Cytotoxic Effects during Knock Out of Multiple Porcine Endogenous Retrovirus (PERV) Sequences in the Pig Genome by Zinc Finger Nucleases (ZFN)

Abstract

Xenotransplantation has been proposed as a solution to the shortage of suitable human donors for transplantation and pigs are currently favoured as donor animals. However, xenotransplantation may be associated with the transmission of zoonotic microorganisms. Whereas most porcine microorganisms representing a risk for the human recipient may be eliminated by designated pathogen free breeding, multiple copies of porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated this way. PERVs are released as infectious particles and infect human cells. The zinc finger nuclease (ZFN) technology allows knocking out specifically cellular genes, however it was not yet used to eliminate multiple integrated proviral sequences with a strong conservation in the target sequence. To reduce the risk of horizontal PERV transmission and to knock out as many as possible proviruses, for the first time the powerful tool of the ZFN technology was used. ZFN were designed to bind specifically to sequences conserved in all known replication-competent proviruses. Expression and transport of the ZFN into the nucleus was shown by Western blot analysis, co-localisation analysis, PLA and FRET. Survival of transfected cells was analysed using fluorescent ZFN and cell counting. After transfection a strong expression of the ZFN proteins and a co-localisation of the expressed ZFN proteins were shown. However, expression of the ZFN was found to be extremely toxic for the transfected cells. The induced cytotoxicity was likely due to the specific cutting of the high copy number of the PERV proviruses, which is also commonly observed when ZFN with low specificity cleave numerous off-target sites in a genome. This is the first attempt to knock out multiple, nearly identical, genes in a cellular genome using ZFN. The attempt failed, and other strategies should be used to prevent PERV transmission.

Transspecies Transmission of Gammaretroviruses and the Origin of the Gibbon Ape Leukaemia Virus (GaLV) and the Koala Retrovirus (KoRV)

“…Recently, attempts were undertaken through genome editing to inactivate all PERVs in the genome, either using a zinc finger nuclease (ZFN) [69], or clustered regularly interspaced short palindromic repeats (CRISPR) acting with CRISPR-associated nuclease 9 (Cas9) [70]. Using CRISPR/Cas, 62 proviruses were inactivated in immortalized pig cells and the question is, whether this strategy can be used to generate PERV-free animals suitable for xenotransplantation [71].…”

Gene Editing, Gene Therapy, and Cell Xenotransplantation: Cell Transplantation Across Species

“…Small interfering RNAs have been successfully used to generate transgenic pigs with decreased PERV expression compared to wild-type animals [58,59] and this inhibitory effect lasted up to 3 years [60]. Zinc finger nuclease-driven knockout of PERV proviral sequences proved to be difficult to achieve since it was accompanied by severe cytotoxicity possibly due to the high number of PERV inserts in the genome [61]. Gene editing using clustered regularly interspaced short palindromic repeat-associated system CRISPR/Cas9 successfully inactivated 62 genomic copies of PERV in a porcine kidney cell line [62•] suggesting that generation of completely PERV-free pigs might be possible.…”

Recent Progress in Xenotransplantation, with Emphasis on Virological Safety

“…Fifth, since PERV is present in up to approximately 100 copies in the genome, it is a challenge to knock out all proviruses in the genome using gene editing. When gene editing was performed using the zinc finger nuclease (ZFN), the expression of ZFN was very high, inducing a toxic effect when the nucleases were cutting in multiple sites and destabilizing the genome [99]. When the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas9) technology [100][101][102] was used, a breakthrough was achieved.…”