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 and Tuberculosis (MISRA et al., 2010; 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 main pulmonary diseases are caused by viruses. 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).
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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 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 pulmonary 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 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 success of gene silencing in the lungs by siRNA depends on the proper release in the sites of action, be stable, cellular uptake and be present in satisfactory concentrations in the cytoplasm (DURCAN et al., 2008). Direct chemical modification of siRNA have been studied to improve the aforementioned features, though this approach have offer minimal enhancement on overall delivery efficiency (GHOSN et al., 2010).
The main limitation for efficient cellular uptake of naked siRNA is the anatomy and morphology of the lung epithelium (GUTBIER et al., 2010). So, many studies have been developed 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). The viral vectors have been demonstrated high efficient to carrier nucleic acid, but worries with inherent safety like cytotoxicity, oncogenicity and immunogenicity have bounded the use (GHOSN et al., 2010).
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MASSARO et al., (2004) in their study used lipid-based and pulmonary surface active material (InfaSurf) to delivery siRNA intranasally, demonstrating an efficient silence GADPH protein expression within the lungs. Bikto et al. (2005) used a transfection reagent (transIT TKO), a cationic polymer/lipid combination, in their study with 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 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 (BITKO et al., 2005). The used of lipid-based delivery system have provided efficient delivery for siRNA, although there are concern about systemic cytotoxicity (GHOSN et al., 2010).
Delivery system to siRNA based on polymers have won attention owing the biocompatibility, modifiability, and ease of production. An especial interest has been on cationic polymers that decrease serum degradation and improve the cellular uptake (GHOSN et al., 2010).
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 capacity of this chitosan nanoparticles be uptake by the cells can be due to the diminutive particle size and excess positive charge that help interaction with cellular membranes. The characteristic of mucoadhesive and permeation of chitosan was conceived as a way to improve the delivery of siRNA at respiratory tract, could be an attractive approach to treatment of diseases in the lung. 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 (HOWARD et al., 2006). To improve pulmonary siRNA delivery and gene silencing at the mucosa surface the next step of this study it was develop an aerosol forms for the chitosan/siRNA system (NIELSEN et al., 2010).
The aerosol of chitosan/siRNA nanoparticles were pneumatically created using a nebulising catheter and sized by laser diffraction. This process did not considerably change the suitable size distribution of the non-aerolised chitosan/siRNA particles, and the particle size is an important property to determinate the cellular uptake. The in vivo study was done through intratracheal and intranasal administration, which permit higher doses compared to intratracheal method, but is limited because of the variable dosage achieving the lungs as a consequence of gastrointestinal ingestion or adhesion of nanoparticles in the nasal cavity (NIELSEN et al., 2010).
Ghosn et al. (2010) evaluated the use of imidazole-modified chitosan (chitosan-IAA) as delivery system for siRNA to lung and liver through the i.n. and i.v. route. The results show that chitosan-IAA is very efficient in delivery siRNA in vivo enhanced gene silencing. The improvement of the transfection efficacy was fundamentally credited to the introduction of secondary and tertiary amines to the polymer backbone as well as better solubility at physiological pH (GHOSN et al., 2010).
There are many studies showing the potential applications of siRNA local delivery to the lung, encouraging following with the studies to find treatments to main lung diseases like SARS, RVS and influenza.
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.
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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). A very interesting approch for the prevention and treatment of diseases caused by RSV is the use of siRNA therapy administered intranasally, Table X shows how these studies has evolved and how its results are promising.
NG042-siNS1 (siRNA complexed with a nanochitosan polymer (NG042) targeting the NS1 gene)
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.
(ZHANG et al., 2005)
ALN-RSV01 (siRNA targeting the mRNA of the RSV nucleocapsid (N) protein)
The 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)
The evaluated of the prophylactic and therapeutic effect of ALN-RVS01 show that it have 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.
(ALVAREZ et al., 2009)
(Adults experimentally infected with wild-type RSV)
This research demonstrated that ALN-RSV01 is effective against the virus when the RSV is inoculated. The search in this study 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
(DEVINCENZO et al., 2010)
Human (lung transplant patients infected naturally with RSV)
The study concluded that ALN-RSV01 was safe and well tolerated and has a rate of new or progressive bronchiolitis obliterans syndrome (BOS) considerably lower than placebo group
(ZAMORA et al., 2010)
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).
Tuberculosis is another disease that the treatment using formulations drugs for topical delivery in the lung is very significant. The most recent candidate among those that may be used in inhaled TB therapies is siRNA targeting the host chemokine XCL-1 or lymphotactin. The results show that 10 mg of a naked, 19-base RNA sequence delivered to the lungs of mice sustained to exhibit up to 50% pull-down of the target RNA up to 3 days after administration (MISRA et al., 2010)
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).