Challenges facing gene therapy have forced some researchers toretreat from initial declarations of its potential curative powers, but aUniversity of California scientist may have devised a way of rescuingthe frustrating science by tapping into cells' ability to heal their ownnaturally occurring mutated genes.

Dieter Gruenert, co-director of the Gene Therapy Core Center at theUniversity of California at San Francisco, has been working fiveyears on what he describes as second generation gene therapy.

Rather than trying to deliver to epithelial cells of the lung an entirenormal copy of the mutated gene that causes cystic fibrosis, Gruenertand his colleagues dispatched a DNA fragment that replaces thedefectiveportion of the natural gene, allowing it to operate correctly.

His cystic fibrosis laboratory studies, published in the October issueof Gene Therapy, revealed corrections of mutated genes in 1 to 10percent of epithelial cells treated in vitro with the normal DNAfragments.

The analysis also showed the corrected genes properly expressed thecystic fibrosis transmembrane conductance regulator (CFTR) protein,whose malfunction is the cause of the disease.

In human patients, Gruenert noted, correction of the mutated genes infrom five to 10 percent of lung cells would be considered an effectivetreatment.

Cystic fibrosis affects about 30,000 people in the U.S. and is the mostcommon deadly inherited genetic disease among Caucasians. Withouta properly working CFTR gene, salt and water movement inepithelial cells is interrupted, causing a build up of mucous, whichleads to lung obstruction and spawns infections. The life-span ofpeople with cystic fibrosis is about 30 years.

The first wave of gene therapies already in clinical development forcystic fibrosis use various viral vectors, such as adenoviruses andadeno-associated viruses, to deliver a healthy CFTR gene to cells.The treatment also includes DNA to promote expression of the gene.

Hindering the success of those "classical" approaches are variouscomplications, Gruenert said. The body's potential immune responseto the viral vector can neutralize the therapy. Or the inappropriatestimulation of the gene can be dangerous, resulting in excessivelevels of its protein and expression in cells where it's not normallyactive.

Another challenge, Gruenert noted, has been integrating the deliveredgene into its proper position on the chromosome and sustainingnormal expression.

In his approach, called "small fragment homologous replacement,"Gruenert started with the specific mutation most common in defectiveCFTR genes. Fifty percent of people with cystic fibrosis have twocopies of the CFTR gene with that specific mutation and 85 to 95percent have at least one copy. Regardless of whether the CFTR genecontains any of the other 600 mutations known to occur, correctingthe defective portion targeted by Grunert would help restoreproduction of normal salt- and water-regulating proteins in the lungs.

To counter the mutation, pieces of DNA with the correct nucleic acidsequence for a healthy CFTR gene were made and the fragment wasencapsulated in a liposome or polyaminoamine carrier.

Epithelial cells containing the mutated CFTR gene were treated with1 million therapeutic DNA fragments per cell. After entering the cell,the DNA makes its way into the nucleus and locates the mutatedsequence it is supposed to replace.

"We're operating in somewhat of a black box," Gruenert observed intrying to describe how the mutation is repaired.

The cell's enzymatic machinery, he said, exchanges the incomingDNA for the defective portion. Just how that works is the subject ofGruenert's continuing research.

Experiments on animals also will have to precede tests on humans.

But if Gruenert's approach is effective, it avoids inappropriate CFTRgene expression, because the only CFTR genes affected will be thoseactive in epithelial cells. In addition, liposomes may solve theproblem of immune system reaction to viral vectors. And if the CFTRgene mutation is corrected in the original gene, in theory, it should bea permanent cure.

So far, Gruenert said, his research has been funded by the CysticFibrosis Foundation and the National Institutes of Health. But thatcould change soon. Several companies have shown interest in thescience and the University of California is in negotiations with oneparticular firm, whose identity was not disclosed. n

-- Charles Craig

(c) 1997 American Health Consultants. All rights reserved.

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