The significant findings of this report include the development of an adeno-associated virus-based siRNA approach for downregulating gene expression. A recombinant AAV-siDEN3UT was utilized to induce significant decreases in DEN infection compared to control in both Vero cells and human DCs. The results indicate that siRNAs may be used to attenuate DEN infection in human DCs and may have therapeutic value.
Interference of gene expression by siRNAs is a novel strategy to knock down specific genes in cells or tissues, and the specific silencing of pathogen genes using siRNA is a very attractive approach for the clinical treatment of infectious diseases. Long dsRNAs (of >30 nt in length) activate a dsRNA-dependent protein kinase and 2', 5'-oligoadenylate synthetase in mammalian cells, which leads to a non-specific reduction in levels of mRNAs . The endogenous expression of siRNAs from introduced DNA templates is thought to overcome some limitations of exogenous siRNA delivery, in particular its transient effects on silencing specific genes and loss of phenotype . AAV vectors have been proven to be safe and efficacious in Phase I clinical trials for gene therapy of cystic fibrosis and hemophilia B and are regarded as a potential alternative to retroviral and adenoviral vectors for gene therapy in humans. The AAV vectors have a number of advantages over other vectors. They are not pathogenic and do not induce production of neutralizing antibodies that could reduce transgene function. They possess a broad-range of tissue tropism and the capability of inducing long-term transgene expression . In this study, we utilized a novel AAV system to deliver DEN siRNA into mammalian cells and estimated its anti-DEN effect in vitro. In this AAV system, we incorporated the mouse U6 promoter, which is important for transcription and folding of the suppressor RNA, into a plasmid pCMV-U6.
The choice of appropriate target genes is necessary for the success of the siRNA strategy, and two siRNAs derived from either the pre-M or the 3' NCR region of DEN-2 were used in our study. An internal deletion of 3' NCR nucleotide sequences was found to be lethal for DEN virus replication in an in vivo study . The 3' NCR of the flavivirus genome, which presumably functions as a promoter for minus-strand RNA synthesis, is predicted to form a stem-and-loop secondary structure. Computational analyses have revealed that there is conserved sequence in all flaviviruses within the 3' end [18, 19]. Thus, two siRNA cassettes were tested in this study that included the 3'NCR sequence common to all four DEN serotypes. The other siRNA cassette is from the gene encoding the preM protein which is important for maturation of the virus into an infectious form. Our test of anti-DEN efficiency showed that siDEN3UT attenuated DEN Infection better than siDENpreM. Knocking down viral genes at the earlier stage of the viral multiplication cycle rather than in the structural protein synthesis phase may provide better antiviral protection, although the limited plasmid transfection ratio appeared to influence the suppression efficiency of siDEN to DEN-2 infection in Vero cells in the present study (Fig. 1C). The other DEN serotypes will be investigated with our 3'NCR cassette.
DEN is transmitted through Aedes aegypti mosquito bite, and resident skin DCs are an early target of DEN infection . Immature DCs are the most permissive for DEN infection and serve as a source of DEN replication and production . Replication in the early target cells may be essential for dengue pathogenesis in the human host. In this study, we also utilized peripheral blood iDCs as a cell model to test our AAV system. Similar to results in Vero cells, AAV-mediated siDEN3UT delivery down-regulated DEN-2 protein expression in iDCs. However, the magnitude of suppression in iDCs at the same infectious titer of AAV-siDEN was less compared to that found in Vero cells. Previous data showed that variations in the efficiency of transduction among DCs derived from different normal blood donors is between 2% and 50% , and we found that the infectious ratio for AAVEGFP is about 45%~50% in Vero cells. That may be due to limited expression of the AAV receptor or differential activation of the mouse U6 promoter in Vero cells compared to DCs . Increasing the AAV infection titer or utilizing a more effective promoter within the AAV vector backbone might elevate the suppression for DEN replication in iDCs. Nevertheless, DCs treated with recombinant AAV showed a significant reduction in DEN virus titer compared to control. This is important as viral titer is the gold standard for measuring antiviral activity.
DCs are one of the most powerful of APCs. After infection with virus in the periphery, iDCs process viral antigens, then differentiate into mature DCs and migrate from peripheral tissues to lymph nodes where they prime naïve CD4 and CD8 T lymphocytes to maintain protective antiviral cytotoxic T cell memory [23, 24]. Thus, DCs play an important role in the initiation of antiviral immunity and provide a crucial step in the development of adaptive antiviral immunity. Previous data showed that DEN infection induces apoptosis of DCs , which leads to a state of temporary immune-suppression during DEN fever. An important observation in our study is that AAV-siDEN treatment resulted in a significant decrease in apoptotic iDCs. The attenuation of apoptosis in iDCs following AAV-mediated siRNA delivery suggests that AAV-siRNA may be immunologically protective. After the primary DEN infection, most patients appear viremic in the early febrile phase, but the viruses are quickly cleared from the blood system after defervescence . The activation of both a humoral and cellular immune response is considered to be involved in DEN clearance. The most severe outcome in DEN infection is development of DHF/DSS, which is associated with secondary infections by heterotypic DEN serotypes. It is postulated that the preexisting, cross-reactive, adaptive immune response leads to excessive cytokine production, complement activation, and the release of other inflammatory factors that produce DHF/DSS . Therefore, it should be imperative for prophylaxis of DHF/DSS to eliminate DEN infection by different serotypes in the early target cells. Attenuation of DEN infection in DCs and protection of infected DCs from apoptosis would be a benefit for the elimination of the early DEN infection and the development and maintenance of antiviral innate/adaptive immune response in vivo.
One of the important features of AAV vectors is the lack of inflammation following infection. We failed to detect significant IFNγ or IL-12 production in the supernatants of AAV-siDEN-infected DCs. This is in accordance with previous data [26–28], which demonstrated our AAV delivery system did not induce significant acute inflammatory responses and, therefore, is useful in gene therapy for DEN infection in humans.
In conclusion, we developed a novel AAV-mediated siRNA delivery system. Our results demonstrate significant downregulation of DEN protein expression in Vero cells and human DCs, which strongly suggest that our AAV vector can be useful for siRNA delivery and that this AAV system may be applied in clinical settings to attenuate DEN infection.