Rohn Troy T, Radin Dean, Brandmeyer Tracy, Linder Barry J, Andriambeloson Emile, Wagner Stéphanie, Kehler James, Vasileva Ana, Wang Huaien, Mee John L, Fallon James H
Department of Biological Sciences, Boise State University, Boise, ID 83725, USA.
Cognigenics, Eagle, ID 83616, USA.
PNAS Nexus. 2023 Jun 20;2(6):pgad170. doi: 10.1093/pnasnexus/pgad170. eCollection 2023 Jun.
The expanding field of precision gene editing using CRISPR/Cas9 has demonstrated its potential as a transformative technology in the treatment of various diseases. However, whether this genome-editing tool could be used to modify neural circuits in the central nervous system (CNS), which are implicated in complex behavioral traits, remains uncertain. In this study, we demonstrate the feasibility of noninvasive, intranasal delivery of adeno-associated virus serotype 9 (AAV9) vectors containing CRISPR/Cas9 cargo within the CNS resulting in modification of the receptor gene. In vitro, exposure to primary mouse cortical neurons to AAV9 vectors targeting the gene led to a concentration-dependent decrease in spontaneous electrical activity following multielectrode array (MEA) analysis. In vivo, at 5 weeks postintranasal delivery in mice, analysis of brain samples revealed single base pair deletions and nonsense mutations, leading to an 8.46-fold reduction in mRNA expression and a corresponding 68% decrease in the 5HT-2A receptor staining. Our findings also demonstrate a significant decrease in anxiety-like behavior in treated mice. This study constitutes the first successful demonstration of a noninvasive CRISPR/Cas9 delivery platform, capable of bypassing the blood-brain barrier and enabling modulation of neuronal 5HT-2A receptor pathways. The results of this study targeting the gene provide a foundation for the development of innovative therapeutic strategies for a broad range of neurological disorders, including anxiety, depression, attentional deficits, and cognitive dysfunction.
利用CRISPR/Cas9的精准基因编辑领域不断拓展，已展现出其作为一种变革性技术在治疗多种疾病方面的潜力。然而，这种基因组编辑工具是否可用于修饰中枢神经系统（CNS）中与复杂行为特征相关的神经回路，仍不明确。在本研究中，我们证明了经鼻非侵入性递送携带CRISPR/Cas9的腺相关病毒9型（AAV9）载体至CNS内，从而实现对受体基因的修饰是可行的。在体外，将原代小鼠皮层神经元暴露于靶向该基因的AAV9载体后，经多电极阵列（MEA）分析显示，自发电活动呈浓度依赖性降低。在体内，小鼠经鼻给药5周后，对脑样本的分析揭示了单碱基对缺失和无义突变，导致mRNA表达降低8.46倍，5HT - 2A受体染色相应减少68%。我们的研究结果还表明，治疗后的小鼠焦虑样行为显著减少。本研究首次成功展示了一种非侵入性CRISPR/Cas9递送平台，该平台能够绕过血脑屏障并实现对神经元5HT - 2A受体通路的调控。针对该基因的研究结果为开发针对包括焦虑、抑郁、注意力缺陷和认知功能障碍在内的广泛神经疾病的创新治疗策略奠定了基础。
