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The Gene Therapy Journey: From Bench to Bedside

Gene Therapy for Childhood Blindness

By Anita Manning
Freelance Writer

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Nearly 40 years ago, scientists began exploring the concept of curing genetic diseases by replacing damaged genes with healthy ones. Since then, hundreds of studies have attempted to turn theory into practice, but despite progress in animal models, the goal of successfully reversing genetic disease in humans has been frustratingly elusive.

Now, the years of investment in basic and applied science are close to paying off in the form of the first therapy in humans to reverse a form of childhood blindness called Leber congenital amaurosis, or LCA.

LCA is a group of degenerative diseases of the retina, and is the most common cause of congenital blindness in children. It is an autosomal recessive condition: when both parents carry the specific genetic mutation, each child has a 25 percent chance of being affected. Symptoms usually are noticed in early infancy and include loss of vision, light sensitivity, and wobbly eye movement (nystagmus).

One type of LCA is caused by a mutation in the RPE65 gene, whose function is to process a type of vitamin A needed to keep light-sensing photoreceptor cells - the rods and cones of the retina - in operating order. The disorder is rare, affecting about 2,000 patients, but it is untreatable and severe, causing blindness early in life.

Scientists set out to replace the nonfunctioning gene with one that works - and to restore vision.

A National Eye Institute (NEI)-sponsored clinical study led by investigators at the University of Pennsylvania, Philadelphia and the University of Florida, Gainesville, is testing the safety of the gene transfer procedure in humans. The Investigators include Samuel G. Jacobson, M.D., Ph.D., Shalesh Kaushal, M.D., Ph.D., William W. Hauswirth, Ph.D., Barry J. Byrne, M.D., Ph.D., Artur V. Cideciyan, Ph.D. and Tomas S. Aleman, M.D.

In October and December, 2007, and late January, 2008, three young adults with the form of LCA caused by RPE65 mutation underwent gene transfer surgery. Using a hair-thin needle, the vitreous fluid was removed from inside the patients’ eyes and millions of copies of healthy RPE65 genes were injected just behind their retinas.

If results are positive, the study will be expanded to include more patients and will involve delivering healthy RPE65 genes to a wider area of the retina, to confirm the effectiveness of the therapy. A similar trial in children is being conducted at Children’s Hospital of Philadelphia by researchers at the University of Pennsylvania; a third study is under way in England at Moorefields Eye Hospital, London.

“Most patients with diseases like LCA have very few options, and gene therapy is one of the only options,” says Dr. C. Barry Byrne, director of the Powell Gene Therapy Center at the University of Florida, and co-principal investigator of the LCA trial there. A success in this study will show “the concept of molecular medicine is sound,” he says. If LCA can be treated effectively with gene therapy, it will point the way toward treatment of other diseases caused by single, malfunctioning genes. “It would open the door for many previously untreatable conditions,” Byrne says.

The eye is an especially good target for gene therapy, says Paul A. Sieving, M.D., Ph.D., director of the National Eye Institute, National Institutes of Health. About 500 genes have been found that affect the eye and visual system, he says, accounting for 20 percent% of all the genes identified so far.

What makes the eye “a wonderful place to test therapy ideas,” he says, is that while it is obviously connected to the rest of the body by blood and nerve tissue, “the eye is a separate compartment. We only need to deliver a microscopic quantity of gene vector in order to try a treatment,” unlike other gene therapy experiments, in which the gene and the virus that carries it, known as a vector, can circulate throughout the whole body system.

Another advantage is that in gene transfer trials involving the eye, outcomes will be crystal clear. “The visual system has very precise, quantifiable measures of function, and even tiny measures of success can be documented,” he says.

Sieving says the groundbreaking clinical trials to restore vision in patients with LCA rest on 15 years of NEI-sponsored research. Long before the gene transfer procedure could be tested in people, four critical milestones had to be met: the discovery of the RPE65 gene; creation of a mouse model that illustrates the gene’s functions and what happens when it’s missing; development of a safe way to carry healthy replacement genes to the target within the eye; and studies of the procedure in a large animal model.


The gene

In 1993, T. Michael Redmond of the National Eye Institute’s Laboratory of Retinal Cell and Molecular Biology, and colleagues identified a gene in the thin layer of cells behind the retina known as the retinal pigment epithelium (RPE). Suspecting the gene was important to the physiology of the eye, they began testing people who had severe early-onset blindness, and found that many of them had mutations in this gene, which they called RPE65.

