Falcicchia Chiara, Trempat Pascal, Binaschi Anna, Perrier-Biollay Coline, Roncon Paolo, Soukupova Marie, Berthommé Hervé, Simonato Michele
Department of Medical Science, Section of Pharmacology, Neuroscience Center, University of Ferrara and National Institute of Neuroscience, Ferrara, Italy.
Bioviron, Université Claude Bernard Lyon 1, Villeurbanne, France.
PLoS One. 2016 Mar 8;11(3):e0150995. doi: 10.1371/journal.pone.0150995. eCollection 2016.
Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.
脑源性神经营养因子（BDNF）已被发现具有促癫痫和抗癫痫两种作用。因此，必须使用旨在阻断或增强其信号的先进工具来验证其作为治疗靶点的有效性。本研究的目的是开发使BDNF信号沉默的工具。我们构建了1型单纯疱疹病毒（HSV-1）衍生的扩增载体，即含有由表达盒的串联重复序列构成的152 kb基因组的病毒颗粒，能够使感兴趣的基因以多拷贝形式表达。基于HSV-1的扩增载体无致病性，过去已成功用于将基因导入活体动物大脑。因此，扩增载体应该是表达沉默盒的合理选择，预期多拷贝的沉默盒会导致靶基因表达的有效敲低。在此，我们采用了两种基于扩增载体的BDNF沉默策略。第一种是反义策略，旨在靶向并降解BDNF的细胞质mRNA池，而第二种基于收敛转录技术，旨在抑制BDNF基因的转录。这两种扩增载体在体外表达BDNF的中胚层血管母细胞中均被证明能有效下调BDNF表达。然而，在癫痫持续状态模型中接种到海马体后，只有反义策略在体内有效，在该模型中BDNF mRNA水平会大幅升高。有趣的是，尽管BDNF反义诱导的BDNF水平敲低仅在单个海马体的一部分中产生，但足以产生显著的行为效应。总之，本研究证明了扩增载体在体外和体内敲低基因表达方面具有可靠的效果。因此，这种方法可能在神经生物学研究中得到广泛应用。
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