Combined stem cell-gene therapy approach seen as potential treatment for cystic fibrosis
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Public release date: 20-Dec-2004
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Contact: Lisa Rossi, University of Pittsburgh
RossiL@upmc.edu
412-647-3555
Melanie Finnigan, Children's Hospital of Pittsburgh
melanie.finnigan@chp.edu
412-692-5016
University of Pittsburgh Medical Center
Combined stem cell-gene therapy approach seen as potential treatment for cystic fibrosis
PITTSBURGH, Dec. 20 – Patients with cystic fibrosis (CF) could potentially be treated using their own stem cells that have been manipulated by gene therapy, suggests a study reported in this week's Proceedings of the National Academy of Sciences (PNAS). The authors, who represent five institutions, including the University of Pittsburgh School of Medicine and Children's Hospital of Pittsburgh, demonstrate for the first time that human bone marrow-derived adult stem cells can be coaxed to differentiate into airway epithelial cells and that encoding these cells with the gene that is defective in CF restores an important cellular function essential for keeping the airways clear of mucus and air-borne irritants.
"Our results provide proof of principle that a cell-based therapy using marrow stromal stem cells is both a feasible and promising clinical approach. We plan to further investigate its potential and are hopeful that we can perform a small clinical trial within the next two to three years," noted senior author Jay K. Kolls, M.D., professor of pediatrics, immunology and molecular genetics and biochemistry at the University of Pittsburgh School of Medicine and chief, Division of Pediatric Pulmonology and Laboratory of Lung Immunolgy and Host Defense at Children's Hospital of Pittsburgh.
CF is the most common, fatal genetic disorder affecting Caucasians and is characterized by a mutation of a gene that sits on the surface of epithelial cells, including those that line the airways in the lungs. The gene, transmembrane conductance regulator (CFTR), is responsible for channeling chloride out of cells, an essential function that influences the level of airway surface liquid. This fluid sits atop the airway cells and provides support for the hair-like projections called cilia that sweep mucus and dirt away from the cell. When the level of liquid is too low, as is the case in CF, mucus builds up and the area becomes an attractive environment for bacteria to colonize and cause infection.
Finding ways to correct the gene defect responsible for CF has been the focus of much research and some clinical studies are even looking at gene therapy approaches. To date, no studies have evaluated the potential of adult stem cells or the use of stem cells in combination with ex vivo gene therapy, whereby the CFTR gene is delivered to the cells in a laboratory setting before their introduction into the patient.
While some studies have suggested the potential for bone marrow derived stem cells to differentiate into airway epithelial cells, Dr. Kolls and his co-authors have provided significant proof. Using a method they developed and report in PNAS, stem cells taken from normal subjects and placed in a culture with mature epithelial cells for 14 days differentiated into the specialized cells, taking on their shape and displaying their characteristic protein markers. Importantly, stem cells aspirated from the bone marrow of three CF patients and co-cultured in the same manner also produced airway epithelial cells, but, as expected, were deficient for the CFTR gene. To correct the genetic flaw, the researchers used the Maloney murine leukemia virus as a vector to shuttle the CFTR gene into the stem cells.
A technique called polymerase chain reaction, or PCR, confirmed the gene was expressed in the newly differentiated airway epithelial cells, reported Guoshun Wang, M.D., D.D.M, of Louisiana State University (LSU), where Dr. Kolls previously had worked, and Bruce A. Bunnell, Ph.D, of Tulane University, the study's first authors. Moreover, the gene therapy-treated cells from CF patients had the same developmental potential as bone marrow-derived stem cells from healthy people.
To determine whether the gene therapy actually corrected the CFTR defect, the researchers exposed the cells to a medium containing labeled chloride and a molecule that regulates the intake and secretion of salts and water necessary for cellular function. If the CFTR gene were not functioning properly, the chloride taken in by the cell would not be able to be secreted. Yet, as the authors reported, the CFTR-corrected stem cells secreted significantly more chloride than stem cells from CF patients without the inserted gene.
"Being able to demonstrate that the gene permitted enhanced chloride channeling was critical to determining the potential of the stem cell-based therapy in the clinical setting," said Dr. Kolls.