“That made the link between RPE65 and blindness,” Redmond says. RPE65 was the second gene found to be associated with LCA, so the form of disease caused by mutations in this gene is known as LCA2. Since then, some 14 or 15 genes in the retina have been found to be associated with LCA, Redmond says, “and it’s likely to be closer to 30 when all is said and done.”

His research showed that RPE65 is critical for metabolism of a form of vitamin A that allows photoreceptor cells to function after exposure to light. When the gene isn’t doing its job, the patient’s world grows darker and darker, as if a dimmer switch were being turned down. This mutation accounts for 10 percent of all LCA or about 2,000 patients each year, all of them legally blind.


The mouse

Once the gene was identified, Redmond and colleagues set about genetically engineering a “knockout” mouse, one in which RPE65 genes had been blocked, allowing scientists to see exactly what happens in the eye when there is no RPE65 present.

“That is about as crucial to the whole story as discovering the gene in the first place,” Redmond says. “Having the gene got us so far, but we had to establish the function. It wasn’t until we did the actual experiment, making the knockout mouse, that everything was made clearer.”

What they found was that, starved of the vitamin A factor normally made by RPE65, photoreceptor cells stopped working - but they remained essentially intact.

“They’re just waiting for something the RPE65 is supposed to supply them,” Redmond says. “It’s like having a car that has run out of gas. As soon as you put in the gas, you can drive away.”


The vector

Getting the gas into the car - or healthy RPE65 genes into the eye - requires a delivery device, in this case, a harmless virus that most people already carry around inside them.

In the lab of William W. Hauswirth at the University of Florida, Gainesville, scientists have engineered an RPE65-carrying virus vector that can be used to carry the gene safely to its target. Adeno-associated virus, or AAV, was discovered in the mid-1960s as a non-disease-causing relative of adenovirus, one of the germs that cause the common cold, says Hauswirth. He has been working with the virus for 30 years, helping to discover the processes involved in AAV replication and sequence the DNA. In 1984, other University of Florida scientists developed a way to make a recombinant virus, by removing two genes from the AAV and substituting a contracted version of normal RPE65, derived from human cells, along with a piece of DNA that regulates it, called a promoter.

The recombinant AAV vector has advantages over other candidate virus vectors, Hauswirth says, because AAV is already present in the body, so it is not seen by the immune system as a foreign invader. “This is one of the safest, if not the safest, vectors, because it doesn’t elicit an immune response,” he says, and once it’s placed into the eye, it doesn’t move elsewhere in the body.

The idea of gene transfer therapy, he says, “is not rocket science, it’s very simple. We’re just treating these patients that are missing this function by supplying a normal copy, a good copy of the gene that they can use.”

The trick, he says, is “to put it inside the vector and target it to the right place surgicallyThe actual injection takes seconds.”


The dogs

Before it could be tested in people, the gene transfer process had to be tried out in animals.

“In 1996 we were the first to show you could use AAV in an animal in the retina and actually deliver the gene to the retina,” Hauswirth says. That was in mice and rats. Then, veterinary ophthalmologist Gus Aguirre, formerly at Cornell University and now at the University of Pennsylvania, began working with Briards, a breed of sheepdogs predisposed to a form of blindness similar to LCA. “It was a no-brainer that they went back to look at this dog, to check out the dog’s RPE65, and lo and behold, they found it had a mutation in it that blocked RPE65 protein,” Redmond says.

The dogs made ideal candidates for the experimental gene therapy, which occurred in 2001, because their eyes are similar in size and structure to those of humans. Electroretinography, which measures eye function, was performed on the dogs three months after the surgery, but Hauswirth recalls that even before the tests confirmed improved vision, animal caretakers knew they could see out of the treated eyes. How? “They started spinning around, doing a little dance,” Hauswirth says. “If you treat one eye and gain some vision in one eye, the dog wants to see his whole field, so he moves around.” One of the dogs, Lancelot, became a media darling when he visited Congress and shook paws with legislators to help increase awareness of the potential of gene therapy. He retains vision today, seven years after treatment, Hauswirth says. Since then, the procedure also has been tested successfully in non-human primates.

The significance of the current clinical trials and the promise they hold goes well beyond treatment of a single disease, says NEI director Paul Sieving. While LCA2 is rare and can be considered an orphan disease, “the person who is blind, who is rescued, is not an orphan. This has a very immediate, sweeping human impact,” he says. “The magic of this trial, and I think there is magic, is the possibility of restoring visual function.”

