Gene Therapy for Lung Diseases

Introduction

Somatic gene therapy, a therapeutic strategy of transferring exogenous genetic information into patient cells, has opened many exciting possibilities for curing or controlling disorders in humans [1]. Application of gene transfer vector systems enabled to test the physiological functions of newly identified genes in vitro and in vivo, and the data is essential for developing novel therapeutic concepts targeting disorders. Although many preclinical studies in vitro and experimental animals suggest that the concept of gene therapy is promising, relatively few human protocols have successfully demonstrated that somatic gene therapy is superior to the conventional therapeutic approaches in humans. Apparently, this is mainly because currently available vector systems are still in their infancy. During the past decade, however, an enormous amount of data on the refinement, modification, and development of novel gene transfer techniques that are promising for clinical application has been accumulated. Moreover, understanding of molecular mechanisms of disorders in humans is remarkable. In this manuscript, therefore, we aim to review outcomes of clinical human gene therapy protocols targeting lung diseases, clarify the hurdles to overcome for successful therapy, and present recent progresses in gene transfer technology targeting the lung. Finally, feasibility of vector-mediated gene transfer strategies for various common disorders of the lung, such as cancer, asthma, lung fibrosis and acute lung injury are discussed.

Status of human gene therapy trials targeting lung disorders

To evaluate the feasibility of transferring exogenous genes as a novel therapeutic strategy to various disorders, lung represents an appropriate target in that, (1) it is easily accessible by vectors using fiberscope or aerosols, (2) most frequent hereditary disorders, cystic fibrosis (CF) and alpha1-antitrypsin deficiency (a1AT), occur in the lung, and (3) carcinoma of the lung is one of the most common causes of death in humans. As of April 2000, 368 protocols for human gene therapy trials have been approved in the U.S., and at least 36 of them are targeting lung diseases [2]. Twenty-three of those 36 protocols are for the respiratory manifestations of cystic fibrosis (CF), and 10 protocols are for the treatment of small cell, or non-small cell lung cancer. Two protocols have been approved for malignant mesothelioma.

Gene Therapy for CF

Gene therapy for CF is based on the hypothesis that transfer and overexpression of the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA in the airway epithelium may result in the improvement of the respiratory manifestations of the disease [3-14]. As an alternative for the airway epithelium of the lower airway, approximately half of the CF trials use nasal epithelium as therapeutic targets. This is primarily because of easy evaluation of the electrophysiological aspects of the CFTR function in nasal epithelium. Replication-deficient adenovirus (Ad), cationic liposomes, and adeno-associated virus (AAV) have been used to deliver the CFTR cDNA [3-14]. Use of Ad containing CFTR cDNA in patients with CF revealed that transfer and expression of the transgene has been demonstrated at a level theoretically sufficient to correct impaired chloride secretion lasting <30 days, however, results on the electrophysiological study to evaluate the CFTR function were not consistent. Strategy of repetitively administering the Ad for persistent CFTR expression demonstrated that, transgene expression was not detected after third administration of the vector, but that was not clearly correlated with humoral responses in the serum [4-6, 13-14]. Successful CFTR cDNA transfer has been also demonstrated by using cationic liposomes in the nasal and respiratory epithelial cells of CF patients lasting at least 1 week, with no apparent inflammatory reactions. Correction of the chloride permeability has also been reported using liposomes [10-12]. A recent study using AAV has demonstrated that administration of the vector into the maxillary sinus of patients with CF results in transgene expression lasting more than 2 weeks, with minimal inflammatory or immune responses [7-9].

Results of these CF gene therapy trials demonstrated that transfer and overexpression of exogenous genes in the airway by viral- or nonviral vectors is relatively safe and potentially promising, but that is not sufficient to provide therapeutic effects in humans at the present time. Based on the low-copy numbers of endogenous CFTR mRNA transcripts in the airway epithelium, and in vitro data showing that transfer of exogenous CFTR cDNA into only 10 % of the cells is sufficient to correct the defective chloride secretion in CF, all of the human protocols hypothesizes that currently available vectors can deliver sufficient amount of exogenous normal CFTR cDNA into human airway [15-17]. In contrast, recent studies have demonstrated that approximately all of the cells should be transduced with normal CFTR cDNA to correct sodium absorption of the airway epithelium in CF, and it still remains a matter of concern how much of the cells should be corrected in human airways [17-18]. Moreover, all of the ongoing CF gene therapy utilizes vector administration via airway route to transduce columnar, ciliated airway epithelial cells, but it is not known whether these cells are appropriate targets to be corrected. Importance of serous cells of the secretory glands in the development of respiratory manifestations is not established, however, these cells express abundant endogenous CFTR mRNA and protein [19]. If the serous cells are to be targeted, administration of the vector via airway route may not be appropriate, and vector administration via systemic circulation should be considered.

