Clinical Review: Gene-based therapies for ALI/ARDS: where are we now?
© BioMed Central Ltd 2011
Published: 20 June 2011
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© BioMed Central Ltd 2011
Published: 20 June 2011
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) confer substantial morbidity and mortality, and have no specific therapy. The accessibility of the distal lung epithelium via the airway route, and the relatively transient nature of ALI/ARDS, suggest that the disease may be amenable to gene-based therapies. Ongoing advances in our understanding of the pathophysiology of ALI/ARDS have revealed multiple therapeutic targets for gene-based approaches. Strategies to enhance or restore lung epithelial and/or endothelial cell function, to strengthen lung defense mechanisms against injury, to speed clearance of infection and to enhance the repair process following ALI/ARDS have all demonstrated promise in preclinical models. Despite three decades of gene therapy research, however, the clinical potential for gene-based approaches to lung diseases including ALI/ARDS remains to be realized. Multiple barriers to effective pulmonary gene therapy exist, including the pulmonary architecture, pulmonary defense mechanisms against inhaled particles, the immunogenicity of viral vectors and the poor transfection efficiency of nonviral delivery methods. Deficits remain in our knowledge regarding the optimal molecular targets for gene-based approaches. Encouragingly, recent progress in overcoming these barriers offers hope for the successful translation of gene-based approaches for ALI/ARDS to the clinical setting.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) constitute the leading cause of death in pediatric and adult critical care . In the United States alone there are approximately 190,600 cases of ALI/ARDS with a 40% mortality rate, amounting to 75,000 deaths annually . Significant ongoing morbidity, including pulmonary, neuromuscular, cognitive and psychiatric sequelae, is seen in 50 to 70% of ALI/ARDS survivors, and the financial burden on society is considerable [1, 3]. There are no specific therapies for ALI/ARDS, and management remains supportive, focusing on protective mechanical ventilation strategies , restrictive intravenous fluid management approaches , and rescue strategies such as prone positioning  or extracorporeal membrane oxygenation  for severely hypoxemic patients. These issues underline the need to consider nonconventional therapeutic approaches.
Gene-based therapy involves the insertion of genes or smaller nucleic acid sequences into cells and tissues to replace the function of a defective gene, or to alter the production of a specific gene product, in order to treat a disease. Gene therapy can be classified into germline and somatic gene therapies. Germline approaches modify the sperm or egg prior to fertilization and confer a stable heritable genetic modification. Somatic gene approaches use gene therapy to alter the function of mature cells. Commonly used somatic gene therapy strategies include the overexpression of an existing gene and/or the insertion of smaller nucleic acid sequences into cells to alter the production of an existing gene.
ALI/ARDS may be suitable for gene-based therapies as it is an acute but relatively transient process , requiring short-lived gene expression, obviating the need for repeated therapies and reducing the risk of an adverse immunological response. The distal lung epithelium is selectively accessible via the tracheal route of administration, allowing targeting of the pulmonary epithelium . The pulmonary vasculature is also relatively accessible, as the entire cardiac output must transit this circulation. Antibodies that bind antigens selectively expressed on the pulmonary endothelial surface can be complexed to gene vectors to facilitate selective targeting following intravenous administration . It is also possible to use gene-based strategies to target other cells central to the pathogenesis of ALI/ARDS, such as leukocytes and fibroblasts . Furthermore, gene-therapy-based approaches offer the potential to selectively target different phases of the injury and repair process. The potential to target specific aspects of the injury and repair processes such as epithelial-mesenchymal transition, fibrosis, fibrinolysis, coagulopathy and oxidative stress with these approaches is also clear.
