Delivery of Gene Transfer Vectors to Lung: Obstacles and the Role of Adjunct Techniques for Airway Administration
Authors: Daniel J. Weiss
Journal: Molecular Therapy (2002)

Physical Barriers and Innate Immune ResponsesThe delivery of exogenous DNA or genes directly to the lung airways is a uniquely appealing opportunity to specifically target gene expression to airway and alveolar epithelium. Large bodies of literature and experience support the feasibility of this approach. However, airway-directed gene delivery is not as simple as was originally anticipated. The lung has both physical and immunologic barriers that hinder effective transduction of airway and alveolar epithelial cells [1Look D.C. Brody S.L. Engineering viral vectors to subvert the airway defense response.Am. J. Respir. Cell Mol. Biol. 1999; 20: 1103-1106Crossref PubMed Scopus (30) Google Scholar, 2Bromberg J.S. Debruyne L.A. Qin L. Interactions between the immune system and gene therapy vectors: bidirectional regulation of response and expression.Adv. Immunol. 1998; 69: 353-409Crossref PubMed Google Scholar]. Physical barriers that can impede vector access to airway and alveolar epithelium include mucins and surfactants lining the airways and alveolar spaces, and glycocalyceal proteins on the apical surface of airway epithelial cells. Tight junctions between epithelial cells additionally limit access of vectors to receptors on basolateral cell surface membranes [3Walters R.W. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia.J. Biol. Chem. 1999; 274: 10219-10226Crossref PubMed Scopus (309) Google Scholar]. The lung also has a highly developed innate immune response to the introduction of foreign vector particles into the airways. For example, alveolar macrophages phagocytose and inactivate a large proportion of adenovirus and retrovirus vectors following intratracheal administration to normal lung [4Worgall S. Role of alveolar macrophages in rapid elimination of adenovirus vectors administered to the epithelial surface of the respiratory tract.Hum. Gene Ther. 1997; 8: 1675-1684Crossref PubMed Scopus (121) Google Scholar, 5McCray P.B. Alveolar macrophages inhibit retrovirus-mediated gene transfer to airway epithelia.Hum. Gene Ther. 1997; 8: 1087-1093Crossref PubMed Scopus (32) Google Scholar]. Stimulation of inflammatory pathways by the vector can limit both the initial extent as well as the duration of epithelial cell gene expression [1Look D.C. Brody S.L. Engineering viral vectors to subvert the airway defense response.Am. J. Respir. Cell Mol. Biol. 1999; 20: 1103-1106Crossref PubMed Scopus (30) Google Scholar, 2Bromberg J.S. Debruyne L.A. Qin L. Interactions between the immune system and gene therapy vectors: bidirectional regulation of response and expression.Adv. Immunol. 1998; 69: 353-409Crossref PubMed Google Scholar].Physical and other barriers that can impede effective airway gene delivery have been studied mostly for adenovirus vectors. Less information is available for other viral vectors and for nonviral vectors, but it is likely that many of the same barriers affecting adenovirus vector-mediated transduction of epithelial cells will also be applicable to other vectors. In particular, nonviral vectors may be more affected by physical barriers than viral vectors. For example, normal mucins can significantly impede cationic liposome-mediated transfection, but have less effect on transduction mediated by adenovirus vectors [6Kitson C. The extra- and intracellular barriers to lipid and adenovirus-mediated pulmonary gene transfer in native sheep airway epithelium.Gene Ther. 1998; 6: 534-546Crossref Scopus (124) Google Scholar]. Similarly, surfactants can decrease cationic liposome-mediated transfection by disruption of the liposomes with a subsequent decrease in gene transfer [7Duncan J.D. Whitsett J.A. Horowitz A.D. Pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cells in vitro.Hum. Gene. Ther. 1997; 8: 431-438Crossref PubMed Scopus (83) Google Scholar], whereas administration of adenovirus vectors in surfactant can enhance gene expression [8Jobe A.H. Ueda T. Whitsett J.A. Trapnell B.C. Ikegami M. Surfactant enhances