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Lung owing to your location and physiological function is in contact with many pollutants and viruses that become it susceptible to many diseases such as asthma, cancer, influenza, SARS (THOMAS et al., 2007). Diseases lung have high lethality and prevalence, hence many research to find the effective treatment or a vaccine to these disease is being studied (BITKO et al., 2005; THOMAS et al., 2007).
The viruses have some features that become difficult the prevention and therapy. To solve the problems faced with traditional anti-viral therapies, researches are being done using siRNA (BITKO et al., 2005). The siRNA has some advantages compared to other gene/antisense therapies, they are 10-100-fold more potent for gene silencing, specific inhibition and low risk of toxic effects (DURCAN et al., 2008; OZPOLAT et al., 2010).
The therapy with RNAi have several advantages to be used in the treatment of diseases caused by respiratory virus: (1) there are excellent knowledge about the sequence of these virus, so is easy to find potential target sequence, (2) the studies with virus have taught which are the fundamental viral genes for replication, (3) the viral genes are exogenous to the human genome, than can decrease chances of side effects, (4) the development of RNAi therapeutics is potentially more fast than traditional small molecule approaches, which is important in respiratory viruses that can cause sudden epidemics, (5) it is possible select siRNA against sequences that is more conserved in the different variants of virus unlike the traditional molecule antivirals that are mainly designed to inhibit surface proteins, that are often most genetically variable, (6) the delivery of siRNA is facilitated because the infections are topical, the respiratory syncytial virus (RSV) essentially infects ciliated respiratory epithelial cells (DEVINCENZO, 2008).
The therapeutics agents can be administrated by multiples routes to treat lung disease: intravenous (i.v.), intranasal (i.n.), intratracheal (i.t.), subcutaneous, intratumor, intramuscular or oral. These routes of administration determine which are extracellular barriers to siRNA delivery to the lung (THOMAS et al., 2007).
The systemic deliveries of siRNA have some important obstacles comprising rapid degradation by nucleases, systemic toxicity, rapid excretion and inefficient target to the illness organ or cell type (DURCAN et al., 2008).
The delivery by intranasal and intratracheal route is interesting and practicable route for the specific delivery of siRNA to lung (THOMAS et al., 2007). The advantages of the intranasal delivery are a noninvasive method of delivery, lower systemic toxicity and immediate availability, that can be explain due the lungs be highly vascularized composed by capillary beds that permit the rapid uptake cell (DURCAN et al., 2008). The airway administration so is favorable because the degradation of siRNA by nucleases is not significant in the airway (THOMAS et al., 2007). Nevertheless, the delivery through airway is not easy, after pulmonary administration, the gene delivery encounter physical barriers such as cilia beating and mucociliary clearance and others important barriers like airway surface liquid (ASL) that cover the airway epithelial cells and the cell membrane surface is negatively charged (GUTBIER et al., 2010; ROSENECKER et al., 2003; THOMAS et al., 2007). The barriers to nucleic acid transfer in airway ephitelial cells can also affect the efficiency of the cell uptake in vivo (GRIESENBACH et al., 2006). Griensenbach et al. (2006) in your study about new strategies for the treatment of cystic fibrosis tested siRNA complexed with the cationic lipid Genzyme lipid (GL) 67 and concluded that although siRNA can be inhibit gene expression in culture systems and determined organs in vivo, the barriers to delivery to the lung can damage the efficiency of uptake in vivo.
The main limitation for efficient cellular uptake of naked siRNA is the anatomy and morphology of the lung epithelium, so many studies have been developed delivery system to siRNA (GUTBIER et al., 2010). There are researches to develop viral and non-viral delivery systems to outgrow extracellular and intracellular barriers that limit therapeutic use of nucleic acid-based drugs (HOWARD et al., 2006). Howard et.al. (2006) developed a new delivery system of chitosan/siRNA nanoparticle based on the use of an active siRNA agent to form nanoparticles through electrostatic bridges between chitosan polymeric chains. The studies in vitro and in vivo, using transgenic EGFP mouse model, showed that this original system can be used for both in vitro and in vivo RNA interference protocols and can in the future to be used in the treatment of systemic and mucosal disease.
The success of gene silencing in the lungs by siRNA depends on the proper release in the sites of action, be stable, uptake celular and be present in satisfactory concentrations in the cytoplasm (DURCAN et al., 2008).