Prolonged expression of the transgene is another challenge to treat chronic disorders including CF. Liposome-mediated gene expression is associated with little or no inflammatory reactions in humans, and will allow repetitive administration of the vector [10-12]. In contrast, consistent with previous studies in experimental animals, high doses of Ad administration in the human can evoke inflammatory reactions, and efficiency of the repetitive administration of the same vector was limited by unknown mechanisms other than neutralizing antibody in the serum [4]. AAV-mediated gene expression could persist, but currently persistence of CFTR expression in human airway has not been completely understood [7-9].

Gene Therapy for Lung Cancer and Malignant Mesothelioma

Human gene therapy protocols for both non-small cell lung cancer and small cell lung cancer have been approved, and retrovirus, Ad, liposomes and vaccinia virus have been utilized to deliver exogenous genes into tumors of the lung [20-27]. Strategies to treat the cancer cells in the ongoing clinical protocols can be divided into two categories, (1) induction of cancer cell apoptosis by introducing normal p53 or other onco-suppressing genes into targets (mutation compensation), (2) treatment of cancer by inducing tumor-specific anti-tumor immune responses by interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-12 [21, 29-31]. Among the protocols performed, results from retrovirus or Ad-mediated p53 overexpression, together with or without co-administration of chemotherapeutic reagents (i.e. cisplatin) in non-small cell lung cancer, and outcome of ex vivo transfer of IL-2 gene into tissue infiltrating lymphocytes from patients with SCLC have been reported [20-27]. Using vectors expressing normal p53 cDNA, gene expression was detected in the tumor cells. In general, regardless of the vector employed, approximately 30% of the treated patients showed reduction in the tumor size, and another 30% showed stabilization in the clinical coarse. These phase I–II trials suggest that the concept of overexpressing p53 or other tumor-suppressive genes in the lung cancer is safe and potentially promising. The phase I trial of retroviral IL-2 gene transfer was also demonstrated safe, and resolution of pleural effusion was observed in some patients [21].

Malignant mesothelioma of the lung is also an attractive target for gene therapy based on the following: (1) tumor is localized within the thoracic cavity, and the vector can be easily administered simply via drainage tube, which also makes it easy to monitor the status of the lesion treated, (2) it is an aggressive neoplasm with poor prognosis (median survival of 1-2 years after initial diagnosis) [32]. To date, a total of four clinical protocols have been approved in the U.S. and Europe. The HSVtk gene can be categorized as “suicide gene” strategy. The transgene product, thymidine kinase phosphorylates gancyclovir (GCV), an antiviral drug for herpes simplex virus that is relatively nontoxic to humans. When GCV is injected into host where the HSVtk gene is expressed, the GCV is phosphorylated by the activity of the trangene product and that is rapidly converted to a toxic substance, GCV triphosphate by mammalian kinases. Results of phase I clinical trials of Ad-mediated HSVtk transfer have demonstrated that direct, intrathroracic injections of the Ad is safe, but that elicits strong humoral and cellular immune responses against the vector and transgene product [33-34]. Since results of phase II trials have not been reported, efficacy of the HSVtk plus GCV prodrug therapy strategy on mesothelioma has not been established.

In designing gene therapy for solid tumors of the lung, potential problem of strategies to introduce tumor-suppressor genes including p53 is that almost all of the tumor cells should be transfected with the expression cassette for maximal effectiveness. Again, limitation in the ability of Ad to repetitively transduce tumors in the host is another difficulty to use for clinical applications. Not only the refinement of the vector systems currently available, potentials of other therapeutic strategies to kill human lung cancer cells should be also evaluated. Following strategies may be promising in humans; (1) induction of tumor-specific immune reactions, (2) induction of “bystander effect”, that enable to kill untransduced cells by producing permeable toxic substances by the function of the transgene product, (i.e. HSVtk gene plus GCV in mesothelioma and glioma, adenosine deaminase plus 5-fluorocytosine in colon cancer, deoxycytidine kinase plus cytarabine in breast cancer), (3) disruption of angiogenic activity of cancer, (4) controlling metastatic activity of the cancer cells.

