Overview of Research in Gene Therapy at the University of Iowa

By its very nature, gene therapy of genetic diseases is a technology of tremendous potential in relieving human suffering, but it is at the same time a massive and dauntingly complex scientific endeavor. That we currently stand on the threshold of successfully curing genetic diseases is a monument to the unprecedented scientific breakthroughs achieved in recent years, in mapping the genetic mutations underlying inherited metabolic diseases and in precisely defining the pathophysiology of the resulting cellular defects. These accomplishments have required the concerted effort of scores of dedicated clinical and basis science researchers, many of whom are faculty members at the University of Iowa. However, despite the recent advances, the science for gene therapy of human genetic diseases is still currently in its infancy and much work remains to be done. The Iowa Center for Gene Therapy has a particular emphasis on cystic fibrosis, but extends as well to other areas in which the University of Iowa has particular strengths. Historically, the academic culture at The University of Iowa has been one of collaboration and the sharing of resources and expertise, and gene therapy research for CF has benefited from the active gene therapy programs directed at other target organs and diseases. It is the goal of this Center to further facilitate such interactions among researchers investigating gene therapy for diseases affecting the CNS, muscle, vessels, skin, liver, and lung.

Steps for Gene Therapy Research

There are three broad research areas underlying the development of successful gene therapy approaches for the treatment of inherited metabolic diseases:

(1) The first is the identification of disease-causing gene mutations.
(2) The pathophysiology arising from the genetic mutation must then be studied in the context of gene function and the basic cell biology of the affected organ system(s), so that the appropriate cellular targets for gene therapy can be identified.
(3) Lastly, suitable and effective gene therapy vector systems for targeting the specific affected system and providing long-term amelioration of the disease must be developed.

Cystic Fibrosis: The Disease

Cystic Fibrosis is the most common fatal genetic disease in the Caucasian population, with a frequency of about one in 2,500 live births a year. Currently, the median age of survival for a patient with CF is 31 years. Although disseminated throughout other organs, the most life-threatening clinical feature of CF is pulmonary obstruction caused by abnormally thick mucus secretions and chronic infection by opportunistic bacteria, such as Pseudomonas and Staphylococcus, which lead to respiratory failure. Treatment today consists of a comprehensive approach, including postural drainage and percussion, replacement of pancreatic enzymes and proper nutrition, administration of antibiotics, mucus-thinning and anti-inflammatory drugs, and newer drugs aimed at symptomatic correction. In 1989, the defective gene underlying CF was identified as Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), opening a new era of investigation into the pathophysiological mechanisms of CF disease, and leading to the promise of gene transfer as the ultimate therapeutic intervention. CFTR is a cAMP-regulated Cl- channel and also has been implicated in the regulation of other ion channels localized in the apical membranes of airway epithelial cells. Mutations in CFTR result in defective ion transport, leading to thick mucus, impaired mucociliary clearance and decreased bacterial killing. Despite recent progress in associating CF defects with CFTR dysfunction, there remain many unanswered questions concerning the roles of CFTR in normal airway biology and CF pathology, and the identification of the relevant cellular targets for gene therapy.

Gene Therapy of Cystic Fibrosis

The University of Iowa has a long-standing history as a leader in the field of CF research and applied gene therapies for this disorder. In this regard the University of Iowa has several funded CF research programs, including a Research Development Program (RDP) funded by the Cystic Fibrosis Foundation, a CF Scientific Center of Research (SCOR) funded by NHLBI, and a Gene Therapy for Cystic Fibrosis program project grant (PPG) funded by NIH, all under the direction of Michael Welsh. These programs are an integral part of the CF research base involved in the Iowa Center for Gene Therapy of Cystic Fibrosis. In three Clinical Trials for gene therapy of CF, Dr. Michael Welsh’s group and colleagues in the CF Clinical Center have evaluated the safety and efficacy of CFTR gene delivery to the nasal mucosa of CF patients with recombinant adenoviral vectors and cationic liposomes (1, 2, 3, 4, 5). Importantly, the CF research community at the University of Iowa has also made significant contributions to increase understanding of lung development and the identification of putative stem cell targets for gene therapy, as well as the recent identification of new hypotheses regarding the pathogenesis of bacterial infection in CF airways. This group of investigators has also been instrumental in developing animal and in vitro model systems critical for studying disease pathogenesis and for testing treatment strategies and surrogate endpoints. Ongoing research programs in vector development, vector-host interactions and virology have aided in understanding some of the present limitations in current vectors. Studies are now directed toward improving the efficacy of several vector systems (adenoviruses, adeno-associated viruses [AAV], retroviruses, and non-viral vectors) for gene transfer to multiple organs. Development of an effective gene therapy for cystic fibrosis (CF) requires further advancement in areas of basic research on airway biology, CF pathophysiology, CFTR function, and vector design and evaluation.

