新型 AAV 工具未能检测到 Neurod1 介导的体内 Müller 胶质细胞和星形胶质细胞的神经元转化。
New AAV tools fail to detect Neurod1-mediated neuronal conversion of Müller glia and astrocytes in vivo.
机构信息
Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
Department of Molecular Biology and Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
出版信息
EBioMedicine. 2023 Apr;90:104531. doi: 10.1016/j.ebiom.2023.104531. Epub 2023 Mar 20.
BACKGROUND
Reprogramming resident glial cells to convert them into neurons in vivo represents a potential therapeutic strategy that could replenish lost neurons, repair damaged neural circuits, and restore function. AAV (adeno-associated virus)-based expression systems are powerful tools for in vivo gene delivery in glia-to-neuron reprogramming, however, recent studies show that AAV-based gene delivery of Neurod1 into the mouse brain can cause severe leaky expression into endogenous neurons leading to misinterpretation of glia-to-neuron conversion.
METHODS
AAV-based delivery systems were modified for improved in vivo delivery of Neurod1, Math5, Ascl1, and Neurog2 in the adult mouse retina and brain. To examine whether bona fide glia-to-neuron conversion occurs, stringent fate mapping experiments were performed to trace the lineage of glial cells.
FINDINGS
The neuronal leakage is prevalent after AAV-GFAP-mediated delivery of Neurod1, Math5, Ascl1, and Neurog2. The transgene-dependent leakage cannot be corrected after lowering the AAV doses, using alterative AAV serotypes or injection routes. Importantly, we report the development of two new AAV-based tools that can significantly reduce neuronal leakage. Using the new AAV-based tools, we provide evidence that Neurod1 gene transfer fails to convert lineage traced glial cells into neurons.
INTERPRETATION
Stringent fate mapping techniques independently of an AAV-based expression system are the golden standard for tracing the fate of glia cells during neuronal reprogramming. The newly developed AAV-based systems are invaluable tools for glia-to-neuron reprogramming in vivo.
FUNDING
The work in Chen lab was supported by National Institutes of Health (NIH) grants R01 EY024986 and R01 EY028921, an unrestricted challenge grant from Research to Prevent Blindness, the New York Eye and Ear Infirmary Foundation, and The Harold W. McGraw, Jr. Family Foundation for Vision Research. The work in Zhang lab was supported by NIH (R01 NS127375 and R01 NS117065) and The Decherd Foundation.
背景
将内源性胶质细胞重编程为神经元代表了一种有潜力的治疗策略，可以补充丢失的神经元、修复受损的神经回路并恢复功能。基于腺相关病毒（AAV）的表达系统是胶质细胞向神经元重编程中体内基因传递的有力工具，然而，最近的研究表明，将 Neurod1 通过 AAV 递送到小鼠大脑中会导致严重的内源性神经元渗漏表达，从而导致对胶质细胞向神经元转化的误解。
方法
对基于 AAV 的递送系统进行了修饰，以改善成年小鼠视网膜和大脑中 Neurod1、Math5、Ascl1 和 Neurog2 的体内递送。为了检查是否真正发生了胶质细胞向神经元的转化，进行了严格的谱系追踪实验来追踪神经胶质细胞的谱系。
结果
Neurod1、Math5、Ascl1 和 Neurog2 通过 AAV-GFAP 递送至胶质细胞后，神经元渗漏普遍存在。降低 AAV 剂量、使用替代 AAV 血清型或注射途径均不能纠正依赖转基因的渗漏。重要的是，我们报告了两种新的基于 AAV 的工具的开发，这两种工具可以显著减少神经元渗漏。使用新的基于 AAV 的工具，我们提供了证据表明，Neurod1 基因转移不能将谱系追踪的胶质细胞转化为神经元。
解释
严格的谱系追踪技术是胶质细胞在神经元重编程过程中追踪其命运的金标准，不依赖于基于 AAV 的表达系统。新开发的基于 AAV 的系统是体内胶质细胞向神经元重编程的宝贵工具。
资金
Chen 实验室的工作得到了美国国立卫生研究院（NIH）R01EY024986 和 R01EY028921 资助、Research to Prevent Blindness 的无限制挑战赠款、纽约眼耳医院基金会和 Harold W. McGraw, Jr. 家族基金会的资助。Zhang 实验室的工作得到了 NIH（R01 NS127375 和 R01 NS117065）和 Decherd 基金会的资助。
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