Evolving gene transfer techno-logies

Adenovirus Vectors (Ad)

One strategy to improve therapeutic potentials of human gene therapy would be to enhance safety and transduction efficiency of currently available vectors, and numerous data have been accumulated on the refinement of Ad. Despite the high transduction efficiency of Ad in vitro, Ad administration to the human airway is far less efficient in transferring exogenous CFTR cDNA in vivo [35]. This is primarily based on the low number of receptor molecules for Ad (Coxsackievirus and adenovirus receptor: CAR) on the apical surface of the ciliated, columnar epithelium. In vitro experiment has shown that by overexpressing CAR on the surface of target cells, transduction efficiency was improved [36-38]. Roelvink et. al. have demonstrated that cellular attachment of Ad serotype 5 is mediated by binding of the carboxyl-terminal knob of its fiber coat protein to the CAR, and modification of CAR- binding region enables redirection of the vector to a new receptor [39]. Such strategies of modulating vector tropism seem promising in enhancing transduction efficiency of the Ad [40-41]. However, even when the transduction efficiency of the vector is greatly improved, immune reaction of the host against the vector particle remains another hurdle for persistent transgene expression using Ad. Yang and other investigators have demonstrated that transduction of adenoviral genome sequences to the target, together with the transgene expression cassette, is responsible for development of class I-restricted CD4⁺ and CD8⁺ T cells that eliminate target cells transduced with the virus [42-45]. To prevent vector replication within host cells, E1 region of genome sequences is deleted in the currently used, so-called “first generation vectors” [46]. Expression of E1 gene is the initial step for the activation of other early and late genes necessary for genomic DNA replication and amplification of viral capsids, thus deletion in the E1 renders Ad replication-deficient. However, the low-level expression of the remaining adenoviral genome in the absence of E1 activation results in the MHC class I-restricted antigen presentation, and that eventually induces cytotoxic T lymphocytes. Based on these observations, Ad with further modification/deletion in vector genome (i.e.: mutated E2a, E4-deleted) have been developed, and resulted in some success [47-52]. Interestingly, Ad lacking its original genomic sequence, termed as “gutless”, “high-capacity Ad (HC-Ad)” or “gutted” vector has been developed [53-56]. Since the vector does not contain any adenoviral genome, there is no chance of viral capsids or proteins essential for DNA replication to be generated. Theoretically, inflammation mediated by the viral genome products-specific CTL responses will not be induced, thus the safety and persistence of Ad will be greatly improved. Using a HC-Ad containing the 19-kb human a1-antitrypsin (a1AT) gene locus, physiologic level of serum a1AT was detected in mice >1year [55]. Importantly, the transcription of the transgene was initiated from the liver specific promoter and not from the macrophage specific promoter of the genomic sequences delivered. Although HC-Ad administration does not prevent activation of the innate immune response that occurs immediately after the vector administration and development of anti-vector neutralizing antibody, gene transfer by the HC-Ad did not evoke significant hepatotoxicity [55-56].

Another strategy to improve persistence of Ad-mediated transgene expression is to modulate antigen-specific T cell responses by immunosuppressive agents. Effects of cyclosporine, FK506, inhibitors of T cell costimulation, anti-CD40 ligand, and inducing anergy to the Ad by coadministration of CTLA4 ab or injecting anti-CD4 antibody to suppress the adoptive host immune responses against the vector have been reported [57-61]. Development of neutralizing antibody to the Ad has been reported in experimental animals and humans as barrier for efficient gene transfer. In this context, Yang and Wilson have demonstrated that pre-administration of IL-12, a cytokine known to down-modulate proliferation of IL-4-mediated, type I helper T cells, suppresses antibody production, and that allows efficient, second gene transfer by the vector in mice [62]. Other strategies that decrease humoral immune response, such as cyclophosphamide, FK 506, and anti-CD4 have also been tested in experimental animals [63]. In this regard, it has been demonstrated that use of Ad based on different serotypes and species of Ad have been proposed to improve efficient, repetitive vector administration (“sero-switch” strategies) [64-65]. Induction of tolerance to the Ad may be an option to reduce toxicity and enhance persistent transgene expression in the target organ. It has been also demonstrated by intrathymic injection, or oral ingestion of viral antigens [66-67].

