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Assessment of Risks and Benefits of Using Antibiotics Resistance Genes in Mesenchymal Stem Cell-Based Ex-vivo Therapy
International Journal of Stem Cells
Published online June 30, 2023;  
© 2023 Korean Society for Stem Cell Research.

Narayan Bashyal1,*, Young Jun Lee2,3,*, Jin-Hwa Jung1, Min Gyeong Kim2,3, Kwang-Wook Lee2, Woo Sup Hwang2, Sung-Soo Kim2,3, Da-Young Chang1, Haeyoung Suh-Kim1,2,3

1Research Center, CELLeBRAIN, Ltd., Jeonju, Korea
2Department of Anatomy, Ajou University School of Medicine, Suwon, Korea
3Department of Biomedical Sciences, Graduate School, Ajou University School of Medicine, Suwon, Korea
Correspondence to: Da-Young Chang
Research Center, CELLeBRAIN, Ltd., 152 Anjeon-ro, Deokjin-gu, Jeonju 54871, Korea
Tel: +82-70-8025-1518, E-mail: dychang@cellebrain.com
Co-Correspondence to Haeyoung Suh-Kim
Department of Biomedical Sciences, Graduate School, Ajou University School of Medicine, 164 WorldCup-ro, Yeongtong-gu, Suwon 16499, Korea
Tel: +82-31-219-5036, Fax: +82-31-219-5039, E-mail: hysuh@ajou.ac.kr
*These authors contributed equally to this work.
Received April 27, 2023; Revised May 11, 2023; Accepted May 23, 2023.
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background and Objectives: Recently, ex-vivo gene therapy has emerged as a promising approach to enhance the therapeutic 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 efficacy 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 microorganism, 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 clinically available antibiotics.
Keywords : 5-fluorocytosine, Mesenchymal stem cell, Puromycin resistance gene, Puromycin, Cytosine deaminase, Gene therapy

August 2023, 16 (3)
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