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One of the most noninvasive approaches to drug delivery is via inhalation. The delivery of genes via aerosol holds promise for the treatment of a broad spectrum of pulmonary disorders and offers numerous advantages over more invasive modes of delivery. Delivery of genes expressing secretory therapeutic proteins or peptides may even have application to a number of nonpulmonary diseases. After the cloning of the cystic fibrosis gene, there was great interest in the delivery of genes directly to the lung surfaces via inhalation and most early efforts focused on the use of nonviral vectors, particularly cationic lipids. Early on, nebulization shear forces, inefficient penetration of mucous barriers and inhibitory effects of surfactant and other lung specific features generally resulted in a lack of therapeutic effect. But in recent years, a number of other nonviral and even viral vectors have been delivered successfully in this manner. Polyethyleneimine (PEI)-based formulations have proven stable during nebulization and result in transfection of a very large proportion of epithelial cells throughout the airways (though the level of transgene expression per cell may be relatively low), as well as significant, though lower levels of transfection throughout the lung parenchyma. Most importantly, therapeutic responses have been obtained in several animal lung tumor models when PEI-based complexes of p53 and IL-12 genes were delivered by aerosol. This approach may also prove useful as a means of localized genetic immunization. In addition, inhalation delivery of some formulations seems to be associated with surprisingly low toxicity and has resulted in little or no immunostimulatory response to the unmethylated CpG sequences in bacterially-produced plasmid DNA, which has presented a challenge to repeated gene therapy via many other modes of delivery.

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BACKGROUND: Conventional vaccine development for newly emerging pandemic influenza virus strains would likely take too long to prevent devastating global morbidity and mortality. If DNA vaccines can be distributed and delivered efficiently, genetic immunization could be an attractive solution to this problem, since plasmid DNA is stable, easily engineered to encode new protein antigens, and able to be quickly produced in large quantities. METHODS: We compared two novel genetic immunization methods in a mouse model of influenza to evaluate protective effects: aerosol delivery of polyethylenimine (PEI)-complexed hemagglutinin (HA)-expressing plasmid and intravenous (IV) delivery of the plasmid complexed with macroaggregated albumin/PEI. Serial serum samples were obtained for assay of neutralizing antibodies against HA. Mice were then challenged in the airway with influenza virus, and production of infectious virus in the lungs was titered. RESULTS: Most mice immunized with HA plasmid alone by aerosol and all mice immunized IV developed protective immune responses, whereas none administered control plasmid were protected. Aerosol co-administration of HA plasmid with plasmids encoding the cytokines interleukin 12 (IL12) and granulocyte-macrophage colony stimulating factor (GM-CSF) markedly increased neutralizing antibody responses, so that all aerosol immunized mice were protected from high level virus proliferation. CONCLUSIONS: Cytokine-enhanced aerosol delivery of plasmid vaccines can elicit robust protective immune responses against influenza. Thus, aerosol delivery has the potential to address the need for rapid widespread immunization against new influenza virus strains, and may have applications for other infectious and toxic disease processes.

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The lung represents an important target for gene therapy: for correction of genetic abnormalities such as cystic fibrosis, for lung cancer therapy, and for vaccination. Genes in the form of expression plasmids can be delivered both by the intravenous route and via the airways. So-called "naked" DNA can be delivered by both of these methods, but gene expression is low. Successful delivery is usually accomplished by complexing the DNA with cationic lipids or with polycations. This review will discuss the efficacy of delivery for particular purposes by various methods and complexing agents, as well as issues of biodistribution, inflammatory reactions, and improvements in formulations. Non-viral gene delivery to the lung has a long history of development, and it is now poised to represent a significant addition to the medical arsenal.

