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Pulmonary Genetics, Genomics, and Gene Therapy*: Conference Summary FREE TO VIEW

Steven M. Albelda, MD
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*From the Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania Medical Center, Philadelphia, PA.

Correspondence to: Steven M. Albelda, MD, University of Pennsylvania Medical Center, Pulmonary, Allergy, and Critical Care Division, 856 BRB II/III, 426 Curie Blvd, Philadelphia, PA 19104; e-mail: Albelda@mail.med.upenn.edu

Chest. 2002;121(3_suppl):105S-110S. doi:10.1378/chest.121.3_suppl.105S
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The format of the 44th Annual Thomas L. Petty Aspen Lung Conference was somewhat different than those of previous meetings in that the organizers, led by Drs. David Rodman and Mark Geraci, chose to focus on new approaches rather than on a specific disease process. This seemed especially appropriate given the recent excitement relating to the announcement of the completion of the sequencing of the human genome and the virtual explosion of microarray “chip” technology. My goal is to briefly summarize some of the accomplishments, limitations, and possible future directions of pulmonary genetics, genomics, and gene therapy that were discussed at this conference. For details, the reader is referred to the excellent articles describing each of the presentations in this supplement issue of CHEST.

Great progress has been made in the identification of the genetic causes of most of the “simple” mendelian genetic pulmonary diseases (Table 1 ) using either traditional approaches (ie, after the protein defect in α1-antitrypsin [ α1AT] deficiency was determined, the messenger RNA was isolated followed by identification of the gene) or using linkage analysis techniques (ie, isolation of the gene, followed by the identification of the messenger RNA and protein). Thus, over the past decade, the genetic defects for cystic fibrosis, α1AT deficiency, primary ciliary dyskinesia (PCD), lymphangiomyomatosis (LAM), and familial primary pulmonary hypertension (FPPH) have been identified.

One of the most exciting aspects of the conference was the discussion surrounding the genetics of primary pulmonary hypertension (PPH) and the recently identified gene for FPPH. As described by Dr. J. Lloyd from Vanderbilt University and Dr. J. Morse from Columbia University, one gene locus located on chromosome 2, the bone morphogenic protein receptor (BMPR)-2, was identified by two independent groups using linkage analysis techniques that were based on data from large families with FPPH. Defects in this gene appear to account for > 50% of cases of FPPH and also may be involved in a sizable percentage (up to one third) of cases of sporadic PPH, as well as in other diseases associated with PPH such as anorectic-induced PPH. Some of the most interesting questions arising from this discovery relate to the mechanisms by which mutations in BMPR-2 lead to the clinical disease of PPH. BMPR-2 is a member of the transforming growth factor-β receptor family and has been implicated previously in developmental processes. A number of laboratories are now exploring such questions as the following: (1) why does a global gene defect lead to lesions in only very specific regions of the pulmonary vascular system? (2) what are the normal ligands and receptor-signaling molecules in the BMPR-2 pathway? and (3) is BMPR-2 involved in non-FPPH and secondary PPH, and how could this discovery lead to new treatment pathways?

Despite this major advance, however, it seems fairly clear that BMPR-2 will not provide all the answers for treating patients with FPPH or PPH. Data presented by Dr. B. Janssen from the University of Heidelberg suggested that at least one other genetic locus on chromosome 2 also exists. This locus may be a second gene resulting in FPPH or may be a modifying gene. Dr. S. Adnot’s group from Creteil, France, showed strong data suggesting that the overexpression of a serotonin transporter in vascular smooth muscle cells may play an important role in PPH. The gene for this transporter thus could function in a primary or modifier role.

