Zahur Muzna, Tolö Johan, Bähr Mathias, Kügler Sebastian
Department of Neurology, University Medical Center GöttingenGöttingen, Germany.
Center for Nanoscale Microscopy and Molecular Physiology of the Brain at Department of Neurology, University Medical Center GöttingenGöttingen, Germany.
Front Mol Neurosci. 2017 May 23;10:142. doi: 10.3389/fnmol.2017.00142. eCollection 2017.
Gene editing tools like TALENs, ZFNs and Crispr/Cas now offer unprecedented opportunities for targeted genetic manipulations in virtually all species. Most of the recent research in this area has concentrated on manipulation of the genome in isolated cells, which then give rise to transgenic animals or modified stem cell lines. Much less is known about applicability of genetic scissors in terminally differentiated, non-dividing cells like neurons of the adult brain. We addressed this question by expression of a pair of ZFNs targeting the murine cathepsin D gene in CNS neurons by means of an optimized AAV viral vector. We show that ZFN expression resulted in substantial depletion of cathepsin D from neuronal lysosomes, demonstrating a robust gene deletion. Importantly, long-term ZFN expression in CNS neurons did not impair essential neuronal functionality and did not cause inflammation or neurodegeneration, suggesting that potent genetic scissors can be expressed safely in the mouse brain. This finding opens up new venues to create novel research models for neurodegenerative disorders.
像转录激活样效应因子核酸酶（TALENs）、锌指核酸酶（ZFNs）和规律成簇间隔短回文重复序列/CRISPR相关蛋白（Crispr/Cas）这样的基因编辑工具，如今为几乎所有物种的靶向基因操作提供了前所未有的机会。该领域最近的大部分研究都集中在对分离细胞中的基因组进行操作，这些细胞随后会发育成转基因动物或经过修饰的干细胞系。对于基因剪刀在终末分化的、不分裂的细胞（如成年大脑中的神经元）中的适用性，人们了解得要少得多。我们通过优化的腺相关病毒（AAV）载体，在中枢神经系统（CNS）神经元中表达一对靶向小鼠组织蛋白酶D基因的锌指核酸酶，解决了这个问题。我们发现，锌指核酸酶的表达导致神经元溶酶体中的组织蛋白酶D大量减少，证明了有效的基因敲除。重要的是，在中枢神经系统神经元中长期表达锌指核酸酶不会损害基本的神经元功能，也不会引起炎症或神经退行性变，这表明强效的基因剪刀可以在小鼠大脑中安全表达。这一发现为创建神经退行性疾病的新型研究模型开辟了新途径。
