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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳佑宗 | zh_TW |
| dc.contributor.advisor | You-Tzung Chen | en |
| dc.contributor.author | 梁本裕 | zh_TW |
| dc.contributor.author | Ben-Yu Liang | en |
| dc.date.accessioned | 2024-12-26T16:07:21Z | - |
| dc.date.available | 2024-12-27 | - |
| dc.date.copyright | 2024-12-26 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-08-01 | - |
| dc.identifier.citation | 1. Swaiman, K.F., Hallervorden-Spatz syndrome. Pediatric Neurology, 2001. 25(2): p. 102-108.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/96364 | - |
| dc.description.abstract | PKAN (Pantothenate Kinase-Associated Neurodegeneration，泛酸激酶相關神經退化性疾病)，是一種罕見的神經退化性疾病，屬於腦鐵沉積相關的神經退化性疾病(NBIA, Neurodegeneration with brain iron accumulation)的一種。該病首次於1922 年發現。其特徵是PANK2 基因的突變，所轉譯的產物為泛酸鹽激活酵素 (Pantothenate kinase 2)在粒線體中作用，其功能為調控輔酶 A 的生成。此疾病患者大腦蒼白球內鐵的積累，在核磁共振成像 (MRI)上呈現出獨特的“虎眼＂標誌。本研究之目的在於運用引導編輯 (Prime editing)技術修正了源自患者的誘導多能幹細胞 (iPSCs, Induced pluripotent stem cell)中的PANK2 基因突變，並探討了其對分化後的神經幹細胞群體的影響。在方法學上，通過優化的引導編輯系統，我成功修正了PKAN 患者誘導多能幹細胞中的PANK2 exon 1 突變。隨後，這些修正後的同源性誘導多能幹細胞被分化為神經幹細胞 (NSCs, neural stem cells)。我的實驗中發現基因修正後的神經幹細胞相比於PKAN 的神經幹細胞顯示出較高的輔酶 A 濃度。這些結果初步發現了通過基因編輯來糾正PKAN 相關神經缺陷的治療潛力。 | zh_TW |
| dc.description.abstract | PKAN (pantothenate kinase-associated neurodegeneration), a rare neurodegenerative disorder categorized under neurodegeneration with brain iron accumulation (NBIA), was first identified in 1922. Mutations in the PANK2 gene characterize the disease. It is characterized by a mutation in the PANK2 gene, which encodes the enzyme pantothenate kinase 2. This enzyme functions in the mitochondria and regulates the production of coenzyme A. This leads to iron accumulation in the globus pallidus, presenting a distinctive "eye-of-the-tiger" sign on magnetic resonance imaging (MRI). This study aims to correct the PANK2 mutation in patient-derived induced pluripotent stem cells (iPSCs) using gene editing technology and explore its effects on differentiated neural cell populations. Using optimized engineered pegRNA (epegRNA), I successfully corrected the PANK2 exon 1 mutation in PKAN patient-derived iPSCs. These corrected isogenic iPSCs were then differentiated into neural stem cells (NSCs). The corrected NSCs demonstrated higher coenzyme A concentration than PKAN NSCs. These findings underscore some initial signs of the therapeutic potential of gene correction via prime editing in mitigating neural deficits associated with PKAN. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-12-26T16:07:21Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2024-12-26T16:07:21Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 中文摘要 ii
Abstract iii 1. Introduction 1 1.1 Pantothenate kinase-associated neurodegeneration (PKAN) 1 1.2 Models of PKAN 3 1.3 Therapies for pantothenate kinase-associated neurodegeneration 5 1.4 The prime editing system is suitable for creating precise gene editing models 6 1.5 Current progress in neuron differentiation from iPSCs 7 2. Materials and methods 10 2.1 Cell culture 10 2.2 Prime editing- epegRNA design 10 2.3 epegRNA cloning-Golden Gate Assembly 11 2.4 Transient transfection 13 2.5 Cell genomic DNA extraction and amplification 13 2.6 Analysis of Sanger sequencing data and epegRNA editing efficiency 14 2.7 Generation of NSCs from iPSCs 15 2.8 Differentiation of NSCs to neuron cells, astrocytes, and oligodendrocytes 15 2.9 Characterization of NSCs and Differentiated Subtypes of Neural Cells 16 2.10 Cell viability assay 17 2.11 Coenzyme A Detection 17 2.12 Determination of mitochondrial membrane potential 18 3. Results 19 3.1 Design the prime editing system plasmids for creating mutant PANK2 gene 19 3.2 Establish the PANK2 exon 1 (c.332 T > A) mutant HEK293T cell line 20 3.3 Design the prime editing system plasmids for correcting mutant PANK2 gene in PKAN patient-derived iPSCs 21 3.4 Identify the optimal prime editing designs for correcting mutant PANK2 gene 21 3.5 Correction of the PKAN patient-derived iPSCs by prime editing 22 3.6 Induction of neural stem cells from hiPSCs 22 3.7 Differentiation of neural cells from NSCs 23 3.8 Gene correction by prime editing enhances CoA level in NSCs 24 4. Discussion 25 5. References 29 6. Figures 38 Figure 1. Characterizations of prime editing introduced PANK2 exon1 mutant HEK293T 38 Figure 2. Characterizations of prime editing introduced PANK2 exon1 mutant correction HEK293T 39 Figure 3. Genetic correction of PKAN patient-derived PANK2 mutant iPSC 40 Figure 4. Immunofluorescence staining of differentiated NSCs from PKAN and gene-corrected iPSCs 42 Figure 5. Immunofluorescence staining of differentiated neuron cells from WT NSCs 43 Figure 6. Investigation of WT, PKAN, and corrected NSCs 44 7. Supplementary Figures 45 Figure S1. The design of the prime editing system for introducing PANK2 mutant gene 45 Figure S2. Immunofluorescence staining of differentiated neuron cells from PKAN and gene-corrected NSCs 47 8. Tables 48 Table 1. Oligo designs for epegRNA 48 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 引導編輯 | zh_TW |
| dc.subject | 神經幹細胞 | zh_TW |
| dc.subject | 泛酸激酶相關神經退行性疾病 | zh_TW |
| dc.subject | neural stem cells | en |
| dc.subject | pantothenate kinase-associated neurodegeneration | en |
| dc.subject | prime editing | en |
| dc.title | 引導編輯系統於泛酸激酶相關神經退化性疾病細胞模型的應用 | zh_TW |
| dc.title | Application of prime editing systems on PKAN cell model | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 黃祥博;林靜嫻;周祐吉;周志銘 | zh_TW |
| dc.contributor.oralexamcommittee | Hsiang-Po Huang;Chin-Hsien Lin ;Yu-Chi Chou;Chih-Ming Chou | en |
| dc.subject.keyword | 泛酸激酶相關神經退行性疾病,引導編輯,神經幹細胞, | zh_TW |
| dc.subject.keyword | pantothenate kinase-associated neurodegeneration,prime editing,neural stem cells, | en |
| dc.relation.page | 48 | - |
| dc.identifier.doi | 10.6342/NTU202402995 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2024-08-02 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 基因體暨蛋白體醫學研究所 | - |
| 顯示於系所單位： | 基因體暨蛋白體醫學研究所 | |
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