Gene therapy approaches used in preclinical ALI/ARDS models
Viral vector-delivered gene therapy
Adenoviral vectors (dsDNA genome)
Adenoviral transfer of genes for a surfactant enzyme , angiopoietin-1 , HSP-70 , apolipoprotein A-1 , and Na+,K+-ATPase pump  genes attenuate experimental ALI Adenoviral delivery of IL-10 gene attenuates zymosan ALI at low doses, but is harmful at high doses 
Adeno-associated virus vectors (ssDNA genome)
Good safety profile; less immunogenic Inherently replication deficient AAV-5 and AAV-6 lung epithelial tropism [10, 11] Transduce nondividing cells Long-lived gene expression Used in clinical trials for CF [12, 13]
Limited transgene size Difficult to produce in large quantities
AAV vector gene transfer demonstrated in multiple lung cell types including progenitor cells in both normal lungs and following naphthalene-induced ALI 
Lentivirus vectors (RNA genome)
Transduce nondividing cells  Integrate stably but randomly into the genome
Lentiviral transfer of shRNA to silence CD36 gene expression suppresses silica-induced lung fibrosis in the rat 
Nonviral gene-based strategies
Plasmid transfer (closed dsDNA circles)
Easily produced at low cost
No specific cell targeting Very inefficient
Electroporation-mediated gene transfer of the Na+,K+-ATPase rescues endotoxin-induced lung injury 
Nonviral DNA complexes
(lipoplexes or polyplexes)
Complexes protect DNA Complexes facilitate cellular targeting 
Less efficient than viral vectors
Cationic lipid-mediated transfer of the Na+,K+-ATPase gene ameliorated high-permeability pulmonary edema  Lipoplex-delivered IL-10 gene decreased CLP-induced ALI  Systemic cationic polyethylenimine polyplexes incorporating indoleamine-2,3-dioxygenase decreased ischemia-reperfusion ALI 
DNA and RNA oligonucleotides (siRNA, shRNA, decoy oligonucleotides)
Easily produced at low cost Smaller molecules that can easily enter cells Target regulation of specific genes
No specific cell targeting
Cell-delivered gene therapy
Mesenchymal stem/stromal cells
Systemic or intrapulmonary delivery Strategy used in human studies 
MSCs expressing angiopoeitin-1 attenuate endotoxin-induced ALI  Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced lung injury 
Systemic delivery Less expensive
Fibroblasts expressing angiopoeitin-1 attenuate endotoxin induced ALI 
Adenoviruses have double-stranded DNA genomes, have demonstrated promise in preclinical models [12, 13] and are well tolerated at low to intermediate doses in humans [14, 15]. Advantages include their ease of production, the high efficiency at which they can infect the pulmonary epithelium [14, 16] and that they can deliver relatively large transgenes. A disadvantage of adenoviruses is their immunogenicity, particularly in repeated doses . Newer adenoviral vectors, in which much of the immunogenicity has been removed, hold promise . While adenovirus-mediated gene transfer in the absence of epithelial damage is relatively inefficient , this may be less of a problem in ALI/ARDS that is characterized by widespread epithelial damage.
Adeno-associated viruses (AAVs) are single-stranded DNA parvoviruses that are replication deficient . A substantial proportion of the human population has been exposed to AAVs but the clinical effects are unknown. AAV vectors have a good safety profile, and are less immunogenic compared with other viruses, although antibodies do develop against AAV capsid proteins that can compromise repeat administration. AAV vectors can insert genes at a specific site on chromosome 19. The packaging capacity of the virus is limited to 4.7 kb, restricting the size of the transgene that can be used. AAVs are less efficient in transducing cells than adenoviral vectors. Successful AAV vector gene transfer has been demonstrated in multiple lung cell types including lung progenitor cells, in both normal and naphthalene-induced ALI lungs . AAV serotypes have specific tissue tropisms, due to different capsid proteins that bind to specific cell membrane receptors. AAV-5  and AVV-6  exhibit enhanced tropism for the pulmonary epithelium [21, 22]. AAVs can transduce nondividing cells and result in long-lived transgene expression. AAV vectors have been used in clinical trials in cystic fibrosis patients, underlining their safety profile [23, 24].