adenovirus-mediated gene expression in rabbit lungs.Gene Ther. 1996; 3: 775-779PubMed Google Scholar, 9Katkin J.P. Husser R.C. Langston C. Welty S.E. Exogenous surfactant enhances the delivery of recombinant adenoviral vectors to the lung.Hum. Gene Ther. 1997; 8: 171-176Crossref PubMed Scopus (52) Google Scholar]. In contrast, work with adeno-associated virus (AAV) vector serotypes that bind to apical glycoproteins demonstrates that not all barriers, in this instance tight junctions between epithelial cells, are always important [10Kaludov N. Brown K.E. Walters R.W. Zabner J. Chiorini J.A. Adeno-associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity.J. Virol. 2001; 75: 6884-6993Crossref PubMed Scopus (329) Google Scholar].Physical barriers may be increased in acute lung injury in which edema, increased mucin production, and inflammatory cellular debris can further impede effective access of vectors to epithelial cells [11van Heeckeren A. Ferkol T. Tosi M. Effects of bronchopulmonary inflammation induced by pseudomonas aeruginosa on adenovirus-mediated gene transfer to airway epithelial cells in mice.Gene Ther. 1998; 5: 345-351Crossref PubMed Scopus (50) Google Scholar]. The inflammatory environment in an acutely injured lung may also hinder vector access and expression. Inflammatory cytokines such as tumor necrosis factor-α and interferon-7, as well as nitric oxide generated during acute lung injury, can decrease adenovirus-vector mediated expression [12Zhang H.-G. Inhibition of tumor necrosis factor α decreases inflammation and prolongs adenovirus gene expression in lung and liver.Hum. Gene Ther. 1998; 9: 1875-1884Crossref PubMed Scopus (97) Google Scholar]. Bronchial secretions and mucus from cystic fibrosis patients can interfere with adenovirus and cationic liposome-mediated transduction and contain proteolytic activity that can degrade AAV2 vectors [7Duncan J.D. Whitsett J.A. Horowitz A.D. Pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cells in vitro.Hum. Gene. Ther. 1997; 8: 431-438Crossref PubMed Scopus (83) Google Scholar, 13Virella-Lowell I. Poirier A. Chesnut K.A. Brantly M. Flotte T.R. Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients.Gene Ther. 2000; 7: 1783-1789Crossref PubMed Scopus (79) Google Scholar]. Moreover, sputum from cystic fibrosis patients contains anti-adenovirus antibodies that may also interfere with adenovirus vector-mediated transduction [14Perricone M.A. Inhibitory effect of cystic fibrosis sputum on adenovirus-mediated gene transfer in cultured epithelial cells.Hum. Gene Ther. 2000; 11: 1997-2008Crossref PubMed Scopus (34) Google Scholar]. Similar studies have not been done with secretions from acutely injured lung, but it is likely that these contain substances that can interfere with transduction or increase degradation of vectors.There have been several attempts to develop methods to overcome some of these physical barriers. Removal of the transmembrane mucin glycoprotein MUC-1 by treatment with neuraminidase was shown to improve subsequent adenovirus-mediated gene transfer to cultured human airway epithelial cells [15Arcasoy S.M. MUC1 and other sialoglycoconjugates inhibit adenovirus-mediated gene transfer.Am. J. Respir. Cell Mol. Biol. 1997; 17: 422-435Crossref PubMed Scopus (80) Google Scholar]. Similarly, in cultured cells coated with sputum from cystic fibrosis patients, treatment with recombinant human DNase (but not mucolytic agents including N-acetylcysteine, alginase, or lysine) improved adenovirus and cationic liposomal-mediated gene transfer expression [16Stern M. The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro.Gene Ther. 1998; 5: 91-98Crossref PubMed Scopus (107) Google Scholar]. In a related study using sheep trachea in organ culture, removal of mucin resulted in a 25-fold increase in cationic liposomal-mediated gene expression [6Kitson C. The extra- and intracellular barriers to lipid and adenovirus-mediated pulmonary gene transfer in native sheep airway epithelium.Gene Ther. 1998; 6: 534-546Crossref Scopus (124) Google Scholar]. These studies illustrated the role mucin lining layers may play in impeding adenovirus