Stem Cell-based Therapy Investigated to Treat Cystic Fibrosis
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 the 22 December 2004 early online edition of the Proceedings of the National Academy of Sciences.
The authors demonstrated that human bone marrow-derived adult stem cells could be coaxed to differentiate into airway epithelial cells. They further showed that delivering to these cells the gene that is defective in CF restores an important cellular function essential for keeping the airways clear of mucus and airborne 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.
CF is the most common genetic disorder affecting Caucasians. It is characterized by a mutation of the gene encoding the CF transmembrane conductance regulator (CFTR), which 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 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.
Using a method they developed and report in the article, stem cells taken from normal subjects and placed in a culture with mature epithelial cells for two weeks 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.
To determine whether the gene transfer actually corrected the CFTR defect, the researchers exposed the cells to a medium containing labeled chloride. 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.
Kolls 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. The ex vivo approach to gene therapy may offer several advantages, including the ability to screen the cells before actually delivering them to patients.
To further study the potential of the stem cell approach, including in CF patients, Dr. Kolls and colleagues 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.
The Proceedings of the National Academy of Sciences, online, doi: 10.1073/pnas.0406266102
Human ES Cells Drive Beat of Animals’ Hearts
New research suggests that genetically engineered heart cells derived from human embryonic stem (ES) cells might one day provide a biological alternative to the electronic pacemakers used by hundreds of thousands of people worldwide.
Electronic pacemakers are used in patients with heart conditions that interfere with a normal heartbeat. However, these life-saving devices do not react the way the heart's own pacemaker normally does—raising the heart rate to help us climb stairs, for example.
In the new study, described in the 20 December 2004 advance online edition of Circulation, human ES cells were genetically engineered to express green fluorescent protein (GFP), and then expanded and induced to differentiate into heart cells. The genetic engineering of the ES cells inserted GFP so that the human cells would be easily distinguished from the animal cells.
The researchers then selected clusters of the cells that beat on their own accord, indicating the presence of pacemaking cells. These clusters triggered the unified beating of heart muscle cells taken from rats, and, when implanted into the hearts of guinea pigs, triggered regular beating of the heart itself. The researchers had destroyed the animals’ own pacemaking cells, located near the point of injection, by freezing them.
To prove that the human heart cells were controlling the beat of the guinea pigs’ hearts, workers conducted experiments that showed exactly where the electrical signal originated and followed the signal's conduction across the heart's surface. The new beat was coordinated by the implanted human cells and was slower than the animals’ normal heart rate—likely reflecting humans’ lower heart rate.
“These implanted cells also responded appropriately to drugs used to slow or speed the heart rate, which electronic pacemakers can't do,” says study leader Ronald Li. “But many challenges remain before this technique could be used for patients. We want to bring this to the clinic as fast as possible, but we need to be extremely careful. If this process isn't done properly, it could jeopardize a very promising field.” Since the engineered cells survived and worked properly, other more clinically important genetic engineering of the cells also will probably not interfere with the cells’ fate, say the researchers.
New questions have come up because of these promising results, notes Li. For instance, the researchers don't know why the animal's immune system failed to attack and kill the human cells. One possibility is that the cluster of cells did not connect well enough with the animal's circulatory system to trigger an immune response, but more experiments will be necessary to determine whether that was the case and, if so, how that might affect the implanted cells’ long-term survival.
Circulation, online, doi: 10.1161/01.CIR.000015131318547.A2
SCID Gene Transfer Continues to Encourage
A study in the 17 December 2004 issue of THE LANCET provides further evidence that gene therapy can be effective in creating a functional immune system for infants with X-linked severe combined immunodeficiency (SCID).
SCID is a genetic disease characterized by a block in the differentiation of T and natural-killer cells, due to defective expression or function of the γc cytokine receptor subunit. This leaves individuals at high risk of infection. The new report confirms data from a previous report that described correction of immunodeficiency in nine of ten patients with SCID.
In the latest study, four children with SCID had stem cells removed from their bone marrow. Autologous CD34+ haemopoietic bone marrow stem cells were transduced ex vivo with a leukemia virus carrying the corrective gene. The transduced cells were then returned to the patients without previous cytoreductive therapy.
The patients were monitored for integration and expression of the gene vector and for functional immunological recovery. Nearly normal immune function was established in all four patients after the procedure. Lead investigator Adrian Thrasher commented that, “At last follow up, all patients were at home in normal family and social environments, without restrictions on activities or exposure.”
An accompanying commentary by Marina Cavazzana-Calvo and Alain Fischer discussed all clinical research done in this field, noting that seventeen of these eighteen patients had their immunodeficiencies corrected with clear and sustained clinical benefits, demonstrating that the results of this gene-therapy strategy are reproducible.
THE LANCET, online, doi: 10.1016/S0140-6736(04)17590-9
SARS Vaccine Trial Underway
Human tests have begun for a preventive vaccine against the respiratory disease SARS. The disease killed hundreds of people around the world before it was brought under control in 2003 with aggressive conventional public health measures.
Researchers at the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID) will conduct the trials. The experimental vaccine against SARS, or severe acute respiratory syndrome, will be tested on ten healthy volunteers at the NIH Clinical Center in Bethesda, MD. The clinic will do periodic follow-up exams on each volunteer for 32 weeks.
The primary goal of the study is to determine if the experimental vaccine is safe in people. A secondary goal is to assess how well the vaccine stimulates the immune system to produce antibodies and cellular immunity, in this case, focusing on the SARS spike protein. Instead of using weakened or inactivated virus, which is typical for vaccine development, the new vaccine is composed of DNA that encodes the spike protein. The spike protein protrudes from the virus’ outer envelope and helps it bind to cells it infects.
Just 21 months passed from when international health officials recognized SARS as a new infectious disease to the opening of the NIAID human clinical vaccine trial. It often takes decades for scientists to develop a successful vaccine against an infectious disease. “In the case of SARS, we have dramatically cut vaccine development time with powerful new tools from two different fields, molecular biology and information technology,” says Anthony S. Fauci, director of NIAID.
Under a contract with NIAID, Vical Inc. of San Diego, CA, is producing the SARS vaccine for the NIAID clinical trial.
Chinese researchers began human testing of a SARS vaccine in May of this year. The Chinese vaccine trial uses an inactivated SARS virus vaccine developed through conventional vaccine technology.