Engineering of mesenchymal stem cells to express cytosine deaminase gene with a selecable marker gene

e14050 Background: Suicide genes induce apoptosis not only in the cell itself but also in surrounding cells. The former is called the suicide effect and the latter is called the bystander effect. Two major suicide gene strategies currently pursued, cytosine deaminase (CD)/5-fluorocytosine (5-FC) and the herpes simplex virus-thymidine kinase/ganciclovir. Both genes have similar potential to induce suicide effects, but bystander effect is higher in CD/5-FC. Here we show that the level of CD expression/activity influence the anti-cancer activity of CD-expressing cells and further demonstrate the need to use selectable markers during manufacturing of CD-expressing cells. Finally we test whether live microbes harboring the shed selectable marker genes are controllable by commonly used antibiotics. Methods: We introduced a microbial CD gene and puromycin-N-acetyltransferase ( pac) gene to bone-marrow derived mesenchymal stem cells MSCs) using a retroviral vector and selected the transduced cells with 4 ug/mL puromycin for one week. To determine the effect of purity, we varied the ratio of CD-expressing MSCs with naïve MSCs and 5-FC concentration in the culture and on co-cultured glioma cells. To mimic the situation in which the shed pac gene is transmitted to live microbes in vivo, we introduced the pac gene to E. coli and tested the growth of the pac-expressing E. coli using VITEK following the standard protocol M100-S32 in the presence of diverse antibiotics. Results: We found that selection of CD-expressing cells with puromycin during manufacturing process enhanced the bystander (anti-cancer effect). Lowering the purity of CD-expressing MSCs required more 5-FC with higher IC50. Growth of pac-expressing E. coli was completely inhibited by commonly used antibiotics in the hospital. Conclusions: We proved that antibiotic selection is necessary during manufacturing process. We also showed that the risk of transmission of shed pac gene can be manageable with commonly used antibiotics. We propose that using pac selectable marker gene can be beneficial to maintain the purity of engineered cells.