Gene therapy progress and prospects: alpha-1 antitrypsin.

Over the 2 years covered here, there has been one clinical study in which a normal alpha-1 antitrypsin (AAT) gene was delivered to the nasal epithelium of AAT-deficient subjects using plasmid-liposome complexes; a second study using an adeno-associated vector should begin soon. Although progress in clinical studies has been slow, advances in both viral and nonviral vector designs show considerable promise. Strategies that combine liposome technology with imaginative vector design may permit long-term expression of a normal transgene that is sufficient to achieve therapeutic serum AAT concentrations. While reproducing the normal physiology by targeting normal AAT gene expression to the liver is logical, local expression in lung cells may be less demanding of the technology and offers therapeutic benefits that are produced neither by AAT protein therapy nor by AAT gene therapy targeted to the liver. Developing technologies may permit direct correction of the mutant AAT gene using innovative approaches to in vivo gene repair.

Dysregulated cytokine production in human cystic fibrosis bronchial epithelial cells.

Although pulmonary inflammation is an important pathologic event in cystic fibrosis (CF), the relationship between expression of the CF gene and the inflammatory response is unclear. We studied tumor necrosis factor (TNF) alpha and IL-1beta stimulated production of IL-6 and IL-8 by CF, corrected CF, and normal human bronchial epithelial cells in culture. During the first 24 hours of TNFalpha stimulation, CF cells produced significantly more IL-8 than normal or corrected CF cells. In the second 24 hours of TNFalpha stimulation, IL-6 and IL-8 generation ceased in normal and corrected CF cells but accelerated in CF cells, resulting in marked IL-6 and IL-8 accumulation in CF cells. Similar results were found when cells were stimulated with IL-1beta. Finally, when CF cells were grown at 27 degrees C (a culture condition which results in transport of CF transmembrane conductance regulator, CFTR, to the cell membrane and normalization of chloride conductance) TNFalpha-stimulated production of IL-6 and IL-8 reverted to normal. We conclude that dysregulation of cytokine generation by CF bronchial epithelial cells is directly related to expression of mutant CFTR and these observations provide a potential mechanism for persistence of airway inflammation in CF.

Exaggerated activation of nuclear factor-kappaB and altered IkappaB-beta processing in cystic fibrosis bronchial epithelial cells.

In cystic fibrosis (CF), inflammatory mediator production by airway epithelial cells is a critical determinant of chronic airway inflammation. To determine whether altered signal transduction through the nuclear factor (NF)-kappaB pathway occurs in CF epithelial cells and results in excessive generation of inflammatory cytokines, we evaluated tumor necrosis factor (TNF)-alpha-induced production of the NF-kappaB-dependent cytokine interleukin (IL)-8 and activation of NF-kappaB in three different human bronchial epithelial cell lines: (1) BEAS cells that express wild-type CF transmembrane conductance regulator (CFTR), (2) IB3 cells with mutant CFTR, and (3) C38 cells, which are "corrected" IB3 cells complemented with wild-type CFTR. Treatment of cells with TNF-alpha (30 ng/ml) resulted in markedly elevated NF-kappaB activation and production of IL-8 by IB3 cells compared with BEAS and C38 cells. Despite the differences in NF- kappaB activation, no differences in basal levels of IkappaB-alpha or TNF-alpha- induced IkappaB-alpha processing and degradation were detected among the cell lines. In contrast, the basal level of IkappaB-beta was increased in the IB3 cells. Treatment with TNF-alpha resulted in increased formation of hypophosphorylated IkappaB-beta and increased nuclear localization of IkappaB-beta in IB3 cells compared with the other cell types. These findings provide additional evidence of a dysregulated inflammatory response in CF.

Hepatic cryoablation-induced acute lung injury: pulmonary hemodynamic and permeability effects in a sheep model.

