A NOVEL STRATEGY TO PREVENT PERV TRANSMISSION TO HUMAN CELLS BY REMODELING THE VIRAL ENVELOPE GLYCOPROTEIN

Abstract

P1065 Aims: Xenotransplantation offers a potential solution to the shortage of available organs for transplantation. The swine represents an ideal source of such organs. However, the discoveries that pig endogenous retroviruses (PERV) can infect human cells in vitro and SCID mouse tissue in vivo have stimulated considerable discussion concerning infectious risk in xenotransplantation. In addition, in pigs, at least 50 proviral copies of PERVs are present in the genome. Therefore, pigs that are completely devoid of all PERV related elements will never be a possibility.vIn our previous work, we demonstrated that the high mannose type of N-glycan of the envelope glycoprotein is closely related to PERV infectivity with respect to human cells. In this study, we addressed the effects of the remodeling pig cell-surface glycoproteins, especially high mannose type N-glycan, on the susceptibility of PERV by alpha-1,2 mannosidase IB (Man-IB), which converts Man9GlcNAc to Man5GlcNAc, and alpha- 3-D-mannoside-beta-1,2-N-acetylglucosaminyltransferase I (GnT I) which catalyzes the first step in the conversion of oligomannose to complex or hybrid N-glycans of glycoproteins. Methods: Pig endothelial cells (PEC) were transduced with the LacZ gene with the packaging signal of murine leukemia virus (MuLV) under the control of the long terminal repeat of MuLV by a pseudotype infection to produce PEC(Z). The PEC(Z)s were then further infected with PERV subtype B (PERV-B) to produce PEC(Z)/PB.The PEC(Z)/PBs were next trasnfected to the Man IB or GnT-I gene by the lipofection method in order to reduce the levels of high mannose type of N-glycans. Culture supernatants of infected cells were inoculated to HEK293 cells. The inoculated cells were histochemically stained and the lacZ-positive blue foci (Blue forcus Forming Unit: BFU) were counted. Results: PERV transmission from the PEC(Z)/PB with Man IB to HEK 293 cells was reduced in comparison with control PEC(Z)/PB in each of the three independent cell lines (277.9±42.5 vs. 91.6±15.0, 373.0±65.7 vs. 101.9±65.7, 215.1±25.7 vs.63.2±8.6 BFU/well; p<0.01). The reduction rate of infectivity was approximately 30% (32.6±2.8, 30.1±2.5, 30.1±2.5%). On the other hand, GnT-I transfection to the PEC(Z)/PB downregulated the PERV transmission in three independent cell lines (766.0±80.7 vs. 191.4±32.2, 919.1±69.9 vs. 404.2±54.4, 386.5±95.9 vs.69.5±7.7 BFU/well; p<0.01). The reduction rate of infectivity was also approximately 30% (24.8±3.2, 40.9±5.1, 29.4±4.5%). The co-transfection of Man IB and GnT-I experiments are under investigation. Conclusions: The transfection of the Man IB and GnT-I genes to pig cells is quite effective in reducing PERV infectivity to human cells. The results suggest that our strategy could be useful as a new approach for solving PERV infections in clinical xenotransplantation.