From his laboratory in the wood-sided Surge building high in the woods above Parnassus Avenue, Dieter Gruenert, PhD, speaks with a sense of urgency.

He is explaining the work he and his colleagues are doing to refine a novel form of gene therapy he developed and reported last year.

Dieter Gruenert

The technique, called "small fragment homologous replacement," involves inserting a minuscule segment of DNA into certain lung cells to stimulate repair of the most common mutation in the gene that causes cystic fibrosis.

Gruenert, an associate professor of laboratory medicine and investigator at the Cardiovascular Research Institute, reported the study, conducted in lung epithelial cell cultures, in the October 1996 issue of Gene Therapy. He believes the procedure may some day offer treatment to some of the millions of people with the disease.

But Gruenert, the co-director of the National Institutes of Health-sponsored Gene Therapy Core Center at UCSF, isn't focused just on cystic fibrosis. He believes the therapy could someday be used to treat numerous genetically based diseases. Nor is he focused strictly on his own novel approach. Gruenert wants to make gene therapy, in all its potential forms, work.

"Gene therapy definitely holds promise," says Gruenert, "but a lot more work needs to be done before it is a reality. And UCSF should be a key player in this movement."

A handwritten letter is tacked on a bulletin board outside of Gruenert's lab. "Thank you for the work you are doing on behalf of our children," it says. "We are counting on you."

"Gene therapy" has rolled into the lay person's lexicon. And cystic fibrosis, the most prevalent lethal genetic disorder in Caucasians, is one of the most talked about, and principal targets, of therapeutic investigation.

But classic gene therapy for inherited disorders, which involves a much larger, more complex segment of DNA than Gruenert's approach, has not yet produced a cure for cystic fibrosis or any other inherited disorder. Moreover, while it does have potential as a treatment for cystic fibrosis, numerous concerns surround its use.

This is one reason, says Gruenert, that he is promoting investigations of alternative forms of gene therapy. His effort has gained momentum, as was evident in a symposium, entitled "Gene Therapy, The Next Generation," which Gruenert chaired and directed in October in Paris.

While scientists from leading academies around the world congregated, their numbers were relatively still small. "There are only a handful of labs in the world right now working on alternative forms of gene therapy," says Gruenert.

The goal of gene therapy for inherited disorders is to replace or correct defective genes. Mutated genes produce malfunctioning proteins, and it is these proteins that ultimately cause disease. In theory, a corrected gene will begin producing working protein.

In patients with cystic fibrosis, a defective protein known as CFTR (cystic fibrosis transmembrance conductance regulator) is unable to control salt and water movement in lung epithelial cells. As a result, patients experience lung obstruction and infections, and typically die by age 30. Thus, gene therapy for cystic fibrosis focuses on correcting the mutated gene that spawns the defective CFTR protein.
Classical gene therapy takes a broad approach to this reengineering effort. An entire protein-encoding sequence of DNA, flanked with regulatory DNA from another gene or virus, is injected into a cell. The DNA sequence, known as complementary DNA, or cDNA, contains only the genetic elements directly involved in protein coding. The regulatory DNA fragments, known as "promoters" or "enhancers," are added to the sequence to help activate the DNA and ultimately produce the protein.

There is concern, however, that the regulatory elements, which operate independently of the cell's own control machinery, will disrupt cell metabolism. This could lead to altered levels of gene expression and alterations in the timing of gene expression.

These regulatory elements would likely initiate protein production in a cell that doesn't naturally make the protein, says Gruenert, possibly leading to a pathology even worse than the disease itself.

On the positive side, in clinical phase I trials of the traditional therapy, some cells have been successfully transfected with the new gene. But in many cases the expression has been transient or limited. And in genetic disorders ongoing expression is needed.

Gruenert and his colleagues have developed an approach for introducing gene sequence that is intended to avoid the potential problem of cell-inappropriate gene expression. In contrast to classical therapy, the technique is designed to target only the mutated portion of the gene's DNA.

The small fragment homologous DNA replacement strategy is made up of DNA that is almost identical, or "homologous," to the naturally occurring, but mutated, DNA.

The hope is that the cell's own control machinery will recognize small fragments of homologous DNA and, with this recognition, initiate enzymatic genetic engineering by removing the mutated gene and splicing the new, therapeutic DNA into place. The homologous segment targets the mutated portion of the gene by some as yet unknown enzymatic mechanism.

The theory is that the newly engineered gene will begin producing a working version of the salt-regulating protein CFTR -- but only in cells where it is naturally expressed. "If we initiate repair in a cell that doesn't normally express the gene it won't make any difference," says Gruenert, "because what we do to the cell won't change the way the CFTR gene is expressed."

Preliminary studies indicate some success. As Gruenert reported last year, about one percent of the airway cells grown in lab cultures showed correction of the CFTR (cystic fibrosis transmembrance conductance regulator) protein and restoration of normal salt regulation for up to three weeks. Cell culture evidence suggests that only six percent of the lung epithelial cells needs to be altered to change the ion transport characteristics to approach that of normal, says Gruenert.

If the same degree of correction can be obtained in human airways -- and on a permanent basis -- a few applications of this process could get the ion transport characteristics to approach normal levels, says Gruenert.

Another alternative form of gene therapy reported at the Paris symposium takes the opposite approach to genetic reengineering. In this case, parts of chromosomes, as opposed to mere fragments of DNA, are injected into cells. But the goal here is, again, to provide the cell's regulatory machinery with the genetic information that pertains to the mutated gene of interest.

The technique, says Gruenert, could be particularly appropriate for such diseases as Duchennes muscular dystrophy. Eighty percent of patients with the disease have very large DNA deletions.

Regardless, he says, "while these therapies may not be the complete answer, they are pointing in a particular direction and hopefully are causing people to reflect on other possibilities and potentially other means to solve the problem."

Such inquiry needs to be intensified at UCSF, Gruenert says. In fact, he says, the whole field of gene therapy research on campus could be galvanized by greater integration of different lines of investigation.

He says he wants to expand the Gene Therapy Core Center into a comprehensive core center that involves not only gene therapy of inherited diseases, but infectious diseases and cancer. The NIH-funded center currently supports a small group of scientists to look at gene therapy for cystic fibrosis, certain hematological disorders and inherited metabolic disorders.

"By utilizing the nucleus established by the NIH core center, and by integrating these currently disparate programs, we could make more progress," he says. "A comprehensive gene therapy program could provide the structure for a graduate program and a fellowship program, and could provide the mechanisms to establish a comprehensive core facility that would be available to a lot of different researchers in cross disciplines."

Gruenert says he envisions such a program as somewhat aligned and parallel with the human genetics program recently started by Charles Epstein, MD, professor of pediatrics, and Ira Herskowitz, PhD, professor of biochemistry.

"Gene therapy is going to be one of the medicines of the 21st century. It is not necessarily going to be the answer to all diseases but it is going to be a type of medicine that an institution like UCSF should be involved with, and it should devote a certain amount of time energy and money to make sure it is part of that effort."

By Jennifer O’Brien

1st appeared 1/20/98