HK022 bacteriophage Integrase mediated RMCE as a potential tool for human gene therapy.

HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active 'attB' sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native 'attB' sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native 'attB' sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.

Anti-cancer binary system activated by bacteriophage HK022 integrase.

The binary system presented in this work is based on the bacteriophage HK022 integrase recombinase that activates the expression of a silenced Diphtheria toxin gene, both controlled by the cancer specific hTERT promoter. Using a lung cancer mice model, assays of different apoptotic and anti-apoptotic factors have demonstrated that the Integrase based binary system is highly specific towards cancer cells and more efficient compared to the conventional mono system whose toxin is directly expressed under hTERT. In a mice survival test, this binary system demonstrated longer persistence compared to the untreated and the mono treated ones. The reason underlying the advantage of this binary system over the mono system seems to be an overexpression of various hTERT suppressing factors induced by the mono system.

Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase.

Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system.