Semaan Marwan, Ivanusic Daniel, Denner Joachim
Robert Koch Institute, Nordufer 20, Berlin, Germany.
Robert Koch Institute, Nordufer 20, Berlin, Germany; Freie Universität Berlin, Kaiserswerther Str. 16-18, Berlin, Germany.
PLoS One. 2015 Apr 24;10(4):e0122059. doi: 10.1371/journal.pone.0122059. eCollection 2015.
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.
异种移植被认为是解决移植用合适人类供体短缺问题的一种方法，目前猪是首选的供体动物。然而，异种移植可能与动物源性微生物的传播有关。虽然大多数对人类受体有风险的猪源微生物可以通过无特定病原体繁殖来消除，但猪内源性逆转录病毒（PERV）的多个拷贝整合在所有猪的基因组中，无法通过这种方式消除。PERV以感染性颗粒的形式释放并感染人类细胞。锌指核酸酶（ZFN）技术可以特异性敲除细胞基因，但尚未用于消除在靶序列中具有高度保守性的多个整合前病毒序列。为了降低PERV水平传播的风险并尽可能多地敲除前病毒，首次使用了ZFN技术这一强大工具。设计ZFN使其特异性结合所有已知具有复制能力的前病毒中保守的序列。通过蛋白质免疫印迹分析、共定位分析、PLA和FRET显示ZFN在细胞核中的表达和转运。使用荧光ZFN和细胞计数分析转染细胞的存活率。转染后显示ZFN蛋白有强烈表达且表达的ZFN蛋白有共定位现象。然而，发现ZFN的表达对转染细胞具有极高的毒性。诱导的细胞毒性可能是由于PERV前病毒的高拷贝数被特异性切割，这在低特异性ZFN切割基因组中大量脱靶位点时也很常见。这是首次尝试使用ZFN敲除细胞基因组中的多个几乎相同的基因。该尝试失败了，应采用其他策略来防止PERV传播。