Another intriguing set of observations remains to be connected with the new genetic data. Investigators from the University of Colorado have postulated that cells within the characteristic plexiform lesions of PPH behave in ways that are very similar to malignant cells. This hypothesis is based on a number of pieces of data, including previous observations from Dr. R. Tudor’s group that the characteristic plexiform lesions of PPH are areas of monoclonal endothelial cell proliferation that have lost their ability to stay in monolayers and that they overexpress growth factors such as vascular endothelial growth factor. New work supporting this hypothesis was presented by Dr. M. Yeager showing that these endothelial cells had mutations in mismatch repair enzymes leading to alterations in the transforming growth factor-β type II receptor and in the antiapoptotic protein, Bax. In addition, work from Drs. M. Geraci and N. Voekel using microarray technology suggested that gene expression patterns may be different in cases of spontaneous PPH and FPPH, and that many of the altered genes are similar to that seen in malignancy (ie, down-regulation of DNA repair enzymes).

Another new area of interest stems from genetic information relating to LAM. LAM is an unusual disease that affects women almost exclusively and results in cystic lung disease. Recently, it has been recognized to be closely related to the tuberous sclerosis complex of diseases, which are due to a mutation in the TSC1 or TSC2 genes (the function of which remains unknown). One key genetic factor in this disease appears to be the proliferation of a modified smooth muscle cell called the “LAM cell.” Dr. J Moss from the National Institutes of Health described studies defining the incidence of LAM in patients with tuberous sclerosis and his efforts to grow and characterize LAM cells. Dr. F. McCormack from the University of Cincinnati also has been carefully characterizing the clinical and radiographic manifestations of a disease the symptoms of which are consistent with LAM in women with tuberous sclerosis. Like many of the newly identified pulmonary genetic diseases, however, the link between the genetic defect and the clinical phenotype is still not known.

Unlike the rapid progress in the single-gene disorders, our understanding of the genetic basis of more complex genetic diseases and of gene-gene and gene-environment interactions remains more limited. One powerful technique being used by a number of the presenters was the analysis of strain differences in mouse models of disease using quantitative locus analysis. By defining differential sensitivities to specific damaging agents in different mouse strains and by using genetic analyses of a series of back-crosses, disease-sensitivity genes were being sought for lung fibrosis sensitivity after bleomycin exposure (Dr. R. Barth, University of Rochester), susceptibility to emphysema after smoking (Dr. S. Shapiro, Washington University), sensitivity to endotoxin (Dr. D. Schwartz, Duke University), and susceptibility to lung injury after oxidant stress (Dr. G. Leikauf, University of Cincinnati). Although a specific gene has been found only for endotoxin sensitivity to date, candidate chromosomal areas have been defined in each of the other models.

Much attention has been focused on human diseases that appear to have more complex genetic etiologies such as COPD, asthma, interstitial pulmonary fibrosis, bronchiectasis, and acute lung injury. The following two approaches are being taken: (1) positional cloning (linkage analysis) by the collection of DNA from members of affected families followed by chromosome mapping and gene identification; and (2) the analysis of candidate genes. It was evident from the discussions that both approaches were needed. Dr. E. Silverman (Brigham and Women’s Hospital) is leading the Boston COPD study that is aimed at collecting data from families in which the early onset of severe emphysema occurs. In the preliminary analysis, a few chromosomal regions of interest have been identified, but no highly significant associations have been found yet. In contrast, as described by Dr. W. Cookson (Wellcome Trust Center for Human Genetics, UK), there is good agreement among many groups about certain relatively large chromosomal regions linked to asthma. To date, however, fine mapping to identify individual genes has not yet been accomplished. In addition to linkage studies, Dr. Cookson described a number of candidate genes that have been studied. One of the most promising of these is the Fcε receptor (the IgE receptor) gene. The ultimate task of finding the “asthma genes” is going to be complicated by a number of factors including complex environmental interactions, epigenetic factors (such as maternal imprinting), and the fact that it appears that different components of asthma (ie, airway reactivity, eosinophilia, atopy, and eczema) may map to very different regions.