HYPOTHESIS: Hepatic cryoablation of 30% to 35% or more of liver parenchyma in a sheep model results in eicosanoid and nuclear factor-kappaB (NF-kappaB)-mediated changes in pulmonary hemodynamics and lung permeability. SETTING: Laboratory. INTERVENTIONS: At initial thoracotomy, catheters were placed in the main pulmonary artery, left atrium, right carotid artery, and efferent duct of the caudal mediastinal lymph node for subsequent monitoring in adult sheep. After a 1- to 2-week period of recovery, animals underwent laparotomy and left-lobe cryoablation (approximately 35% by volume) with subsequent awake monitoring and on postoperative days 1 to 3. MAIN OUTCOME MEASURES: Cryoablation-induced lung permeability and hemodynamic changes were compared with baseline values in sheep that underwent instrumentation. Similarly handled sheep underwent resection of a similar volume of hepatic parenchyma or had pulmonary artery pressure increases induced by mechanical left atrial obstruction. Activation of NF-kappaB was assessed with electrophoretic mobility shift assay, and serum thromboxane levels were measured with mass spectroscopy. RESULTS: Cryoablation resulted in acutely increased mean pulmonary (20 to 35 cm water) and systemic pressures, which returned to baseline at 24 hours with no change in cardiac output. Serum thromboxane levels increased 30 minutes after cryoablation (9-fold) and returned to baseline at 24 hours. Activation of NF-kappaB was present in liver and lung tissue by 30 minutes after cryoablation. Lung lymph-plasma protein clearance markedly exceeded the expected increase from pulmonary pressures alone, and increased lymph-plasma protein ratio persisted after pulmonary artery pressures normalized. Similar changes were not associated with 35% hepatic resection. CONCLUSIONS: This study demonstrates that 35% hepatic cryoablation results in an acute but transient increase in pulmonary artery pressure that may be mediated by increased thromboxane levels. Increases in pulmonary capillary permeability are not accounted for by pressure changes alone, and may be a result of NF-kappaB-mediated inflammatory mechanisms. These data show that cryosurgery causes pathophysiological changes similar to those observed with endotoxin and other systemic inflammatory stimuli.

Transfection of nasal mucosa with a normal alpha1-antitrypsin gene in alpha1-antitrypsin-deficient subjects: comparison with protein therapy.

We sought to determine whether a normal alpha1-antitrypsin (AAT) gene could be expressed in respiratory epithelium and whether local expression would have antiinflammatory effects. In an unblinded study, we delivered a normal AAT gene in a plasmid-cationic liposome complex to one nostril of each of five subjects with AAT deficiency; the other, untreated nostril served as a control. AAT protein concentration in nasal lavage fluid (NALF) increased in the transfected nostril (TN), but not in the control nostril (CN), of every subject, peaking on day 5 at levels about one-third normal (baseline CN, 4.1 +/- 1.2 microg/mg of protein; baseline TN, 4.3 +/- 1.3; day 5 CN, 4.0 +/- 0.5 [p = NS versus baseline]; day 5 TN, 9.0 +/- 1.7 [p < 0.5 versus baseline]); isoelectric focusing identified the transgene-generated protein (M) in the only two patients in whom the measurement was possible. The reverse transcriptase-polymerase chain reaction (RT-PCR), performed on NALF from TN and CN of four of the five subjects, was positive for transgene message in TN in all cases and negative in NALF from CN except for one time point in one subject. Interleukin 8 (IL-8) concentrations in NALF were elevated at baseline (normal [N = 10] = 2.5 +/- 0.5 ng/mg of protein; baseline TN = 5.5 +/- 0.8, p < 0.05 versus normal) and decreased after AAT transfection (TN = 2.9 +/- 0.6, p < 0.05 versus baseline) but not in the control nostril (CN = 6.5 +/- 2.2, p = NS versus baseline). NALF samples taken from four of the patients while receiving intravenous AAT protein showed normal concentrations of AAT, but IL-8 concentrations (10.5 +/- 4.2 ng/mg of protein, p = NS versus baseline) were not decreased from baseline. We conclude that plasmid-cationic liposome delivery of a normal AAT gene to the respiratory epithelium of deficient patients produces potentially therapeutic local AAT concentrations and that AAT gene therapy, unlike AAT protein therapy, is antiinflammatory.

Gene therapy for acute lung injury.

