Neuman Samuel S, Metzger Jeanette M, Bondarenko Viktoriya, Wang Yuyuan, Felton Jesi, Levine Jon E, Saha Krishanu, Gong Shaoqin, Emborg Marina E
Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, United States.
Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, United States.
Front Bioeng Biotechnol. 2023 Jul 26;11:1237613. doi: 10.3389/fbioe.2023.1237613. eCollection 2023.
Targeted gene editing is proposed as a therapeutic approach for numerous disorders, including neurological diseases. As the brain is organized into neural networks, it is critical to understand how anatomically connected structures are affected by genome editing. For example, neurons in the substantia nigra pars compacta (SNpc) project to the striatum, and the striatum contains neurons that project to the substantia nigra pars reticulata (SNpr). Here, we report the effect of injecting genome editors into the striatum of Ai14 reporter mice, which have a LoxP-flanked stop cassette that prevents expression of the red fluorescent protein tdTomato. Two weeks following intracerebral delivery of either synthetic nanocapsules (NCs) containing CRISPR ribonucleoprotein targeting the tdTomato stop cassette or adeno-associated virus (AAV) vectors expressing Cre recombinase, the brains were collected, and the presence of tdTomato was assessed in both the striatum and SN. TdTomato expression was observed at the injection site in both the NC- and AAV-treated groups and typically colocalized with the neuronal marker NeuN. In the SN, tdTomato-positive fibers were present in the pars reticulata, and SNpr area expressing tdTomato correlated with the size of the striatal genome edited area. These results demonstrate anterograde axonal transport of reporter gene protein products to the SNpr following neuronal genome editing in the striatum.
靶向基因编辑被提议作为治疗多种疾病的方法，包括神经疾病。由于大脑是由神经网络组成的，了解基因组编辑如何影响解剖学上相连的结构至关重要。例如，黑质致密部（SNpc）的神经元投射到纹状体，而纹状体包含投射到黑质网状部（SNpr）的神经元。在这里，我们报告了将基因组编辑器注射到Ai14报告基因小鼠纹状体中的效果，该小鼠具有一个侧翼为LoxP的终止盒，可阻止红色荧光蛋白tdTomato的表达。在脑内递送含有靶向tdTomato终止盒的CRISPR核糖核蛋白的合成纳米胶囊（NCs）或表达Cre重组酶的腺相关病毒（AAV）载体两周后，收集大脑，并在纹状体和黑质中评估tdTomato的存在情况。在NC处理组和AAV处理组的注射部位均观察到tdTomato表达，并且通常与神经元标记物NeuN共定位。在黑质中，网状部存在tdTomato阳性纤维，并且表达tdTomato的SNpr区域与纹状体基因组编辑区域的大小相关。这些结果表明，在纹状体进行神经元基因组编辑后，报告基因蛋白产物可向SNpr进行顺行轴突运输。