and cationic liposomal-mediated gene delivery to normal and injured lung, but have not yet resulted in a clinically useful strategy for enhancing vector delivery.Methods of Airway Gene DeliveryCurrent techniques of delivering gene transfer vectors to airway and alveolar epithelium include nasal deposition or instillation, direct instillation into the trachea or lower airways using a catheter or bronchoscope, and aerosolization and inhalation of vectors. Nasal deposition in obligate nasal breathers such as rodents can be effective in distributing vector throughout the lower airways [17Halbert C.L. Standaert T.A. Wilson C.B. Miller A.D. Successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure.J. Virol. 1998; 72: 9795-9805Crossref PubMed Google Scholar]. However, in humans, gene expression resulting from nasal deposition of vector is generally limited to nasal epithelia [18Zabner J. Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis.J. Clin. Invest. 1996; 97: 1504-1511Crossref PubMed Scopus (240) Google Scholar]. There is little experience with nasal deposition and delivery of vectors to acutely injured lungs.There are several approaches to direct vector instillation into the trachea or lower airways. In small animal models, transtracheal puncture or direct tracheal cannulation is commonly used, whereas in larger animal models (sheep, primates) and in patients bronchoscopic administration of vector is usually performed. Whatever the method, direct vector instillation more often results in heterogenous deposition in limited portions of lung. Multiple instillations are required to achieve more widespread vector distribution. Moreover, the resulting transgene expression is usually more evident in epithelium of larger proximal airways. Catheter-directed instillation, particularly when using a bronchoscope, generally also requires topical analgesia and sedation to be performed comfortably and safely. Recent techniques using spray devices directed through the bronchoscope seem to improve vector distribution and deposition, particularly in distal airways [19Harvey B.G. Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus.J. Clin. Invest. 1999; 104: 1165-1166Crossref PubMed Scopus (201) Google Scholar].Inhalation of aerosolized vector solutions can result in more diffuse transgene distribution throughout the lung and in increased distribution to distal airway and alveolar epithelium [20Katkin J.P. Gilbert B.E. Langston C. French K. Beaudet A.L. Aerosol delivery of a β-galactosidase adenoviral vector to the lungs of rodents.Hum. Gene Ther. 1995; 6: 985-995Crossref PubMed Scopus (44) Google Scholar, 21Sene C. Aerosol-mediated delivery of recombinant adenovirus to the airways of non-human primates.Hum. Gene Ther. 1995; 8: 411-422Google Scholar]. This approach is also easier to use clinically than direct intratracheal instillation. However, there are significant limitations to current aerosol techniques. Large amounts of vector are required as material is lost by deposition in the aerosolization equipment and on oro- or nasopharyngeal, laryngeal, or upper airway mucosa. Moreover, cationic liposomes may be disrupted during the aerosolization process [22Brown A.R. Chowdhury S.I. Propellant-driven aerosols of DNA plasmids for gene expression.J. Aerosol Med. 1997; 10: 129-146Crossref Scopus (16) Google Scholar]. Additionally, there is no standardized approach to nebulization of vector solutions and administration of the resulting aerosol. Several different methods and nebulization devices have been described. A reasonable goal of the nebulization equipment is to produce homogenously sized aerosol droplet particles of an appropriate size to deposit in bronchiolar and distal airways. Larger droplets will tend to deposit in the nares, mouth, or pharynx and are less likely to result in useful gene transfer to lower airways. In addition to particle size, density, and hygroscopicity, other factors including mode of inhalation and depth and rate of breathing influence where droplets are deposited [23Muir D.F.C. Particle Deposition.The Lung: Scientific Foundations. 