The remarkable transition of biological science into the age of molecular biology held great promise for development of new therapies for treatment of human disease. The fact that the technology exists for analyzing genetic material in exquisite detail and constructing DNA in virtually any desired form was the basis for promising rapid translation into clinical medicine and the final cure for genetically determined diseases; cystic fibrosis is the prime example of such a lung disease. The promise was not kept, at least not in a time frame which was expected. That result is neither because the rationale was faulty nor because the tools of molecular biology were wanting. The devil was and is in the details. How do you deliver DNA to the desired cell targets in amounts sufficient to accomplish the desired effect? Viral vectors have received the most attention, but viral vectors have proven to have both theoretical and practical problems. In the lungs, these vectors have not fulfilled their original promise. Non-viral based strategies work in a general sense, but efficiency of gene delivery in vivo has been a limitation. In addition, the experimental end points in both clinical and preclinical investigation have been most often designed to demonstrate phenomenology rather than potential efficacy. And, why limit the potential of gene therapy to inherited disease? In fact, treatment of acquired diseases by increasing or decreasing expression of a given gene in the lungs that would hasten recovery from an acquired disease might be easier than treating inherited disease because the requirements for duration of transgene expression would be less stringent. Over the past two decades, we have learned enough about the pathogenesis of acute lung injury to predict that increased (or decreased) production of certain biologically active mediators should be beneficial. Genes encoding some of these mediators have been cloned and constructs made which express the genes. It is now possible using either viral or non-viral strategies to deliver expression constructs to the lungs and, since acute lung injury has a dismal prognosis and no effective drugs have been identified, this seems a good clinical target for gene therapy. In preclinical studies, we have shown that increased expression of the gene encoding the constitutive form of the cyclooxygenase gene (COX-1) results in increased production of prostacyclin and PGE2 by the lungs and inhibits endotoxin induced pulmonary hypertension and edema. Additional studies demonstrate that increased expression of the alpha-1 antitrypsin gene in human respiratory epithelium in culture and in vivo has anti-viral and anti-inflammatory effects that are not predicted by extracellular concentrations of the transgene product. Thus, acute lung injury is a reasonable target for gene therapy, and evidence to date indicates that current technology is sufficiently robust to pursue this novel area for treatment of this devastating disease.

Optimization of cationic liposome-mediated gene transfer to human bronchial epithelial cells expressing wild-type or abnormal cystic fibrosis transmembrane conductance regulator (CFTR).

We determined optimum conditions for delivering DNA to transformed human bronchial epithelial cells expressing wild-type (BEAS) or abnormal (2CF) cystic fibrosis transmembrane conductance regulator (CFTR) using cationic liposomes (Lipofectin, [N-(N,N-dimethylaminoethane)carbamyl] cholesterol[DC-Chol]/dioleoylphosphatidylethanolamine[DOPE], or LipofectAMINE) and reporter genes which measured overall transgene expression (luciferase) or the fraction of cells transfected (heat-stable alkaline phosphatase). All liposomes showed dose-related toxicity. Optimal liposome and lipid: DNA ratios were different for BEAS than for 2CF cells. For all 3 liposome preparations, small particle size and net cationic charge related to transfection efficiency. Both LipofectAMINE and DC-Chol/DOPE transfected a maximum of 3% of BEAS cells, but luciferase expression could be increased without increasing the fraction of cells transfected. LipofectAMINE transfected a maximum of 6% of 2CF cells, and luciferase expression could be increased with no further increase in fraction of transfected cells. DC-Chol/DOPE transfected over 12% of 2CF cells with relatively small increases in luciferase expression. We conclude that an optimal cationic liposome and lipid: DNA ratio for transfecting bronchial epithelial cells depends on: (1) small particle size and net cationic charge, (2) whether the cells have the cystic fibrosis defect, and (3) whether the desired outcome is transfection of the maximum fraction of the cells or maximum total expression of the transgene.

Plasmid-liposome transfer of the alpha 1 antitrypsin gene to cystic fibrosis bronchial epithelial cells prevents elastase-induced cell detachment and cytokine release.