Although he acknowledges that more work needs to be conducted, including developing a way to identify the specific subpopulation of bone marrow stem cells that are key to successful engraftment, Dr. Kolls is optimistic about the possibility that a patient's own stem cells – which would not be subject to immune system attack – could be manipulated by gene therapy and used successfully as a treatment for CF. The ex vivo approach to gene therapy may offer several advantages, including the ability to screen the cells before actually delivering them to patients. In addition, the virus vector being studied by Dr. Kolls is bigger than others being used in trials and has the capacity for both the gene and its accessory apparatus, which may help ensure long-term function.
To further study the potential of the stem cell approach, including in CF patients, Dr. Kolls and colleagues from Children's Hospital, in collaboration with the University of Pittsburgh, are seeking funding from the National Institutes of Health (NIH) to establish a center focusing on the development of adult stem cell-based therapies for diseases affecting the lung, heart and blood vessels.
In addition to Drs. Kolls, Wang and Bunnell, other authors of the PNAS study include Richard G. Painter, Ph.D., Nicholas A. Lanson, Jr., Ph.D., and Donna Bertucci, from LSU; Blesilda C. Quiniones, M.D., Susan Tom, Jeffrey L. Spees, Ph.D., Alexandra Peister, Ph.D., and Darwin J. Prockop, M.D., Ph.D., from Tulane; Daniel J. Weiss, M.D., Ph.D., of the University of Vermont College of Medicine; and Vincent G. Valentine, M.D., of the Ochsner Clinic Foundation in New Orleans.
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Their research was supported by the Louisiana Gene Therapy Research Consortium and the National Heart, Lung and Blood Institute of the NIH.
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pnas.org
Published online before print December 22, 2004
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0406266102
PubMed
PubMed Citation
Articles by Wang, G.
Articles by Kolls, J. K.
Medical Sciences
Adult stem cells from bone marrow stroma differentiate into airway epithelial cells: Potential therapy for cystic fibrosis
Guoshun Wang *, Bruce A. Bunnell , Richard G. Painter *, Blesilda C. Quiniones , Susan Tom , Nicholas A. Lanson Jr. *, Jeffrey L. Spees , Donna Bertucci *, Alexandra Peister , Daniel J. Weiss ¶, Vincent G. Valentine ||, Darwin J. Prockop , and Jay K. Kolls **
*Departments of Medicine and Genetics, Gene Therapy Program, Louisiana State University Health Sciences Center, New Orleans, LA 70112; Departments of Pharmacology and Medicine, Center for Gene Therapy, School of Medicine, Tulane University, New Orleans, LA 70112; ||Lung Transplantation Program, Ochsner Clinic Foundation, New Orleans, LA 70121; ¶Pulmonary and Critical Care, Vermont Lung Center, University of Vermont College of Medicine, Burlington, VT 05405-0075; and **Department of Pediatrics, Division of Pulmonology, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
Contributed by Darwin J. Prockop, November 19, 2004
Cystic fibrosis (CF), the most prevalent, fatal genetic disorder in the Caucasian population, is caused by mutations of CF transmembrane conductance regulator (CFTR). The mutations of this chloride channel alter the transport of chloride and associated liquid and thereby impair lung defenses. Patients typically succumb to chronic bacterial infections and respiratory failure. Restoration of the abnormal CFTR function to CF airway epithelium is considered the most direct way to treat the disease. In this report, we explore the potential of adult stem cells from bone marrow, referred to as mesenchymal or marrow stromal stem cells (MSCs), to provide a therapy for CF. We found that MSCs possess the capacity of differentiating into airway epithelia. MSCs from CF patients are amenable to CFTR gene correction, and expression of CFTR does not influence the pluripotency of MSCs. Moreover, the CFTR-corrected MSCs from CF patients are able to contribute to apical Cl- secretion in response to cAMP agonist stimulation, suggesting the possibility of developing cell-based therapy for CF. The ex vivo coculture system established in this report offers an invaluable approach for selection of stem-cell populations that may have greater potency in lung differentiation.
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G.W. and B.A.B. contributed equally to this work.
To whom correspondence should be addressed at: Louisiana State University Health Sciences Center, Gene Therapy Program, 642 CSRB, 533 Bolivar Street, New Orleans, LA 70112.
Guoshun Wang, E-mail: gwang@lsuhsc.edu
www.pnas.org/cgi/doi/10.1073/pnas.0406266102 |
