Gene Therapy for Lung Disease: Hype or Hope?

Approximately 10 years ago, advances in molecular genetics and gene transfer technology made possible the development of gene therapy, modification of the genetic makeup of cells for therapeutic purposes. Although gene therapy was originally proposed for treatment of inherited recessive disorders in which transfer of a normal copy of a single defective gene might prevent the development of disease or slow its progression (1), it soon became clear that reasonable targets for gene therapy extended beyond traditional genetic disorders into the realm of acquired diseases, such as vascular diseases and cancer. The concept of gene therapy now encompasses the treatment of any pathophysiologic state on the basis of the transfer of genetic material, including complementary DNA, full-length genes, RNA, or oligonucleotides. Lung diseases are prominent among the disorders for which human gene therapy protocols have been approved (2). From a technical perspective, delivery to the lung can be accomplished by multiple approachesthrough the airway, by direct transthoracic injection, or through the dual pulmonary blood supply. From a disease perspective, the genetic basis for the two most common fatal inherited lung disorders, cystic fibrosis and 1-antitrypsin deficiency, is known, and current treatment regimens are supportive but not curative (3, 4). Several other pulmonary disorders, such as lung cancer, mesothelioma, pulmonary vascular diseases, radiation-induced lung injury, transplantation-induced lung injury, asthma, and pulmonary inflammation, are potential targets for gene therapy. Study Selection English-language articles were identified by searching the MEDLINE database from 1966 to the present using the terms gene therapy, lung, lung cancer, cystic fibrosis, 1 -antitrypsin deficiency, mesothelioma, retrovirus, adenovirus, adeno-associated virus, lentivirus, vaccinia virus, and liposome. Selected references cited in identified articles were reviewed. We focused on articles that described clinical trials. Because of space limitations, the references include excellent review articles that discuss the many in vitro and animal model experiments that form the scientific background for the clinical studies. Vectors Used in Gene Therapy Efficient gene transfer is a basic requirement for effective gene therapy. Various viral and nonviral gene transfer vectors are currently available (5-8). As summarized in Table 1, each vector has certain advantages in DNA-carrying capacity, types of cells targeted, efficiency of in vivo gene transfer, duration of expression, and induction of inflammation. It has become clear that no one gene delivery strategy will be suitable for all candidate disorders. Table 1. Characteristics of Gene Therapy Vectors The most widely used vector in lung gene therapy has been replication-incompetent recombinant adenovirus . This vector system offers many advantages, including high-efficiency transduction in a wide variety of target cells (including nondividing cells) and high levels of expression of the delivered transgene (8, 9). Of note, these vectors are stable in vivo, permitting direct delivery of the gene to many tissue sites, including the lung parenchyma and pleural space. The two primary disadvantages of adenoviruses are that they result in only transient gene expression and that when virions are used for direct in vivo applications, they elicit a prominent local and systemic inflammatory response. This inflammatory response, which includes an early innate immune response resulting in cytokine release and a late acquired immune response resulting in the generation of neutralizing antiadenoviral antibodies and cytotoxic T lymphocytes (10-15), has been the primary source of adenoviral vector toxicity and has limited the amount of vector that can be delivered. Gene Therapy for Inherited Pulmonary Disorders Cystic Fibrosis Cystic fibrosis results from mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) (4). The CFTR seems to play an important role in regulating the vectorial movement of fluid and electrolytes across various tissues and probably contributes to processes as diverse as mucus secretion, sialylation of surface receptors, sulfation of glycoconjugates, and local host defense (4, 16, 17). An early finding that made gene therapy for cystic fibrosis a potentially achievable goal was the observation that in a polarized epithelial sheet, expression of CFTR in as few as 6% to 10% of airway epithelial cells lacking CFTR restored normal chloride transport properties (18, 19). Because respiratory problems are responsible for most of the morbidity and mortality in persons with cystic fibrosis, the bulk of gene therapy research in cystic fibrosis has focused on methods of transfer to airway epithelium. Three vector systems have been used in clinical trials to date: adenovirus, adeno-associated virus, and liposomal/DNA complexes. Adenovirus To date, results