博碩士論文 111821011 詳細資訊



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姓名 龍庭翔(Ting-Hsiang Lung)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討以RNA修飾為基礎的基因治療對於肌萎縮側索硬化症的應用
(Exploring the Application of RNA Modification-Based Gene Therapy for Amyotrophic Lateral Sclerosis)
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摘要(中) 肌萎縮側索硬化症（Amyotrophic lateral sclerosis, ALS），又稱漸凍症，是一種好發於成年人的運動神經元退化性疾病，控制肌肉動作的大腦以及脊髓神經細胞會在發病後會逐漸死亡，目前ALS的致病機制以及治療方式還有待研究。近期，我們實驗室發現N6-甲基腺嘌呤（N6-methyladenosine, m6A）的RNA化學修飾在細胞以及動物的ALS疾病模型中有下降的趨勢。先前實驗中，我們發現利用藥物提升m6A的修飾量，能夠延緩由ALS病人誘導型幹細胞所分化的運動神經元的退化速度。基於上述的在細胞實驗的發現，我們在此研究中想要驗證上述的理論是否也能應用在動物的模型當中，並藉此尋找新的ALS治療方式。首先，我們利用化學化合物和RNA干擾技術並測試在分化的神經母細胞瘤細胞，驗證m6A在神經性退化疾病的甲基化程度。之後，為了建立應用在動物模型的治療方法，我們使用腺相關病毒使其表達針對m6A的去甲基化酶基因「Fto」的short hairpin RNA(scAAV9-shFto)，並利用脊髓鞘內注射的方式將scAAV9-shFto打入ALS的疾病小鼠模型（SOD1G93A 小鼠）。我們的研究結果顯示，在接受scAAV9-shFto注射的SOD1G93A小鼠在運動行為有較好的表現；通過電生理的測試，我們也發現SOD1G93A 小鼠在肌肉收縮能力方面也有改善的效果。更重要的是，scAAV9-shFto的注射能夠延緩了SOD1G93A小鼠的發病時間並且延長其壽命。此外，實驗室先前利用Nanopore定序找出了一些帶有m6A修飾的ALS致病基因，為了更進一步的驗證這些數據，我們利用了m6A的抗體進行RNA的免疫沈澱反應並結合定量反轉錄聚合酶連鎖反應實驗。結果顯示，利用此方式發現大部分的ALS致病基因接受到m6A的甲基修飾，提供了m6A修飾在ALS疾病中的重要性，並結合先前實驗結果，也為ALS找到一個新的治療策略的方向。
摘要(英) Amyotrophic lateral sclerosis (ALS) is an adult-onset, irreversible motor neuron (MN) degenerative disease affecting brain and spinal cord nerve cells that control voluntary muscle movement. Our recent finding observed decreased N6-methyladenosine (m6A) RNA modification in both the in vitro and in vivo ALS disease models. Surprisingly, restoring the m6A level in ALS iPSC-derived MNs will delay the MN degeneration process. Based on those findings and rationales, we aim to investigate whether this phenomenon can be proven in vivo and identify a new therapeutic approach for ALS. Before that, we investigated the role of m6A and its writer, which can deposit the m6A on mRNA in the differentiated neuroblastoma cell line using chemical compounds and RNA interference. To establish the therapy tool, we utilized scAAV9 expressing short hairpin RNA to target one of the m6A eraser enzymes, Fto, which can demethylate the methyl group on m6A. We perform the intrathecal injection to deliver scAAV9-shFto into the SOD1G93A mice. Our result found that SOD1G93A mice with scAAV9-shFto intrathecal delivery demonstrated improved motor activity performance and better neuromuscular function measured by motor behavior tests and electrophysiology tests, respectively. Furthermore, the administration of scAAV9-shFto resulted in a notable delay in the disease onset and prolongation in overall survival in SOD1G93A mice. In addition, we have discovered some ALS-related genes with m6A modification by using m6A nanopore sequencing. To further confirm these data, we performed m6A immunoprecipitation combined with RT-qPCR (m6A-RIP-qPCR) and found that several ALS risk genes are m6A-modified. These findings provide a new therapy perspective on ALS disease and the importance of m6A RNA modification in ALS disease.