Some progress has been made in the search for genes impacting on the development and severity of interstitial lung diseases (Dr. R. du Bois, Royal Brompton Hospital, London, UK). A key advance in this area has been the ability of the group to define homogeneous disease phenotypes. The use of serologic subgroups has been especially helpful in this regard. The du Bois group has used the candidate gene approach with heavy emphasis on the analysis of single-nucleotide polymorphisms. Associations have been found with human leukocyte antigen (HLA) class II genes in patients with scleroderma and sarcoidosis. Dr. M. Rossman (University of Pennsylvania) also presented data linking specific alleles and specific amino acid epitopes of the HLA class II genes with sarcoidosis. Other human diseases in which progress has been made include the identification of a mutation in the Toll-like receptor 4 gene that correlates with human responses to endotoxin (Dr. D. Schwartz, Duke University) and associations of angiotensin-converting enzyme alleles and polymorphisms of the interleukin-6 promoter with the development of acute lung injury (Dr. R. Marshall, Centers for Respiratory Research and Cardiovascular Genetics, London, UK).

A number of problems in ongoing genetic studies were identified. Defining precise disease phenotypes is critical and will determine the best groups to study. Despite the publicity, the sequence of the human genome is still not available in an organized and user-friendly form. It became evident that we need new statistical tools and larger well-defined patient populations to analyze complex genetic disorders. Given these limitations, areas that will advance the field will be in the development of more detailed single-nucleotide polymorphism maps, a more fully characterized human genome, and the development of informatic, statistical, and epidemiologic techniques that will allow more sophisticated analyses of gene-gene and gene-environmental interactions. The identification of new and better candidate genes will be fostered by careful clinical studies (ie, the identification of surfactant protein C deficiencies in interstitial lung disease as presented by Dr. S. Wert from the University of Cincinnati) or from genomics studies (ie, the up-regulation of matrix metalloproteinase-7 as presented by Dr. G. Cosgrove of the University of Colorado and Dr. N. Kaminski of the Sheba Medical Center, Israel), or secreted frizzled related protein-1 (as presented by Dr. J. D’Armiento of Columbia University) in emphysema. Finally, and perhaps most importantly, cooperative group studies that collect genetic and environmental information from large numbers of patients will be required. As an example of the power of this approach, Dr. M. Rossman presented data from the ACCESS study, a cooperative group studying sarcoidosis, investigating HLA class II gene interactions with environmental factors.

Functional genomics, and especially the use of gene expression arrays, has emerged as an extremely powerful technology for both hypothesis generation and hypothesis testing. The full gamut of uses of this technique were presented at the meeting (Table 2 ).

Arrays can be used to study changes in gene expression patterns after defined perturbations. As described by Dr. S. Lory (Harvard Medical School) and Dr. M. Saavedra (University of Colorado), microarrays can be used to monitor the change in epithelial cells after interactions with bacteria. In addition, changes in bacterial gene expression can also be examined. Dr. D. Sheppard (University of California, San Francisco) provided a striking example of gene expression changes in lung cells after exposure to the cytokine interleukin-13. Interestingly, the changes in gene expression were completely different for epithelial cells, smooth muscle cells, and fibroblasts.

There is a great deal of interest in using array technology to determine altered patterns of gene expression in specific lung disease tissues with the goal of improving diagnosis and in better understanding pathogenesis. Examples were provided for lung cancer (by Dr. C. Powell of Columbia University and Dr. N. Kaminski of Sheba Medical Center), emphysema (Dr. D’Armiento of Columbia University), IPF (Dr. Cosgrove of the University of Colorado and Dr. Kaminski), and bronchiolitis obliterans (Dr. S. Shapiro of Washington University).

One growing use of arrays is to discover previously unexpected pathways in disease development. Using such an approach, Dr. Sheppard presented data showing how his group determined a role for matrix metalloproteinase-12 in emphysema seen in αvβ6 knockout mice. Another example from my group (by Drs. S. Perkowski and J. Sun, University of Pennsylvania) was the discovery that the endothelial cell anticoagulant protein thrombomodulin was markedly down-regulated after 24 h of 100% oxygen exposure. This finding raises new questions about the role of the coagulant system in hyperoxia.

