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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 117-121, 2002
© 2002 American Thoracic Society

Perspective

Gene Therapy for Airway Diseases

Continued Progress toward Identifying and Overcoming Barriers to Efficiency

Joseph M. Pilewski

Departments of Medicine, Cell Biology and Physiology, and Pediatrics, University of Pittsburgh, PIttsburgh, Pennsylvania

Address correspondence to: Joseph M. Pilewski, M.D., NW 628 MUH, 3459 Fifth Avenue, Pittsburgh, PA 15213. E-mail: pilewskijm{at}msx.upmc.edu

Abbreviations: adeno-associated virus, AAV • cystic fibrosis, CF • {alpha}-1 antitrypsin, A1AT • Coxsackie and adenovirus receptor, CAR • vesicular stomatitis virus G protein, VSV-G,


   Introduction
Top
 Introduction
References
 
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 {alpha}-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 {alpha}vß3 and {alpha}vß5) (11) as critical to efficient binding and internalization of adenovirus. As shown schematically in Figure 1 , the CAR and {alpha}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 replication-incompetent adenoviruses, as well as other viral and nonviral vectors.



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  		Figure 1. Barriers to airway epithelial gene transfer. Shown schematically are two differentiated airway epithelial cells. The steps of adenovirus-mediated gene transfer are indicated in the cell on the left. Viral particles must overcome a mucus gel and other secreted proteins (1), then bind to cellular receptors and co-receptors (2). For adenovirus, these receptors (CAR and {alpha}v integrin receptors) are located on the basolateral membrane beneath the tight junction complex (TG); the apical membrane is minimally permissive for viral entry. Virions that gain access to necessary receptors undergo endocytosis (3), uncoating of DNA (4), and transport to the nucleus (5). Inefficiency at one or more of these steps limits gene transfer.

 
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 epithelial cell barrier or by identifying alternative cellular entry pathways that circumvent the epithelial barrier. The mucus layer on the apical surface of respiratory epithelium, by entrapping gene transfer vectors, has been presumed to limit access of the vector to target cells. This has been demonstrated for cationic liposome:DNA complexes, but, at least in some model systems, does not appear to significantly influence adenovirus- or polymer/DNA-mediated gene transfer (12, 13). The effect of a mucus layer has not been carefully examined for other vectors, but appears likely to be important. Other secreted components of the airway surface liquid, including antibodies, surfactant, and unidentified inflammatory mediators, also appear to inhibit gene transfer with adenoviruses (12, 14–16) and cationic lipids (12, 17). In agreement with these observations, altering sputum properties appears to improve gene transfer efficiency with both adenoviruses (18) and cationic lipids (19). Thus, extracellular barriers to gene transfer appear important but not insurmountable.


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  		table 1 Strategies to overcome barriers to efficient gene transfer to human airway epithelium

 
More formidable barriers for most vectors are the apical membrane, tight junctions, and restricted expression of viral receptors. For adenoviruses, localization of CAR and {alpha}v integrin receptors appears to contribute to inefficient gene transfer to polarized airway epithelium (20, 21), with additional contributions from inefficient nonspecific apical membrane endocytic pathways (22) and apical membrane glycoconjugates (23). The contribution of cell surface glycoconjugates remains to be defined, as current data is conflicting. Studies in a polarized cell line created to express CAR in the apical membrane revealed that surface glycoconjugates significantly diminished gene transfer efficiency (24). In contrast, in human airway cells engineered to express an apically-targeted CAR, adenovirus-mediated gene transfer was efficient, suggesting that surface glycoconjugates are not prohibitive (25). Successful targeting of other apical receptors (see below) supports this conclusion. An alternative strategy to circumvent apical membrane barriers is to transiently disrupt the tight junctions that limit vector access to the basolateral membrane. Recently, several in vitro studies have supported this strategy by demonstrating that disruption of the tight junction complex via calcium chelators and/or other tight junctional–disrupting agents increases the efficiency of adenovirus-mediated gene transfer (12, 26–28). Other approaches have been to use calcium phosphate coprecipitates or polycations to augment the binding and internalization of adenovirus or DNA complexes (29–31). Whether any of these strategies could prove both effective and safe in human airway remains to be determined. Similar issues related to DNA delivery are relevant to nonviral vectors (32–34; reviewed in Ref. 35), and strategies to improve cellular entry of DNA complexes are being evaluated.