In the lung, approximately 70-80% of the Ad genome is lost within first 24 hr following the vector administration given via airway [68]. Depleting the alveolar macrophages with liposomes prevented the rapid loss of the virus, thus supporting the concept that innate immune responses represent a major hurdle to overcome for efficient gene transfer. In addition to the destruction of the virus, activation of the innate immune systems in response to the Ad result in a prominent inflammatory reactions. In experimental animals, single intratracheal administration of Ad results in a rapid increase in the elevation of proinflammatory cytokines such as IL-1b and TNF-a [69-71]. Patients with CF receiving Ad expressing CFTR cDNA have experienced transient fever that was associated with elevated serum IL-6, a cytokine known inducible by the IL-1b and TNF-a [72]. Consistent with this observation, majority of patients with lung cancer treated with Ad containing normal p53 gene experienced a transient fever that lasts 1-2 days. Wolff et. al. have demonstrated that depletion of Kuppfer cells by liposomes dramatically decreases IL-6 elevation in the serum following intravenous Ad administration, and that was associated with much prolonged transgene expression in the liver [73]. Transient elevation in the body temperature has not been emphasized as major side effect in p53-mediated cancer gene therapy studies, but the use of Ad for inflammatory lung diseases including pulmonary fibrosis, acute lung injury, asthma represent theoretical concerns that superimposing a large amount of inflammatory cytokines on the host may result in exacerbations of the status of the diseases. Activation of the innate immunity by Ad has been demonstrated inducible by vector particles with their original genome sequences destroyed by irradiation. In this regard, a recent study successfully demonstrated that coadministration of neutralizing antibodies to the soluble TNF receptor results in significant reduction in the vector toxicity [74].

In designing cancer gene therapy using tumor-suppressor genes or genes related to cell cycle regulation, therapeutic potentials of Ad would be further improved when the virus is allowed to replicate specifically within tumor to transduce neighboring cells where the transgene was not transferred by the direct Ad injection. In this context, it has been demonstrated that administration of a second vector coding for E1a region of the adenovirus permits localized amplification of the vector in vivo, and trials to treat tumors with p53 expressing adenovirus capable to replicate within tumors are ongoing [75].

Cationic Liposomes

Next to Ad, liposome-mediated gene transfer has been widely utilized for human gene therapy trials for lung disorders. Majority of the protocols using liposomes target CF, with lesser number of protocols for lung cancer where the liposome-DNA complex is directly injected into the lung. The liposome has many advantages over other gene transfer strategies using recombinant viruses. For example, (1) there is no limitation in the size of DNA to be transfected, (2) preparation of the liposome and DNA is easy, and (3) despite cationic liposomes can be toxic when administered at high doses in cell culture, no toxicity or inflammatory reactions have been reported by using liposomes in humans [76]. Cationic liposomes, a form of liposomes most widely used for gene transfer, is positively charged, and efficiently complex negatively changed DNA plasmids into small and colloidally stable particles. Efficiency of liposome-mediated gene transfer is affected by many factors. In vitro, overall charge density determined by DNA:liposome ratio, size, shape and stability of the complex, and lipid composition, are major factors to be optimized in each cationic lipids developed. Many novel cationic lipids are being engineered, however, no clear QSAR (quantitative structure-activity relationships) are established yet. Nevertheless, recent development in the field resulted in more than thousand-fold increase in gene expression over the initial experiments.

Based on the ability of cationic lipid-based DNA complex to transfect lung epithelial cells upon local intratracheal instillation, and endothelial cells in the lung following its systemic administration, it is quite reasonable to use liposomes for gene therapy of various lung disorders. For direct installation of the liposomes to the lung, as in the case of Ad administration, majority of the liposomes are entrapped, and degraded by macrophages [74]. To enhance liposome-mediated gene transfer, the vector should interact readily with epithelial cells and have the ability to destabilize cell membranes to promote intracellular delivery of the DNA. Blocking of the mononuclear phagocytic system by doxorubicin or pre-administration of empty liposomes may be a partial solution, but apparently, these are not appropriate strategies for clinical applications where the vector will be repetitively administered into targets with ongoing disease processes such as various inflammation/immune reactions [77]. In considering systemic injection of the liposome complex, stability in the serum and biodistribution needs to be improved for successful gene transfer. When the stability of the liposome complex in the serum is improved, that allows long-term circulation, and gene transfer efficiency to the target should be enhanced [78]. This is particularly important when designing gene therapy strategies to the metastatic cancer of the lung. Vascularture of the neoplasms is characterized by wide interendothelial gaps, large number of fenestrate and transendothelial channels formed by vesicles, and discontinuous or absent basement membrane, and most importantly, that is more permeable than that of normal tissue. In this regard, liposome-mediated gene transfer may hold great promise. By incorporating PEG derivatives into the liposomal membrane, it has been demonstrated that plasmid DNA accumulates more efficiently in the tumor in a murine model [79]. Such liposomal membranes could be modified to have a targeting ligand attached on the far end of the polymer chain for targeted gene delivery [80].

