Di Maria Valentina, Moindrot Marine, Ryde Martin, Bono Antonino, Quintino Luis, Ledri Marco
Laboratory of Molecular Neurophysiology and Epilepsy, Department of Clinical Sciences, Epilepsy Center, Lund University, Lund, Sweden.
Laboratory of CNS Gene Therapy, Department of Experimental Medical Sciences, Lund University, Lund, Sweden.
Front Mol Neurosci. 2020 Sep 8;13:168. doi: 10.3389/fnmol.2020.00168. eCollection 2020.
Gene therapy approaches using viral vectors for the overexpression of target genes have been for several years the focus of gene therapy research against neurological disorders. These approaches deliver robust expression of therapeutic genes, but are typically limited to the delivery of single genes and often do not manipulate the expression of the endogenous locus. In the last years, the advent of CRISPR-Cas9 technologies have revolutionized many areas of scientific research by providing novel tools that allow simple and efficient manipulation of endogenous genes. One of the applications of CRISPR-Cas9, termed CRISPRa, based on the use of a nuclease-null Cas9 protein (dCas9) fused to transcriptional activators, enables quick and efficient increase in target endogenous gene expression. CRISPRa approaches are varied, and different alternatives exist with regards to the type of Cas9 protein and transcriptional activator used. Several of these approaches have been successfully used in neurons and , but have not been so far extensively applied for the overexpression of genes involved in synaptic transmission. Here we describe the development and application of two different CRISPRa systems, based on single or dual Lentiviral and Adeno-Associated viral vectors and VP64 or VPR transcriptional activators, and demonstrate their efficiency in increasing mRNA and protein expression of the gene, coding for neuronal CB1 receptors. Both approaches were similarly efficient in primary neuronal cultures, and achieved a 2-5-fold increase in expression, but the AAV-based approach was more efficient Our dual AAV-based VPR system in particular, based on dCas9, when injected in the hippocampus, displayed almost complete simultaneous expression of both vectors, high levels of dCas9 expression, and good efficiency in increasing mRNA as measured by hybridization. In addition, we also show significant upregulation of CB1 receptor protein , which is reflected by an increased ability in reducing neurotransmitter release, as measured by electrophysiology. Our results show that CRISPRa techniques could be successfully used in neurons to target overexpression of genes involved in synaptic transmission, and can potentially represent a next-generation gene therapy approach against neurological disorders.
多年来，使用病毒载体进行靶基因过表达的基因治疗方法一直是针对神经疾病的基因治疗研究的重点。这些方法能够实现治疗性基因的强劲表达，但通常仅限于单个基因的递送，并且常常无法调控内源性基因座的表达。在过去几年中，CRISPR-Cas9技术的出现通过提供允许对内源性基因进行简单高效操作的新工具，彻底改变了许多科研领域。CRISPR-Cas9的应用之一，称为CRISPRa，基于使用与转录激活因子融合的无核酸酶Cas9蛋白（dCas9），能够快速有效地增加靶内源性基因的表达。CRISPRa方法多种多样，在使用的Cas9蛋白和转录激活因子类型方面存在不同选择。其中一些方法已在神经元中成功应用， 但迄今为止尚未广泛应用于参与突触传递的基因的过表达。在此，我们描述了基于单或双慢病毒和腺相关病毒载体以及VP64或VPR转录激活因子的两种不同CRISPRa系统的开发和应用，并证明了它们在增加编码神经元CB1受体的 基因的mRNA和蛋白质表达方面的效率。两种方法在原代神经元培养中同样有效，并且 表达增加了2至5倍，但基于腺相关病毒的方法效率更高。特别是我们基于双腺相关病毒的VPR系统，基于 dCas9，当注射到海马体中时，显示出两种载体几乎完全同时表达、高水平的dCas9表达，以及通过 杂交测量在增加 mRNA方面的良好效率。此外，我们还显示CB1受体蛋白 显著上调，这通过电生理学测量的减少神经递质释放能力的增加得到反映。我们的结果表明，CRISPRa技术可以成功地用于神经元，以靶向参与突触传递的基因的过表达，并有可能代表一种针对神经疾病的下一代基因治疗方法。
