Development of viral vector-mediated RNA interference targeting several P2X receptors in the rat central nervous system

RNA interference (RNAi) is an evolutionarily conserved process of sequencespecific post-transcriptional gene silencing that is triggered by double-stranded short interfering RNA (siRNA). At present RNAi has become an important and widely used tool for evaluating target gene function both in vitro and in vivo and has been used for the screening of potential therapeutic targets in pharmaceutical research. This project has been carried out in order to investigate the applicability of RNA interference for target gene suppression in the rat central nervous system (CNS) both in vitro and in vivo. In particular its application in the process of target validation has been evaluated focusing on the purinergic receptor P2X7, a putative target for mood disorders and the two related receptors P2X2 and P2X4. The purinergic receptor P2X7 together with the P2X2 and P2X4 receptors are members of the ATP-gated cation channel family permeable to sodium, potassium and calcium. These receptors are expressed in neurons in many regions of the brain where they play a role in release of neurotransmitters and activation of multiple intracellular signaling pathways. The P2X7 gene is also expressed in several types of glia where it is involved in the modulation of neuroinflammatory processes and cytokine release. In order to obtain P2X receptor (P2XR) suppression, specific siRNAs were designed for each gene and screened for activity by transfecting both primary cells and cell lines expressing the target genes. In this way the most active and specific siRNA sequence for each gene was selected for the production of a short hairpin RNA (shRNA) cassette leading to intracellular production of the targeting siRNA sequence. Viral vectors have been used for long term siRNA expression in neurons and glia in the brain since these vectors can efficiently transduce brain tissue and persist episomally within the infected cell. In order to drive siRNA expression in neuronal cells, Adeno-associated viral vectors (AAV) containing an shRNA cassette were produced for each target gene, since these vectors can infect neurons with high efficiency. Adenovirus 5 (Ad5)-shRNA vectors targeting P2X7 were also produced to investigate the effects of P2X7 suppression in glia since these viral vectors preferentially infect glia. As a filter to select the most active and specific viral vectors that could down-regulate P2XRs in vivo, viral vectors were first tested in vitro in primary cells. The AAV2/6-shRNA vectors were evaluated by infecting hippocampal neurons. A maximum of 80-90% down-regulation of P2XR mRNA was observed by TaqMan analysis of the infected cells. An active Ad5-shRNA vector able to suppress of 80% P2X7 mRNA expression in cultured rat astrocytes was also identified. Preliminary data demonstrated a statistically significant decrease in P2X7 function in Ad5-shRNA infected microglial cells. These data indicate the suppression of target receptor after shRNA viral vector delivery in vitro. Both the AAV2/6- and Ad5-shRNA-P2X7 viral vectors were then microinfused into rat hippocampus though glass needles. Transduction of neurons and glia respectively after infection of the dentate gyrus (DG) region of the hippocampus by AAV2/6- and Ad5-shRNA vectors was demonstrated. Moreover a statistically significant down-regulation of P2X7 mRNA in AAV-P2X7-shRNA injected DG compared to controls was revealed by in situ hybridization. A similar trend was observed in the Ad5-P2X7-shRNA injected samples. In conclusion, in this study active and specific shRNA-viral vectors able to suppress target gene expression in neuronal cells both in vitro and in vivo were generated and validated.