Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-alpha/beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.     

RNA interference-mediated inhibition of Hepatitis B Virus replication

Persistent and recurrent infection of hepatitis B virus (HBV) represents one of the most common and severe viral infections of humans, and has caused a formidable health problem in the affected countries. Currently used antiviral drugs have a very limited success on controlling HBV replication and infection. RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of target mRNA in mammalian and plant cells, has recently been used to knockdown gene expression in various species. In this study, we sought to determine whether RNAi-mediated silencing of HBV viral gene expression could lead to the effective inhibition of HBV replication. We first developed RNAi vectors that expressed small interfering RNA (siRNA) and targeted the HBV core or surface gene sequence. Our results demonstrated that these specific siRNAs efficiently reduced the levels of corresponding viral RNAs and proteins, and thus suppressed viral replication. Treatment with siRNA gave the greatest reduction in the levels of HBsAg (92%) and in HBeAg (85%) respectively in the cultured cell medium. Our findings further demonstrated that the RNAi-mediated antiviral effect was sequence-specific and dose-dependent. Therefore, our findings strongly suggest that RNAi-mediated silencing of HBV viral genes could effectively inhibit the replication of HBV, hence RNAi-based strategy should be further explored as a more efficacious antiviral therapy of HBV infection.     

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.     

Generation of a recombinant herpes simplex virus which can provide packaging function for recombinant adeno-associated

The generation of a recombinant HSV (rHSV) that can provide packaging function for rAAV production is described. A set of cosmids including cos48, cos28, cos6, cos14 and cos56, which represents the HSV-1 genome was used for generation of this rHSV. Rep and cap genes of AAV-2 were inserted into Xba Ⅰ site of UL2 gene on cos6, generating cos6-rcΔUL2. After being digested with Pac Ⅰ , cos6-rcΔUL2 and the other 4 cosmids were cotrans-fected into BHK-21 cells. The recombinant virus HSV1-rc/ΔUL2 carrying rep and cap genes was generated due to the homologous recombination of the 5 cosmids. The results showed that the existence of rep and cap genes on this rHSV was stable from passage to passage and the rHSV could support the packaging of rAAV either in cells transiently transfected with AAV vector or in stable cell line harboring AAV vector. Further modification of this rHSV and optimization of conditions involved in rAAV preparation may lead to a large-scale production of rAAV in the near future.     

Natural killer cells act as rheostats modulating antiviral T cells

Antiviral T cells are thought to regulate whether hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections result in viral control, asymptomatic persistence or severe disease, although the reasons for these different outcomes remain unclear. Recent genetic evidence, however, has indicated a correlation between certain natural killer (NK)-cell receptors and progression of both HIV and HCV infection, implying that NK cells have a role in these T-cell-associated diseases. Although direct NK-cell-mediated lysis of virus-infected cells may contribute to antiviral defence during some virus infections--especially murine cytomegalovirus (MCMV) infections in mice and perhaps HIV in humans--NK cells have also been suspected of having immunoregulatory functions. For instance, NK cells may indirectly regulate T-cell responses by lysing MCMV-infected antigen-presenting cells. In contrast to MCMV, lymphocytic choriomeningitis virus (LCMV) infection in mice seems to be resistant to any direct antiviral effects of NK cells. Here we examine the roles of NK cells in regulating T-cell-dependent viral persistence and immunopathology in mice infected with LCMV, an established model for HIV and HCV infections in humans. We describe a three-way interaction, whereby activated NK cells cytolytically eliminate activated CD4 T cells that affect CD8 T-cell function and exhaustion. At high virus doses, NK cells prevented fatal pathology while enabling T-cell exhaustion and viral persistence, but at medium doses NK cells paradoxically facilitated lethal T-cell-mediated pathology. Thus, NK cells can act as rheostats, regulating CD4 T-cell-mediated support for the antiviral CD8 T cells that control viral pathogenesis and persistence.     

Mediation of virion penetration into vascular cells by association of basic fibroblast growth factor with herpes simplex virus type 1

Herpes simplex virus type-1 (HSV-1) is a ubiquitous pathogen that is associated with considerable morbidity in the general population. Although it is known that the virion uses a basic fibroblast growth factor (FGF) receptor to penetrate vascular cells, it is not known how the viral particle recognizes and binds to this cell surface protein. Here we report that an immunoreactive basic FGF-like protein is associated with the viral particle and that this association appears responsible for viral uptake. Accordingly, HSV-1 infection of Swiss 3T3 cells stimulates the tyrosine phosphorylation of the specific substrate that characterizes the initial cellular response to basic FGF. Antibodies to basic FGF prevent this phosphorylation and inhibit HSV-1 uptake. Because no basic FGF sequence is found in the HSV-1 genome, a model for the infection for some target cells is presented whereby the viral particle uses host cell-derived basic FGF to ensure subsequent infectivity of newly replicated virus.     

