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

Abstract

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.

In vitro and in vivo characteristics of frozen/thawed neonatal pig split-skin strips: A novel biologically active dressing for areas of severe, acute or chronic skin loss

“…Nevertheless, adequate caution, like the exposure of NPSS strips to zidovudine (42), and patient follow-up are in order for the temporary grafting of frozen/thawed NPSS strips onto wounds in vivo. Additional procedures, such as the inhibition of PERV expression by RNA interference or the remodelling of the PERV envelope glycoprotein (86)(87)(88), will further ensure the safety of using frozen/thawed NPSS strips in clinical settings.…”

Recent Progress in Xenotransplantation, with Emphasis on Virological Safety

“…Sensitive and specific methods have been developed to screen for PERV-Cpositive animals [88,89]. Third, RNA interference technology has been successfully used in transgenic pigs to reduce the expression of PERV in vitro [90][91][92] and in vivo [93][94][95]. Fourth, vaccination is an excellent tool to prevent virus transmission.…”

Methods and Tools for Detection and Evaluation of the Risks of Porcine Endogenous Retrovirus in Porcine to Human Xenotransplantation

“…Proof of concept studies have been performed in vitro to identify candidate siRNAs; (Karlas, Kurth et al 2004;Miyagawa, Nakatsu et al 2005;Dieckhoff, Karlas et al 2007). Two groups have generated transgenic pigs that express the interfering RNAs (Dieckhoff, Petersen et al 2008;Ramsoondar, Vaught et al 2009).…”