1991; : 1839-1843Google Scholar]. Administration of aerosolized vector solutions is inefficient, even with the most sophisticated currently available devices and techniques. It is estimated that at best only 10–30% of aerosolized particles reach the lower airways [23Muir D.F.C. Particle Deposition.The Lung: Scientific Foundations. 1991; : 1839-1843Google Scholar].View Large Image Figure ViewerDownload Hi-res image Download (PPT)Most studies of direct instillation or aerosol administration of vectors have been done either in normal animal models or patients or in cystic fibrosis patients with mild disease and no acute inflammatory exacerbation [24Aitken M.L. A phase 1 study of aerosolized administration of tgAAVCF to cystic fibrosis patients with mild lung disease.Hum. Gene Ther. 2001; 12: 1907-1916Crossref PubMed Scopus (198) Google Scholar]. There is less experience with direct instillation or aerosol administration to more severely diseased lungs.Adjunct Methods for Airway Gene DeliverySeveral techniques have been demonstrated to augment expression following direct airway instillation of vectors, particularly viral vectors, in animal models. Whereas the clinical applicability or feasibility of some of these approaches is unclear, methods using surfactant and perfluorochemical liquid are based on clinical experience with each of these for treatment of acute lung injury.CAPO4Precipitating adenovirus or AAV2 vectors with CaPO4 results in increased nonspecific uptake via the apical surface of airway epithelial cells [25Lee J.H. Zabner J. Welsh M.J. Delivery of an adenovirus vector in a calcium phosphate coprecipitate enhances the therapeutic index of gene transfer to airway epithelia.Hum. Gene Ther. 1999; 10: 603-613Crossref PubMed Scopus (46) Google Scholar, 26Fasbender A. Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo.J. Clin. Invest. 1998; 102: 184-193Crossref PubMed Scopus (134) Google Scholar, 27Walters R.W. Duan D. Engelhardt J.F. Welsh M.J. Incorporation of adeno-associated virus in a calcium phosphate coprecipitate improves gene transfer to airway epithelia in vitro and in vivo.J. Virol. 2000; 74: 535-540Crossref PubMed Scopus (34) Google Scholar]. This approach abrogates the need for vectors to transverse tight junctions to access receptors located on basolateral cell surfaces. Enhanced gene expression was observed in cultured lung epithelial cells and in mouse lung in vivo with no apparent toxicity. Precipitation of vectors with CaPO4 may also increase release of virus particles from endosomes following vector internalization by cells [25Lee J.H. Zabner J. Welsh M.J. Delivery of an adenovirus vector in a calcium phosphate coprecipitate enhances the therapeutic index of gene transfer to airway epithelia.Hum. Gene Ther. 1999; 10: 603-613Crossref PubMed Scopus (46) Google Scholar]. The efficacy of this approach has not yet been demonstrated in larger animal models or in injured lung.EgtaThe rationale for use of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) is that localized calcium chelation will disrupt calcium-dependent proteins in tight junction complexes and increase permeability of tight junctions to vectors. Application of EGTA to cultured lung epithelial cells and to rabbit tracheal and human nasal epithelium in vivo increased tight junction permeability as measured by changes in transepithelial resistance and by electron micrographic analysis [28Wang G. Increasing epithelial junction permeability enhances gene transfer to airway epithelia in vivo.Am. J. Respir. Cell Mol. Biol. 2000; 22: 129-138Crossref PubMed Scopus (127) Google Scholar]. Adenovirus and retrovirus vector-mediated gene expression was increased both in cultured lung epithelial cells and in rabbit trachea in vivo [28Wang G. Increasing epithelial junction permeability enhances gene transfer to airway epithelia in vivo.Am. J. Respir. Cell Mol. Biol. 2000; 22: 129-138Crossref PubMed Scopus (127) Google Scholar]. Use of EGTA also enhanced adenovirus vector mediated transgene expression in mouse lung [29Chu Q. EGTA enhancement of adenovirus-mediated gene transfer to mouse tracheal epithelium in vivo.Hum. Gene Ther. 2001; 12: 455-467Crossref PubMed Scopus (62) Google Scholar]. This promising approach also has not yet been demonstrated to have efficacy in larger animal models or in injured lung.Detergents and Thixotropic SolutionsAirway administration of a phospholipid detergent can also disrupt tight junctions between mature ciliated airway epithelial cells and has been demonstrated to increase adenovirus-mediated transgene expression in murine nasal epithelium [30Parsons D.W. Grubb B.R. Johnson L.G. Boucher R.C. Enhanced in vivo airway gene transfer via transient modification of host barrier properties with a surface-active agent.Hum. Gene Ther. 1998; 9: 2661-2672Crossref PubMed Scopus (77) Google Scholar]. Whether this approach is applicable in either normal or injured lung remains to be determined. More recent work demonstrates that formulating adenovirus vectors in a thixotropic solution (a gel that becomes liquid with agitation) increased gene expression in cultured human airway epithelium and in murine airway epithelium in vivo [31Seiler M.P. Thixotropic solutions enhance viral-mediated gene transfer to airway epithelia.Am. J. Resp. Mol. Cell Biol. 2002; PubMed Google Scholar] (Joseph Zabner, pers. comm.). The likely mechanism is decreased mucociliary clearance of vector particles. Importantly, these studies also demonstrate enhanced correction of the chloride current defect in cultured human cystic fibrosis epithelial cells transduced with an AdCFTR vector in thixotropic solution compared with cells transduced with the vector administered in PBS.SurfactantSurfactant can be safely administered to the airways and has become a mainstay of treatment for respiratory failure in premature infants [32Greenough A. Expanded use of surfactant replacement therapy.Eur. J. Pediatrics. 2001; 159: 635-640Crossref Scopus (24) Google Scholar]. Administering adenovirus vectors in either 10 or 25 mg/ml solutions of the synthetic surfactant Survanta increased gene expression in rabbit lung parenchyma [8Jobe A.H. Ueda T. Whitsett J.A. Trapnell B.C. Ikegami M. Surfactant enhances adenovirus-mediated gene expression in rabbit lungs.Gene Ther. 1996; 3: 775-779PubMed Google Scholar, 9Katkin J.P. Husser R.C. Langston C. Welty S.E. Exogenous surfactant enhances the delivery of recombinant adenoviral vectors to the lung.Hum. Gene Ther. 1997; 8: 171-176Crossref PubMed Scopus (52) Google Scholar]. Expression in trachea and main bronchi was decreased, suggesting that the surfactant vehicle resulted in more effective distribution of vector to the lower airways [8Jobe A.H. Ueda T. Whitsett J.A. Trapnell B.C. Ikegami M. Surfactant enhances adenovirus-mediated gene expression in rabbit lungs.Gene Ther. 1996; 3: 775-779PubMed Google Scholar]. Similar findings have been observed delivering adenovirus vector in a 50% Survanta solution to rat lungs [33Factor P. Augmentation of lung liquid clearance via adenovirus-mediated transfer of a Na, K-ATPase β-l subunit gene.J. Clin. Invest. 1998; 102: 1421-1430Crossref PubMed Scopus (115) Google Scholar]. In contrast, gene expression mediated by naked DNA in mouse lungs was decreased when the DNA was administered with a different synthetic surfactant solution (Exosurf) [34Raczka E. Kukowska-Latallo J.F. Rymaszewski M. Chen C. Baker J.R. The effect of synthetic surfactant Exosurf on gene transfer in mouse lung in vivo.Gene Ther. 1998; 5: 1333-1339Crossref PubMed Scopus (45) Google Scholar]. The mechanism by which gene expression was inhibited remains unclear. Survanta had no effect on naked DNA-mediated gene expression in cultured lung epithelial cells or cultured lung fibroblasts, but did inhibit expression mediated by cationic liposomes [35Tsan M.F. Tsan G.L. White J.E. Surfactant inhibits cationic liposome-mediated gene transfer.Hum. Gene Ther. 1997; 8: 817-825Crossref PubMed Scopus (46) Google Scholar]. Naturally occurring bovine and rabbit surfactants also inhibited cationic liposomal-mediated gene expression in cultured lung epithelial cells [7Duncan J.D. Whitsett J.A. Horowitz A.D. Pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cells in vitro.Hum. Gene. Ther. 1997; 8: 431-438Crossref PubMed Scopus (83) Google Scholar]. In these