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The purpose of this study was to characterize performance of a miniaturized AERx((R)) Pulmonary Delivery System designed for aerosol administration to large animal models. The miniaturized AERx System was developed through a systematic scaling down of the AERx System used for humans to allow for operation in certain animal models with lower inspiratory flow rates and inhaled volumes than those used for humans. We used gamma scintigraphy to characterize the in vivo particle deposition achieved with the miniaturized AERx System in two dogs. The dogs were 3-4 years old, and weighed 10.4 kg and 13.6 kg. Acepromazine was used as pre-anesthetic medication. Anesthesia was induced with 5% isoflurane. The trachea was intubated using an endotracheal tube (internal diameter 8.5 mm), and the dogs were ventilated using positive pressure during the exposure using the LRRI puff generator. An inhalation of aerosol was initiated by activation of the puff generator though the computer-controlled interface. Each dog inhaled approximately 0.8 L per puff, of which the aerosol volume comprised approximately 0.25 L, at a target flow rate of 15 L/min. The dogs were exposed to 10 AERx Strips in 10 puffs. The mass median aerodynamic diameter of the aerosolized formulation was approximately 1.25 microm with a fine particle fraction <3.5 microm of 0.976. The scintigraphic images showed uniform bilateral lung deposition following aerosol delivery with the AERx System. Total lung deposition for the two dogs was 10.7% and 18% of the loaded dose from the AERx Strip. The corresponding peripheral lung: inner lung (P/I) ratios were 0.83 and 0.75, suggestive of deposition in the deep lung. Only 0.1% to 0.2% of the loaded dose was exhaled. These results show the miniature AERx System can efficiently deliver aerosols to the deep lung of dogs. The miniaturized AERx System would be a valuable tool for conducting proof-of-concept studies as well as safety and tolerability analysis of inhaled drug candidates in large animal models.

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The delivery of genes by inhalation holds promise for the treatment of a wide range of pulmonary and non-pulmonary disorders and offers numerous advantages over more invasive modes of delivery. Subsequent to the cloning of the cystic fibrosis gene, there was great interest in the delivery of genes directly to the lung surfaces by aerosol, and most early efforts focused on the use of non-viral vectors, particularly cationic lipids. Unfortunately, nebulisation shear forces, inefficient penetration of mucous barriers and inhibitory effects of surfactant and other lung-specific features have generally resulted in a lack of therapeutic effect, and much of this work has diminished in recent years as a consequence. Polyethyleneimine (PEI)-based formulations have proven stable during nebulisation and result in nearly 100% efficient transfection throughout the airways, as well as significant, although lower, levels of transfection throughout the lung parenchyma. Most importantly, therapeutic responses have been obtained in several animal lung tumour models when PEI-based complexes of p53 and IL-12 genes were delivered by aerosol. This approach may also prove useful as a means of localised genetic immunisation. In addition, this mode of delivery seems to be associated with surprisingly low toxicity, and results in little or no CpG immunostimulatory response, which has presented a challenge to repeated gene therapy via other modes of delivery.

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PURPOSE: We determined whether polyethylenimine (PEI), a polycationic DNA carrier, can be used to deliver the interleukin (IL) 12 gene by aerosol to treat established osteosarcoma (OS) lung metastases in a nude mouse model. EXPERIMENTAL DESIGN: Tumor response was assessed using our OS lung metastases model. Treatment with aerosolized PEI containing the murine IL-12 gene (PEI:IL-12; 600 microl PEI and 2 mg IL-12) was given twice weekly for 5-6 weeks. RESULTS: Aerosol therapy for 2 weeks resulted in high expression of both the p35 and p40 subunits of IL-12 in the lungs but not in the livers of mice. Peak IL-12 mRNA expression was seen 24 h after a single aerosol PEI:IL-12 treatment. This expression gradually decreased with continued detection for up to 7 days. IL-12 protein was not detectable in plasma even after 6 weeks of aerosol therapy. The number of lung metastases in mice treated with aerosol PEI:IL-12 was decreased significantly (median, 0; range, 0-33) compared with mice that received PEI alone (median, 37.5; range, 11-125; P = 0.002). Nodule size was also significantly smaller in the aerosol PEI:IL-12 group with 87% of the nodules measuring 1 mm. Weekly aerosol PEI:IL-12 therapy was as effective as twice weekly therapy. CONCLUSIONS: Aerosol therapy resulted in selective gene expression and protein production in the tumor area. Aerosol PEI:IL-12 may avoid the systemic toxicities described previously in patients treated with i.v. IL-12. Because OS metastasizes almost exclusively to the lung, aerosol PEI:IL-12 therapy may provide a therapeutic option, which may be especially valuable.