1) Adenovirus-Mediated Gene Transfer Transiently Corrects the Chloride Transport Defect in Nasal Epithelia of Patients with Cystic Fibrosis. Cell, 75:207-16, 1993.
2) Cystic Fibrosis Gene Therapy Using an Adenovirus Vector: In Vivo Safety and Efficacy in Nasal Epithelium. Human Gene Therapy, 5:209-19, 1994
3) Adenovirus-Mediated Gene Transfer for Cystic Fibrosis: Part A. Safety of Dose and Repeat Administration in the Nasal Epithelium. Part B. Clinical Efficacy in the maxillary Sinus. Human Gene Therapy, 6:205-18,1995
4) Repeat Administration of an Adenovirus Vector Encoding CFTR to the Nasal Epithelium of Patients with Cystic Fibrosis. J. Clinical Invest. 97:1504-1511,1996.
5) Comparison of DNA/Lipid Complexes and DNA Alone for Gene Transfer to Cystic Fibrosis Airway Epithelia In Vivo. J. Clinical Invest. 100: 1529-1537, 1997.

Lung Biology and CF Pathophysiology

Michael Apicella, M.D. Pathogenesis of several types of bacterial infections, innate immune responses to microbial infection, and mucosal responses to lipooligosaccharides. Most relevant to CF includes work on Haemophilus influenzae infections and the molecular mechanisms responsible for the transition of this usually commensal organism to the pathogenic state.

John F. Engelhardt, Ph.D. Identification of stem cells in the airway as targets for in utero gene therapy of CF lung diseases; submucosal gland involvement in the pathogenesis of CF lung disease; mechanisms of fluid and electrolyte abnormalities in CF airway disease.

Aloysius J. Klingelhutz, Ph.D. Generation of conditionally transformed airway cell lines capable of polarizing into airway epithelia in vitro.

Fred S. Lamb, M.D., Ph.D. Ontogeny and function of CLC chloride channel expression in the airway.

Dwight C. Look, M.D. Epithelial cell responses to H. Influenzae in the airway.

Rama Mallampalli, M.D. Gene transfer of novel surfactant enzymes in the lung models of P. aeruginosa infection. Regulation of acute lung injury by TNF-alpha and lipoproteins in response to infection.

Paul B. McCray Jr., M.D. Cell biology of retroviral receptor and beta-defensin expression in the airway; ontogeny of ENaC expression during development.

Lynda Ostedgaard, Ph.D. Structure-function of the CFTR R-domain.

Mark Sinski, Ph.D. Regulation of human cytomegalovirus gene expression; viral RNA's, proteins and DNA sequencing and their uses in viral vector system and gene delivery.

John B. Stokes III, M.D. Regulation of ion transport by epithelial cells with an emphasis on sodium channels and their importance in CF epithelial dysfunction.

Paola Vermeer (Drapkin), Ph.D. Mechanisms of airway epithelial regeneration and repair in response to injury.

Michael J. Welsh, M.D. Structure/function of CFTR and pathogenesis of CF airways disease; antibacterial defenses in the airway.

Joseph Zabner, M.D. Mechanisms of electrolyte transport in the airways.

Gene Therapy Vector Development

Vector development is a highly critical component of this Gene Therapy Center that enhances applications for gene therapy of all genetic diseases. Researchers within this Center and the affiliated Gene Therapy Vector Core Facility have a wide range of expertise with various vectors including adenovirus, adeno-associated virus, retroviruses, and liposomes. Results from basic vector research and previous clinical trials have demonstrated that a concrete program in immunology is critical for the development and testing of viral vectors for gene therapy. In this regard several investigators have research programs studying the mechanisms of immune tolerance, immune responses to prokaryotic DNA by virtue of altered methylation, and immunologic therapies to block both cellular and humoral immunity to recombinant adenovirus. Investigators at the University of Iowa were the first to identify that unmethylated CpG dinucleotides, which are more frequent in the genomes of bacteria than of vertebrates CpG motifs, are critical activators of the immune response (1). This finding may be instrumental in the development of improved gene therapy vectors for treatment of both CF and other genetic diseases.

Beverly Davidson, Ph.D. Applications of rAAV and the development of novel pseudotyped FIV and MMLV-based retroviral vectors for gene transfer to the airway.

John F. Engelhardt, Ph.D. Biology of rAAV transduction with an emphasis on the intracellular trafficking and barriers to transduction in polarized airway epithelia. Development of novel gene correction technologies to target airway stem cells and correct CFTR defects.

Gary W. Hunninghake, M.D. Regulation of cytokine gene expression and animal models of sepsis, pulmonary fibrosis, and acute lung injury. Signal transduction of activated alveolar macrophages. Immunology of adenoviral infection in animal models using recombinant and wild type adenovirus.

