Editor's note: Doug Modlin, Ph.D., chairperson of the Research Advisory Committee for CFRI, compiled the following research proposal summaries from biomedical research projects funded by CFRI during 1994. Many of the projects are still underway. They are organized into three kinds of research: basic, clinical and gene therapy. Basic research attempts to answer fundamental questions about the molecular processes underlying cellular function (and dysfunction in the case of cells affected by cystic fibrosis). It guides all other scientific work. Clinical research tests proposed therapies on real people, so it may yield approaches that we can use in the near future. Gene therapy research has become possible since the discovery of both the gene and the knowledge of precise genetic differences between people with and without CF. While it is among the newest areas of research, we at CFRI are hopeful that it will provide the basis for human gene therapy or cell transfer in patients having cystic fibrosis.
1. "Regulation of CFTR's Gating and Effects of the x- F508 Mutation," Principal Investigator: Terry E. Machen, Ph.D., Professor of Cell Biology and Developmental Biology, Department of Molecular and Cell Biology, University of California at Berkeley.
Dr. Machen continues his study of the gating and regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel by a variety of powerful methods including patch clamping. He will examine both wild-type and mutant CFTR molecules expressed in NIH 3T3 fibroblast cells. Fibroblast cells are a type of connective tissue cell that grows well in culture.
2. "New Mechanisms of Fluid Secretion from Identified Secretory Cells in Cystic Fibrosis," Principal Investigator: Madireddi M. Reddy, Ph.D., Assistant Research Physiologist, Division of Biomedical Sciences, University of California at Riverside.
The sweat gland is composed of three cell types: myoepithelial cells and dark and clear secreting cells. Dr. Reddy and Dr. Paul Quinton have observed that the secreting cells in the sweat gland are either sensitive or insensitive to stimulation by beta-adrenergic drugs. Unfortunately, their electrophysiological technique does not allow them to see the cells they are testing. Dr. Reddy wants to link the cell type (dark or clear) to its relative sensitivity to beta-adrenergic drugs. He attempts this by injecting a fluorescent dye into a cell immediately after it has been tested for drug sensitivity and then examines it under a fluorescent microscope (it will glow). Once found and examined, Dr. Reddy can then determine the cell type. His goal is to ascertain whether the clear cells are abnormal in CF which is what he and Dr. Quinton suspect.
3. "Small GTP Binding Proteins and Trafficking of CFTR," Principal Investigator: Jonathan Widdicombe, Ph.D., Cardiovascular Research Institute, Professor of Physiology, University of California at San Francisco.
This project is a continuation of research begun last year. The original goal of Dr. Widdicombe's work was to shed light on the involvement of small GTP binding proteins in the trafficking of CFTR to the apical membrane of primary cultures of human airway cells. This was to be achieved by testing the effects of lovastatin which, hypothetically, block the GTP binding proteins and subsequently the transport of CFTR to the plasma membrane. In the first year, Dr. Widdicombe confirmed that lovastatin did greatly inhibit the transport of CFTR to the plasma membrane. cAMP-dependent chloride secretion was effectively eliminated. Furthermore, it was shown that the effects of lovastatin could be reversed by mevalonate, geranylgeranyl, or farnesil. His 1994 research is focused on studying the effects of other man-made compounds (peptides) on the intracellular trafficking of CFTR. Improving CFTR trafficking may be a potential direction for therapeutic intervention in CF as it is suspected that some forms of mutant CFTR may function once it can be delivered to the plasma membrane.
4. Cystic Fibrosis Research Laboratory, Basic and Gene Therapy Research, Principal Investigator: Jeffrey Wine, Ph.D., Director, Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California.
The Cystic Fibrosis Research Laboratory scientists have primarily been involved in basic research on how CFTR works and how its dysfunction leads to disease. However, they have also focused their efforts on gene therapy for CF and providing tools and methods that should greatly speed the development of therapy for CF. Dr. Jeff Wine is currently working on a plan that allows him to manipulate DNA directly. The bulk of CFRI's money was used to purchase a Perken-Elmer 9600 Thermal Cycler to carry out polymerase chain reaction amplifications of DNA. The remainder was used to buy a variety of molecular biological reagents, supplies, and minor equipment. On the basis of preliminary data obtained with the CFRI funds, Dr. Wine was awarded a much larger grant from another well-established funding source to pursue these studies.