studies, mixing of surfactant components with those in the cationic liposomes likely resulted in disruption of the liposomes with subsequent decrease in gene transfer.Administering adenovirus vector in a (50%) Survanta solution enhanced gene expression in rat lungs with experimentally induced pulmonary edema and lung injury [35Tsan M.F. Tsan G.L. White J.E. Surfactant inhibits cationic liposome-mediated gene transfer.Hum. Gene Ther. 1997; 8: 817-825Crossref PubMed Scopus (46) Google Scholar]. This finding suggests that use of surfactants during airway vector delivery may be effective in other forms of acute lung injury as well.Perfluorochemical LiquidPerfluorochemical (PFC) liquids are chemically inert fluorinated carbon chain liquids that are excellent solvents for O2, CO2, and other gases, yet are immiscible with aqueous and lipid solutions [37Shaffer T.H. Wolfson M.R. Clark L.C. Liquid ventilation.Pediatric Pulmonol. 1992; 14: 102-109Crossref PubMed Scopus (196) Google Scholar]. Other properties include high density and low surface tension, which allow PFC liquids to distribute throughout communicating airways and spread over alveolar walls following instillation into lungs [37Shaffer T.H. Wolfson M.R. Clark L.C. Liquid ventilation.Pediatric Pulmonol. 1992; 14: 102-109Crossref PubMed Scopus (196) Google Scholar]. Clinical experience with the PFC liquid perflubron (LiquiVent Alliance Pharmaceuticals, San Diego) in both children and adults demonstrated that this compound can be safely instilled and used for partial liquid ventilation in critically ill patients [38Leach C.L. Partial liquid ventilation with perflubron in premature infants with severe respiratory distress syndrome.N. Engl. J. Med. 1996; 335: 761-767Crossref PubMed Scopus (405) Google Scholar, 39Hirschl R.B. Prospective, randomized, controlled pilot study of partial liquid ventilation in adult acute respiratory distress syndrome.Am. J. Resp. Crit. Care Med. 2002; 165: 781-787Crossref PubMed Scopus (170) Google Scholar]. PFC liquids also have anti-inflammatory effects in injured lung. Clinical studies of liquid ventilation as well as observations in animals with experimentally induced lung injury undergoing liquid ventilation demonstrate that in addition to improving oxygenation and lung mechanics, use of perflubron decreased edema, bleeding, and neutrophil influx into injured lungs as well as decreasing histopathologic lung injury [40Papo M.C. Perfluorocarbon-associated gas exchange improves oxygenation, lung mechanics, and survival in a model of adult respiratory distress syndrome.Crit. Care Med. 1996; 24: 466-474Crossref PubMed Scopus (94) Google Scholar, 41Colton D.M. Neutrophil accumulation is reduced during partial liquid ventilation.Crit. Care Med. 1998; 26: 1716-1724Crossref PubMed Scopus (70) Google Scholar, 42Rotta A.T. Gunnarsson B. Hernan L.J. Fuhrman B.P. Steinhorn D.M. Partial liquid ventilation influences pulmonary histopathology in an animal model of acute lung injury.J. Crit. Care. 1999; 14: 84-92Abstract Full Text PDF PubMed Scopus (60) Google Scholar]. More recent studies have demonstrated a variety of anti-inflammatory effects of PFC liquids in clinical and animal models of lung injury and lung infection [43Croce M.A. Partial liquid ventilation decreases the inflammatory response in the alveolar environment of trauma patients.J. Trauma Inj. Infect. Crit. Care. 1998; 45: 273-282Crossref PubMed Scopus (98) Google Scholar, 44Dickson E.W. Liquid ventilation with perflubron in the treatment of rats with pneumococcal pneumonia.Crit. Care. Med. 2002; 30: 393-395Crossref PubMed Scopus (22) Google Scholar, 45Haeberle H.A. Nesti F. Dieterich H.