The treatment offers hope for humans with a similar condition.

The achievement, with young dogs suffering from congenital stationary night blindness, which is similar to a childhood disease called Leber congenital amaurosis, is reported in the May 2001 issue of the journal Nature Genetics.

Co-authors include Gregory M. Acland, Gustavo D. Aguirre, Jharna Ray, Qi Zhang and Susan E. Pearce-Kelling, all at the Cornell University College of Veterinary Medicine; Tomas S. Aleman, Artur V. Cideciyan, Vibha Anand, Yong Zeng, Albert M. Maguire, Samuel G. Jacobson and Jean Bennett, all at the University of Pennsylvania, and William W. Hauswirth at the University of Florida.

"We have shown that gene therapy can restore vision in dogs with one of the most clinically severe retinal degenerations," says Acland, a research veterinarian at Cornell's James A. Baker Institute for Animal Research.

"Many safety and efficacy studies must be performed before we can begin clinical trials, but this [gene therapy] could correct defects in humans with RPE65 mutations," Acland says. RPE65 is the gene associated with the retinal pigment epithelium (RPE).

The RPE cell layer in the eyes of humans, dogs and other mammals supports the retina by providing nourishment and removing waste products while supplying vitamin A to the photoreceptors. Puppies and human infants with defective RPE65 genes produce a mutant form of the RPE65 protein, resulting in early vision loss, degeneration of the retinas and near-total blindness later in life.

The canine form of this retinal degenerative disease has been found only in the briard dog breed. In the gene therapy experiments, researchers used RPE65 genes that were cloned from dogs without the disease, together with a viral vector (recombinant adeno-associated virus, or AAV) to carry the normal dogs' DNA.

They injected the combination into the subretinal space of the eyes of 3-month-old briard-beagle mix dogs that were known to have the defective RPE65 gene and had been blind since birth.

Within six weeks, the treated eyes were producing the correct form of RPE65 protein. By three months, a series of tests (electroretinography, pupillometry and obstacle-avoidance tests in a dimly lit room) demonstrated that vision was restored to the treated eyes.

"This is a perfect example of how research in animals helps both the animal -- in this case the dog -- and the human patient," says Aguirre, who is the Caspary Professor of Ophthalmology at Cornell and whose laboratory discovered the RPE65 gene defect in briard dogs in 1998.

Since then, DNA tests developed at Cornell are allowing breeders of briard dogs to screen for the defective RPE65 gene and, by selective breeding, essentially to eliminate congenital stationary night blindness in the canine population outside the laboratory.

"Regardless of how successful the treatment has been for dogs, it is essential that more studies are carried out to establish the long-term safety and efficacy of gene therapy for human patients," Aguirre adds.

Previous experiments had used gene therapy to slow degeneration of retinas in mice, but the treatment reported in Nature Genetics is believed to be the first cure for retinal degenerative disease in a "large animal."

Jacobson, the director of the Center for Hereditary Retinal Degenerations at Pennsylvania's Scheie Eye Institute, says the gene therapy study in dogs "signals that the time has finally arrived to stop theorizing about treatment for these devastating human eye diseases and to start preparing seriously. We will now be assessing patients who could potentially benefit from this discovery."

At the F.M. Kirby Center for Molecular Ophthalmology in the Scheie Institute, Bennett says, "We have worked hard for many, many years trying to develop a treatment for retinal degeneration, and this is the biggest leap forward yet. The results will expand the possible treatment strategies for a diverse set of retinal diseases."

She attributes the gene therapy achievement to "an exceptional and talented group of investigators" at the Retinal Disease Studies Facility, a resource managed by the investigators on behalf of Cornell for various canine breeds with different forms of inherited blindness.

Bennett also credits Hauswirth, a member of the University of Florida's Department of Ophthalmology and Powell Gene Therapy Center, for designing the viral vector used in the experiments.

Whatever the future holds for human patients with retinal degeneration, gene therapy for dogs with the RPE65 defect is not likely to be performed on a routine basis, Acland says.

For one thing, the procedure would be prohibitively expensive to dog owners and must be performed during a narrow window of opportunity before puppies' retinas are destroyed by the disease.

"And, thanks to the RPE65 gene test -- and to selective breeding of dogs, an ethical option we do not have in humans -- this is well on its way to becoming a non-disease in dogs."

However, Aguirre adds, "as medical advances are made both in human and veterinary medicine, "treatments such as gene therapy, which now are done on an experimental basis in both animals and man, will likely become accepted treatment modalities in both species."

Acland, Maguire and Jacobson will give more details of the gene therapy procedure and discuss possible treatments for human patients tomorrow in Fort Lauderdale, Fla., at the annual meeting of the Association for Research in Vision and Ophthalmology.

"In gene therapy with other eye diseases, we've been able to prevent worsening of the condition in animals, but this is the first time we've been able to recover a lost function," said William W. Hauswirth a contributing author who is the Rybaczki-Bullard professor of molecular genetics and microbiology at UF's College of Medicine. "This is also really the first study that shows you can use gene therapy to get a response in a human-sized eye, that you can effectively treat enough of the retina to have an effect on the animal's visual function."