There are many studies showing the potential applications of siRNA local delivery to the lung, and the experiments with animals encouraging following with the studies to find treatments of lung disease.
Severe acute respiratory syndrome (SARS) is a disease that has the SARS coronavirus (SCV) as the causative pathogen, those infected with SCV generally develop high fever followed by severe clinical symptoms including acute respiratory distress syndrome with a diffuse alveolar damage (DAD) at autopsy. SARS is a new disease so, safe and effective vaccine is not exist yet, though the searches for its development have been advanced (LI et al., 2005; THOMAS et al., 2007).
Li et al. (2005) using like animal model Rhesus macaque, administrated the siSC2-5 (a mixture of two SCV-specific siRNA duplexes, siSC2 and siSC5), a potent inhibitor of SCV RNA in solution D5W (5% D-glucose in water, wt/vol, made in-house) that showed to be very effective carrier through intranasal instillation to mimicking the natural route of SCV infection of humans with SARS. The siSC2-5 was administrated with prophylactic, concurrent or early postexposure treatments within a period of 5 d.p.i., and all the treatment regimens showed potent suppression of SCV. This study support that the intranasal administration is preferable under proinflammatory cytokine treatments, because allow a high-specificity inhibition of SCV with minimal induction of a proinflammatory cytokine antiviral response that can assist prevent the exacerbation of symptoms and lung injury.
Respiratory syncytial virus (RSV), is a member of the Pneumovirinae subfamily in the Pneumovirus genus, there are two major groups of human RSV, serotypes A and B (ALVAREZ et al., 2009). It is an enveloped, non-segmented, negative-stranded RNA virus, is the pathogen responsible for the most serious respiratory infections like bronchiolitis and pneumonia in infants and young children (THOMAS et al., 2007; ZHANG et al., 2005). RSV infected basically all the children with less two years old and in the elderly is an important cause of morbidity and mortality (BITKO et al., 2005). There is no vaccine to prevent RSV actually (BITKO et al., 2005; ZHANG et al., 2005) and the only accepted therapy (ribavirin) is seldom used due to its potential teratogenicity, limited antiviral effect, and controversial clinical effectiveness (DEVINCENZO et al., 2010).
Bikto et al. (2005) studied siRNA against P protein, an essential subunit of the viral RNA-dependent RNA polymerase. The siRNA was efficient in vitro and in vivo. They used BALB/c mouse like a well-established animal model for RSV infection, and siRNA was administered with and without transfection reagent (transITTKO) by intranasal route. At a dose of 5 nmol intranasal siRNA per mouse, siRNA reduced pulmonary RSV titers by about 99.98%. The siRNA without transfection reagent also reduced pulmonary viral titers, this results is interesting because intranasal siRNA without any carrier reduce the possible side effect that this transfection reagent can cause. The finding results showed that siRNA administrated accordingly can be useful to prevent and to treat the respiratory infection.
Zhang et.al. (2005) also studied siRNA against RSV, but their target were NS. NS (NS1 and NS2) is a protein and has an important role in viral replication and blocking them can mitigate the RSV replication. To prove this, Zhang and co-workers check the potency of siNS1 to inhibit RSV replication in vitro and in vivo. The in vitro test was performed using cultured human epithelial cells. To examine whether siNS1 also is effective in vivo it was used BALB/c mice, the siNS1 was complexed with a nanochitosan polymer, referred to as Nanogene 042 (NG042). The nanoparticles were administrated as a nasal drop 2 d before viral inoculation. Also they tested the therapeutic potential of NG042-siNS1, for this they administered the NG042-siNS1 complex to mice at day 0 along with RSV inoculation or at day 2 or 3 after infection. The application of NG042-siNS1 either before or after RSV infection significantly attenuates RSV infection and showed a considerable diminish in lung inflammation, goblet cell hyperplasia and infiltration of inflammatory cells compared to control mice. These studies showed that siNS1 nanoparticles can be used both prophylactically as a therapeutic agent in infections with RSV in humans.