Because of their ability to integrate into the host genome and provide long-term gene expression, recombinant retroviruses have a theoretical advantage over adenoviruses and nonviral vectors, as repeated delivery should not be necessary if the retrovirus infects an airway stem cell population. Murine leukemia viruses have limited application to pulmonary gene transfer, because cell replication is required for infection and integration. In addition, receptors used for viral entry are expressed at low levels in pulmonary epithelium, surfactant inactivates these viruses (36), and alveolar macrophages inhibit retrovirus-mediated gene transfer in vitro (37). In contrast to conventional retroviruses, lentiviruses do not require cell division for entry and gene expression and have been pseudotyped with surface proteins that permit binding and entry to airway epithelium (reviewed in Refs. 38–40). Similar to adenoviruses, cellular barriers to viral binding and internalization appear relevant to lentiviruses, as basolateral delivery appears necessary for gene transfer for viruses that are pseudotyped with vesicular stomatitis virus-G envelope (41). Alternative envelope proteins are being developed that may overcome this limitation by facilitating entry via the apical membrane of polarized airway epithelium.

Pathways for infection of airway epithelium by adeno-associated viruses have not been as well-defined as those for adenoviruses; however, both cellular entry mechanisms and post-entry viral processing appear to limit the efficiency of gene transfer. Like adenovirus and liposome–DNA complexes, immortalized and undifferentiated airway cells are more susceptible to gene transfer with AAV than primary or well-differentiated cells (42, 43), and AAV-mediated gene transfer to airway epithelial cells is more efficient from the basolateral than the apical membrane (44, 45). This appears related to basolateral localization of heparan sulfate proteoglycan receptor, a receptor for some serotypes of AAV, and coreceptors fibroblast growth factor receptor 1 and {alpha}vß5 integrin receptor (45), and the presence of apical membrane glycoconjugates that inhibit viral entry (44). Unlike adenovirus, there also appear to be inefficiencies related to processing of adeno-associated virus in the endocytic pathway, as inhibition of ubiquitination or proteosome function significantly increases gene transfer efficiency to mouse airway with AAV 2 (46). Similar inefficiencies with nuclear delivery and translocation appear critical for nonviral gene transfer vectors (reviewed in Ref. 47). Collectively, these studies indicate that epithelial barriers are relevant to each of the carefully evaluated gene transfer vectors, and they also emphasize the importance of choosing appropriate in vitro and animal model systems for the evaluation of gene transfer efficiency. Efficiency of gene transfer to immortalized cell lines and rodent lower airways has, in general, been poorly predictive of efficiency in polarized human airway epithelium.

In this issue of the AJRCMB, Seiler and coworkers (48) present evidence for an additional barrier to adenovirus-mediated gene transfer and a potential solution that likely has implications for other vectors. Several studies have previously shown that limited contact time of recombinant adenovirus with the target epithelium contributes to the low efficiency of gene transfer to differentiated human airway epithelium (49, 50). Rapid dispersion of the administered vector by mucociliary clearance has been presumed to account for limited contact time. A logical approach to improve efficiency is therefore to at least transiently slow mucociliary clearance. Seiler and coworkers (48) reasoned that a thixotropic solution would accomplish this, and studies in physiologically relevant airway models demonstrated that the solution slowed clearance, altered the spatial relationship of the mucus layer(s) with the cilia, and increased the percentage of cells expressing the reporter gene more than tenfold.