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This report describes the time-dependent biodistribution of human p53 plasmid delivered in aerosol with polyethyleneimine in mice compared to the distribution of this material following intravenous injection. Area-under-the-curve values for p53 plasmid after inhalation were 2.8-fold greater than values after intravenous administration, despite the fact that the delivered aerosol dose was one-fifth the intravenous dose. After aerosol administration, pulmonary concentrations of p53 plasmid were high and other organs showed amounts not distinguishable from untreated control. High concentrations of p53 plasmid in the lungs remained with negligible reduction for at least 24 h. Shortly after intravenous injection, organs exhibited the following relative levels of exogenously administered p53: liver > spleen > blood > or = lungs > heart > kidney. These results demonstrate effective pulmonary delivery of DNA in complex with PEI by aerosol, without significant systemic dissemination. In contrast, intravenous administration caused a prompt systemic distribution of DNA with a shorter half-life of the administered gene in the lungs.

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The long-term survival of lung cancer patients treated with conventional therapies (surgery, radiation therapy and chemotherapy) remains poor and has changed little in decades. The need for novel approaches remains high and gene therapy holds promise in this area. A number of genes have been shown in vitro, in animal studies and most recently, in human clinical trials, to have antitumor actions. However, a number of problems still exist and success in human patients to date has been marginal. Among the numerous considerations are the efficiency of delivery of the gene to the tumor or, if an indirect effect is the aim, possibly nontumor tissues, the efficiency and persistence of expression of the therapeutic gene, the specificity of the gene action against the tumor, potential toxic or pathogenic consequences of either the genes or the delivery vectors used, convenience of the therapy and how likely the therapy will compliment or complicate other conventional anticancer therapies. After the cloning of the cystic fibrosis gene, there was great interest in the noninvasive delivery of genes directly to the pulmonary surfaces by aerosol. Clearly, this approach could have application to some pulmonary cancers as well and most early efforts focused mainly on the use of nonviral vectors, primarily cationic lipids. Unfortunately, nebulization shear forces and inefficient pulmonary uptake and expression of plasmid DNA-cationic lipid formulations have generally resulted in a lack of therapeutic effect, so much of this work has diminished in recent years. Polyethyleneimine (PEI)-based formulations have proven stable during nebulization and result in nearly 100% efficient transfection throughout the airways and lung parenchyma. Therapeutic responses have been obtained in several animal lung tumor models when PEI-based formulations of p53 and other antitumor genes were delivered by aerosol. In addition, this mode of delivery seems to be associated with low toxicity and results in little or none of the immunostimulatory response typically associated with the delivery of bacterially produced plasmid DNA containing unmethylated CpG motifs, which has presented a challenge to repeated gene therapy via other modes of delivery. Other potential applications of PEI aerosol gene delivery include the treatment of asthma, lung alveolitis and fibrosis and a variety of monogeneic diseases such as cystic fibrosis and alpha-1-antitrypsin deficiency. In addition, a wide range of conditions treatable via genetic immunization could benefit from this approach to gene delivery as well.

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Gene therapy is a novel field of medicine that holds tremendous therapeutic potential for a variety of human diseases. Targeting of therapeutic gene delivery vectors to the lungs can be beneficial for treatment of various pulmonary diseases such as lung cancer, cystic fibrosis, pulmonary hypertension, alpha-1 antitrypsin deficiency, and asthma. Inhalation therapy using formulations delivered as aerosols targets the lungs through the pulmonary airways. The instant access and the high ratio of the drug deposited within the lungs noninvasively are the major advantages of aerosol delivery over other routes of administration. Delivery of gene formulations via aerosols is a relatively new field, which is less than a decade old. However, in this short period of time significant developments in aerosol delivery systems and vectors have resulted in major advances toward potential applications for various pulmonary diseases. This article will review these advances and the potential future applications of aerosol gene therapy technology.

