys into nonimmunosuppressed baboons clearly revealed a control of complement activation and an inhibition of hyperacute rejection (Loveland et al, 2004). The CD46 transgenic pigs were further modified by a homozygous GGTA1 knockout (Hara et al, 2008) and are now distributed by Revivicor Inc., Blacksburg, USA.…”