Joel N. Kline, M.D. Inflammatory mediators limiting gene transfer to the airway; CpG DNA and its effect on the pathogenesis of airway inflammation.

Dwight C. Look, M.D. Interactions between adenoviral E1A and multiple proteins in the FN-gamma-dependent signal transduction pathway that enable adenovirus to more fully subvert the immune response.

Rama Mallampalli, Ph.D. Development of the CCT promoter for airway expression with rAAV.

Paul B. McCray Jr., M.D. Retroviral and lentiviral gene transfer to the airway with an emphasis on the identification of novel pseudotyping strategies to increase apical infection.

Lynda Ostedgaard, Ph.D. Generation of R-domain deleted CFTR cDNAs for use in rAAV and generation of short promoters for use with CFTR delivery.

Kevin Rice, Ph.D. Development of DNA-complex delivery systems.

Colleen S. Stein, Ph.D. Establishment of immune tolerance to viral vectors.

Christie P. Thomas, MRCP. Development of airway epithelial specific promoters for gene targeting.

Paola Vermeer (Drapkin), Ph.D. Retargeting of adenoviral vectors to the urokinase plasminogen activator receptor on the apical surface of polarized airway epithelia.

Daniel Weeks, Ph.D. Gene targeting using triplex oligonucleotides.

Michael J. Welsh, M.D. Viral and non-viral mediated gene transfer to the airway.

Chung-Fang Wu, Ph.D. Evaluation of deleterious effects of rAAV vector enhancer sequences on the expression of endogenous gene loci using a developmental mutagenesis approach in drosophila.

Ziying Yan, Ph.D. Dissection of rAAV gene conversion biology and application in novel hybrid ITR vectors and dual vector systems.

Joseph Zabner, M.D. Serotype 17 adenovirus for gene transfer to the airway.

Clinical Surrogate Endpoints for Gene Therapy of CF Lung Disease

The development of clinically relevant endpoints for determining the efficacy of gene therapy of CF is also a critical feature of any program directed at the treatment of CF lung disease. This Center, which has a strong history of gene therapy and other clinical trials for CF lung disease is uniquely positioned to address the challenges of developing surrogate endpoints for clinical trials of gene therapy. The Cystic Fibrosis Clinical Center, which interfaces with both the General Clinical Research Center and the Cystic Fibrosis Research Center, has facilitated clinical trials for gene therapy of CF under the context of SCOR and PPG center grants. Although these clinical trials used molecular endpoints for CFTR functional complementation, several investigators within this Center have expertise in the evaluation of intact lung functions and radiological diagnosis of CF lung disease, which may, with further development, provide additional means of evaluating the success of gene therapy. Such efforts highlight the major emphasis of this Center to evaluate in vivo efficacy of gene therapy in CF.

Richard Ahrens, M.D. High resolution CT for measuring lung injury and inflammation in CF. Active involvement in clinical trials assessing changes in lung function.
Frederick E. Domann, Ph.D. The development of non-invasive radiological imaging of gene transfer in vivo using reporter genes which transport radioactive ions.
Eric A. Hoffman, Ph.D. Image and model based analysis of lung disease Lung imaging and analysis for multi-center pulmonary related clinical trials. Volumetric high resolution CT scanning to evaluate intrapulmonary airway reactivity for outcomes assessment and evaluation of emerging therapies for lung disease
Paul B. McCray Jr., M.D. In vivo imaging of luciferase gene expression in the lung using recombinant FIV.
Geoffrey McLennan, Ph.D. The development of computer-aided methods for objective analysis of abnormal lung parenchyma, and development of in vivo laser technologies for bronchoscopic determination of GFP reporter gene expression.
Joseph Zabner, M.D. In vivo imaging of luciferase gene expression in the lung using recombinant AAV and adenovirus.

Gene Therapy of Other Genetic Diseases

Although the primary focus of the Gene Therapy Center is on Cystic Fibrosis, the University of Iowa has a strong and broad research base for gene therapy for other genetic diseases. For example, there is an exceptionally large multidisciplinary research group affiliated with the University of Iowa Cardiovascular Center. This Center studies mechanisms of neurovascular regulation, cerebrovascular disease, atherosclerosis, and the application of gene therapy strategies for treating inherited disorders of these organ systems. The Vector Core and its Director, Beverly Davidson, interact with diverse researchers from all over the University of Iowa campus with interests in investigation and therapy for genetic diseases. These diseases range from genetic disorders of the skin to muscular dystrophies. Lastly, the laboratories of Jeff Murray, Bento Soares, Val Sheffield and Edwin Stone provide a strong scientific base in the identification of new disease-causing genes.

