Abstract
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.
Highlights
The neurotrophin brain-derived neurotrophic factor (BDNF) is widely expressed in the brain, where it exerts a key role in neuronal survival, differentiation, and plasticity [1,2]
We generated a plasmid in which the BDNF complementary DNA (cDNA) is inserted between two cytomegalovirus (CMV) promoters oriented in opposing directions (Fig 1B), such that the resulting convergent transcription could elicit down-regulation of the BDNF gene transcription through chromatin remodeling associated with epigenetic silencing marks [14]
We generated two types of amplicon vectors to locally knock down the levels of BDNF: a classical antisense approach and an approach based on convergent transcription
Summary
The neurotrophin brain-derived neurotrophic factor (BDNF) is widely expressed in the brain, where it exerts a key role in neuronal survival, differentiation, and plasticity [1,2]. BDNF is viewed as a promising therapeutic target for central nervous system disorders, for example in the transformation of a normal brain in epileptic, a phenomenon in which cell death, neurogenesis and morphological and functional changes are thought to be involved [3,4]. We present novel tools to pursue this aim: amplicon vectors derived from Herpes simplex virus type 1 (HSV-1) capable to down-regulate BDNF expression in vitro and in vivo. HSV-1 amplicon vectors hold considerable promise as gene-transfer vehicles because of their very high capacity to host foreign DNA with high number of repeats of the transgene [5], a feature that, in principle, makes them suited to efficiently knock-down a target gene
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