關鍵字(中) ★ RNA修飾
★ 基因治療
★ 肌萎縮側索硬化症		 關鍵字(英) ★ RNA modification
★ Gene Therapy
★ Amyotrophic Lateral Sclerosis
論文目次 Table of Contents
摘要 v
Abstract vi
誌謝 vii
Table of Contents viii
Abbreviation xi
Chapter I Introduction 1
1-1 N6‐methyladenosine (m6A) 1
1-2 m6A detection methods 3
1-3 Amyotrophic lateral sclerosis (ALS) 5
1-4 ALS disease model 8
1-5 Therapy for ALS 10
1-6 The relationship between m6A and neural development/degeneration 11
1-7 Overview of this project 12
Chapter II Research Context and Methods 14
2-1 Generation of an inducible METTL14-knockdown SH-SY5Y cell line 14
2-2 Inducible METTL14-knockdown SH-SY5Y cell line differentiation 14
2-3 RNA extraction 14
2-4 Western blot 15
2-5 m6A ELISA assay and quantification 15
2-6 Reverse transcription and real-time quantitative PCR (RT-qPCR) 16
2-7 m6A dot blot assay 17
2-8 Immunofluorescence staining 17
2-9 scAAV9 plasmid construction 18
2-10 scAAV9 virus preparation and injection 18
2-11 Spinal motor neuron quantification 18
2-12 GFAP-positive cell quantification 19
2-13 Mouse breeding 19
2-14 Survival analysis 19
2-15 Behavioral and Electrophysiologic Assay 19
2-15-1 Rotarod 20
2-15-2 Grip strength 20
2-15-3 Compound muscle action potential (CMAP) 20
2-16 Mouse motor neuron differentiation from mouse embryonic stem cell (mESC) 21
2-17 m6A immunoprecipitation and quantitative real-time PCR 21
2-17-1 RNA preparation 21
2-17-2 RNA immunoprecipitation 22
2-17-3 m6A enrichment calculation by RT-qPCR 22
Chapter III Results 23
3-1 The chemical compound-induced m6A hypomethylation caused neuron degeneration in differentiated SH-SY5Y 23
3-2 Establish the METTL14 knockdown-inducible system in SH-SY5Y cell line 23
3-3 The neurites of SH-SY5Y cells showed no prominent defects upon induction of METTL14 knockdown 24
3-4 Generation of scAAV9-shFto and the functional test 25
3-5 Explore the gene therapy function of scAAV9-shFto in SOD1G93A mice 27
3-6 Verify the m6A-modified sites in ALS-related gene 29
Chapter IV Discussion 31
Chapter V Future Perspective 36
5-1 Investigate the relationship between SNPs of m6A-related genes and MN degeneration 36
5-2 Dissect the effect of FTO knockdown on ALS disease 37
5-3 Identification and verification of potential m6A-modified transcript linked to ALS disease from m6A-RIP -seq and nanopore sequencing 37
Chapter VI Figure and Figure Legends 39
Figure 1. METTL3 inhibitor causes differentiated SH-SY5Y degeneration. 39
Figure 2. The METTL14 knockdown-inducible system can successfully regulate the m6A level. 41
Figure 3. Differentiated SH-SY5Y-Tet-shMETTL14 demonstrate no prominent defect upon doxycycline induction. 43
Figure 4. The shRNA targeting Fto effectively knocked down Fto expression and led to a notable increase in the m6A level. 45
Figure 5. The AAV delivery through intrathecal injection successfully transmits to motor neurons and decreases the Fto expression in the mouse spinal cord. 46
Figure 6. The intrathecal injection of scAAV9-shFto mitigates the pathology of SOD1G93A mice. 48
Figure 7. The intrathecal injection of scAAV9-shFto protects neuromuscular function and delays the disease onset of SOD1G93A mice. 50
Figure 8. To verify the m6A-modified sites in ALS-related genes in differentiated mouse motor neuron 53
Appendix 1: Previous data from Dr. Ya-Ping Yen 54
Appendix 2: Plasmid, Primer, Antibody list, the Recipe of Buffer and Medium 55
Reference 60
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指導教授 陳俊安 陳盛良(Jun-An Chen Shen-Liang Chen) 審核日期 2024-7-22
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