These RNA viruses can transfect nondividing cells such as mature airway epithelial cells . The virus stably but randomly integrates into the genome and expression is likely to last for the lifetime of the cell (~100 days). The transgene can be transmitted post mitosis, and there is also a risk of tumorigenesis if the transgene integrates near an oncogene. The development of leukemias in children following gene therapy for severe combined immunodeficiency highlights this risk [26, 27]. While lentiviral vectors may be useful to correct a gene deficiency associated with increased risk of ALI, the long-lived gene expression of lentiviral delivered genes may be more suitable for chronic diseases than for ALI/ARDS.
Nonviral delivery systems, while generally less efficient than viral vectors in transfecting the lung epithelium, are increasingly used to deliver smaller DNA/RNA molecules (Table 1). Strategies include the use of DNA-lipid and DNA-polymer complexes and naked DNA/RNA oligo-nucleotides, such as siRNA , decoy oligonucleotides  and plasmid DNA . Nonviral delivery systems are less immunogenic than viral vector-based approaches, and can be generated in large amounts at relatively low cost.
Plasmid vectors are composed of closed circles of double-stranded DNA. As naked and plasmid DNA contain no proteins for attachment to cellular receptors, there is no specific targeting to different cell types and thus it is essential that the DNA is placed in close contact with the desired cell type. These limitations make this approach less relevant clinically.
The therapeutic DNA is held within a sphere of lipids, termed a lipoplex, or within a sphere of polymers, such as polyethyleneimine, termed a polyplex. Lipoplexes and polyplexes act to protect the DNA, facilitate binding to the target cell membrane and also trigger endocytosis of the complex into the cell, thereby enhancing gene expression. These systems can be modified to include a targeting peptide for a specific cell type, such as airway epithelial cells . These complexes efficiently and safely transfect airway epithelial cells , and they have demonstrated promise in human studies .
siRNAs are dsRNA molecules of 20 to 25 nucleotides that can regulate the expression of specific genes. Specific siRNAs reduce inflammation-associated lung injury in humans  and in animal models [28, 34]. shRNA is a single strand of RNA that, when introduced into the cell, is reverse transcribed and integrated into the genome, becoming heritable. During subsequent transcription, the sequence generates an oligonucleotide with a tight hairpin turn that is processed into siRNA. shRNAs have reduced lung injury in animal models [35, 36]. Decoy oligonucleotides are double-stranded DNA molecules of 20 to 28 nucleotides, which bind to specific transcription factors to reduce expression of targeted genes, and have been successfully used in animal models [37, 38].
An alternative approach is to use systemically delivered cells to deliver genes to the lung. This approach has been used to enhance the therapeutic potential of stem cells - such as mesenchymal stem/stromal cells, which demonstrate promise in preclinical ALI/ARDS models . Fibroblasts have also been used to successfully deliver genes to the lung to attenuate ALI . Preliminary data from a clinical trial in pulmonary hypertension show that endothelial progenitor cells overexpressing endothelial nitric oxide synthase (NOS3) decrease pulmonary vascular resistance , highlighting the potential of cell-delivered gene therapy for ALI/ARDS.
Nebulization of genetic material into the lung is effective , safe and well tolerated [32, 43, 44]. The integrity of AAV vectors [9, 43] and adenoviral virus vectors  are maintained post nebulization, as are cationic lipid vectors  and DNA and RNA oligonucleotides . A number of gene therapy clinical trials have utilized nebulization to deliver the transgene to the lung [23, 43], but without clear clinical benefit to date [43, 44].
Intravascular delivery approaches target the lung endothelium. These approaches have been successfully used in preclinical studies of cell-based gene therapies [39, 40], and also with vectors that incorporate components such as antibodies to target antigens on the lung endothelium .
Technical challenges to gene-based therapies for ALI/ARDS models
Pulmonary defense mechanisms against inhaled particles
Airway mucus and epithelial lining fluid
Tight intercellular epithelial junctions
Limited endocytosis at luminal surface
Difficulties in transducing the acutely injured lung
Loss of alveolar epithelium
Collapsed and/or consolidated alveoli
Bronchial plugging by mucus and debris
Limitations of vector systems
Imunogenicity of viral vectors particularly in repeated doses
Limitations regarding transgene size
Limited transfection efficiency of nonviral vectors
Knowledge deficits regarding the optimal molecular targets
The lung has evolved effective barriers to prevent the uptake of any inhaled foreign particles . While advantageous in minimizing the potential for uptake of external genetic material (for example, viral DNA), these barriers make it more difficult to use gene-based therapies in the lung. Barriers to entry of foreign genetic material into the lung include airway mucus and the epithelial lining fluid, which traps and clears inhaled material. The glycocalyceal barrier hinders contact with the cell membrane, while the tight intercellular epithelial junctions and limited luminal endocytosis further restrict entry of foreign material into the epithelial cells.