Generation of a recombinant herpes simplex virus which can provide packaging function for recombinant adeno-associated virus

The generation of a recombinant HSV (rHSV) that can provide packaging function for rAAV production is described. A set of cosmids including cos48, cos28, cos6, cosl4 and cos56, which represents the HSV-1 genome was used for generation of this rHSV.Rep andcap genes of AAV-2 were inserted intoXba I site ofUL2 gene on cod, generating cos6rcΔUL2. After being digested withPac I, cos6-rcΔUL2 and the other 4 cosmids were cotransfected into BHK-21 cells. The recombinant virus HSV1-rc/ΔUL2 carryingrep andcap genes was generated due to the homologous recombination of the 5 cosmids. The results showed that the existence ofrep andcap genes on this rHSV was stable from passage to passage and the rHSV could support the packaging of rAAV either in cells transiently transfected with AAV vector or in stable cell line harboring AAV vector. Further modification of this rHSV and optimization of conditions involved in rAAV preparation may lead to a large-scale production of rAAV in the near future.     

A resource for large-scale RNA-interference-based screens in mammals

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.     

Delayed resistance to Cucumber mosaic virus mediated by 3’UTR-derived hairpin RNA

RNA silencing has been shown to function in the plant antivirus defense response, leading to viral RNA degradation induced by vsiRNA-containing RISC cleavage activity. Cucumber mosaic virus （CMV） 3′UTR sequences share a high conservation of nucleotide sequence and secondary structures that are important for CMV replication. Here, in an attempt to simultaneously target the multiple genomic and subgenomic RNAs of CMV for degradation, CMV 3′UTR were used to design hairpin RNA （hpRNA） to transform tobacco （Xanthi. nc） so as to constitutively produce viral siRNAs. Most of the transgenic plants expressing CMV Q strain （Q-CMV, subgroup Ⅱ strain） RNA3 3′UTR-derived hpRNA showed delayed resistance to Q-CMV infection and exhibited recovery phenotypes. Compared with Q-CMV-inoculated leaves, the upper leaves showed weak or no disease symptoms and a reduced accumulation level of viral RNAs. Together with transient assays, our results indicate that the 3′UTR-derived siRNAs were biologically active in targeting viral RNA for degradation. Recovery resistance in transgenic plants was also observed against subgroup IB strain SD-CMV infection, indicating a broad-spectrum anti-CMV effect of the 3′UTR-based antiviral silencing. Northern blot assays indicated that there was no strong correlation between the degree of resistance and the accumulation level of 3′UTR-derived siRNAs, suggesting that to target a highly structured RNA, such as the CMV 3′UTR, the quantity of siRNAs may not be the only determinant of silencing efficiency. Target RNA secondary structures may also affect target accessibility, siRNA-containing RISC-target recognition and the consequent antiviral effect.     

Short interfering RNA confers intracellular antiviral immunity in human cells

Gene silencing mediated by double-stranded RNA (dsRNA) is a sequence-specific, highly conserved mechanism in eukaryotes. In plants, it serves as an antiviral defence mechanism. Animal cells also possess this machinery but its specific function is unclear. Here we demonstrate that dsRNA can effectively protect human cells against infection by a rapidly replicating and highly cytolytic RNA virus. Pre-treatment of human and mouse cells with double-stranded, short interfering RNAs (siRNAs) to the poliovirus genome markedly reduces the titre of virus progeny and promotes clearance of the virus from most of the infected cells. The antiviral effect is sequence-specific and is not attributable to either classical antisense mechanisms or to interferon and the interferon response effectors protein kinase R (PKR) and RNaseL. Protection is the result of direct targeting of the viral genome by siRNA, as sequence analysis of escape virus (resistant to siRNAs) reveals one nucleotide substitution in the middle of the targeted sequence. Thus, siRNAs elicit specific intracellular antiviral resistance that may provide a therapeutic strategy against human viruses.     