Thixotropic solutions have been utilized for drug delivery; however, the mechanism(s) whereby they increase gene transfer remain to be fully defined. Thixotropy refers to the ability of a solution (in this case carboxymethyl cellulose) to exhibit reduced viscosity when subjected to a shearing force, then regain viscosity when the shear force is removed. For approved nasal steroids, delivery in a thixotropic solution has been proposed to increase the dwell time of the drug in the nasal cavity. Studies of solution mechanics confirm that carboxymethyl cellulose solutions exhibit thixotropy; however, intrinsic viscosity of the cellulose solutions may be mechanistically more important than thixotropy (51). Seiler and coworkers provide compelling evidence that carboxymethyl cellulose solutions reversibly impair mucociliary clearance in an in vitro human airway model and in monkey trachea (48). Electron micrographs reveal alterations in ciliary orientation that may reflect effects of the thixotropic solution on ciliary movement. This is a reasonable explanation for the effects on gene transfer, as some important alternative mechanisms, such as loosening of tight junctional complexes by the carboxymethyl cellulose solution, were excluded experimentally. However, effects of the solution on surface tension or adhesiveness (52) or volume of airway surface liquid remain considerations beyond any direct effect on ciliary function. It will be important to determine which components of the thixotropic solution mediate the effects on vector delivery, and, in particular, to define the cellular entry pathway mediating the apparent delivery. Irrespective of mechanism, studies such as that of Seiler and colleagues (48) demonstrate how delineation of various cellular barriers to gene transfer has generated potential solutions. In addition, they demonstrate that mucociliary clearance can be transiently modified; this has implications for both small and large molecule delivery to conducting airway epithelium.

A number of important issues remain with respect to increasing gene transfer efficiency with current vectors. Among these are whether one or more of the recently identified modifications of the epithelial barriers could increase efficiency in primate and human airway sufficiently for these vectors to have a physiologic effect. Based on existing work, it appears that some of the cellular barriers exist in series rather than in parallel. For cystic fibrosis, overcoming any one barrier may not sufficiently increase efficiency to the proposed 6–10% of surface epithelial cells that must express a normal copy of the CF gene to restore a normal cyclic adenosine monophosphate–mediated chloride secretory response (53), and almost certainly not to the nearly 100% of epithelial cells necessary to restore normal sodium transport (54). For applications based on secreted proteins, such as A1AT, improvements in gene transfer efficiency in recent years may be adequate; however, studies of physiologic effect in clinically relevant model systems should be performed before initiating human studies. Future preclinical studies should determine whether combining two or more epithelial barrier modifications yields additive increases in efficiency, and how this impacts on the host response. For example, increased viral delivery to the epithelium may be accompanied by increases in antigen load and/or processing by intraepithelial dendritic cells that may unacceptably increase inflammatory responses.

In addition to modification of cellular barriers to airway epithelial gene transfer, several promising new vectors have been identified. One approach has been to re-target existing vectors by intentionally modifying tropism (reviewed in Ref. 55) or by identifying mutations of previously evaluated vectors that are more efficient at viral entry (56). Examples of the former are modifying the viral capsid proteins or linking virus to a peptide that targets an apical membrane receptor (57, 58), such as the urokinase plasminogen activator receptor (59), serpin enzyme receptor complex (60), or an apically expressed purinergic receptor (61). These studies suggest that, at least in cell culture systems, the apical membrane glycoconjugates and endocytic rate are not insurmountable obstacles for airway gene transfer. For enveloped viruses, incorporation of alternative viral proteins that mediate efficient entry across the apical membrane without altering endosomal processing and nuclear targeting appears to be a promising approach. One example is pseudotyping of lentivirus with the Filovirus envelope protein EboZ (62) that, in contrast to VSV-G or influenza hemagglutinin proteins, mediated efficient lentiviral entry. Also, other viral serotypes are being investigated and found to have differences in efficiency of viral entry; for example, AAV 5 and AAV 6 appear significantly more efficient than AAV 2 (63, 64). Lastly, novel replication-incompetent viruses that have greater tropism for airway epithelial cells than adenovirus or AAV, such as respiratory syncytial virus (65) and Sendai virus (66), have been developed and reported to mediate efficient gene transfer to human airway. These studies suggest that the progress toward identifying barriers to gene transfer will lead to one or more efficient vectors, advance the field to issues of safety and immunogenicity, and ultimately produce gene transfer techniques that may revolutionize therapy for airway diseases.

Received in original form June 10, 2002


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