While the implications of that for vision are “very important, deep and broad,” he says, “the implications beyond that, for all science, are also very deep and broad.”

By providing a clear example of success in the area of vision restoration, he says, this work can provide a welcome burst of support for the whole field of gene therapy, building public confidence in the science and interest in further research. That is critical to the future of scientific discovery. “Ultimately, the American people have to be in back of science and medicine for it to succeed,” he says.


Human studies

During 2008, the work of a generation of researchers produced the most promising advances in treatment of LCA ever seen.

In groundbreaking studies published in September 2008 in Human Gene Therapy and in the October issue of Proceedings of the National Academy of Sciences, scientists reported for the first time that gene therapy restored both day and night vision in three young adults who were treated. These new reports, sponsored by the National Eye Institute and conducted at the University of Pennsylvania, Philadelphia, and the University of Florida, Gainesville, added weight to findings of two others published earlier this year in the New England Journal of Medicine.

Converging results from three independent and contemporaneous clinical trials support the idea that RPE65 gene therapy has biological activity in the human eye and can lead to increased vision,” said Artur V. Cideciyan, Ph.D., research associate professor of ophthalmology at the University of Pennsylvania and lead author of the PNAS paper. What remains to be teased out are the reasons why some of the improvements are modest while others are more dramatic.”

Dr. Cideciyan and colleagues reported that patients experienced a 50-fold improvement in day vision and 63,000-fold improvement in night vision. The restored vision was localized to the area of treatment, and Dr. Cideciyan said, We now know that one of the contributors for visual success is retinal location, and refined targeting of the treatment to optimal regions is a must.”

The researchers also learned that while the speed of restored daytime vision was near normal, patients’ adjustment to darkness took three to five hours longer than expected, something they had no way of knowing from the earlier work done with animals.

[Our] first clues came while interviewing patients about their visual experiences after treatment,” Cideciyan said. In future studies, doctors will allow added time before assessing restored night vision.

The gene replacement therapy did not affect vision lost as a result of degeneration of photoreceptor cells. However, it is the first to determine the power of gene therapy to reverse vision impairment due to blockage of the enzyme normally made by the gene RPE65.

These early studies involving small numbers of patients were aimed at assuring the safety of the procedure, and so far there have been no red flags,” said Dr. Samuel G. Jacobson of the University of Pennsylvania’s Scheie Institute, a co-author on both papers. We will continue to watch all patients very carefully,” he said, which includes reporting any abnormal reactions to the treatment.

As exciting as the research is for those interested in the future of gene therapy, it means even more to study participant Dale Turner, 22, of Toronto, who underwent gene replacement surgery on one eye on Jan. 31, 2008.

Born with LCA, Turner said that his vision gradually diminished throughout childhood. He could read aloud at the front of the classroom in sixth grade; by seventh grade, he could no longer see well enough to do that. I was so scared,” he said. For a child trying to understand his world, it was more than overwhelming.”

By using assistive technologies that allowed him to read, Turner was able to graduate high school, earn a business degree at Trent University, Ontario, and has started working toward a law degree at the prestigious Osgoode Hall Law School of York University. He also volunteers with the Foundation Fighting Blindness in Canada.

He hasn’t let blindness stop him, he said, but that doesn’t mean it hasn’t affected me.”

Following the surgery at the University of Florida, he said it took a few days of recovery before he was able to go outdoors—but what he saw was spectacular. I saw blue sky for the first time,” he said. I was just standing in disbelief, thinking that the sky was always this blue, and I could never see it. I couldn’t believe it.”

Since then, he has emailed Dr. Jacobson, the researcher who has been working with Turner for 14 years, to thank him and the many other scientists who have brought him to this day. I said that every morning I wake up and look at the sun rise, and every day it goes down, and I think these things are so priceless. And they gave me that gift,” Turner says.


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The National Eye Institute (NEI) is part of the National Institutes of Health (NIH) and is the Federal government’s lead agency for vision research that leads to sight-saving treatments and plays a key role in reducing visual impairment and blindness. For more information, visit the NEI Website at www.nei.nih.gov.

The National Institutes of Health (NIH) - The Nation’s Medical Research Agency - includes 27 Institutes and Centers and is a component of the U. S. Department of Health and Human Services. It is the primary Federal agency for conducting and supporting basic, clinical, and translational medical research, and it investigates the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.