Adeno-associated Virus (AAV) Vector

AAV is a family of the Parvoviridae, which are among the smallest DNA viruses with simplest structure. AAV contains a linear, single-strand DNA genome of 4.6kb. AAV is a defective virus (adenovirus or herpes virus) that requires a helper virus for replication. In the absence of helper virus, AAV integrates preferentially to a specific locus on human chromosome 19, and in the presence of helper virus, AAV genome is rescued from genome and formation of AAV is initiated [81-85]. Recombinant AAV (rAAV) is an attractive gene transfer vector in that; (1) infection with wild AAV is nontoxic to humans, although AAV is widespread, and (2) AAV has the ability to infect dividing or quiescent cells. Recombinant AAV lacks any coding sequences for viral structural proteins, therefore, vector does not express any protein that evokes prominent immune responses in the host. To amplify the rAAV, conventional protocols of preparing rAAV requires helper virus infection, and the generated rAAV requires purification from the helper virus by cesium chloride (CsCl) ultracentrifugation. However, preparation of rAAV is now possible by using a novel helper-free method that is free from contamination of helper viruses, and can be relatively easily scaled up for human trials [86]. Persistent expression of exogenous gene using rAAV has been demonstrated in the muscle, brain, and lung [87-92].

Hybrid Vector

Based on the fact that retrovirus vector can be rapidly inactivated in vivo, Ad has been most widely used in clinical trials for cancer gene therapy. However, exogenous genes transferred by Ad locate in epichromosomal fashion, and together with inflammatory reactions targeting the transduced cells with the Ad genome, transgene expression is transient. As a novel approach, a chimeric vector that has favorable aspects of both Ad and retrovirus has been developed. Ad induces target cells to function as transient retroviral producer cells in vivo. The progeny of retroviral vector particles can then effectively achieve stable transduction of neighboring cells [93-94].

Lentivirus Vector

Lentivirus is one of the three classifications of Retroviridae, and that include a variety of primate (human immunodeficiency viruses [HIV-1 and HIV-2] and simian immunodeficiency viruses [SIV]) and non-primate (for example, maedi-visna virus [MVV], feline immunodeficiency virus [FIV]) viruses. The ability to integrate into the genome of non-dividing cells makes lentivirus vector an attractive gene transfer vehicle for gene therapy. It has been demonstrated that lentivirus vector can transduce macrophages and airway epithelial cells [95]. A recent report by Wang et. al. demonstrated that terminally differentiated nondividing airway epithelial cells allow exogenous CFTR cDNA transfer and expression by a FIV-based lentivirus in vitro, and reporter gene expression was observed in 1-14% of adult rabbit airway epithelia in vivo [96]. Importantly, the gene expression persisted and cells with progenitor capacity were also targeted. These data clearly demonstrates that lentivirus vector may be feasible for human gene therapy for respiratory disorders including CF. Other potential targets of non-dividing cells for gene therapy include hepatocytes, neurons, hematopoietic stem cells and myocytes.

Epstein-barr Virus (EBV) Vector

Vectors based on components of EBV have ability to establish virus in the nucleus in a latent state as a circular, multicopy, extrachromosomal replicating plasmid. The vector replicates once per cell cycle, in synchrony with the host chromosomes, by using the host replication apparatus. Retention rates of the vector within nucleus ranging 92-98% per cell generation are far superior to the rapid loss of conventional plasmid vector system [97-100]. EBV vector has been demonstrated to chronically express normal human CFTR cDNA in transformed dividing airway epithelial cells >2 months. Based on the ability to stably transfect respiratory cells, and to replicate one copy per cell cycle, EBV vector is an attractive gene transfer vehicle for cancer gene therapy [101-102].