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Inhibition of pulmonary metastases poses a difficult clinical challenge for current therapeutic regimens. We have developed an aerosol system utilizing a cationic polymer, polyethyleneimine (PEI), for topical gene delivery to the lungs as a novel approach for treatment of lung cancer. Using a B16-F10 murine melanoma model in C57BL/6 mice, we previously demonstrated that aerosol delivery of PEI-p53 DNA resulted in highly significant reductions in the tumor burden (P < .001) in treated animals, and also lead to about 50% increase in the mean length of survival of the mice-bearing B16-F10 lung tumors. The mechanisms of this antitumor effect of p53 are investigated in this report. Here, we demonstrate that the p53 transfection leads to an up-regulation of the antiangiogenic factor thrombospondin-1 (TSP-1) in the lung tissue and the serum of the mice. Furthermore, there is a down-regulation of vascular endothelial growth factor (VEGF) in the lung tissue and serum of the B16-F10 tumor-bearing mice treated with PEI-p53 DNA complexes, compared with untreated tumor-bearing animals. In addition, staining for von Willebrand factor (vWF), a marker for the angiogenic blood vessels, revealed that p53 treatment leads to a decrease in the angiogenic phenotype of the B16-F10 tumors. Immunohistochemistry for transgene expression reveals that the PEI-p53 aerosol complexes transfect mainly the epithelial cells lining the airways, with diffuse transfection in the alveolar lining cells, as well as, the tumor foci in the lung tissue. There was also some evidence of apoptosis in the lung tumor foci of animals treated with p53. The data suggest that aerosol delivery of PEI-p53 complexes leads to inhibition of B16-F10 lung metastases, in part by suppression of angiogenesis.

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The use of adenoviral vectors for therapeutic delivery of genes via pulmonary application poses several problems in terms of immune responses. The purpose of this study was to determine whether polyethylenimine (PEI), a polycationic DNA carrier, can be used to deliver the IL-12 gene into the lungs of mice having microscopic osteosarcoma (OS) lung metastases. Incubation of SAOS-LM6 cells in vitro with PEI containing the murine IL-12 (mIL-12) gene (PEI:IL-12) resulted in expression of both the p35 and p40 subunits of IL-12 mRNA and production of mIL-12 protein. Using our newly developed OS nude mouse model, we demonstrated that treatment of mice using intranasal PEI:IL-12 resulted in significant IL-12 mRNA expression in the lung but not the liver. Furthermore, plasma IL-12 was undetectable after up to 4 weeks of intranasal PEI:IL-12 therapy given twice weekly. No IL-12 expression was seen following intranasal PEI therapy alone. The number of lung metastases in animals that received intranasal PEI:IL-12 twice weekly for 4 weeks starting 6 weeks after tumor inoculation was significantly decreased (median, 11; range, 0-47) compared with those that received PEI alone (median, 89; range, 2 to >200; P=.012). Also, the size of the nodules was significantly smaller in the PEI:IL-12-treated animals, with 90% measuring < or =0.5 mm in diameter compared with 56% in the PEI-alone group. Animals that received PEI alone also had numerous large nodules (3-6 mm) throughout the lungs. Intranasal therapy is a noninvasive way to administer agents and has the advantage of targeting the pulmonary region, resulting in higher concentrations in the tumor area. Additionally, delivery of IL-12 to the lung via the airway using PEI may avoid systemic toxicity. Because OS metastasizes almost exclusively to the lung, this may be a novel approach to the treatment of pulmonary OS metastases.

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Delivery of therapeutic genes to the lungs is an attractive strategy to correct a variety of pulmonary dysfunctions such as cystic fibrosis, alpha-1 antitrypsin deficiency, pulmonary hypertension, asthma, and lung cancer. Different delivery routes such as intratracheal instillation, aerosol and intravenous injection have been utilized with varying degrees of efficiency. Both viral and non-viral vectors, with their respective strengths and weaknesses, have achieved significant levels of transgene expression in the lungs. However, the application of gene therapy for the treatment of pulmonary disease has been handicapped by various barriers to the delivery vectors such as serum proteins during intravenous delivery, and surfactant proteins and mucus in the airway lumen during topical application of therapeutic genes. Immune and cytokine responses against the delivery vehicle are also major problems encountered in pulmonary gene therapy. Despite these shortcomings much progress has been made to enhance the efficiency, as well as lower the toxicity of gene therapy vehicles in the treatment of pulmonary disorders such as cystic fibrosis, lung cancer and asthma.

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