Assessment of Risks and Benefits of Using Antibiotics Resistance Genes in Mesenchymal Stem Cell-Based Ex-vivo Therapy

Background and Objectives: Recently, ex-vivo gene therapy has emerged as a promising approach to enhance the ther-apeutic potential of mesenchymal stem cells (MSCs) by introducing functional genes in vitro. Here, we explored the need of using selection markers to increase the gene delivery efficiency and evaluated the potential risks associated with their use in the manufacturing process. Methods and Results: We used MSCs/CD that carry the cytosine deaminase gene (CD) as a therapeutic gene and a puromycin resistance gene (PuroR) as a selection marker. We evaluated the correlation between the therapeutic effi-cacy and the purity of therapeutic MSCs/CD by examining their anti-cancer effect on co-cultured U87/GFP cells. To simulate in vivo horizontal transfer of the PuroR gene in vivo, we generated a puromycin-resistant E. coli (E. coli/PuroR) by introducing the PuroR gene and assessed its responsiveness to various antibiotics. We found that the anti-cancer effect of MSCs/CD was directly proportional to their purity, suggesting the crucial role of the PuroR gene in eliminating impure unmodified MSCs and enhancing the purity of MSCs/CD during the manufacturing process. Additionally, we found that clinically available antibiotics were effective in inhibiting the growth of hypothetical micro-organism, E. coli/PuroR. Conclusions: In summary, our study highlights the potential benefits of using the PuroR gene as a selection marker to enhance the purity and efficacy of therapeutic cells in MSC-based gene therapy. Furthermore, our study suggests that the potential risk of horizontal transfer of antibiotics resistance genes in vivo can be effectively managed by clin-ically available antibiotics.