Evaluation Of Biologic Safety With Porcine Endogenous Retrovirus In Xenotransplantation

The number of candidates waiting for organ transplantation has greatly outpaced the supply of human organs available in recent years, which leads to a renewed interest in pig to human xenotransplantation as an alternative. Though tissue engineering and technology of stem cell provide a feasible solution to organ shortage, they have many difficulties in construction of the functional organs. The size of pig organs is similar with human beings, and pig can be bred largely and easy to gene modify, so it has been the best organ supply for researchers of xenotransplantation. Otherwise, pig to human xenotransplantation provide not only a substitutive therapy for patiencts suffering from intractable neurologic disorders and type I insulin-dependent diabetes mellitus, but also a interim method for patients suffering from acute fulminant hepatitis with acute and chronic liver and renal failure.There are two major problems in xenotransplantation, one is immunological reject, the other is biological safety. Especially American scientist Clive Patience reported porcine endogenous retrovirus (PERV) could infect human cells in vitro in 1997, safety concerns over the risk of xenozoonosis has become the focus in xenotransplantation. PERV are proviruses, inherited in a stable Mendelian fashion and could not be removedby normal technology.It has been proved that PERV is a typical mammalian C-type retrovirus with genomic structure of gag, pol, env and 3', 5'long terminal repeat (LTR). PERV is classified to two subtypes, named 7(1-5) and 0(1-4). So far PERV provirus intergrated in all tested foreign pigs of different species, and virus mRNA expressed. PERV can infect many cell lines and primary cells of human beings in vitro, but the results of PERV infectivity in animal models are different, and clinical retrospective studies abroad showed different conclusions with animal studies. Therefore, the infectivity of PERV in vivo is unknown, which is essential to further investigated. Otherwise, the risk of inducing disease by PERV has not been reported.In this study a series of screening experiments were performed to evaluate the potential risk in pig-to-human xenotransplantation. PERV provirus, mRNA and reverse transcriptase (RT) were detected in four species of Chinese pigs. Some specific methods for PERV detection were established, such as PCR, RT-PCR, Real-Time PCR, ELISA, Western Blot. Otherwise, the long-term effects of PERV infection on human cells were detected and some biological characteristics of infected human cells were reported. At last, PK15 and SMMC-7721 cells were transplanted to nude mice to evaluate the infectivity of PERV in vivo. The results are acquired from these studies as following:1. PERV gag, pol and env genes are intergrated and expressed in all detected Chinese pigs, including BMI, WZSP, NJP and SWP. The subtype of PERV in BMI and WZSP is PERV-A,-B, and no envC is detected in all tested pigs.2. BMI genome had more copies of PERV-gag, pol, envB gene, and there was no significant different between WZSP, NJP and GDP. The copy numbers of PERV-envAin three species of domestic pigs were ranged from 1 to 5, which was in agreement with that of BMI.3. The average concentration of reverse transcriptase in three subspecies of BMI—JS111, JS133, and JS151 was far lower than that of HIV-1 and PK15, and there was no apparent difference among the three subspecies.4. PERV particles in the harvested supernatant of PK15 were same in morphology under electron microscopy, which were round and about 120 nm in size. Virus particles had dense virus core and capsid, belonging to typical mammalian C-type viruses.5. PERV pro virus and viral RNA were detected in most of tested human cells, including seven cell lines and four primary cells. However, Chang Liver cell line and human embryonic cartilage cell were not infected by PERV in vitro.6. Further evaluation of effects on human cells after PERV six-month infection in vitro was performed in this study. As the results, there were no significant differences in morphology, cell apoptosis, total DNA/cell and thymidine incorporation between uninfected and infected HEK-293, only infected HEK-293 cells doubled a little earlier than the control. Western blot showed there was no distinct band with apparent molecular masses of lOKDa in infected HEK-293 cells.7. The mutation of LTR region after PERV infection had no apparent significance. Besides, semi-quantitative analysis demonstrated PERVinfection had no effect on transcription of HERV-K genes.8. Some tissues in nude mice after PK15 and SMMC-7721 transplantation were found PERV infection, but no evidence of human cell infection was detected.Above all, PERV-A,-B existed in all tested Chinese pigs, and no PERV-C was detected. BMI genome had more copies of PERV-gag, pol, envB gene than other pigs, which indicated copy number of PERV increased during inbreeding. The average concentration of reverse transcriptase in BMI was far lower than that of HIV-1 and PK15, indicating the low infectivity of PERV. We also examined a few aspects of the biology of PERV infected human cells by 6 months culture, and the results suggested PERV had no apparent effect on human cells. Human cells showed no acute changes of growth after PERV infection. No significant effects were showed on transcription of HERV-K genes after six-month infection with PERV, and the mutation of LTR region after PERV infection had no apparent significance, and these results provided more safety information to xenotransplantation. Results of nude mice model indicated PERV could transmit from pig to nude mice, but no evidence of human cell infection was detected. Therefore, considering results of clinical retrospective studies abroad we considered it was reasonable to carefully perform clinical xenotansplantation as immunologic obstacle was overcome, and it was essential to keep our eyes on potential risk of PERV infection once xenotransplantation is performed, and patients must be seriously detected and tracked regularly. This viewpoint was also supported by Jay A. Fishman and Clive Patience. Otherwise, study for the characteristics of PERV causing disease should be performed and moresensitive and specific methods are also to be developed to meet the progress of clinical xenotransplantation.