In new gene therapy approach, UF researchers destroy mutant message while inserting corrective genes

University of Florida geneticists have hit on an improved way to deliver a genetic “one-two punch” to cells that could someday help patients with an often-fatal hereditary lung-liver disorder.

The investigative approach uses a modified virus to deliver corrective genes to cells along with an enzyme that acts like a pair of molecular scissors, snipping apart mutant genes before they order the creation of liver-damaging proteins. Scientists from UF’s Genetics Institute conducted their experiments in cultures of hamster ovary cells, models for alpha-1-antitrypsin deficiency in humans.

Unlike classic gene therapy, which solely aims to ferry corrective genes into the body to compensate for those that are malfunctioning, this method also seeks to muzzle destructive genes by removing their ability to command protein production. To do so, researchers package the genetic material in a viral vehicle known as the adeno-associated virus, or AAV, which appears to usher it in to a larger percentage of cells than other gene delivery mechanisms. The findings were reported this week at the joint meeting of the Pediatric Academic Societies and the American Academy of Pediatrics.

“It’s the delivery technique that sets this apart,” said Dr. Terry Flotte, an associate professor of pediatrics, molecular genetics and microbiology and co-director of the Powell Gene Therapy Center at UF's College of Medicine. “This solves the problem of devising a vector that can deliver genes to a high percentage of liver cells, which has been the major task at hand. And of course, AAV does all the other things that are required – it is long-lasting and nontoxic.”

The genetic disorder, the second most-common among Caucasians, causes early emphysema and severe liver disease. It affects an estimated 100,000 Americans. Alpha-1-antitrypsin is produced by the liver and protects the lungs from injury by a common enzyme that normally fights bacteria and cleans up dead tissue. Those affected do not generate enough of the protein to adequately protect the lungs, and permanent and irreversible damage results. Many people with the disorder also battle liver problems, apparently caused by a defective version of the protein that accumulates in the liver.

“If you look at alpha-1-antitrypsin-deficient individuals when they are born, about 20 percent have some liver problems, and 20 percent of that 20 percent go on to have severe liver problems and die or get transplanted,” said Dr. Mark Brantly, a professor of medicine and of molecular genetics and microbiology at UF’s College of Medicine.

“The caveat is all alpha-1 patients have a lifelong risk of increased liver problems. So though we tend to think about this sometimes as a problem among children (with alpha-1), it really is a problem of all alpha-1 patients and becomes more of a significant problem over time.”

Brantly is part of a research team that includes Flotte; Alfred S. Lewin, a professor of molecular genetics and microbiology in UF's College of Medicine; and graduate student Thomas Conlon, who presented their data at this week’s meeting. Their studies were funded with the support of the Powell Gene Therapy Center at UF and the National Institutes of Health, and took place in Flotte’s laboratory.

Current treatment for the disorder includes avoiding exposure to cigarette smoke and weekly injections of alpha-1-antitrypsin. Gene therapy could someday replace the need for the shots.

In cells, ribonucleic acid, or RNA, is copied from strands of DNA and typically acts as a courier, delivering DNA’s genetic instructions. UF scientists studied a specialized type of RNA that operates like an enzyme, a substance that spawns chemical reactions within cells. In this form it is known as a ribozyme. UF researchers evaluated four different ribozymes to see which most effectively clobbered the abnormal RNA, while sparing the form carrying the good genes. They packaged the approach with standard gene therapy techniques to insert normal copies of the alpha-1-antitrypsin gene into cells.

One of the four – aptly named WAR – reduced the concentration of mutant genes by 91 percent, according to Conlon. The scientists also were able to protect the normal copies of the alpha-1-antitrypsin gene they were replacing by genetically altering them to shield them from the ribozyme’s chopping action.

“When there’s a mistake in the gene sequence it confers a change in the building blocks [of the cell’s RNA], and that change is associated with production of a protein that misfolds and is improperly disposed of within the cell. We think this improper disposal of or handling of the misformed protein causes damage in the liver cells,” Brantly said.

“If we can just ‘swim upstream’ a little bit and prevent the production of the protein, that may actually reduce the chances of an individual developing liver disease. So no bad protein, no liver disease, is the hope.”

But this is no easy task, researchers concede.

“The bigger trick is to make the ribozyme so good that it can see the bad RNA and basically leave the good RNA alone,” Brantly said. “The idea in that particular circumstance is if we have a ribozyme that’s so specific it only cuts up the bad RNA, we can actually at the same time replace the bad gene. The ribozyme is targeted to destroy the abnormal RNA and also produce a normal RNA that will then make a normal protein.”

Next, UF scientists plan to test the approach in culture in human cells that express the alpha-1-antitrypsin defect. These cells will be obtained through blood samples or lung biopsies.

“We’re working hard, but it’s going to be awhile before we study this in humans,” Brantly said. “We need to make sure we’re not causing any kind of toxicity with these procedures. After these early experiments, we’re going to be looking at mice that have been given the abnormal alpha-1 gene to see if we can fix it in them.”