Gene Therapy For Lung Disease Research Articles

Telomere Shortening and Its Association with Cell Dysfunction in Lung Diseases.

Telomeres are localized at the end of chromosomes to provide genome stability; however, the telomere length tends to be shortened with each cell division inducing a progressive telomere shortening (TS). In addition to age, other factors, such as exposure to pollutants, diet, stress, and disruptions in the shelterin protein complex or genes associated with telomerase induce TS. This phenomenon favors cellular senescence and genotoxic stress, which increases the risk of the development and progression of lung diseases such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, SARS-CoV-2 infection, and lung cancer. In an infectious environment, immune cells that exhibit TS are associated with severe lymphopenia and death, whereas in a noninfectious context, naïve T cells that exhibit TS are related to cancer progression and enhanced inflammatory processes. In this review, we discuss how TS modifies the function of the immune system cells, making them inefficient in maintaining homeostasis in the lung. Finally, we discuss the advances in drug and gene therapy for lung diseases where TS could be used as a target for future treatments.

Optimization of adeno-associated virus vector-mediated gene transfer to the respiratory tract.

An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken β-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.

Analysis of cis and trans Requirements for DNA Replication at the Right-End Hairpin of the Human Bocavirus 1 Genome.

Parvoviruses are single-stranded DNA viruses that use the palindromic structures at the ends of the viral genome for their replication. The mechanism of parvovirus replication has been studied mostly in the dependoparvovirus adeno-associated virus 2 (AAV2) and the protoparvovirus minute virus of mice (MVM). Here, we used human bocavirus 1 (HBoV1) to understand the replication mechanism of bocaparvovirus. HBoV1 is pathogenic to humans, causing acute respiratory tract infections, especially in young children under 2 years old. By using the duplex replicative form of the HBoV1 genome in human embryonic kidney 293 (HEK293) cells, we identified the HBoV1 minimal replication origin at the right-end hairpin (OriR). Mutagenesis analyses confirmed the putative NS1 binding and nicking sites within the OriR. Of note, unlike the large nonstructural protein (Rep78/68 or NS1) of other parvoviruses, HBoV1 NS1 did not specifically bind OriR in vitro, indicating that other viral and cellular components or the oligomerization of NS1 is required for NS1 binding to the OriR. In vivo studies demonstrated that residues responsible for NS1 binding and nicking are within the origin-binding domain. Further analysis identified that the small nonstructural protein NP1 is required for HBoV1 DNA replication at OriR. NP1 and other viral nonstructural proteins (NS1 to NS4) colocalized within the viral DNA replication centers in both OriR-transfected cells and virus-infected cells, highlighting a direct involvement of NP1 in viral DNA replication at OriR. Overall, our study revealed the characteristics of HBoV1 DNA replication at OriR, suggesting novel characteristics of autonomous parvovirus DNA replication. Human bocavirus 1 (HBoV1) causes acute respiratory tract infections in young children. The duplex HBoV1 genome replicates in HEK293 cells and produces progeny virions that are infectious in well-differentiated airway epithelial cells. A recombinant AAV2 vector pseudotyped with an HBoV1 capsid has been developed to efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to human airway epithelia. Here, we identified both cis-acting elements and trans-acting proteins that are required for HBoV1 DNA replication at the right-end hairpin in HEK293 cells. We localized the minimal replication origin, which contains both NS1 nicking and binding sites, to a 46-nucleotide sequence in the right-end hairpin. The identification of these essential elements of HBoV1 DNA replication acting both in cis and in trans will provide guidance to develop antiviral strategies targeting viral DNA replication at the right-end hairpin and to design next-generation recombinant HBoV1 vectors, a promising tool for gene therapy of lung diseases.

Recent Developments in Recombinant AAV-Mediated Gene Therapy for Lung Diseases

Recent studies have shed light on a number of important obstacles to safe and effective gene transfer to the respiratory tract with recombinant AAV vectors. Among these are blocks at the level of receptor binding and internalizations, evasion of proteasomal degradation, inefficiency of nuclear entry, and nuclear factors that inhibit the conversion of rAAV genomes into active double-stranded DNA form. Other important issues have been the size constraints of the vector, the lack of retention of episomal forms of the vector genome, and immune responses which may limit the efficiency of repeated doses of rAAV. Each of these potential obstacles has been addressed with new vector designs. In addition, the availability of an abundance of novel rAAV serotypes, each with its own receptor tropism, has expanded the range of possibilities for long-term success of gene therapy in the respiratory tract.

Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung.

High-molecular-mass polyethylenimines (PEIs) are widely used vectors for nucleic acid delivery. We found that removal of the residual N-acyl moieties from commercial linear 25-kDa PEI enhanced its plasmid DNA delivery efficiency 21 times in vitro, as well as 10,000 times in mice with a concomitant 1,500-fold enhancement in lung specificity. Several additional linear PEIs were synthesized by acid-catalyzed hydrolysis of poly(2-ethyl-2-oxazoline), yielding the pure polycations. PEI87 and PEI217 exhibited the highest efficiency in vitro: 115-fold and 6-fold above those of the commercial and deacylated PEI25s, respectively; moreover, PEI87 delivered DNA to mouse lung as efficiently as the pure PEI25 but at a lower concentration and with a 200-fold lung specificity. These improvements stem from an increase in the number of protonatable nitrogens, which presumably results in a tighter condensation of plasmid DNA and a better endosomal escape of the PEI/DNA complexes. As a validation of the potential of such linear, fully deacylated PEIs in gene therapy for lung diseases, systemic delivery in mice of the complexes of a short interfering RNA (siRNA) against a model gene, firefly luciferase, and PEI25 or PEI87 afforded a 77% and 93% suppression of the gene expression in the lungs, respectively. Furthermore, a polyplex of a siRNA against the influenza viral nucleocapsid protein gene and PEI87 resulted in a 94% drop of virus titers in the lungs of influenza-infected animals.

Chairman's Summary

The airways are exposed to a torrent of alien DNA in inspired air ranging from human nasal and bronchial secretions to pollens, house dust mite feces, animal danders, and flying insects. It is no surprise that the airways are well defended against the risk that alien DNA may reach the nuclei of airway cells and induce them to express the proteins that it encodes. These defense mechanisms are obstacles that must be overcome in inhalational gene therapy for lung disease. The barriers are many and include: (1) delivery to a distal lung site in a congested and obstructed airway—a topic covered in more depth under particulates; (2) access to the target cells, some of which lie in mucosal glands, not on the surface; (3) entry into the cytoplasm of a large and insoluble piece of DNA; (4) passage from the cytoplasm into the nucleus; and (5) stable, and ideally controllable, expression of the encoded protein. At a symposium I attended 10 years ago a leading molecular biologist declared, roundly, that we were “nowhere” in achieving any of these objectives. It is in some ways disappointing how little progress has been made in the interval—but there has been some. It has proved possible to transfect the nasal mucosa of mice with cystic fibrosis DNA using liposomes as a delivery vehicle, but the efficiency is so low that most discussants at the TAC doubted that this was a viable approach. Mechanical, electrical, or chemical techniques that cause a transitory increase in permeability can increase efficiency, but their practicality for use deep in the human lung is doubtful. Viral vectors including adenoviruses, adeno-associated viruses, and paramyxoviruses can achieve higher efficiency because they carry the biochemical machinery to deliver a relatively large payload across both the cell membrane and the nuclear membrane. But viruses have serious problems. The human immune response makes repeated administration problematic—although the extent of this problem varies with the virus. Virus-transfected cells may have reduced viability or become the target of killer T cells. Barring a tangential discovery—never to be discounted in science—a step-by-step approach will be necessary, probably using viral mechanisms for crossing the cell and nuclear membranes, but not a complete viable virus. Another suggestion was that ligands to internalized G protein–coupled receptors could be used, although there was doubt about the size of the possible payload. One discussant suggested moving the gene through the membrane in pieces and reassembling it in the nucleus—akin to the building of the space station in orbit and probably fraught with comparable levels of difficulty. Another discussant commented that we need something like a hundredfold improvement in transfection efficiency before we shall have an effective therapy.

