Prevention of PERV Infections in Pig to Human Xenotransplantation by the RNA Interference Silences Gene

• Published in: Journal of biochemistry (Tokyo) 2005-04, Vol.137 (4), p.503-508
• Main Authors: Miyagawa, Shuji, Nakatsu, Shino, Nakagawa, Takatoshi, Kondo, Akihiro, Matsunami, Katsuyoshi, Hazama, Kenji, Yamada, Junko, Tomonaga, Keizo, Miyazawa, Takayuki, Shirakura, Ryota
• Format: Article
• Language: English
• Subjects: Animals; Animals, Genetically Modified; BFU; blue focus forming unit; Cells, Cultured; Endogenous Retroviruses - pathogenicity; GFP; green fluorescence protein; Humans; PEC; PERV; PERV-B; pig endothelial cell; pig endothelial cells; Porcine endogenous retrovirus; porcine endogenous retroviruses; pseudotype infection; Retroviridae Infections - prevention & control; Retroviridae Infections - transmission; RNA Interference; RNA, Small Interfering - therapeutic use; short interference RNA; siRNA; Swine; Transplantation, Heterologous - adverse effects; xenotransplantation
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvi059

The possibility of preventing the transmission of porcine endogenous retrovirus (PERV) to human cells using short interfering RNAs (siRNA) was investigated. The siRNA for the p30 of PERV gag region was cloned into pSUPER, the polymerase-III H1-RNA gene promoter. A green fluorescence protein (GFP) was also cloned into pSUPER to establish pSXGH. Pig endothelial cells (PEC) were transduced with the LacZ gene by pseudotype infection, and infected with PERV subtype B, resulting in the formation of PEC(LacZ)/PB. The PEC(LacZ)/PB was next transfected with pSXGH-siRNA. The expression of siRNA was provisionally checked by determining the level of expression of GFP. Culture supernatants of infected cells were then inoculated into HEK293 cells. The siRNA clearly destroyed the PERV infectivity of PEC(LacZ)/PB in both transient cell lines and stable clones. Moreover, the decreased levels of mRNA and gag protein were evidenced in the stable clones by real-time PCR and Western blotting, respectively. The final goal of our study was to establish a transgenic pig expressing the siRNA for PERV. The results suggest that siRNA represents a novel approach for controlling PERV infections in clinical xenotransplantation.