Human neutrophil elastase (NE) stimulates release of neutrophil chemotactic activity by a bronchial epithelial cell line and from nasal epithelial cells. In this article, we show that NE stimulates the production of neutrophil chemotactic activity by 2CFSMEo-cells, a transformed cystic fibrosis bronchial epithelial cell line. The production of chemotactic activity is dose- and time-dependent and can be blocked by preincubation of NE with alpha 1 antitrypsin (alpha1AT). Incubation of the NE-stimulated culture supernatant with neutralizing concentrations of rabbit anti-human interleukin 8 antibody completely neutralizes the chemotactic activity. Transfection of 2CFSMEo- cells with the eukaryotic expression vector pCMV4alpha1AT, complexed to cationic liposomes in a 1:3 wt/wt ratio, results in at least a 10-fold increase in measured human alpha1AT protein in culture supernatant. Detection of human alpha1AT mRNA by reverse transcriptase polymerase chain reaction in total RNA from transfected, but not untransfected cells, confirms successful gene transfer. Compared with untransfected cells, transfer of the human alpha1AT gene decreases chemotactic activity in culture supernatant and prevents cell detachment after NE exposure. Our data indicate that alpha1AT gene transfer is capable of blocking at least some of the biological effects of free elastase on cultured epithelial cells.

Secretory leukoprotease inhibitor attenuates lung injury induced by continuous air embolization into sheep.

Continuous air embolization (CAE) into the pulmonary arterial circulation of sheep results in functional and structural changes of chronic pulmonary hypertension. Release of elastin peptides into lung lymph during CAE and attenuation of CAE-induced pulmonary hypertension by neutrophil depletion suggest that neutrophil elastase may contribute to these changes. To investigate this notion, we treated awake sheep with a potent neutrophil elastase inhibitor, recombinant secretory leukoprotease inhibitor (SLPI) (100 mg/day by aerosol), during 12 days of CAE (CAE+SLPI; n = 7). Controls included sheep receiving CAE + vehicle (VEH) (n = 6), VEH alone (n = 3), and SLPI alone (n = 3). SLPI significantly attenuated the CAE-induced increases in lung lymph flow (day 8; 2.3 +/- 0.5 vs. 5.6 +/- 1.7 ml/15 min), protein clearance (day 8; 1.36 +/- 0.32 vs. 3.08 +/- 0.84 ml/15 min), and elastin peptide concentration (day 8; 243 +/- 41 vs. 398 +/- 44 ng/ml). SLPI delayed the onset of sustained pulmonary hypertension from day 8 to day 12. Both CAE groups showed similar structural changes in the pulmonary arteries. SLPI was well tolerated in control sheep and did not affect hemodynamics or structure. We conclude that serine proteases may contribute to the early initiation of chronic pulmonary hypertension but do not play a striking role in its eventual development.

Gene therapy in acute critical illness.

The development of techniques for manipulating nucleic acids and strategies for delivering DNA to humans has made gene therapy a reality. Although mostly focused on genetically based diseases so far, there is every reason to expand the concept to include acquired diseases. Critical illness may be a good target for gene therapy because of the high mortality and need for only transient treatment. Genes can be delivered in vivo using viral vectors (replication-deficient adenovirus and adeno-associated virus most often). Viral vectors have some negatives, mainly the triggering of an inflammatory and an immune response. Nonviral DNA delivery systems include liposomes (cationic or anionic), direct DNA injection, and polycation-DNA-glycoconjugates. Combining liposomes with viral components to deliver plasmids with a transgene may improve efficiency of delivery without causing toxicity. In a model of acute lung injury, in vivo delivery of a vector hyperexpressing the prostaglandin synthase gene using cationic liposomes resulted in increased production of prostaglandin E2 and prostacyclin in the lungs, and protected the lungs from the effects of endotoxin. This end-result demonstrates the feasibility of this approach. A similar rationale for the treatment of sepsis could be used. Other promising therapeutic genes would include those encoding antioxidant enzymes or antiproteases. The logistics for moving to initial studies of gene therapy in critically ill humans have been worked out for other diseases; such steps should expedite the exploration of this new category of therapies.

No lung toxicity after repeated aerosol or intravenous delivery of plasmid-cationic liposome complexes.

