S. Zucchelli

We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8(+) T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.

Antisense (AS) transcripts are RNA molecules that are transcribed from the opposite strand to sense (S) genes forming S/AS pairs. The most prominent configuration is when a lncRNA is antisense to a protein coding gene. Increasing evidences prove that antisense transcription may control sense gene expression acting at distinct regulatory levels. However, its contribution to brain function and neurodegenerative diseases remains unclear. We have recently identified AS Uchl1 as an antisense to the mouse Ubiquitin carboxy-terminal hydrolase L1 (Uchl1) gene (AS Uchl1), the synthenic locus of UCHL1/PARK5. This is mutated in rare cases of early-onset familial Parkinson's Disease (PD) and loss of UCHL1 activity has been reported in many neurodegenerative diseases. Importantly, manipulation of UchL1 expression has been proposed as tool for therapeutic intervention. AS Uchl1 induces UchL1 expression by increasing its translation. It is the representative member of SINEUPs (SINEB2 sequence to UP-regulate translation), a new functional class of natural antisense lncRNAs that activate translation of their sense genes. Here we take advantage of FANTOM5 dataset to identify the transcription start sites associated to S/AS pair at Uchl1 locus. We show that AS Uchl1 expression is under the regulation of Nurr1, a major transcription factor involved in dopaminergic cells' differentiation and maintenance. Furthermore, AS Uch1 RNA levels are strongly down-regulated in neurochemical models of PD in vitro and in vivo. This work positions AS Uchl1 RNA as a component of Nurr1-dependent gene network and target of cellular stress extending our understanding on the role of antisense transcription in the brain.