Here we describe transplantation of olfactory ensheathing cells (OECs) or Schwann cells derived from transgenic pigs expressing the human complement inhibitory protein, CD59 (hCD59), into transected dorsal column lesions of the spinal cord of the immunosuppressed rat to induce axonal regeneration. Non-transplanted lesion-controlled rats exhibited no impulse conduction across the transection site, whereas in animals receiving transgenic pig OECs or Schwann cells impulse conduction was restored across and beyond the lesion site for more than a centimeter. Cell labeling indicated that the donor cells migrated into the denervated host tract. Conduction velocity measurements showed that the regenerated axons conducted impulses faster than normal axons. By morphological analysis, the axons seemed thickly myelinated with a peripheral pattern of myelin expected from the donor cell type. These results indicate that xenotranplantation of myelin-forming cells from pigs genetically altered to reduce the hyperacute response in humans are able to induce elongative axonal regeneration and remyelination and restore impulse conduction across the transected spinal cord.

Hyperacute rejection (HAR) is the first critical immunological hurdle that must be addressed in order to develop xenogeneic organs for human transplantation. In the area of cell-based xenotransplant therapies, natural antibodies (XNA) and complement have also been considered barriers to successful engraftment. Transgenic expression of human complement inhibitors in donor cells and organs has significantly prolonged the survival of xenografts. However, expression of complement inhibitors without eliminating xenogeneic natural antibody (XNA) reactivity may provide insufficient protection for clinical application. An approach designed to prevent XNA reactivity during HAR is the expression of human alpha1, 2-fucosyltransferase (H-transferase, HT). H-transferase expression modifies the cell surface carbohydrate phenotype of the xenogeneic cell, resulting in the expression of the universal donor O antigen and a concomitant reduction in the expression of the antigenic Galalpha1,3-Gal epitope. We have engineered various transgenic pig lines that express HT in different cells and tissues, including the vascular endothelium. We demonstrate that in two different HT transgenic lines containing two different HT promoter constructs, expression can be differentially regulated in a constitutive and cytokine-inducible manner. The transgenic expression of HT results in a significant reduction in the expression of the Galalpha1,3-Gal epitope, reduced XNA reactivity, and an increased resistance to human serum-mediated cytolysis. Transgenic pigs that express H-transferase promise to become key components for the development of xenogeneic cells and organs for human transplantation.

Research in pig-to-primate xenotransplantation aims to solve the increasing shortage of organs for human allotransplantation and develop new cell- and tissue-based therapies. Progress towards its clinical application has been hampered by the presence of xenoreactive natural antibodies that bind to the foreign cell surface and activate complement, causing humoral graft rejection. Genetic engineering of donor cells and animals to express human complement inhibitors such as hCD59 significantly prolonged graft survival. Strategies to decrease the deposition of natural antibodies were also developed. Expression of human alpha1,2-fucosyltransferase (H transferase, HT) in pigs modifies the cell-surface carbohydrate phenotype resulting in reduced Galalpha1,3-Gal expression and decreased antibody binding. We have developed transgenic pigs that coexpress hCD59 and HT in various cells and tissues to address both natural antibody binding and complement activation. Functional studies with peripheral blood mononuclear cells and aortic endothelial cells isolated from the double transgenic pigs showed that coexpression of hCD59 and HT markedly increased their resistance to human serum-mediated lysis. This resistance was greater than with cells transgenic for either hCD59 or HT alone. Moreover, transgene expression was enhanced and protection maintained in pig endothelial cells that were exposed for 24 h to pro-inflammatory cytokines. These studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograft survival and improving the prospect of clinical xenotransplantation.

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The lack of supply and access to human tissue has prompted the development of xenotransplantation as a potential clinical modality for neural cell transplantation. The goal of the present study was to achieve a better understanding of the immune factors involved in neural xenograft rejection in primates. Initially, we quantified complement mediated cell lysis of porcine fetal neurons by primate serum and demonstrated that anti-C5 antibody treatment inhibited cell death. We then developed an immunosuppression protocol that included in vivo anti-C5 monoclonal antibody treatment, triple drug therapy (cyclosporine, methylprednisolone, azathioprine) and donor tissue derived from CD59 or H-transferase transgenic pigs and applied it to pig-to-primate neural cell transplant models. Pre-formed alphaGal, induced alphaGal and primate anti-mouse antibody (PAMA) titers were monitored to assess the immune response. Four primates were transplanted. The three CD59 neural cell recipients showed an induced anti-alphaGal response, whereas the H-transferase neural cell recipient exhibited consistently low anti-alphaGal titers. Two of these recipients contained surviving grafts as detected by immunohistochemistry using selected neural markers. Graft survival correlated with high dose cyclosporine treatment, complete complement blockade and the absence of an induced PAMA response to the murine anti-C5 monoclonal antibodies.

The herpesvirus saimiri genome encodes a complement control protein homolog (CCPH). Stable mammalian cell transfectants expressing a recombinant transmembrane form of CCPH (mCCPH) or a 5FLAG epitope-tagged mCCPH (5FLAGmCCPH) conferred resistance to complement-mediated cell damage by inhibiting the lytic activity of human serum complement. The function of CCPH was further defined by showing that the mCCPH and the 5FLAGmCCPH transfectants inhibited C3 convertase activity and effectively reduced cell surface deposition of the activated complement component, C3d.

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Human CD59 (hCD59) is a cell surface glycoprotein that regulates the formation of terminal complement proteins, thereby inhibiting the formation of the membrane attack complex (Rollins et al. 1991). Baboon CD59 (babCD59) and African green monkey CD59 (AgmCD59) homologous cDNAs were isolated by cDNA library screening and the polymerase chain reaction. DNA sequence analysis revealed that the predicted primary amino acid sequence of the babCD59 and AgmCD59 molecules exhibited 84% and 82% sequence identity to hCD59, respectively (Fig. 1). Stable Balb/3T3 transfectants generated with the hCD59 (Rother et al. 1994), babCD59, and AgmCD59 cDNAs exhibited cell surface expression and were shown to be tethered to the plasma membrane via a glycosyl-phosphatidylinositol-anchor (data not shown). The complement regulatory activity of hCD59, babCD59, and AgmCD59 was evaluated by comparing the sensitivity of the transfectants to C5b-9 mediated cell lysis. Whole serum complement as well as C8/C9 titration assays revealed that hCD59, babCD59, and AgmCD59 inhibited complement-mediated dye release by human complement (data not shown). These data illustrate the highly conserved sequence and functional activity of the babCD59 and AgmCD59 homologues to hCD59. ReferencesRollins, S. A., Zhao, J., Ninomiya, H., and Sims, R J. Inhibition of homologous complement by CD59 is mediated by a speciesselective recognition conferred through binding to C8 within the C5b-8 or C9 within C5b-9.