Dr. Jean Bennett, a senior author of the paper and a scientist at the F.M. Kirby Center for Molecular Ophthalmology at Penn's Scheie Eye Institute, said the study results expand the possible treatment strategies for degenerative retinal diseases.

"We have worked hard for many, many years trying to develop a treatment for retinal degeneration, and this is the biggest leap forward yet," she said.

UF researchers had established that the apparently harmless adeno-associated virus can carry healthy copies of a gene into the cells of the retina, which is composed of layers of light-sensitive nerve cells. The healthy gene's mission: to produce a protein critical to translating light waves into nerve impulses that can be interpreted as images by the brain.

Scientists from Cornell and Penn, where the experiment was conducted, had identified the eye disease in the dogs and the mutated RPE65 gene responsible. The mutated RPE65 gene causes an estimated 15 percent of LCA cases, with the rest attributed to other mutations. Efforts are under way to develop gene therapy approaches to combat those other mutations.

In the experiment, three Briard puppies had their right eyes treated late last year with a single injection of thousands of copies of the corrective gene in the virus provided by Hauswirth. Their left eyes were untreated as a control. The scientists planned to formally test eye function three months later, when the dogs were 6 months old.

"Well before those tests, the researchers thought the dogs could see because they kept turning their heads to the right to use the eye that had been treated," said Hauswirth, who also holds an appointment in the department of ophthalmology and is affiliated with UF's Genetics Institute and Powell Gene Therapy Center. "But then when the official tests were conducted, I got this very excited phone call, 'Bill, Bill, the dogs can see!'"

The tests showed that the treated eyes responded to light. The dogs also were able to navigate through a maze, even when the lighting was dim.

"The dogs bumped into objects on the left side, but would always avoid the objects on the right," Hauswirth said.

Officials from the Foundation Fighting Blindness, which supported the study with grant funds, said the success in reversing blindness in dogs is an important advance.

"With this study, gene therapy has overcome a major hurdle," said Gerald Chader, the organization's chief scientific officer. "The Food and Drug Administration wants to see evidence that a treatment is safe and effective in a large animal model before granting permission to begin clinical trials in humans. Genetic medicine is now making things we could only once dream of a reality."

Hauswirth cautioned that it will take several years before the experimental treatment can be tested in people.

"This experiment showed us that this kind of therapy has potential to work, but now we need to go back and do very structured and rigorous tests to look for any signs of ill effects from the treatment," Hauswirth said. "And then in the early phase of testing in people, we also will be looking first at whether it causes any problems, rather than actually trying to reverse blindness."

LCA is an autosomal recessive disease, meaning that one copy of a mutated gene must be inherited from each parent to cause symptoms. Such mutations also occur in Briards and other dogs that have been inbred to preserve characteristics of their breed.

"The retina initially looks normal," Hauswirth said. "Over time, however, the retina starts losing part of its structure because there is no visual input. By the time a child is 10 or 15 years old, the light-sensitive photoreceptor cells are usually gone. At that point, putting in this gene isn't going to restore vision. So there almost certainly is going to be a window of treatability for LCA patients -- probably the younger the better."

In their next phase of animal testing, the scientists will try to pinpoint the time of life during which treatment might be effective.

"That's such a critical question to deciding what populations in humans might benefit, although there's no guarantee that it will translate exactly to people," Hauswirth said. "No one really knows what dog years versus human years means in the eye."

According to Jean Bennett, MD, PhD, researcher at the F.M. Kirby Center for Molecular Ophthalmology at Penn's Scheie Eye Institute and a senior co-author of the study, previous studies to reverse blindness in rodents have been successful, but this is the first successful outcome using larger animals.

"The results are spectacular -- in fact, they are the sort of findings that a scientist usually only hopes to, but rarely does, see, in the course of a career," she explains. "This study takes a great stride forward in demonstrating that gene therapy does not just slow down a retinal degenerative disease, but can actually provide recovery of vision to an animal that was previously blind."

"However, we are nowhere near the introduction of the missing protein in humans to restore sight," she cautioned.

"While this may prove to be the treatment method used for other forms of retinal disease, such as macular degeneration, this particular therapy is useful only for LCA," adds co-author, Albert Maguire, MD.

Researchers who collaborated with Bennett and Maguire include Penn researcher Samuel Jacobson, MD, PhD; Cornell University researchers Gregory Acland, VMD, and Gustavo Aguirre, VMD, PhD; and University of Florida researcher William Hauswirth, PhD.

The studies were funded by grants from the National Institutes of Health and The Foundation Fighting Blindness. The research effort continues to be supported by grants from the National Institutes of Health, the Foundation Fighting Blindness, Research to Prevent Blindness and the Macular Vision Research Foundation.

Related websites:

Veterinary Medicine at Cornell

Scheie Eye Institute

[Contact: Roger Segelken, Ellen O'Brien, Rosann Giordano Thompson]

30-Apr-2001