122. Expression of hCD40 in the Pulmonary Endothelium Targeted Via a Fiber-Modified Adenovirus Improves Gene Delivery to the Lung

The lung is an attractive target for gene therapy since many diseases such as cystic fibrosis, lung cancer, primary pulmonary hypertension and acute respiratory distress syndrome require novel therapeutic approaches. However, pulmonary gene transfer by adenovirus (Ad) vectors via the vascular route has proven to be limited with respect to efficiency due to low expression of human coxsackie adenovirus receptor (hCAR) in the lung. Conversely, the liver expresses high levels of hCAR leading to ectopic transduction and the risk of liver damage. Therefore, development of CAR-independent gene transfer strategy to the lung is needed. We recently constructed an Ad in which the fiber was replaced by human CD40 ligand (Ad5 Luc FF / CD40L) and showed the utility of this fiber replacement strategy for genetic targeting of the virus to human CD40 (Belousova et al. J. Virol 77:11367–77, 2003). In addition, we recently showed the promoter for vascular endothelial growth factor receptor 1 (fms-like tyrosine kinase-1; flt-1) has high activity in pulmonary endothelial cells, low activity in hepatocytes (Nicklin et al. Hypertension 38:65–70, 2001), and also provided transcriptional targeting to the lung vasculature (Reynolds et al. Nat. Biotechnol.19:838–42, 2001). We hypothesized that hCD40 expression, transcriptionally targeted to the vascular endothelium via the flt-1 promoter, could provide CD40-targeted gene delivery to the lung in a CAR-independent manner. We constructed an E1-deleted recombinant adenovirus expressing hCD40 driven by the flt-1 promoter (Ad5flt1-hCD40). We then analyzed hCD40 expression and flt-1 activity in several cell lines by luciferase assay and fluorescence-activated cell sorting (FACS) analysis. We selected NTERA-2 cells as low hCD40 and flt-1 positive cell line, and HeLa cells as low hCD40 and flt-1 negative cell line as control for further experiments. Forty-eight hours after the first infection with Adflt1-hCD40 and the control virus, we quantitated hCD40 expression on the cell surface by FACS analysis. Adflt1-hCD40 infected NTERA-2 cells had markedly increased hCD40 expression compared to either control virus infected cells or flt-1 negative HeLa cells. Further, we evaluated infectivity enhancement gained by increased hCD40 in the flt-1 positive cell line. Forty-eight hours after the first infection, cells were infected with Ad5 Luc FF / CD40L vectors at an MOI of 1000 viral particles / cell to determine infectivity enhancement accrued via CD40 targeting. Luciferase assays were performed 24 hours post second infection. Ad5 Luc FF / CD40L vector infectivity in Ad flt-1 hCD40 infected NTERA-2 cells showed 13-fold greater luciferase reporter gene activities versus negative control virus infected cells. These data suggest that expression of hCD40 in pulmonary endothelial cells using recombinant Ad vectors containing hCD40 gene under the control of the flt-1 promoter and targeted with a novel fiber modified Ad vector can increase the efficacy and specificity of adenoviral gene therapy for lung disease.

The Status of Gene Therapy for Cystic Fibrosis

Cystic fibrosis (CF) has been a primary focus for gene therapy of lung diseases because the genetic cause is known and the airway epithelium is accessible for direct deoxyribonucleic acid (DNA) delivery. Soon after the mutated gene was identified in 1989, investigators demonstrated that transfer of a normal copy of the CF gene corrected ion transport abnormalities, thus validating the potential for use of gene therapy for this autosomal recessive disease. However, subsequent studies in a variety of in vitro and animal models, and more limited human studies, have revealed several obstacles to gene therapy for CF: (1) The incomplete understanding of CF lung disease pathogenesis, particularly the relative importance of ion transport and other cellular abnormalities (including glycoconjugate processing, pH regulation of intracellular organelles, and membrane trafficking), and of surface epithelial versus submucosal gland CF transmembrane regulator (CFTR) expression, generates uncertainty as to the necessary target cells for gene transfer and the optimum end point(s) for short-term human studies. (2) The airway epithelium has protective barriers against viral infection that impair gene transfer with several vectors, including recombinant viruses and DNA conjugates. Improvement in DNA transfer technology will be necessary for successful gene therapy. (3) Immune responses to recombinant viruses and inflammatory effects of bacterial DNA are only partially understood and appear to limit efficacy, particularly with repeated administration. Identification of these obstacles is prerequisite for progress, and recent studies with novel DNA delivery methods appear promising.

