Gene therapy for airway diseases: continued progress toward identifying and overcoming barriers to efficiency.

Over the last decade, numerous investigators have pursued gene transfer as a definitive treatment for inherited airway diseases, and although the promise of “gene therapy” appears far from being realized, steady progress is clear. The potential for gene transfer to revolutionize therapy for a number of pulmonary diseases has been suggested by in vitro and animal studies (reviewed in Refs. 1– 3). The rationale for this approach is most apparent for monogenetic diseases such as Cystic Fibrosis (CF) and -1 antitrypsin (A1AT) deficiency. For other genetic or acquired diseases, such as asthma, primary pulmonary hypertension, lung cancer, mesothelioma, and acute lung injury, the rationale is less obvious, yet the potential for therapies that will specifically address proximal events in disease pathogenesis is significant. Initial successes related to CF generated tremendous hype, and when human studies failed to fulfill the promise, enthusiasm waned. Fortunately, several groups of investigators remain undeterred and have quite appropriately shifted their focus from potentially harmful and costly human studies to more fundamental studies that seek to delineate the cell and molecular biology of gene transfer to airway epithelium. This has improved the understanding of normal epithelial host defense, and may provide lessons that will be applicable to other target cells in the lung. The principle underlying gene transfer appeared seductively simple, but early physiologically relevant in vitro and animal models quickly identified problems with efficiency of gene transfer and host response to the vehicle used for gene delivery. Numerous approaches to gene delivery have been developed (reviewed in Ref. 4). Most efforts have involved inserting a gene into a plasmid or replication-incompetent virus that would efficiently and safely transfer a normal copy of the gene to the appropriate cells in the lung. In contrast, gene repair approaches attempt to correct the mutant gene (reviewed in Ref. 5). Gene transfer efforts have been directed toward recombinant viruses, particularly adenoviruses, adeno-associated viruses, retroviruses, and complexes of plasmid DNA with carrier molecules, such as cationic lipids or polymers. Adenoviruses appeared to be the ideal vector for gene transfer to airway epithelium: the molecular pathogenesis of adenovirus infection appeared well-defined, recombinant viruses were relatively easy to produce in high titer, and it appeared logical that because adenoviruses infected the upper respiratory tract, viral entry to airway epithelial cells would be efficient. Several in vitro and rodent studies supported these premises; however, it soon became obvious that in relevant human airway cell models and in primate airway, adenovirus-mediated gene transfer to conducting airway was very inefficient and elicited a significant inflammatory response. Human studies of adenovirus delivered to the nasal or bronchial epithelium confirmed problems with gene transfer efficiency and revealed an acute inflammatory response (reviewed in Refs. 6–8), and subsequent efforts have therefore focused on defining the barriers to efficient gene transfer. Efforts to understand the barriers to gene transfer have successfully identified a series of epithelial cell defenses against viral infection and large molecule delivery to the nucleus. The phases of adenovirus infection were well-defined in undifferentiated cell lines (9); however, only after several years did it become apparent that airway epithelium had evolved mechanisms to inhibit the initial steps of adenovirus-mediated gene transfer:binding and subsequent internalization of the vector. The adenovirus receptor was unknown when adenovirus-mediated gene transfer studies were begun. Subsequent work identified the Coxsackie and Adenovirus Receptor (CAR) (10) and specific vitronectin-binding cell adhesion receptors (integrins v 3 and v 5 ) (11) as critical to efficient binding and internalization of adenovirus. As shown schematically in Figure 1, the CAR and v integrin receptors are expressed in the basolateral membrane of differentiated airway epithelial cells, making them inaccessible to viral particles that are delivered to the apical surface. Thus, the tight junctional complex that separates the apical from basolateral membrane of polarized epithelia provides an important barrier to binding and cellular entry of wild-type and replicationincompetent adenoviruses, as well as other viral and nonviral vectors. Several other epithelial barriers to gene transfer have been identified, including secreted and cell surface mucins, other secretory products at the airway surface, and limited apical membrane endocytic capacity. Many of these barriers have been shown to impact gene transfer with both viral and nonviral vectors, as summarized in Table 1. As each of these barriers has been identified, strategies have been developed to overcome them by modifying the epi( Received in original form June 10, 2002 )