of seven phase I clinical trials that used an adenoviral vector carrying the CFTR transgene have been published (Table 2). In the initial studies, the CFTR transgene was delivered by direct instillation in the nose or lung (20-22). In a randomized, vehicle-controlled, blinded study in 12 patients, Knowles and coworkers (23) studied gene transfer to nasal epithelium. Although no significant side effects occurred, gene transfer was inefficient and significant inflammation was noted at a dose of 2 1010 plaque-forming units, an estimated multiplicity of infection of 1000 (that is, 1000 infectious units of virus per cell). Furthermore, the investigators could not detect correction of sodium or chloride transport as measured by using nasal electrical potential difference. Zabner and colleagues (24) reported on the effect of repeated administration of recombinant adenoviral vector CFTR applied to nasal epithelium. Six patients received five escalating doses of adenovirus over at least 28 days. A humoral immune response was stimulated, gene transfer was inefficient, and functional correction was variable and seemed to be attenuated by subsequent administration of vector. To improve gene transfer, Bellon and coworkers (25) used an adenoviral vector CFTR (identical to that used by Crystal and associates [21]) to deliver aerosolized recombinant CFTR sequentially to the nasal epithelium and lung in 6 patients. Recombinant transcripts and CFTR protein were detected up to 15 days later in epithelial cells harvested from the nose and lung, although the amounts detected were low. In an effort to reduce immunogenicity, Zuckerman and colleagues (26) delivered a more crippled form of recombinant adenovirus, in which the E1 and E4 regions were deleted, to the lower respiratory tracts of 11 patients. As in the previous studies, gene transfer efficiency was low; gene transfer was detected in less than 1% of harvested bronchial epithelial cells at 4 days and 0% at 42 days. Systemic humoral immune responses to adenovirus were relatively mild, but cell-mediated responses (measured by lymphoproliferative responses to adenovirus) were stimulated in all patients. Dose-limiting toxicity was noted at 1011 viral particles. Table 2. Clinical Trials of Gene Therapy for Cystic Fibrosis Adeno-Associated Virus Adeno-associated viruses have been considered as gene therapy for cystic fibrosis for some time, but they were only recently used in human trials because of technical constraints related to production issues (32, 33). Adeno-associated virus is attractive because preclinical studies have suggested that long-term (up to 6 months) transgene expression can be obtained (32, 34) with little inflammatory response (35). Two phase I clinical studies have been approved by the National Institutes of Health, and to date, a summary of the results of one has been published. The first study (27, 28) involved a randomized, nonblinded, dose-escalation protocol in which a vector suspension was instilled into the maxillary sinuses of 10 patients with cystic fibrosis who had previously undergone bilateral endoscopic antrostomies (Table 2). Vector-derived DNA was detected in all participants who received more than 104 replication units. Although vector-derived RNA could not be detected in any of the specimens, transepithelial potential difference responses consistent with CFTR-mediated chloride transport were observed up to 14 days after instillation in patients receiving more than 5 104 replication units. The second study, which is ongoing, is examining dose-escalated administration of adeno-associated virus CFTR, both intranasally and to a single lobe of the lung (by bronchoscope); toxicity is the primary end point (36). Although the results from the sinus study are encouraging, questions remain about the efficiency of transduction of differentiated respiratory epithelium (37-39) and the long-term safety of adeno-associated virus vectors (40). Liposomes The liposomal vectors in clinical use are generally composed of cationic lipids mixed with cholesterol and dioleoylphosphatidylethanolamine. The liposome/DNA complexes were originally thought to be less immunogenic than viral vectors, although as more efficient complexes have been designed, it seems that these vectors can also generate significant inflammatory responses, probably because of the inherent immunogenicity of bacterial-derived DNA (41, 42). The results of three clinical trials (Table 2) have been published to date. Caplen and colleagues (29) conducted a double-blind, placebo-controlled study in which 9 patients with cystic fibrosis received nasal spray application of liposome/DNA complex and 6 such patients received control liposomes. The investigators observed vector-derived RNA in 5 of 8 patients and a transient, partial normalization of the response to low chloride perfusion during nasal potential difference measurements in the treated patients with cystic fibrosis (compared with controls