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

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。

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.