Transducing the acutely injured lung may be difficult, due to the presence of pulmonary edema, consolidated or collapsed alveoli, and additional extracellular barriers such as mucus. Gene-based therapies targeted at the pulmonary epithelium may be less effective where there is extensive denudation of the pulmonary epithelium, as may occur in primary ARDS. Encouragingly, there is some evidence to suggest that ALI may not substantially impair viral gene transfer to the alveolar epithelium .
The key limitation of nonviral vector approaches has been their lack of efficiency in mediating gene transfer and transgene expression in the airway epithelium. Viral vectors are immunogenic, due to the protein coat of the viral vector, and the immune response is related to both vector dose and number of administrations. The potential to limit administration to a single dose in ALI/ARDS may reduce this risk. However, the development of an inflammatory response resulting in death following administration of a first-generation adenoviral vector highlights the risks involved . Additional limitations of viral vectors include transgene size, which is limited by the size of the capsid that encloses the viral genes.
The therapeutic potential of gene therapy for ALI/ARDS is underlined by a growing body of literature demonstrating efficacy in relevant preclinical models. In considering the clinical implications of these studies, it is important to acknowledge that animal models of ARDS do not fully replicate the complex pathophysiological changes seen in the clinical setting. This is highlighted by the fact that many pharmacologic strategies demonstrating considerable promise in preclinical studies were later proven ineffective in clinical trials. Nevertheless, these studies provide insights into the clinical potential of these strategies.
An early murine study demonstrated that cationic lipid-mediated transfer of the Na+,K+-ATPase gene ameliorated high-permeability pulmonary edema . Electroporation-assisted gene transfer of plasmids encoding for Na+,K+-ATPase reverses endotoxin-induced lung injury . The lipoplex-delivered IL-10 gene decreased lung and systemic organ injury induced by cecal ligation and puncture in mice . Systemically administered cationic polyethylenimine polyplexes incorporating indoleamine-2,3-dioxygenase transduced pulmonary endothelial cells and decreased lung ischemia-reperfusion injury .
Future directions for gene-based therapies
Capsid protein modification to reduce immunogenicity 
Capsid protein modification to enhance tissue specificity 
Envelope protein pseudotyping
Use of targeting peptides on lipoplexes and polyplexes 
Strategies to enhance gene transfer; for example, electroporation, ultrasound, gene gun delivery
Gene expression strategies
Development of promoters that regulate gene expression 
Enhanced therapeutic targeting
Nebulization technologies 
Strategies to target the pulmonary endothelium 
Improved cellular uptake of vector
Surface active agents to enhance vector spread 
Reduce ubiquitination of viral capsid proteins 
Better therapeutic targets
Enhancement or restoration of lung epithelial and/or endothelial cell function 
Strengthening lung defense mechanisms against injury 
Speeding clearance of inflammation and infection
Enhancement of the repair process following ALI/ARDS .
Viral vectors remain the focus of intensive research to optimize their efficiency, to minimize their immunogenicity and to enhance their tissue specificity [19, 31, 69, 70]. Strategies to develop less immunogenic vectors have focused on modifying the naturally occurring proteins in the viral coat . Much research has been devoted to searching and characterizing both naturally occurring  and engineered capsid variants from mammalian species . Capsid protein modification has also been used to enhance tissue specificity . Envelope protein pseudotyping involves encapsulating the modified genome from one virus, such as simian immunodeficiency virus, with envelope proteins from another virus, such as vesicular stomatitic virus. This encapsulation can enhance the therapeutic potential of viral vectors, by combining the advantages of one viral genome (for example, bigger payload or site-specific integration) with the tissue tropism of another virus.
