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Preclinical Efficacy and Biosafety Evaluation of Genetically Modified Mesenchymal Stem Cell
- Author(s)
- BASHYAL NARAYAN
- Advisor
- Haeyoung Suh-Kim
- Department
- 일반대학원 의생명과학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2022-02
- Language
- eng
- Keyword
- Ajou Thesis
- Alternative Abstract
- Human mesenchymal stem cells (MSCs) are multipotent stem cells that have been intensively studied as therapeutic tools for a variety of disorders. To enhance the efficacy of MSCs, therapeutic genes are introduced using adenovirus, retroviral and lentiviral vectors. We generated lentivirus vectors encoding three genes such as red fluorescence protein (RFP), Copepod green fluorescence protein (GFP) and puromycin resistance gene (PuroR) in a single vector which can efficiently expressed all three genes in MSCs (MSC-Lenti-RGP). We evaluated stemness properties, long term transgenes expression, and biosafety in the transduced MSCs. In other hand, it has been known that serious adverse events (SAEs) such as tumorigenesis can be induced by insertional mutagenesis by the introduced viral vector. To mitigate this, the wild-type herpes simplex virus thymidine kinase (HSV-TK) gene and a gene containing a point mutation that results in an alanine to histidine substitution at residue 168 was introduced by replacing PuroR and used for transducing MSCs (MSC-TK and MSC-TK A168H). Transduction of lentiviral vectors encoding the TK A168H mutant did not alter the proliferation capacity, mesodermal differentiation potential, or surface antigenicity of MSCs. The MSC-TK A168H cells were genetically stable, as shown by karyotyping. MSC-TK A168H responded to ganciclovir (GCV) with an IC50 value 10-fold less than that of MSC-TK. Because MSC-TK A168H cells were found to be non-tumorigenic, U87-TK A168H brain tumor cells were used for mimicking a SAE-like condition and we evaluated the effect of valganciclovir (vGCV), an oral prodrug for GCV. U87- TK A168H tumors were more efficiently ablated by 200 mg/kg vGCV than U87- TK tumors. These results indicate that MSC-TK A168H cells appear to be pre-clinically safe for therapeutic use owing to reducing the amount of the drugs for complete removal. Accordingly, we propose that genetic modification with HSV-TK A168H makes allogeneic MSC-based ex vivo therapy safer by eliminating transplanted cells during SAEs such as uncontrolled cell proliferation. Furthermore, this vector can be used to modify MSCs by incorporating disease specific therapeutic genes for therapeutic purpose.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Biomedical Sciences > 4. Theses(Ph.D)
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