By Lisa Seachrist
BETHESDA, Md. - It's been more than 10 years since researchers as the National Institutes of Health (NIH) first tested gene therapy as a means to treat patients who had inherited ineffective immune systems.
Since that time, so many gene-therapy protocols have been proposed and attempted that the Recombinant DNA Advisory Committee (RAC) no longer reviews and approves each attempt. But, despite numerous attempts and general enthusiasm for such approaches, no patient with a genetic disease has ever been cured by gene therapy.
That statistic has not deterred scientists from trying to correct the DNA misspellings that cause disease, and aiming to treat the problem earlier - even before the affected individual is born.
Much of the difficulty with gene therapy has been in delivering the gene to the appropriate area and getting therapeutic levels of expression of the corrected gene.
Amniotic Fluid Posited As Gene Delivery Medium
Janet Larson, staff scientist at the Ochsner Medical Foundation, in New Orleans, posited that amniotic fluid provided the ideal means to deliver a gene to correct cystic fibrosis (CF).
CF results when a child inherits two defective copies of the cystic fibrosis transmembrane regulator (CFTR) gene. Without a functioning CFTR gene, an abnormal, sticky mucus builds up, clogs the lungs and blocks the pancreas. Because the major affected areas in CF are the airways and the intestines, Larson decided to try to use the fetus's normal breathing and swallowing of amniotic fluid to deliver a replication incompetent adenovirus with a normal CFTR gene to the affected cells.
Larson and her colleagues injected the adenovirus vector into pregnant rats and mice that were destined to bear pups with two defective copies of CFTR - a fatal phenotype in rodents. The mothers were injected with the viral vector at a time that would correspond to 10-20 weeks human gestation.
At that time of development, rodent airways are lined with multipotential columnar and cuboidal stem cells. These stem cells grow and differentiate after the infection with the adenovirus vector, and are the target for the vector.
By treating the pups in utero, the researchers were able to rescue the animals from the fatal phenotype, and the lung and intestinal tissue showed normal development patterns.
"We were hoping to have the fetuses breathe and swallow up the virus and see if we could correct CF," Larson said. "As it turned out, timing was the most important factor. The more immature the lung, the better."
Curiously, though, the researchers found that there were no detectable levels of CFTR expression in either the lung or the intestine after birth.
"The dominant paradigm is that CFTR encodes for a chloride channel whose replacement will result in better ion balance and alleviate the symptoms of CF," Larson told members attending the RAC's Gene Therapy Policy Conference on Prenatal Gene Transfer here. "Our idea is that CFTR plays a critical role in the normal development of the secretory epithelium."
CFTR clearly codes for a cyclic adenosine monophosphate dependent chloride channel. When the channel is active, it causes a flux of adenosine triphosphate (ATP). Larson and her colleagues decided to see if a direct injection of ATP into the amniotic fluid could rescue pups just as well as the adenovirus vector.
"We found that we could rescue the pups by directly or indirectly increasing the concentration of ATP in the amniotic fluid," Larson said.
The researchers are moving into primate models of CF in order to establish the necessary timing for delivering the adenovirus in larger animals. Larson predicts there is a four-week to five-week window in which to act and they need to pinpoint that window before moving to human studies.
"We hope to move into human transfers sometime in the year 2000," Larson told BioWorld Today.
However, many scientists and ethicists, along with the general public, have reservations about the safety and ethics of prenatal gene transfers. As a result, Larson said that she would also be looking methods to directly increase ATP in the amniotic fluid.
"My preferred method would be to do a gene transfer," Larson said. "Directly increasing ATP would likely mean multiple injections or a pump or something like that. It will be technically more difficult." n