Another types of siRNA against RSV were studied by Alvarez et.al. (2009). In their studies they tested 70 siRNA against RSV N, P e L genes, they performed this study in an in vitro RSV plaque inhibition assay. Of these 70 siRNA, 19 inhibited plaque formations in more than 80% compared with a PBS control. And of these 19, the siRNA that show the biggest antiviral activity was ALN-RSV01 (ALVAREZ et al., 2009). This siRNA target the mRNA of the RSV nucleocapsid (N) protein, this protein has role at many critical steps in the viral replication cycle including RNA polymerase function (DEVINCENZO et al., 2010). The N-protein gene is among the most conserved across the various circulating RSV isolates, this characteristic is so important because allow a broad-spectrum activity of siRNA (ALVAREZ et al., 2009). By intranasal administration to healthy volunteers at doses up to 150 mg daily for 5 days was well tolerated, establishing that ALN-RSV01 is safe (DEVINCENZO et al., 2008). ALN-RSV01 is stable when administrated locally to the lung and unstable in systemic circulation to reduce any potential systemic exposure, and this feature was purposely designed in the molecule (ALVAREZ et al., 2009).
Alvarez et.al. (2009) studied ALN-RSV01 in vivo using BALB/c mouse. They evaluated the prophylactic and therapeutic effect and found that ALN-RVS01 is potent antiviral in both effects, reaching to 3-log-unit viral load reductions compared to the reductions reached with either PBS or nonspecific siRNA controls. DeVicenzo et al. (2010) tested the antiviral activity of ALN-RSV01 in a randomized, double-blind, placebo controlled trial in adults (88 subjects) experimentally infected with wild-type RSV. The experiment was conducted by the administration of a nasal spray of ALN-RSV01 or saline placebo daily for 2 days before and for 3 days after RSV inoculation. The authors discuss that this research demonstrated that ALN-RSV01 is effective against the virus when the RSV is inoculated. The search does not found that ALN-RSV01 will be efficacious in naturally infected patients with established lower respiratory tract disease. So, it is necessary future trials in naturally infected patients.
One of the most predominant infections in human is influenza virus infection. It is caused by an enveloped virus of the Orthomyxovirus family (THOMAS et al., 2007). This infection that resulting in up 40,000 deaths per year in the United State, is worrying because there is the possibility of a new influenza pandemic, because the virulence of influenza A virus is high due to its easy transmission, antigenic shift and drift of the virus, the existing vaccines are not as effective and the use of four drugs approved for treatment are limited because of its side effects and the possibility of resistant virus emerging (GE et al., 2003). So, the development of an efficient therapy or vaccine is necessary. An study tested 20 siRNA against influenza A virus, and found that specific siRNA can stop influenza virus production in both cell lines and embryonated chicken eggs (GE et al., 2003). The in vivo studies also show that the lung virus titers in influenza-infected mice can be reducing by the intranasal delivery of plasmids expressing influenza-specific siRNAs (GE et al., 2004).
Another lung disease that your treatment is being studied through the application of siRNA is Pulmonary Fibrosis. In many acute and chronic pulmonary diseases is commom to happen the fibrin accumulation. It was observed that the expression level of plasminogen activator inhibitor-1 (PAI-1) is directly correlated with the extent of collagen accumulated that follows lung injury, so PAI-1 is the main molecule related to the development of pulmonary fibrosys (SENOO et al., 2010). Senoo et al. (2010), studied the effect of the administration of siRNA against PAI-1 (PAI-1-siRNA) through intranasal route in murine model of bleomycin-induced pulmonary fibrosys. They chosen this route because previous studies showed to be easy and safe route. The finding results demonstrate that the direct administration of PAI-1-siRNA, in the absence of transfection agents, could reduce the PAI-1 level in BAL fluid from mice with bleomycin-induced lung injury. Moreover, the PAI-1-siRNA when administrated repeatedly, at the beginning of the fibrotic phase of bleomycin-induced lung injury, was also effective in limiting the accumulation of collagen in the lungs. So, this strategy seems prevent the development of pulmonary fibrosis and enhance the survival rate in mice with bleomycin-induced lung injury and is an interesting therapeutical approach because it will avoid systemic side effects that may be caused by oral administration of a PAI-1 inhibitor.
Therapy based on siRNA also is being used to treat disease in other animals, is being tested siRNA delivered intranasally against an equine pathogen (Equine herpesvirus type 1 - EHV-1). This virus is spread via nasal secretions and causes respiratory disease, neurological disorders and abortions. The in vitro and in vivo studies demonstrated that the treatment with siRNA is effective as a prophylactic and early treatment of EHV-1 infections (FULTON et al., 2009).