In addition, during in vivo responses, arrays provide a way to identify groups of coordinately regulated genes. As examples, interesting gene clusters were identified in lung tissue after exposure to bleomycin (Dr. D. Sheppard), during development (Dr. T. Mariani, Washington University), in bronchiolitis obliterans (Dr. S. Shapiro), and after lung oxidant injury (Dr. G. Liekauf, University of Cincinnati). Informative in vitro studies also were performed after the exposure of neutrophils to endotoxin (Dr. J. Arcaroli, University of Colorado) and after the exposure of fibroblasts to lung edema fluid (Dr. M. Olman, University of Alabama, Birmingham).

Despite these exciting new applications, however, functional genomics is still in its early stages and has a number of potential limitations. One obvious drawback is that arrays measure only messenger RNA expression levels, which may or may not correlate with changes in protein levels. A second obvious limitation is that arrays cannot measure post-translational changes, such as protein phosphorylation, that play such an important role in cellular physiology. Another important issue relates to the large amounts of variability arising from many sources including array production, RNA extraction and amplification, and hybridization. Key factors in determining the value of array data are the quality and source of the tissues analyzed. In many instances, tissue represents a mixture of cell types with differing amounts of normal tissue, inflammatory tissue, and pathologic tissue. It is important to remember the importance of biological variability in any experiment. This variability requires that multiple samples be analyzed multiple times for meaningful results to be obtained.

Another consideration is that array results need to be validated using independent means. If messenger RNA expression levels are important (ie, in diagnostic profiling), the array results should be confirmed using quantitative PCR, northern analysis, or RNAse protection assays. If conclusions about a specific gene product are to be made, the confirmation of changes in protein levels using immunoblotting or immunohistochemistry are required. As reported by Dr. M. Fessler (University of Colorado) the correlation between proteomic data and genomic data can be surprisingly low (ie, < 25%)!

A number of other limitations also were highlighted, including the notion that many array data experiments are not hypothesis-driven but are hypothesis-generating (creating potential funding problems), that the current high expense tends to limit replication, that different platforms are not very compatible, that problems remain in analyzing small tissue samples, and that questions still remain about what should be used as an appropriate comparison standard. A major problem acknowledged by all the investigators was the difficulty in handling the large amounts of data generated by arrays, necessitating sophisticated informatics help.

Presented with these challenges, one of the key areas for the future development of genomics will be better ways to “sort the wheat from the chaff.” Advances in bioinformatics and finding better ways to analyze and display data are major directions of the field. A number of other areas are being actively explored by both academic and commercial laboratories. These include the development of bigger, better, more standardized, and more inexpensive array technology. For example, Dr. D. Earle (University of California, San Francisco) presented preliminary results using 70-mer oligonucleotide arrays. The increased use of smaller custom arrays for specific purposes (ie, a lung fibrosis chip) also may allow many more investigators to use array technology. Given the heterogeneity of tissue for analysis, improvements need to be made in improving microdissection techniques along with improved ways to amplify small amounts of RNA with high fidelity. One area of great interest will be the refinement of techniques that will allow high-throughput protein analysis, or proteomics. Some exciting preliminary data using two-dimensional gel electrophoresis coupled with mass spectroscopic analysis was presented by Dr. M. Fessler, however, this technology is in its infancy. The next decade should see dramatic advances in this area. Finally, it will be critical to share data from high-quality experiments to allow others to mine data for gene discovery or cluster analysis. For example, the availability of datasets from emphysema tissue (Dr. D’Armiento), lung development (Dr. Mariani), lung cancer (Dr. Kaminski), lung fibrosis (Dr. Sheppard), or lung oxidant injury (Dr. Leikauf) would be very valuable to the research community.

In the past few years, the field of gene therapy has suffered from a combination of the failure to live up to extremely high levels of expectation coupled with the death of a healthy volunteer in a clinical trial. Nonetheless, the last decade has seen substantial progress. One area in which the value of the field has clearly been established is the use of gene therapy vectors (such as liposomes, adenoviruses, and retroviruses) as excellent tools to address important scientific questions. Two presentations exemplified this approach. Dr. P. Factor (Northwestern University) showed how an adenovirus making the β2-adrenergic receptor could be used to study mechanisms of pulmonary edema, and Dr. G. Jenkins (University College, London, UK) used a modified liposomal vector delivering cyclooxygenase-2 to explore mechanisms of fibroblast proliferation.