The safety aspects of human gene therapy are of paramount importance in developing an ideal system for gene transfer. Lipofection using DNA in the form of a plasmid has been shown to successfully transfect the lungs when administered either intravenously or by aerosol. We have shown that repeated intravenous or aerosol administration of a plasmid containing the recombinant human alpha 1-antitrypsin gene and a cytomegalovirus promoter complexed to cationic liposomes results in no adverse effects on pulmonary histology, lung compliance, lung resistance, or alveolar-arterial oxygen gradient. Immunohistochemistry and Western blot analysis confirm successful gene transfer using this delivery system. We conclude that plasmids complexed to cationic liposomes may be a safe and efficacious delivery system for in vivo gene transfer to the lungs. Using this delivery system, in vivo gene therapy to the lungs can be achieved by either intravenous or aerosol administration of the transgene.

Protection of rabbit lungs from endotoxin injury by in vivo hyperexpression of the prostaglandin G/H synthase gene.

A recombinant prostaglandin G/H (PGH) synthase gene has been expressed in vitro in bovine pulmonary artery endothelial cells and in vivo in rabbits by transfection with a plasmid using cationic liposomes. Transfection of bovine pulmonary artery endothelial cells with the PGH synthase cDNA resulted in increased intracellular PGH synthase protein (determined by Western blot analysis) and increased release of prostacyclin. Rabbits intravenously transfected with the PGH synthase gene had increased plasma levels of prostacyclin and PGE2, and their lungs produced increased amounts of the same eicosanoids. In an in situ, perfused preparation of PGH synthase transfected rabbit lungs, the pressor response to endotoxin was markedly attenuated. In addition, pulmonary edema and release of thromboxane B2 into the perfusate after endotoxin infusion were markedly decreased in transfected lungs compared to controls (animals transfected with a pCMV4 construct that did not contain a cDNA insert). The data suggest that augmented endogenous production of prostacyclin and PGE2, achieved by liposome-mediated gene transfer, protects the lungs from endotoxin. This may be caused in part by suppression of endotoxin-stimulated thromboxane B2 production. Modification of lipid mediator responses by in vivo transfection is a potential approach to the therapy of acute lung injury.

The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination.

The acute respiratory distress syndrome (ARDS), a process of nonhydrostatic pulmonary edema and hypoxemia associated with a variety of etiologies, carries a high morbidity, mortality (10 to 90%), and financial cost. The reported annual incidence in the United States is 150,000 cases, but this figure has been challenged, and it may be different in Europe. Part of the reason for these uncertainties are the heterogeneity of diseases underlying ARDS and the lack of uniform definitions for ARDS. Thus, those who wish to know the true incidence and outcome of this clinical syndrome are stymied. The American-European Consensus Committee on ARDS was formed to focus on these issues and on the pathophysiologic mechanisms of the process. It was felt that international coordination between North America and Europe in clinical studies of ARDS was becoming increasingly important in order to address the recent plethora of potential therapeutic agents for the prevention and treatment of ARDS.

Report of the American-European Consensus conference on acute respiratory distress syndrome: definitions, mechanisms, relevant outcomes, and clinical trial coordination. Consensus Committee.

The acute respiratory distress syndrome (ARDS), a process of nonhydrostatic pulmonary edema and hypoxemia associated with a variety of etiologies, carries a high morbidity rate, mortality rate (10% to 90%), and financial cost. The reported annual incidence in the United States is 150,000 cases, but this figure has been challenged and may be different in Europe. Part of the reason for these uncertainties is the heterogeneity of diseases underlying ARDS and the lack of uniform definitions for ARDS. Thus, those who wish to know the true incidence and outcome of this clinical syndrome are stymied. The European American Consensus Committee on ARDS was formed to focus on these issues and on the pathophysiologic mechanisms of the process. It was felt that international coordination between North America and Europe in clinical studies of ARDS was becoming increasingly important to address the recent plethora of potential therapeutic agents for the prevention and treatment of ARDS.

Gene therapy for inflammatory diseases.