HJV-liposomes

HJV-liposome was developed by combining liposomes with fusion proteins derived from the hemagglutinating virus of Japan (HVJ or Sendai virus) [103-104]. In this delivery system, DNA containing transgenes is packaged in a liposome that comprises phospholipids and cholesterol. The liposome is fused with UV-inactivated HVJ to form the HJV-liposome, which can fuse with the cell surface to deliver the DNA directly to the cytoplasm. Importantly, the HJV-liposome is less immunogenic and less toxic than the currently available Ad. In rodents, detectable levels of antibody against the vector, and induction of cytotoxic T cells against transduced cells can be developed following vector administration, but that was not sufficient to inhibit repetitive administration of the same vector [105]. This indicates HJV-liposome system may be suitable for various inflammatory lung diseases such as lung injury or CF [106-107]. Interestingly, further refinement on transgene expression efficiency has been demonstrated. By utilizing the Epstein-Barr virus replicon apparatus that support stable localization of the transferred DNA in the nucleus, level and persistence of reporter gene expression has been dramatically improved, thus suggesting the concept of using this system for cancer gene therapy is promising [108].

Novel concepts of gene therapy targeting lung diseases

Emerging evidences clearly suggest that development of non-toxic, highly efficient gene transfer vehicles is promising. Together with recent progresses in the understanding of molecular mechanisms of a variety of lung disorders, there are several new concepts that seem appropriate to be tested as new strategies for gene therapy for lung diseases.

Lung Cancer

Cancer gene therapy strategies can be classified as follows, (1) introduction of tumor suppressor genes or genes that inactivate oncogenes, (2) introduction of sensitivity genes (suicide gene therapy), (3) immunotherapy, and (4) strategies targeting tumor vascularture.

Remarkable regression of NSCLC by using p53-expressing vectors in humans, despite poor transduction efficiency, has been explained not only by the apoptosis induced by the transgene expression, but by the “bystander effect”, probably through anti-angiogenetic property of p53 [109]. Upon overexpression of p53, synergistic tumor suppressing effect has been reported by irradiation, coexpressing p21, or treating cells with cisplatin [110-115]. Using antisense strategies, L-myc, c-myc, and K-ras transfer have been effective in vitro. As an alternative to the antisense strategy to inactivate oncogenes, introduction of vectors encoding neutralizing antibody fragment against the oncogenes has been demonstrated feasible, although this strategy requires efficient, long-term expression within cells [116-118].

Similar to the concept of utilizing HSVtk gene plus GCV for mesothelioma, Kojima et.al. have developed a cancer gene therapy strategy for lung cancer to convert Irinotecan, a camptothecin analogue that functions as an inhibitor of mammalian DNA topoisomerase I by Ad-expressing carboxyl esterase (CE) cDNA [119]. Irinotecan is now commercially available, and large-group, randamized trials on the efficacy of using this agent with cisplatin are ongoing [120]. CE converts Irinotecan into SN-38, a 1000-fold more potent compound in inhibiting topoisomerase I, and Ad-mediated expression of CE significantly suppressed growth of human lung tumors established in mice. Based on the favorable preliminary outcomes of clinical data using Irinotecan plus cisplatin, coadministration of cisplatinum plus Irinotecan in the presence of CE overexpression may provide further benefit.

Cancer immunotherapy aims to kill tumor cells in vivo by enhancing anti-tumor immunity of the host, or by abrogating suppression of antitumor immunity mediated by the factors secreted from the tumor. Cytokine therapy is to induce, or up-regulate host immune responses against the established tumors by overexpressing cytokine cDNA. IL-7, 12, interferon (IFN)-g, GM-CSF are examples of cytokines tested in preclinical studies [29-31]. The other strategy of utilizing cytokine overexpression is to inject genetically modified, irradiated tumor cells with cytokine cDNA expression cassette such as GM-CSF as vaccine to induce anti-tumor immunity. It has been suggested that the GM-CSF induces differentiation and proliferation of dendritic cells (DC), most potent, professional antigen presenting cells (APC) [121-122]. Recent studies have also demonstrated that transfer and expression of exogenous genes in the DC is possible, and genetically modified DC has the ability to induce strong CTL activity against the transgene product [123]. This strategy will be useful for human gene therapy when appropriate genes coding for antigenicity of the lung cancer are identified.

As described elsewhere in the text, establishment of vascular network is essential for the growth of tumors in vivo. Among the cytokines with angiogenic activities, preclinical studies have demonstrated that growth of tumor cells can be dramatically suppressed by overexpressing soluble receptor for VEGF, suggesting signaling mediated by VEGF receptor is essential for neovascularizaion in the tumor [124]. Importantly, local administration of Ad expressing soluble form of the VEGF receptor (flt-1) resulted in therapeutic effect that was localized within the organ or tumor where the vector was injected. Therefore, expression of the soluble flt-1 in the systemic circulation will not affect basal, normally required physiologic levels of neovascularization [125].