Strategies to enhance the effectiveness of the lipoplexes used to deliver plasmids and other DNA/RNA oligo-nucleotides involve manipulation of the lipoplex lipid content and the use of targeting peptides. The choice of lipid influences expression efficiency by enhancing release of the genetic material within the target cell [73, 74]. Targeting peptides increases transfection efficiency by directing the lipid to a particular cell membrane or cell type . Physical methods of plasmid delivery such as electroporation  and ultrasound can enhance gene transfer by bringing the plasmid DNA into closer proximity with the cell membrane and/or causing temporary disruption of the cell membrane. Other physical methods can also be used to increase in vivo gene transfer, including pressurized vascular delivery, laser, magnetic fields and gene gun delivery. These systems enable plasmid-based gene delivery to reach efficiencies close to that achieved with viral vectors.
Successful gene therapy relies upon being able to target the injury site, and to control the duration and levels of gene expression. Modifying the transgene DNA to exclude nonmethylated CpG motifs, typical of bacterial DNA, decreases the immune response and may increase transgene expression [75, 76]. High-efficiency tissue-specific promoters may improve the efficiency and specificity of transgene expression. Lung-specific promoters include surfactant promoters  such as the surfactant protein C promoter , a ciliated cell-specific promoter FOXJ1 , the cytokeratin 18 promoter , and the Clara cell 10-kDa protein . Promoters can also be used to target a specific phase of illness, switching on when required to produce an effect at the optimal time point.
A related approach is the development of promoters that allow for transfected genes to be turned on and off. Currently, the tetracycline-dependent gene expression vector  is the most widely used regulated system as it has a good safety profile. Tetracycline is rapidly metabolized and cleared from the body, making it an ideal drug to control gene expression. However, the potential for an activator such as tetracycline to modulate the lung injury should be borne in mind. New-generation transactivators, with no basal activity and increased sensitivity, have now been developed . In an ARDS context, conditional regulation of gene expression by the combined use of a lung-specific promoter and the tetracycline-dependent gene expression system may be a useful approach .
Specific strategies have been developed to maximize uptake of vector into alveolar epithelial cells. It is possible to enhance lung transgene expression with the use of surface-active agents such as perflurocarbon, which enhances the spread of vector and mixing within the epithelial lining fluid . Agents that reduce ubiquitination of AAV capsid proteins following endocytosis, such as tripeptide proteasome inhibitors, dramatically augment (>2,000-fold) AAV vector transduction in airway epithelia .
Ultimately, the success or failure of gene-based therapies for ALI/ARDS is likely to rest on the identification of better gene targets. Ongoing advances in our understanding of the pathophysiology of ALI/ARDS continue to reveal novel therapeutic targets for gene-based approaches. Promising potential approaches include strategies to enhance or restore lung epithelial and/or endothelial cell function , to strengthen lung defense mechanisms against injury , to speed clearance of inflammation and infection, and to enhance the repair process following ALI/ARDS .
Key points regarding gene-based therapies for ALI/ARDS
ALI/ARDS may be amenable to gene-based therapies
Ongoing advances in our understanding of the pathophysiology of ALI/ARDS have revealed multiple therapeutic targets for gene-based approaches
Numerous gene-based approaches have demonstrated promise in relevant preclinical models
The clinical potential for gene-based approaches to ALI/ARDS remains to be realized
Multiple barriers exist to successful gene-based approaches for ALI/ARDS
A greater understanding of the molecular mechanisms underlying injury and repair in
ALI/ARDS, coupled with improvements in gene-based approaches, offer hope for ALI/ARDS
acute lung injury
acute respiratory distress syndrome
small hairpin RNA
small interfering RNA.
The present work was supported by funding from the Health Research Board, Dublin, Ireland (Grant No. RP/2008/193), and by the European Research Council, Brussels, Belgium, under the Framework 7 Programme (Grant No. ERC-2007-StG 207777).