The concept of gene therapy may be broadened to include transient gene therapy (gene therapeutics) as a potential intervention in prevention and treatment of diseases which are a consequence of triggering the inflammatory response. Functioning genes can be delivered in vivo by a variety of technologies. Liposome technology is particularly attractive for gene therapeutics because plasmid DNA constructs can be delivered using liposomes which express transiently and do not readily incorporate into the host genome. In the lungs, DNA may be targeted by either intravenous or airway delivery. Airway delivery may be achieved either by direct injection into the airways or by aerosolizing liposome-DNA constructs. Expression of transgenes might also be targeted to areas of inflammation by choosing DNA constructs which contain the appropriate regulatory regions. Several genes have been cloned which are directly relevant to manipulating the inflammatory response and this technology could, in theory, by using either sense or antisense constructs, provide an opportunity to increase or decrease proteins relevant to the inflammatory response. Because of rapid progress in the technology of molecular biological techniques, and rapid progression of human trials using gene transfer methodologies, it is likely that extension of gene therapy to acute diseases such as those which are characterized by inflammation will open a new vista for pharmacological approaches to these complex diseases.

Report of the American-European consensus conference on ARDS: definitions, mechanisms, relevant outcomes and clinical trial coordination. The Consensus Committee.

The acute respiratory distress syndrome (ARDS), a process of non-hydrostatic pulmonary edema and hypoxemia associated with a variety of etiologies carries a high morbidity, mortality (10-90%) and financial cost. The reported annual incidence in the United States is 150,000 cases, but this figure has been challenged and may be different in Europe. Part of the reason for these uncertainties is the heterogeneity of diseases underlying ARDS and the lack of uniform definitions for ARDS. Thus, those whose wish to know the true incidence and outcome on this clinical syndrome are stymied. The European American Consensus Committee on ARDS was formed to focus on these issues and on the pathophysiologic mechanisms of the process. It was felt that international coordination between North America and Europe in clinical studies of ARDS was becoming increasingly important in order to address the recent plethora of potential therapeutic agents for the prevention and treatment of ARDS.

Aerosol and intravenous transfection of human alpha 1-antitrypsin gene to lungs of rabbits.

In vivo gene transfer to the lungs is possible either by an intravenous or an airway route of administration. A plasmid containing the recombinant human alpha 1-antitrypsin (h alpha 1AT) gene and a cytomegalovirus promoter complexed to cationic liposomes was given either intravenously or by aerosol to New Zealand White rabbits. Both routes of administration resulted in successful transfection and expression of the h alpha 1AT gene. h alpha 1AT mRNA and protein were detected for at least 7 days. Immunohistochemical staining showed h alpha 1AT protein in the pulmonary endothelium following intravenous administration, in alveolar epithelial cells following aerosol administration, and in the airway epithelium by either route. After intravenous injection of radiolabeled plasmids, autoradiographs showed localization of plasmid in endothelial cells, especially at arterial bifurcations, and at the alveolar level. A plasmid-liposome delivery system for gene therapy to the lungs may permit targeting of the DNA to subsets of lung cells by selection of the route of delivery and may permit a broad application of gene therapy to acute as well as chronic diseases.

Expression of human growth hormone fusion genes in cultured lung endothelial cells and in the lungs of mice.

We sought to develop genetic therapy for acute lung diseases by introducing genes into lung cells in vivo that were only transiently expressed. To that end, we introduced a gene encoding a physiologically relevant secreted human protein into bovine lung endothelial cells in culture and into the lungs of mice using the technique of lipofection. We exposed cultured endothelial cells to a plasmid containing the coding region for human growth hormone (hGH) driven by a metallothionein (MT) promoter. In cells lipofected with the plasmid containing the MT promoter, expression of the hGH gene in medium was low (peak = 30 ng hGH/24 h/60-mm dish), but expression was markedly increased by addition of either dexamethasone (peak = 91) or cadmium (peak = 120). Lipofection with the same construct except a thymidine kinase promoter showed no cadmium response. We gave mice 5,000 ppm ZnSO4 in their drinking water and 24 h later injected intravenously plasmid containing the MT promoter complexed to liposomes. Mice were killed 1, 3, and 5 days after injection, and hGH production by minced lung, liver, and kidneys was determined in vitro. Neither kidneys nor liver produced detectable hGH. However, hGH was produced by the lungs, beginning on day 1, peaking on day 3 (approximately 1.0 ng hGH/24 h/g tissue), and declining by day 5. Lungs from mice injected either with DNA alone or with liposome alone did not produce hGH. mRNA specific for hGH was demonstrated in the lungs by polymerase chain reaction amplification of cDNA followed by agarose gel electrophoreses.(ABSTRACT TRUNCATED AT 250 WORDS)