Despite the lack of dramatic cures, a decade of clinical trials has provided important information about the strengths and weaknesses of current vectors. Both adenoviruses and liposomal vectors have been shown to be able to transduce transgenes in patients with a variety of disorders. However, it is now clear that the expression is temporary and is associated with an inflammatory response. Although these features make the use of these vectors suboptimal for genetic diseases such as α1AT, hemophilia, or cystic fibrosis, these characteristics are desirable for the treatment of cancer. A number of groups are using adenovirus in phase I or phase II trials in the treatment of lung cancer and other thoracic malignancies. For example, our group (led by Drs. D. Sterman and L. Kaiser) has treated > 30 patients with malignant mesothelioma using intrapleural administration of an adenovirus encoding the herpes simplex thymidine kinase gene followed by infusion of the prodrug ganciclovir. Only minor side effects were seen, pleural and mesotheliomal gene transfer was documented, and a number of patients had prolonged periods of stable disease or tumor regression. Given the relatively limited amount of tumor cells transduced by the virus, however, it is likely that the antitumor responses were immune-mediated. The use of adenoviruses for immunogene therapy is an area of active interest by many investigators.

Given the limited ability of liposomes and adenoviruses to enable long-term gene expression, and given the poor in vivo performance of retroviruses, other vectors are being developed. One of the most promising of these is adeno-associated virus (AAV). This virus is smaller than the adenovirus and has a relatively low-capacity size, however, it allows for long-term gene expression (ie, months to years) with only minimal induction of inflammation or antiviral immune responses. As discussed by Dr. T. Flotte (University of Florida, Gainesville), a better understanding of the life cycle of this virus, along with improved production techniques, has allowed investigators to conduct clinical trials with AAV in diseases such as hemophilia and cystic fibrosis. Preclinical data in mice injected intramuscularly with an AAV-α1AT vector are very encouraging, and a clinical trial performed by Dr. Flotte’s group is planned within the next year. Another vector system under development, lentiviruses, is based on the HIV virus backbone.

The major problem in gene therapy remains the relative inefficiency of current vectors. Currently, this inefficiency, coupled with a relatively poor specificity of most vectors, requires the delivery of large doses of vector. This is both expensive and more likely to lead to side effects. Questions still remain about which and how many cells need to be transduced to obtain a clinical response. The future of gene therapy, therefore, lies not only in designing improved vectors but also in determining diseases that might be treated with existing vectors. One area of active interest in the cancer field is conditionally replicating vectors, that is vectors that selectively replicate within tumor cells causing either direct lysis or the augmented delivery of a transgene. Another area of exploration is the development of ways to redirect the tropism of vectors to target specific tissues. Finally, gene therapy will benefit tremendously from the identification of new targets from discoveries in genetics, genomics, and other basic science arenas.

One new and very exciting area of gene therapy that has not yet reached clinical trials is “gene correction.” As presented by Dr. R. Metz (ValiGen, Inc), it is possible to design oligonucleotides that bind to areas of single-nucleotide changes that are associated with abnormal functions and to catalyze corrections of the nucleotide errors. This concept clearly has been demonstrated to work in cell cultures and in animal models, although the efficiency is still quite low. With the development of better oligonucleotides and improved delivery methods, this approach will likely be tested first in diseases such as hemophilia and α1AT. The possibility of actually performing gene correction is especially appealing.

The 44th Aspen Lung Conference highlighted an exciting trio of new approaches that will revolutionize the study of lung diseases. For example, the classic approach used to discover the pathophysiology of diseases such as α1AT or PCD, in which abnormal protein function led to the identification of a gene (Fig 1 ), has been replaced by a new paradigm in which positional cloning and genomics are used to isolate abnormal or abnormally expressed genes (Fig 2 ). The mechanisms leading to the clinical manifestations of disease then are uncovered by analyzing the effects of abnormal function of the affected gene. Interestingly, even when the abnormal genes and corresponding proteins have been identified, their contribution to the human disease state still can elude researchers for many years. For example, despite the identification of the “cystic fibrosis gene” > 10 years ago, it is still not clear how this gene defines the pathophysiology of cystic fibrosis. It is likely that the connection between mutations in the BMPR-2 gene and the pathophysiology of PPH also will take years to unravel.