-J. Gatalica Z. Garafalo R.P. Perflubron reduces lung inflammation in respiratory syncytial virus infection by inhibiting chemokine expression and nuclear factor-κB activation.Am. J. Resp. Crit. Care Med. 2002; 165: 1433-1438Crossref PubMed Scopus (48) Google Scholar]. These collected observations suggest that use of PFC liquid may be beneficial in the setting of acute lung injury.Use of perflubron and other PFC liquids during vector administration has been demonstrated to enhance adenovirus vector-mediated gene expression in intubated ventilated rabbits [46Lisby D.A. Enhanced distribution of adenovirus-mediated gene transfer to lung parenchyma by perfluorochemical liquid.Hum. Gene Ther. 1997; 8: 919-928Crossref PubMed Scopus (47) Google Scholar], adenovirus and AAV2 vector-mediated expression in lungs of spontaneously breathing normal rodents [47Weiss D.J. Strandjord T.P. Jackson J.C. Clark J.G. Liggitt D. Perfluorochemical liquid-enhanced adenoviral vector distribution and expression in lungs of spontaneously breathing rodents.Exp. Lung Res. 1999; 25: 317-333Crossref PubMed Scopus (36) Google Scholar, 48Weiss D.J. Bonneau L. Allen J.M. Miller A.D. Halbert C.L. Perfluorochemical liquid enhances adeno-associated virus-mediated transgene expression in lung.Mol. Ther. 2000; 2: 624-630Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar], and adenovirus vector-mediated expression in lungs of spontaneously breathing normal primates [49Weiss D.J. Baskin G.B. Shean M.K. Blanchard J.L. Kolls J.K. Use of perflubron to enhance lung gene expression: safety and initial efficacy studies in non-human primates.Mol. Ther. 2002; 5: 8-15Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar]. Total transgene expression was increased, particularly in mid-sized and distal airways and in alveolar epithelium, and transgene distribution was enhanced within and between lung lobes. Importantly, use of PFC liquids has been demonstrated to enhance adenovirus vector-mediated transgene expression in models of both acute and chronic lung injury in rodents [50Weiss D.J. Strandjord T.P. Liggitt D. Clark J.G. Perflubron enhances adenoviral-mediated gene expression in lungs of transgenic mice with chronic alveolar filling.Hum. Gene Ther. 1999; 10: 2287-2293Crossref PubMed Scopus (36) Google Scholar, 51Weiss D.J. Bonneau L. Liggitt D. Use of perfluorochemical liquid allows earlier detection and use of less adenovirus vector for gene expression in normal lung and enhances gene expression in acutely injured lung.Mol. Ther. 2001; 3: 734-745Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar].Similar to surfactants, PFC liquids likely act by propelling vectors further into and more uniformly throughout the lung than can be accomplished with either catheter-directed or aerosol administration of vectors. The PFC liquids may also transiently displace mucins, allowing better access of vectors to epithelial cells [50Weiss D.J. Strandjord T.P. Liggitt D. Clark J.G. Perflubron enhances adenoviral-mediated gene expression in lungs of transgenic mice with chronic alveolar filling.Hum. Gene Ther. 1999; 10: 2287-2293Crossref PubMed Scopus (36) Google Scholar]. Recent information from our laboratory suggests that PFC liquids may also act by decreasing clearance of vectors by alveolar macrophages and by transiently increasing tight junction permeability (manuscripts submitted). Other actions of PFC liquids may also be important but have not yet been defined. Most recently, we have found that administration of PFC liquid in aerosol form also enhances adenovirus vector-mediated gene expression (results presented at the 2002 American Society of Gene Therapy Meeting, Boston; (D. J. Weiss et al., manuscript submitted). Use of PFC in aerosol rather than liquid form will be a more feasible clinical approach for enhancing lung gene expression.Targeting delivery of gene transfer vectors to the lung airways remains an appealing approach. Although it has much potential, substantial physical barriers remain, particularly in injured lung. Continued development of more effective delivery techniques in conjunction with development of improved vectors that maximize expression while minimizing inflammatory and immune responses will greatly increase the chance of successful lung gene therapy. Physical Barriers and Innate Immune ResponsesThe delivery of exogenous DNA or genes directly to the lung airways is a uniquely appealing opportunity to specifically target gene expression to airway and alveolar epithelium. Large bodies of literature and experience support the feasibility of this approach. However, airway-directed gene delivery is not as s