Gene therapy for lung diseases: development in the vector biology and novel concepts for gene therapy applications.

The lung represents an attractive target organ for somatic gene therapy strategy in that, (1) it is easily accessible by vectors, (2) most frequent hereditary disorders, cystic fibrosis (CF) and alpha1-antitrypsin deficiency (alpha1AT), occur in the lung, and (3) carcinoma of the lung is apparently a most common cause of death in humans. To date, approximately 400 clinical protocols for human gene therapy have been approved, and approximately 10% of the protocols target lung diseases such as cystic fibrosis (CF) and lung cancer. Currently available data from some of these human trials have successfully demonstrated that gene transfer to the human lung is possible, and that the strategy of overexpressing exogenous genes for curing or controlling lung diseases is potentially promising. In this manuscript, focusing on gene therapy of lung disorders, we aim to give an overview of the hurdles of current gene transfer strategies to overcome, then and also we aim to review recent, remarkable progresses in the vector biology that are potentially promising to maximize safety and efficiency of gene therapy. In addition, based on the most recent advances in the understanding of the molecular biological aspects of the pathogenesis of lung cancer, asthma, pulmonary fibrosis, and acute lung injury, novel therapeutic strategies of gene therapy for inflammatory and malignant diseases of the lung are discussed.

Development of cationic liposome formulations for intratracheal gene therapy of early lung cancer.

Regional (intratracheal or aerosol) delivery of cationic liposome-DNA complexes for gene therapy of lung disease offers distinct advantages over systemic (intravenous) administration. However, optimal formulations for early lung cancer treatment have not been established. Therefore, we investigated >50 different liposome and micelle formulations for factors that may affect their transcription efficiency and tested the ideal formulations in an in vivo mouse model. Our data showed that cationic liposomes were generally more effective at transfecting genes than were micelles of the same lipid composition, thus suggesting a role for the bilayer structure in facilitating transfection. In addition, the transfection efficiency of liposome-delivered genes was highly dependent upon the lipid composition, lipid/DNA ratio, particle size of the liposome-DNA complex, and cell lines used. By optimizing these factors in vitro and in vivo, we developed a novel liposome formulation (DP3) suitable for intratracheal administration. Using G67 liposome as control, we found that DP3 was more effective than G67 in vitro and as effective as G67 at both preventing lung tumor growth and prolonging survival in our lung cancer mouse model. We observed a positive correlation between the in vitro p53 function and the in vivo antitumoral activities of liposome-p53 formulations, which had not been reported previously in studies of an intravenous liposome gene delivery system. This correlation may facilitate the development and optimization of a liposome-p53 formulation for aerosol use in lung cancer patients.

Gene therapy for lung disease: hype or hope?

Gene therapy, the treatment of any disorder or pathophysiologic state on the basis of the transfer of genetic information, was a high-priority goal in the 1990s. The lung is a major target of gene therapy for genetic disorders, such as cystic fibrosis and alpha1-antitrypsin deficiency, and for other diseases, including lung cancer, malignant mesothelioma, pulmonary inflammation, surfactant deficiency, and pulmonary hypertension. This paper examines general concepts in gene therapy, summarizes the results of published clinical trials, and highlights areas of research aimed at overcoming challenges in the field. Although progress has been slower than anticipated, gene transfer has been safely achieved in patients with lung diseases. Recent advancements in understanding of the molecular basis of lung disease and the development of improved vector systems make it likely that gene therapy will be an important tool for the 21st-century clinician.

Gene therapy for lung disease.

Gene therapy is a new field of medical research that has great potential to influence the course of treatment of human disease. The lung has been a particularly attractive target organ for gene therapy due to its accessibility and the identification of genetic deficits for a number of lung diseases. Several clinical trials have shown evidence of low levels of gene transfer and expression, but without any benefit to the patients involved. Thus, current studies are focusing on further research and technological improvements to the vectors. Gene therapy is now beginning to benefit from a shift in emphasis from clinical trials to the development of better tools and procedures to deliver gene therapy to the bedside.