1. "A Genetic and Molecular Analysis of the Virulence Factors of Pseudomonas aeruginosa Responsible for Lung Injury in Cystic Fibrosis Patients," Principal Investigator: Joanne N. Engle, M.D., Ph.D., Assistant Professor, Division of Infectious Disease, University of California at San Francisco.
Dr. Engle, using genetic techniques, proposes to learn more about the mechanisms by which Pseudomonas aeruginosa (P. aeruginosa) causes lung damage during pneumonia. She will use both in-vitro (cultured epithelial cells) and in vivo (rats, etc.) models developed by her collaborator, Dr. Jeanine Wiener-Kronish at UCSF. Dr. Engle is attempting to identify strains (mutants) of non-injurious Pseudomonas (unable to cause cell damage) using previously developed genetic methods. These non-injurious mutants of P. aeruginosa will also be tested for their ability to cause cell damage in the in vivo systems. The mutant genes responsible for loss of cell damage in the non-injurious strains will be isolated and characterized and the role of the wild-type genes in the pathogenesis of P. aeruginosa will be studied further.
2. "Characterization of AlgL and its Role in Alginate Synthesis in Pseudomonas aeruginosa," Principal Investigator: Neal L. Schiller, Ph.D., Associate Professor of Biomedical Sciences, Chairman of the Graduate Program in Microbiology and the Associate Dean of the Graduate Division at the University of California at Riverside. Please see Dr. Schiller's article about his research in [article-48].
1. "Correction of CFTR Defects in Airway Gland Cells," Principal Investigator: Walter E. Finkbeiner, M.D., Ph.D., Associate Professor, Department of Pathology, University of California at San Francisco.
Dr. Finkbeiner is developing a gene therapy strategy directed at seromucous gland cells. Seromucous cells are derived from airway gland cells. Since most of the mucus in the airways is thought to come from the glands and it is unknown whether or not the virus-based transfection vectors can reach the glands, Dr. Finkbeiner, in collaboration with Dr. Francis Szoka (Department of Pharmacology, UCSF), is exploring the use of liposomes as the gene delivery system. Dr. Finkbeiner believes these gland cells are a more appropriate target for gene therapy than the surface airway cells currently being targeted by most virus-based strategies. Serous cells in the glands are rich in CFTR and are responsible for providing the water which is mixed with mucus proteins prior to their secretion into the airways.
2. "Use of Leukocytes in Gene Therapy for Cystic Fibrosis," Principal Investigator: Jonathan Widdicombe, Ph.D., Research Scientist, Children's Hospital Oakland Research Institute, and Professor of Physiology, University of California at San Francisco.
Dr. Widdicombe proposes to use Leukocytes (white blood cells) to achieve a blood stream based gene therapy which could readily reach the submucosal gland cells (key to CF pathophysiology). White blood cells will be induced to migrate from the blood vessels into the lungs by inhaling chemotactic agents. When the cells migrate, they temporarily leave behind a pore that is presumably big enough for a gene therapy delivery vehicle such as a liposome to pass through. This "inside out" gene therapy could thus reach tissues that would be difficult or impossible to reach with the inhaled virus delivery vehicles currently being tested.
3. "Gene Therapy of CF using an Episomal Expression Vector containing Wild-type CFTR," Principal Investigator: Dieter C. Gruenert, Ph.D., Cardiovascular Research Institute, Co-director, CF Gene Therapy Center, University of California at San Francisco.
This grant supplements a grant awarded to Dr. Gruenert by CFRI in November, 1993. His project explores an approach to CF gene therapy wherein a circular piece of DNA, called a plasmid, would be delivered to the nucleus of a CF airway cell using liposomes. Once in the nucleus, the plasmid would produce messenger RNA which then tell ribosomes in the cell to synthesize copies of the CFTR protein that do not contain a CF producing defect. A specific feature of this approach is that the plasmid would be designed to be "episomal" and thus would not be incorporated into the host cell's DNA. This is important as there would be less risk of unintended genetic complications during gene therapy trials.
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