Genetic engineering strategies to prevent the effects of antibody and complement on xenogeneic chondrocytes

Abstract

Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from nontransgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.

Xenotransplantation of pig chondrocytes: therapeutic potential and barriers for cartilage repair

“…Accordingly, hormones and decellularized or devitalized tissues of animal origin are already being used in the clinic for therapeutic purposes (Vadori and Cozzi, 2015). New therapies could be developed in the future by using xenogeneic cells and tissues selected by their regenerative capacity and adapted to the intended clinical application by genetic engineering (Costa et al, 2003;Sommaggio et al, 2015). The pig is now considered the best animal source for xenotransplantation because it is domesticated, reproduces in large scale and has a primate-like size of organs and physiology.…”

“…Humoral and cellular responses lead within weeks to months to rejection of pig cartilage transplanted into primates (Stone et al, 1997;Stone et al, 1998). Similar observations have been made in a small animal model (Costa et al, 2003).…”

“…Consequently, pig cartilage is not subjected to a fast rejection process equivalent to HAR of vascularized organs. On the contrary, it is slowly rejected by a delayed process, comprising humoral and cellular responses, that progresses over time from the edges towards the inside (Stone et al, 1997;Costa et al, 2003). Our group has been working during the last decade on elucidating the molecular basis of this cartilage rejection process (Costa et al, 2008;Sommaggio et al, 2009;Sommaggio et al, 2012;Sommaggio et al, 2013).…”

“…This is pending, as additional carbohydrate antigens recognized by human XNA have been recently identified (Vadori and Cozzi, 2015). Nevertheless, our group has clarified to some extent the mechanisms and effects of total antibody and complement activation on rejection of xenogeneic chondrocytes (Sommaggio et al, 2013;Sommaggio et al, 2015). In particular, we have set up an in vitro model by incubating PCs with up to 40 % human serum and determined complement activation by deposition of complement components on the cell surface and release of complement byproducts.…”

“…In particular, we have set up an in vitro model by incubating PCs with up to 40 % human serum and determined complement activation by deposition of complement components on the cell surface and release of complement byproducts. As a result, both the classical and alternative pathways of complement are activated (Sommaggio et al, 2013;Sommaggio et al, 2015), but very little cell death is produced due to an inherent resistance of chondrocytes (Happonen et al, 2012). Instead, exposure of PCs to 20 % human serum leads to PC activation with up-regulation of pig major histocompatibility complex SLA (for swine leukocyte antigen) class I and vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), as well as secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) (Sommaggio et al, 2013).…”