Neurological and Cardiovascular Diseases

Francois M. Abboud, M.B., B.Ch. Paracrine role of endothelial factors in the modulation of baroreceptor activity and the central mediation of the baroreflex with aging and diabetes
Ramesh Bhalla, D.V.M., Ph.D. Mechanisms underlying NO-induced inhibition of VSM cell proliferation and the development of novel approaches to genetic prevention of restenosis using recombinant adenoviruses expressing eNOS.
Mark W. Chapleau, Ph.D. Physiology and cell biology of baroreceptor sensory neurons and the associated reflex control of arterial pressure and circulation; adenoviral mediated gene transfer to cultured baroreceptor neurons and to sites of baroreceptor innervation in vivo.
John M. Dagle, M.D., Ph.D. Synthesis and characterization of modified antisense oligonucleotides to alter gene expression. Involvement of Pitx2 in heart development.
Beverly L. Davidson, Ph.D. Gene transfer to the CNS using FIV and rAAV vectors. Applications in the treatment of beta-glucuronidase deficiency. Understanding and overcoming host responses to encapsidated viruses delivered to brain.
Steven H. Green, Ph.D. Neurotrophic signaling, signal transduction, and mechanisms by which neurotrophic factors promote neuronal survival; use of adenoviral vectors in vivo to regulate neurotrophin expression and study effects on neuronal survival.
Donald Heistad, M.D. Development of gene transfer strategies to cerebral blood vessels and atherosclerotic arteries; gene transfer to study vascular biology in normal arteries with a focus on the importance of intracellular reactive oxygen species.
Alan Kim Johnson, Ph.D. CNS regulation of body fluid and cardiovascular system; adenoviral gene transfer of prevasopressin peptide to forebrain, with goals of restoring antidiuretic function in the Brattleboro rat model of familial diabetes insipidus.
Francis Miller, M.D. Role of reactive oxygen species in vascular disease and the molecular/cellular mechanisms that contribute to the pathophysiology of atherosclerosis; adenoviral-mediated gene transfer to modulate reactive oxygen species important in abnormal endothelial dependent responses.
Curt D. Sigmund, Ph.D. Regulation of genes involved in blood pressure homeostasis and the generation of new transgenic animal models of human cardiovascular disease.
Neil L. Weintraub, M.D. Vascular endothelial and smooth muscle cell biology as it relates to the biological activity of arachidonic acid metabolites under normal and disease conditions (including atherosclerosis and hypertension); adenoviral-mediated gene transfer as a tool to address the importance of free radicals to vascular endothelial and smooth muscle cell function and pathology.

Gene Therapy of Muscular, Liver, Skin and Bone Disorders, and Familial Diabetes

Included in this section are Center Members and Associate Members, many of whom are already using or developing gene therapy methods and investigate a diverse range of genetic diseases. It is one of our goals to unite and recruit those scientists to our Center who have highly relevant research programs, but who are not currently associated with one of the large, cohesive research groups at Iowa. In many ways, the Center can be of benefit to them, such as in the dissemination of advances in vector development and targeting, that widen the scope and extend the potential benefits of gene therapy in relieving human suffering. In return, other Center Members benefit from the affiliation and exchange of ideas and methodology which may have application in Cystic Fibrosis gene therapy research. Represented in this group are research programs centered on muscular, liver, skin and bone disorders, as well as familial diabetes.

Kevin P. Campbell, Ph.D. Molecular and cellular biology of skeletal muscle relating to the pathogenesis of muscular dystrophy and other inherited neuromuscular disorders; gene therapies for these disorders using recombinant adenovirus and adeno-associated virus.
Joseph S. Dillon, BAO Signal transduction mechanisms of the pancreatic beta cell and the genetic basis of type II diabetes in rat models of diabetes.
John F. Engelhardt, Ph.D. Development of redox mediated gene therapies to the liver and hart for ischemia/reperfusion injury and sepsis.

Disease Gene Identification

Of absolute importance to the development of gene therapy for any inherited disease is the identification of the disease-causing mutation. Recent advances in technology have led to the identification of the genetic defects associated with numerous diseases. These findings have arisen in a large part from the efforts of the Human Genome Project. Researchers at the University of Iowa have been instrumental participants in the Human Genome Project, both by contributing technological advances toward facilitating the high resolution mapping project, and by utilizing the genetic markers obtained from the project to track down the genetic causes of both rare and common inherited disorders. Of note for their invaluable contributions to this field are the laboratories of the four investigators in this section. By affiliation with the Iowa Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases as Associate Members, each of them has either initiated or expressed a desire to develop gene therapy strategies for the inherited diseases that they have discovered. It is our goal to foster their entry into this area, by providing the environment and interactions necessary for this step.

Thomas Casavant, Ph..D. Computational aspects of genomics, molecular biology, and human genetics, as well as high performance computing systems, software and networks required for data mining