© 2005 American Thoracic Society doi: 10.1164/rccm.2411003
Pulmonary Edema ClearanceJuicing Up the Sodium PumpUniversity of Minnesota School of Medicine Minneapolis, MN
The quest for successful gene therapy for lung disease has proven more difficult than initially anticipated (1). There are relatively prevalent chronic lung diseases with defined genetic bases, such as cystic fibrosis (CF) and
Viral vectors have commonly been used for gene therapy. Adenoviral vectors cause inflammation with relatively short duration of expression, but newer generation adenoviral vectors may reduce inflammation (3). Adeno-associated viruses induce less inflammation and have been well tolerated in early human CF clinical trials (4, 5). New viral vectors that can transduce nondividing cells and integrate into DNA to provide prolonged expression include modified Lenti, Sendai, and Semliki Forest viruses. Perfluorochemicals, surfactants, or positively charged polymers may augment the efficacy of viral gene transfer. Virally transduced marrow-derived stem cells have delivered genes to the lung (6). Finally, ex vivo transfection of alveolar macrophages can increase Nonviral methods bring the advantages of less inflammation, simpler production, lack of viral shedding and possibly reduced long-term complications, but rarely integrate into DNA, shortening their efficacy. The plasmid DNA used may be naked, in liposomes, or accompanied by polycations (such as poly-L-lysine), or cationic lipids. A new system combines synthetic transposons surrounding the gene of interest with a nonviral Sleeping Beauty fish–derived transposase enzyme that promotes DNA integration of the transposon. This system can yield long term expression in the lung (8) and may allow selective gene expression in particular cells or organs. Airway delivery with nanoparticles offers another new approach. In vitro cell electroporation is a well established gene transfer method, but its use in vivo is just beginning to be explored (9). In vivo electroporation has transferred genes in skin or skeletal muscle. The rapid electrical pulse induces very short-lived micropores in the plasma membrane, allowing DNA to enter the cell. The exciting results of Machado-Aranda and colleagues reported in this issue (pp. 204–211) (10) demonstrate the potential of this approach for achieving exogenous gene expression in the lung. Several groups (11–13) previously demonstrated that increased expression of specific sodium pump (Na,K-ATPase) subunits, particularly the ß1 subunit, augments alveolar epithelial cell ion transport and alveolar fluid clearance (AFC) in animals. Overexpression improves AFC after hyperoxic or ventilator lung injury and in the setting of increased left atrial pressure. After adenoviral transfer Na,K-ATPase expression increases within several days, so it could be used in acute lung disease (14). The current study by Machado-Aranda and coworkers tests whether electroporation with naked plasmid DNA in living rats could increase Na,K-ATPase subunit expression and, in turn, increase AFC (10). Electroporation was used in ventilated rats shortly after intratracheal plasmid DNA instillation. Electrodes placed externally on the lateral chest walls delivered brief electrical pulses of set voltage and time in a specific pattern. Gene expression depended upon the plasmid dose and the pulse time; presumably it also depended upon the applied voltage and the number of pulses. Gene product was detected within 6 hours, with a peak at 3 days and little expression 5 days after electroporation. The gene product was present relatively diffusely in the lungs and, surprisingly, immunochemistry suggested that many different cell types were transfected—well beyond the immediately accessible airway and alveolar epithelial cells. This occurred even though epithelial permeability was not increased, as judged by movement of labeled albumin out of the vascular space into the alveolar compartment. These exciting results also raise several questions. What is the relative efficiency of gene transfer to different lung cell types? The answer will require relatively laborious morphometric studies after this method is optimized. What sensations are caused by "deep" electroporation and how problematic would this be for human therapy? Are there potential long-term side effects of repeated treatment? Can this technique be translated into animals and humans with much larger transthoracic diameters, and how will this affect the location of cells that are transfected? Alveolar fluid resorption occurs principally through the combined action of apical sodium channels (ENaC) and the basolateral sodium pump of the alveolar epithelium (15). Because most cells have more sodium pump capacity than is required for basal transport and because sodium channels are gated and regulated, the dogma for many years was that ENaC was the rate-limiting step and principal point of regulation of transepithelial sodium transport. This was believed to be true in both the adult lung and during perinatal lung development. This study adds to other data (11–13) showing that increasing expression of Na,K-ATPase alone increases AFC. Undoubtedly, ENaC regulation of sodium entry also contributes to control of this system. Thus, ENaC and Na,K-ATPase can be viewed as skilled dancing partners, that usually are regulated in coordinate fashion during development, as well as by glucocorticoids and other factors. Transepithelial sodium transport also can be triggered by activation of apical chloride channels (16, 17). Thus strategic approaches to accelerate AFC can reasonably be targeted to augment activity of the apical sodium or chloride channels or of the basolateral sodium pump. To assess whether the strategy of augmenting AFC will help human lung disease, we need reliable noninvasive measurements of AFC; otherwise we cannot be sure that therapeutic interventions are having their intended physiologic effect. Once this requirement is addressed, testing this strategy using ß-adrenergic agonists, keratinocyte growth factor, and gene transfer will be very important for acute therapy of lung injury. FOOTNOTES Conflict of Interest Statement: D.H.I has received lecture fees from GlaxoSmithKline for sponsored CME and non-CME talks and the University of Minnesota has a patent for lung gene therapy with sleeping beauty transposon system. REFERENCES
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-1 antiprotease deficiency, that are logical targets for gene therapy. Increased understanding of the molecular pathogenesis of acute lung diseases, such as acute respiratory distress syndrome (ARDS), has defined molecular targets for short term gene therapy or drug discovery approaches. For example, overexpression of heat shock protein HSP-70, heme oxygenase, or superoxide dismutase using gene therapy reduces experimental lung injury. Unfortunately, developing simple methods for efficient gene delivery to target cells and achieving adequate protein expression for sufficient periods of time without significant side effects has been elusive. Success has been hampered by: suboptimal delivery to the specific cell types desired; polarized distribution of viral receptors; difficulty in transducing nonproliferating cells; inflammation and short-lived expression (