Cationic Lipids Enhance Cytokine and Cell Influx Levels in the Lung Following Administration of Plasmid: Cationic Lipid Complexes

Administration of plasmid/lipid complexes to the lung airways may be associated, in addition to expression of transgene, with a range of other responses. We report here the induction of cytokines and cellular influx in the lung airway following intratracheal administration of an N-[1-(2-3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol/plasmid positively charged complex in mice. We show that 1) the appearance of the Th1-associated cytokines IFN-gamma and IL-12 in bronchoalveolar lavage fluid is caused by unmethylated CpG dinucleotide sequences present within the plasmid, and is enhanced by the lipid formulation; 2) cationic lipids by themselves do not induce IL-12 or IL-12p40; 3) TNF-alpha is rapidly induced by cationic lipids and plasmid/lipid complex, but not by plasmid alone; 4) an acute cellular influx is induced by cationic lipid alone and by a plasmid/lipid complex, but to a much lesser extent by plasmid alone; and 5) plasmid methylation does not influence the degree of inflammatory cell influx. The induction of the innate immune responses by plasmid/lipid complexes may be advantageous to gene therapy of lung diseases. In particular, induction of the Th1 cell-promoting cytokines by plasmid/lipid complexes could, in conjunction with an expressed transgene, be used to modulate immune responses in the lung airways in disease conditions that are deficient in Th1 cell responses or that have a dominant Th2 phenotype. Alternatively, the elimination of immunostimulatory sequences in plasmids may improve the tolerability and/or efficacy of nonviral gene therapy, especially for diseases requiring chronic administration.

Optimization of plasmid vectors for high-level expression in lung epithelial cells.

Nonviral gene therapy approaches use a plasmid vector to express the desired transgene. We have systematically examined several regulatory elements within plasmid vectors that govern gene expression, e.g., the promoter, enhancer, intron, and polyadenylation signal, by constructing a series of plasmids that differed only in the particular sequence element being evaluated. Of the several promoters and polyadenylation signal sequences that were tested, the human cytomegalovirus (CMV) immediate early gene promoter and the addition of polyadenylation signal sequences from the bovine growth hormone (BGH) gene or rabbit beta-globin gene produced the highest levels of expression in vitro. The inclusion of a hybrid intron 3 to the promoter further increased expression 1.6-fold. The addition of a region of the CMV enhancer 5' to several weak promoters increased expression 8- to 67-fold, and co-transfection with a second plasmid encoding a chimeric transcription factor also enhanced expression. On the basis of these results, the CMV promoter, the hybrid intron, and the BGH polyadenylation signal were selected for consistent high level expression in vitro and in the mouse lung. However, expression was transient, with greater than 60% loss of activity in the first 7 days. This transient expression was not specific to CMV promoter-containing plasmids, because plasmids containing other heterologous promoters showed a similar profile of transient expression in vivo. These comparative analyses begin to provide a basis for the development of optimized expression plasmids for gene therapy of lung diseases.

Inefficient gene transfer by adenovirus vector to cystic fibrosis airway epithelia of mice and humans

The success of adenoviral vectors for gene therapy of lung disease in cystic fibrosis (CF) depends on efficient transfer of the complementary DNA encoding the correct version of the cystic fibrosis transmembrane regulator (CFTR) to the affected columnar epithelial cells lining the airways of the lung. Pre-clinical studies in vitro suggest that low doses of adenovirus vectors carrying this CFTR cDNA can correct defective Cl- transport in cultured human CF airway epithelia. Here we use mice carrying the disrupted CF gene to test the efficacy of this transfer system in vivo. We find that even repeated high doses can only partially (50%) correct the CF defect in Cl- transport in vivo and do not correct the Na+ transport defect at all. We investigated this discrepancy between the in vivo and in vitro transfer efficiency using CF mouse and human samples, and found that it reflects a difference in the susceptibility to adenovirus-5 transduction of the epithelial cell types dosed in vivo (columnar) and in vitro (basal-cell-like). These studies indicate that more efficient adenoviral gene-transfer vectors and/or refinement of dosing strategies are needed for therapy of CF lung disease.