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| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | Haeyoung Suh-Kim | - |
| dc.contributor.author | BASHYAL NARAYAN | - |
| dc.date.issued | 2022-02 | - |
| dc.identifier.other | 31384 | - |
| dc.identifier.uri | https://aurora.ajou.ac.kr/handle/2018.oak/20593 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2022. 2 | - |
| dc.description.tableofcontents | 1. INTRODUCTION 1 2. MATERIALS AND METHODS 5 2.1 Animals 5 2.2 Human MSCs Isolation and Culture 5 2.3 Lentiviral vectors and Cell lines 6 2.4 Lentiviral titration 7 2.5 FACS 8 2.6 Surface antigen analysis 8 2.7 Western blot 9 2.8 Mesodermal differentiation assay 9 2.9 Growth kinetic assay 11 2.10 Quantitative reverse transcription (qRT)-PCR 12 2.11 Chromosomal stability 12 2.12 In vitro suicide effect of HSV-TK 12 2.13 Tumorigenicity assay of lentiviral transduced MSCs 13 2.14 In vivo suicide effect of HSV-TK 13 2.15 Statistical analysis 14 3. RESULTS 15 3.1 Development of lentiviral vector encoding three transgenes, transduction into MSCs and transduced cells characterization 15 3.1.1 Generation of lentiviral transfer plasmids encoding GFP, RFP and PuroR 15 3.1.2 Transducing reagent test in MSCs 19 3.1.3 Generation of RFP, GFP and PuroR expressing MSCs and evaluation of Stem Cell Properties 21 3.1.4 Genomic stability and tumorigenesis test of MSC-Lenti-RGP 26 3.2 Improving the safety of mesenchymal stem cell-based ex vivo therapy using Herpes Simplex virus thymidine kinase 29 3.2.1 Generation of HSV-TK expressing MSCs 29 3.2.2 Stem Cell Properties of MSC- TK A168H 34 3.2.3 Genomic stability and tumorigenesis test of MSC-TK A168H 43 3.2.4 Generation of U87-TK A168H 46 3.2.5 Effect of vGCV on U87-TK A168H tumor as a SAE model 50 4. DISCUSSION AND CONCLUSION 56 5. REFERENCES 63 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Preclinical Efficacy and Biosafety Evaluation of Genetically Modified Mesenchymal Stem Cell | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2022. 2 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.uci | I804:41038-000000031384 | - |
| dc.identifier.url | https://dcoll.ajou.ac.kr/dcollection/common/orgView/000000031384 | - |
| dc.subject.keyword | Ajou Thesis | - |
| dc.description.alternativeAbstract | 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. | - |
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