Telomerase is critical for elongation of telomere that shortens when mitosis occurs. RNA is associated with the telomerase protein, and that binds to the telomere of genome DNA as template, and the enzymatic activity elongates the shortened telomere. Cells cannot divide and proliferate with shortened telomere, therefore, the activity is essential in determining life span of each cell [126-127]. Interestingly, high levels of telomerase activity can be detected in high percentage of tumor cells including lung cancer, while that is at low-levels in normal tissues except for bone marrow and testis. It has been demonstrated that administration of oligonucleotides coding for antisense sequence of the RNA component of the telomerase commits HeLa cells to death after a limited number of cell cycles [128]. Using appropriate gene transfer technology, strategies to reduce telomerase activity may be feasible for clinical applications [129].

As an alternative of using locally replicating Ad to enhance transduction efficiency in vivo, feasibility of human reovirus as cytotoxic agent against cancer has been reported. The human reovirus infection in humans is mild and restricted to the upper airway and gastrointestinal tracts. Interestingly, Coffey et. al. have demonstrated by using the characteristics of the virus to require activated Ras signaling pathway, inoculation of the virus into murine model of tumor that was transformed with ras. Since activating-mutations of Ras has been reported in 40% of lung cancer, and the virus infection seems safe and replicates specifically in the tumor, use of reovirus would be useful for replicating vector for lung cancer in which ras pathway is activated [130].

Asthma

Asthma, one of the most popular airway disorders in man, is manifested by recurrent episodes of airflow limitation. Allergic, eosinophilic inflammation of the airway is considered as the basic mechanisms of the disease [131-132]. For the establishment of allergen-specific immune responses, induction and proliferation of type II helper T cells (Th2) that produce IL-4 and 5 is essential. This is in contrast to the proliferation of type I helper T cells (Th1) under mycobacterial infection that preferentially produce IL-2 and IFN-g, and relative balance of the Th1 and Th2 is critical to determine the magnitude of Th2-mediated, allergic responses against allergens [133-134]. Consistent with this concept, intratracheal administration of IFN-g or IL-12 (Th1 cytokines) has been demonstrated to suppress eosinophils recruitment into the airway following allergen challenges [135-136]. Systemic administration of the IL-12 is also demonstrated capable of preventing allergic sensitizations in mice [137]. In this regard, it has been demonstrated that vaccinia virus vector-mediated overexpression of exogenous IL-12 in the airway results in suppressed eosinophils migration into the murine airway [138]. Overexpression of the Th1 cytokines would be one strategy to suppress allergy using gene transfer strategies.

Nucleotide sequences from bacteria containing repetitive C and G, termed as CpG motif have been characterized as an inducer of strong Th1 responses, and oligonucleotides containing the sequences are known to inhibit development of allergy in experimental animals [139]. Although the mechanisms of CpG to shift the Th1/Th2 balance is not totally understood, it has been recently demonstrated that simultaneous administration of antigen and CpG is essential to induce strong Th1 immune responses [140]. Adenovirus particle or attenuated form of Mycobacterium also have the ability to down-modulate allergic responses in vivo, but components or DNA sequences pivotal to suppress allergy have not been identified. In contrast, a recent report has demonstrated that correction of Th1/Th2 balance did not decrease airway hyperresponsiveness (BHR) that has been established [141]. In this regard, using antisense strategy, blockade of adenosine receptor of the airway epithelial cells results in a significant decrease in airway hyperresponsiveness in rabbits [142].

Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive disorder with poor prognosis, with survival rate of approximately 50% within 5 years after the initial diagnosis [143]. Although causes of IPF has not been identified, recent reports have suggested that apoptosis of alveolar epithelial cells via Fas-mediated pathway is involved in the mechanisms of the disease [144-146]. Strategies to prevent apoptosis of respiratory epithelial cells may be useful to treat IPF. Recently, it has been demonstrated that Ad-mediated hepatic growth factor (HGF) expression in the serum or locally in the lung also prevented collagen deposition following bleomycin treatment. Importantly, post-treatment of the Ad containing HGF cDNA was still capable of suppressing development of fibrosis in the lung [147]. Another group has reported that administration of Ad containing smad gene, an antagonist of transforming growth factor-b (TGF-b), via airway route, resulted in attenuation in the murine model of bleomycin-induced lung fibrosis [148]. These observations suggest that growth factor is a potential target for gene therapy of the disease. Another strategy to treat IPF is to shift Th1/Th2 imbalance in the lung. Interferon-g, a Th1 cytokine, has been demonstrated to suppress expression of TGF-b and connective tissue growth factor (CTGF), cytokines known to induce proliferation of fibroblasts. Defective IFN-g expression in alveolar macrophages from patients with IPF has been demonstrated [149]. Moreover, animal experiments have demonstrated that systemic administrations of Th1 cytokines prevent lung fibrosis induced by bleomycin [150]. Consistent with this, in a preliminary trial on patients with IPF, repetitive administration of IFN-g-1b resulted in improvement of lung volumes, arterial oxygen levels, and decreases in TGF-b and CTGF mRNA transcripts [151]. Strategies to enhance Th1 immune responses described in the former section “Asthma” would be potentially feasible when non-toxic vectors that allow repetitive administration are available for gene therapy of IPF.

Acute Lung Injury

Acute lung injury is caused by many conditions including sepsis, multiple trauma, and pancreatitis manifested by generalized intravascular activation of inflammatory cells with endothelial or parenchymal cell injury [152]. Based on the knowledge that pathophysiology of such inflammatory response is characterized by high levels of pro-inflammatory cytokines, strategies to enhance effects of anti-inflammatory cytokines, or to antagonize pro-inflammatory cytokines have been developed [153]. Overexpression of soluble TNF-a receptor by Ad effectively protected mice from LPS-induced sepsis [154]. Antisense strategies using antisense oligonucleotide against pro-inflammatory cytokines, or adhesion molecule important for the development of inflammation (IL-1, IL-1receptor, TNF-a, ICAM-1) have been also successful in vivo [155-157]. Another strategy to protect lung is based on the fact that high levels of toxic, reactive oxygen species are present in the local milieu where acute inflammation is ongoing. In this regard, transfer of prostaglandin G/H synthase has been demonstrated a potentially promising strategy in an experimental model [158].

Studies on specific signal transduction pathways downstream to cytokine receptors provide wider applications of gene therapy strategies targeting lung injury. For example, a recently identified, mitogen-activated protein kinase (p38MAPK) is activated in cells stimulated with LPS, TNF-a, UV radiation, hydrogen peroxide, and cell wall components from gram-positive bacteriae. P38MAPK is required for activation of a heat shock protein or in the up-regulation of inflammatory cytokines, thus suggesting its essential role in regulating various acute or chronic host inflammatory responses [159-161]. In this regard, carbon monoxide (CO), a gaseous mediator known to attenuate neutrophilic inflammation in response to LPS or hyperoxia, has been demonstrated to modulate p38MAPK activation [162]. When macrophages are cultured under low-concentration of CO, production of TNF-a from the cells under LPS stimulation was decreased, while that of IL-10 was significantly enhanced. CO is a byproduct of degradation process of heme by heme oxygenase (HO), and more importantly, increased survival of rats under hyperoxia was observed by preadministration of Ad expressing the inducible form of HO (HO-1) given intratracheally [163]. Based on these observations, it seems likely that transfer of the HO-1 in the lung may be promising to modify ongoing acute inflammatory processes in humans. Moreover, a recent report showing that pharmacologic inhibition of the p38MAPK dramatically alters IL-12 expression in macrophages or dendritic cells, which is consistent with the concept that the p38MAPK pathway is a logical target for modulating not only innate immune reactions, but also adoptive immunity and allergic disorders including asthma [164].

Conclusions

Current human gene therapy protocols have been resulted in a limited success in demonstrating that human gene transfer strategies to treat human disorders are more beneficial than conventional therapies. However, the concept of using genes as drug has been demonstrated feasible, and vector systems are being evolved at a remarkable rate. Theoretically, it is now obvious to develop less-immunogenic vectors with minimal toxicity that permits chronic expression by combining viral- and non-viral vector systems. For physiological, regulated expression, artificial chromosomes containing gene of interest may be an appropriate choice of expression cassette. Last but not least, complete understanding of disorders that clarify most appropriate molecular targets to manipulate is essential to develop “optimized” therapy strategies to deliver appropriate transgenes into appropriate cells in an appropriate manner. In the next decade, we will be able to see results from clinical trials that are much closer to the clinical application.

Acknowledgement

This work was supported in part by a grant from the Smoking Research Foundation.

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