ИСТИНА

Retroviruses are naturally positioned as a base of choice for constructing gene therapy vectors which is due to their ability to integrate viral DNA into the host cell genome. Unfortunately, insertional mutagenesis caused by random integration catalysed by retroviral integrases remains a considerable obstacle for successful use of such vectors. The reason for random integration lies in the properties of retroviral integrases that show a substantial nonspecific DNA-binding activity. This obstacle can be potentially overcome by a direct modification of retroviral integrase by the addition of a protein domain with specific DNA-binding activity. Such attempts were made before but none of them produced hybrids that would target integration into a specific and safe site in the human genome. Herein, we present hybrid proteins consisting of an artificial “zinc-finger” protein domain (tRL18) attached to either HIV-1 integrase or the integrase of the prototype foamy virus. The tRL18 protein was designed and constructed in such a way that it recognizes an 18 bp site within human leucine tRNA gene, which we consider a safe location taking into account that tRNA genes are presented in tandem copies in the human genome and that several integration events should not undermine the entire cellular protein synthesis. Our hybrids were tested for their catalytic activity, which appeared to be unaffected by the addition of tRL18. Their ability for directed integration in the vicinity of the target site was assayed in an in vitro system. All hybrid proteins were capable of changing the integrational pattern of the wild type enzyme and a preferred integration around the target site. Although the ability of our constructs to direct integration in vivo remains to be tested, we consider our system promising because it is designed for direct integration into a potentially safe region in the human genome and because it has proved worthy for two different retroviral integrases.