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Reduction of the expression of porcine endogenous retroviruses (PERVs) in transgenic pigs by RNA interference: new studies

Marwan Semaan¹, Danny Kaulitz¹, Björn Petersen², Wilfried Kues², Heiner Niemann², Joachim Denner¹

¹HIV and Retrovirology, Robert Koch Institute, Berlin; ²Department of Biotechnology, Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute (FLI), Mariensee; Germany

Background: Transgenic pigs have been generated expressing PERV-specific sh(short hairpin)RNA and showed reduced expression of porcine endogenous retroviruses (PERVs) (Dieckhoff et al., Xenotransplanation, 15, 36, 2008). Since PERVs are integrated in the porcine genome and have been shown to infect human cells in vitro, they pose a risk for xenotransplantation using pig cells, tissues and organs. The shRNA corresponds to a sequence in the polymerase gene of PERV identical in PERV-A, PERV-B and PERV-C. The strategy to prevent PERV transmission by RNA interference would be beneficial for the clinical application of porcine xenotransplants, but the efficacy and long-term maintenance of the inhibition should still have to be evaluated.

Methods: Transgenic pigs were produced by somatic cloning using donor cells expressing a PERV-specific pol2 shRNA or containing the vector alone. Real-time PCRs were developed and used to measure expression of the shRNA and PERV in fibroblasts from ear biopsies and blood cells taken at different time points over three years as well as in different organs from sacrified animals.

Results: Expression of the PERV-specific shRNA and the corresponding inhibition of PERV expression were demonstrated in pol2 shRNA transgenic pigs. The levels of PERV and shRNA expression were different in different organs (PERV expression was highest in spleen and lungs and lower in liver and heart). However, in all organs of the pol2 shRNA transgenic pigs, PERV expression was significantly inhibited compared with the control animals. In addition, the efficacy of dimers and trimers of additional shRNA was shown in vitro.

Conclusion: Long-term characterisation of transgenic pigs expressing PERV-specific shRNA showed maintenance of the RNA interference for more than three years, suggesting that PERV expression in the xenotransplants will be suppressed over extended periods of time.

The work was supported by the Deutsche Forschungsgemeinschaft (DFG).