Inhibition of pathogenic effect of effector T cells by specific suppressor T cells during influenza virus infection in mice

Mice infected with an aerosol of influenza type A virus, or immunized with purified UV-inactivated whole virus or with viral subunits, develop a transient delayed-type hypersensitivity (DTH) which peaks 5-7 days after immunization. The intensity of DTH is greatly enhanced and sustained when mice are pretreated with cyclophosphamide. The reaction is maximal 24 h after elicitation, has classical tuberculin-type histology and is transferable by immune H-21 region restricted Lyt-1+2- T cells (Td) but not by immune serum. These Td cells not only fail to protect mice against influenza virus infection, but increase the mortality rate due to influenzal pneumonia following challenge with homologous lethal virus. On the other hand, antigen-specific suppressor T (Ts) cells which inhibit DTH are readily generated during influenza virus infection, and are detectable for at least 40 days thereafter. The ease with which they are induced and maintained during the infection may be of evolutionary advantage. In support of this, we now report that these Ts cells can reverse the pathogenic effect of Td cells thereby demonstrating a beneficial influence of Ts cells in a viral disease.     

RNAi-mediated gene silencing in non-human primates

The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (ApoB) in non-human primates. APOB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg(-1). A single siRNA injection resulted in dose-dependent silencing of APOB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of APOB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.     

Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion

Human immunodeficiency virus type 1 (HIV-1) continues to spread, principally by heterosexual sex, but no vaccine is available. Hence, alternative prevention methods are needed to supplement educational and behavioural-modification programmes. One such approach is a vaginal microbicide: the application of inhibitory compounds before intercourse. Here, we have evaluated the microbicide concept using the rhesus macaque 'high dose' vaginal transmission model with a CCR5-receptor-using simian-human immunodeficiency virus (SHIV-162P3) and three compounds that inhibit different stages of the virus-cell attachment and entry process. These compounds are BMS-378806, a small molecule that binds the viral gp120 glycoprotein and prevents its attachment to the CD4 and CCR5 receptors, CMPD167, a small molecule that binds to CCR5 to inhibit gp120 association, and C52L, a bacterially expressed peptide inhibitor of gp41-mediated fusion. In vitro, all three compounds inhibit infection of T cells and cervical tissue explants, and C52L acts synergistically with CMPD167 or BMS-378806 to inhibit infection of cell lines. In vivo, significant protection was achieved using each compound alone and in combinations. CMPD167 and BMS-378806 were protective even when applied 6 h before challenge.     

Glycoprotein C of herpes simplex virus 1 acts as a receptor for the C3b complement component on infected cells

Receptors for the Fc portion of immunoglobulins or for the third component of complement (C3) are present on a variety of circulating and fixed tissue cells including granulocytes, monocytes, lymphocytes and glomerular epithelial cells. Cells which lack Fc receptors may express them after infection by herpes simplex virus (HSV)-1, HSV-2, cytomegalovirus or varicella zoster virus. We recently reported that infection by HSV-1 induces both Fc and C3 receptors on human endothelial cells. Glycoprotein E of HSV-1 has been shown to function as an Fc receptor. We now demonstrate that glycoprotein C (gC) of HSV-1 functions as a C3b receptor. This receptor appears following HSV-1, but not HSV-2, infection. Detection of the C3b receptor is blocked by monoclonal antibodies to glycoprotein C (gC) of HSV-1, but not by monoclonal antibodies to other HSV-1 glycoproteins. In addition, the MP mutant of HSV-1, which lacks gC, fails to express a C3b receptor. These results assign a new function of gC of HSV-1 and demonstrate potentially important differences between HSV-1 and HSV-2 glycoproteins.     

An immunologically active chimaeric protein containing herpes simplex virus type 1 glycoprotein D

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) cause both persistent and latent infections, including recurrent cutaneous disease, lethal neonatal disease, central nervous system disease and other clinical syndromes. Modified live vaccines or conventionally prepared subunit vaccines have generally been unsuccessful in the treatment of HSV-1 and HSV-2 infections from the standpoints of safety and efficacy. It has been established that HSV-1 and HSV-2 infectivity may be neutralized in vitro with antisera directed specifically against each of the four major glycoproteins of the virus (gA/gB, gC, gD and gE) and antisera against glycoprotein gD, of either HSV-1 or HSV-2, are capable of neutralizing both HSV-1 and HSV-2 infectivity in vitro and in vivo. We have previously reported on the identification, DNA sequence and expression at low level in Escherichia coli of the gD gene of HSV-1 strain Patton. Here we describe construction of a hybrid gene encoding a chimaeric protein containing HSV-1 gD, bacteriophage lambda Cro and E. coli beta-galactosidase (gD-beta-gal) protein, which is expressed at high level in E. coli. Moreover, the chimaeric protein elicits antibodies in rabbits that not only immunoprecipitate gD from cells infected with HSV-1 and HSV-2 but also neutralize HSV-1 and HSV-2 infectivity in vitro.