The fields of genomics and proteomics are starting to add large amounts of new information about lung diseases. As discussed above, the challenges here will be in identifying the changes that are important in the pathophysiology of disease and in being able to successfully handle large amounts of biological information. I predict, in the near future, that genomics will become a part of all pulmonary research, much the way that investigators now use molecular biology or monoclonal antibodies.

Gene therapy remains the key link between advances in genetics and genomics and the translation of this knowledge into useful outcomes for our patients. Although progress has been slower than hoped for, clear advances are being made, and I have no doubt that gene therapy will find a number of key therapeutic niches.

It is perhaps fitting to end this summary by placing the advances in each section of this year’s area of focus (ie, genetics, genomics, and gene therapy) in the proper context. As with most new approaches or techniques, there is a predictable “curve of enthusiasm” (see Fig 3 ). After discovery or introduction (point A), enthusiasm rises to a high level (point B), resulting in a “suspension of disbelief.” This is the ideal time to submit grant proposals. The possibilities seem endless, and the potential problems seem minimal. Rather quickly, however, reality usually rears its ugly head, and the limitations become apparent. This often leads to an overreaction and to a marked decrease in the initial enthusiasm. Depending on the initial level of hype created by, and sometimes fueled by, spectacular failures, this subsequent fall from grace can be rather dramatic, leading to a reversal of attitudes that results in a “presumption of disbelief” (point C). This is not the time to submit grant proposals. However, at this critical juncture, the true believers continue their work and either find a realistic niche for the approach (point D) or allow it to fall out of favor completely (point E).

This type of roller coaster ride can be most easily pictured for gene therapy, where the initial enthusiasm and unrealistic expectations of the early 1990s has led to feelings of pessimism and cynicism that were exaggerated by the death of a relatively healthy volunteer in 1999. Gene therapy is at an important crossroads now (point C). The field of genomics is at its peak of enthusiasm now, with great expectations and great enthusiasm sweeping almost every field of biological research (point B). Genetics has managed to keep on a bit more level playing field, however, the recent publicity regarding the human genome project has heightened expectations.

The 44th Annual Aspen Lung Conference was a great success and the organizing committee, led by Drs. Geraci and Rodman, is to be congratulated. Let me end with my own personal “bottom line”: “Genomics—be there or be square (but beware). Gene therapy—be there, but take care. Genetics—let lots of other people be there, but be aware.”

Abbreviations: α1AT = α1-antitrypsin; AAV = adeno- associated virus; BMPR = bone morphogenic protein receptor; FPPH = familial primary pulmonary hypertension; HLA = human leukocyte antigen; LAM = lymphangiomyomatosis; PCD = primary ciliary dyskinesia; PPH = primary pulmonary hypertension

Table Graphic Jump Location
Table 1. “Simple” Genetic Diseases for Which the Causative Genes Have Been Identified
Table Graphic Jump Location
Table 2. Applications of Functional Genomics
Figure Jump LinkFigure 1. Classic approach to the discovery of disease pathophysiology.Grahic Jump Location
Figure Jump LinkFigure 2. New paradigm for the discovery of the pathophysiology of disease. CF = cystic fibrosis.Grahic Jump Location
Figure Jump LinkFigure 3. The evolution of new approaches.Grahic Jump Location


Figure Jump LinkFigure 1. Classic approach to the discovery of disease pathophysiology.Grahic Jump Location
Figure Jump LinkFigure 2. New paradigm for the discovery of the pathophysiology of disease. CF = cystic fibrosis.Grahic Jump Location
Figure Jump LinkFigure 3. The evolution of new approaches.Grahic Jump Location


Table Graphic Jump Location
Table 1. “Simple” Genetic Diseases for Which the Causative Genes Have Been Identified
Table Graphic Jump Location
Table 2. Applications of Functional Genomics


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