Gene Therapy

UF is a world-class leader in gene therapy research. This newsletter is about how fundamental discovery at UF has led to important translational research using
gene therapy — both on our campus and around the world.

Recently, Steven Ghivizzani, Ph.D., professor of orthopaedics and molecular genetics and microbiology, received a $900,000, one-year grant for research on gene therapy as a potential one-time treatment for osteoarthritis, a chronic inflammatory condition of joints. This grant, from the NIH Institute of Arthritis, Musculoskeletal and Skin Disease, builds on the previous work of Dr. Ghivizzani and his team, which in turn has been built on path-breaking research at UF. They have had success in using gene therapy to treat osteoarthritis in mice, and the new grant will allow them to expand their clinical trials to the treatment of horses.

The knee joints of equine forelimbs are very similar to human knee joints in size, so the results will hopefully be generalizable to what researchers will find in humans. Osteoarthritis is a very common condition in horses, and represents the primary cause of the end of equine athletic careers, so horse trials fill a significant veterinary need. Patrick T. Colahan, D.V.M., professor of large animal clinical sciences, will supervise all the veterinary aspects of the research. Pending these results, clinical trials in humans are next. Osteoarthritis affects about 27 million Americans, so an effective treatment would have an extraordinary impact on human health.

This latest UF grant in gene therapy builds on years of research in this area at the University of Florida. In the early 1980s, work by Kenneth I. Berns, M.D., Ph.D., distinguished professor of molecular genetics and molecular biology and director of the UF Genetics Institute, and eminent scholar Nicholas Muzyczka, Ph.D., professor of genetics and molecular biology, won international recognition for modifying the adeno-associated virus, or AAV, for use as a gene delivery system (“vector”). In the mid-1990s, UF medical geneticists demonstrated the safety of gene therapy — with use of the AAV vector — in the world’s first gene therapy trial in patients with cystic fibrosis. The AAV vector is now used by scientists worldwide in gene therapy; at recent meetings of the American Society for Gene Therapy, nearly half the presentations involved the use of AAV. Moreover, UF produces the world’s reference standard AAV vector with support from the National Institutes of Health.

In 2001, due to a generous gift from Earl and Christy Powell, the Powell Gene Therapy Center at UF was created, directed by Barry Byrne, M.D., Ph.D., professor of pediatrics and molecular genetics and microbiology. The primary mission of the center is to merge molecular genetics research and health care delivery by developing new therapeutic strategies for the treatment of human diseases that involve gene transfer.

Let’s take a step back, as the idea of curing diseases by replacing damaged genes with healthy ones is a logical and natural progression of the last several decades of research in medical genetics and molecular biology. Most research using gene therapy begins with identification of an abnormal gene responsible for the disease being studied. An attempt is made to insert the normal gene into the genome, so as to replace the abnormal gene. A carrier molecule — or “vector” — must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is, indeed, AAV, in which Drs. Berns and Muzyczka were pioneers in genetically altering the virus to carry normal human DNA. Target cells, such as the patient’s muscle or retinal cells, are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state.

Despite hundreds of studies that attempted to translate theory into clinical practice, and despite progress in animal models, the goal of successfully reversing genetic disease in humans was frustratingly elusive until, for the first time, gene therapy was found to be successful in reversing a form of congenital blindness called Leber congenital amaurosis, or LCA. This work was conducted by William Hauswirth, Ph.D., professor of ophthalmology, Dr. Byrne and other collaborators at the Powell Center, and investigators at the University of Pennsylvania.

LCA, the most common cause of congenital blindness in children, is a group of degenerative diseases of the retina. As an autosomal recessive condition, each child has a 25 percent chance of being affected when both parents carry the specific genetic mutation. Loss of vision and light sensitivity usually are noticed in early infancy.

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.

In late 2007 and early 2008, one woman and two men with this condition, ranging in age from 21 to 24, underwent gene transfer surgery. Using a hair-thin needle, the vitreous fluid was removed from inside the patients’ eyes and tens of billions of copies of healthy RPE65 genes were injected just behind their retinas. The viral vectors used to deliver the corrective genes were manufactured by the Powell Gene Therapy Center. As reported online in August 2009 in Human Gene Therapy and in an August 13 letter to the editor in The New England Journal of Medicine, within months all three study volunteers showed an improvement in vision.

It was truly exciting when the female volunteer came back for her one-year checkup. Dr. Hauswirth recalls that “she said she could read the digital clock in her parents’ car at night with her treated eye. That was something she'd never been able to do before.” He further explained that: “We speculate that her brain grew to recognize that there was a new part of her retina that could be used to see dim objects. What is truly astounding is that the brain, even in an adult, is still adaptable enough to learn to use these regions of the retina.” Since this congenital form of blindness is a disease of children, it is hoped that if the treatment can be delivered at a young age, more function can be restored.

Currently 15 LCA patients have been similarly treated and all have gained visual function: patient light sensitivity improved from 200- to 60,000-fold, pupil response to light improved usually more than 10-fold, most gained in navigation ability, most gained in their ability to read an eye chart, some by over three lines of letters, and four patients have now experienced the additional delayed vision response noted above.

Adding to the excitement about the potential for gene therapy, in the October 8, 2009 issue of Nature, Dr. Hauswirth and collaborators from the University of Washington reported on the use of gene therapy to cure two squirrel monkeys of red/green color blindness — the most common genetic disorder in people. This finding was deemed the No. 3 scientific discovery of 2009 by Time magazine. “We added red sensitivity to cone cells in animals that are born with a condition that is exactly like human color blindness,” explained Dr. Hauswirth. “Although color blindness is only moderately life-altering, we’ve shown we can cure a cone disease in a primate, and that it can be done very safely. That’s extremely encouraging for the development of therapies for human cone diseases that really are severely blinding.”

For his breakthroughs in gene therapy research for vision, Dr. Hauswirth was named “Florida Newsmaker of the Year” for science in the January 2010 issue of Florida Trend and his work was cited in 2011 as one of the 21st century’s first decade of breakthroughs in the journal Science.

Other gene therapy research at the UF Powell Gene Therapy Center is also promising. Dr. Byrne’s research is aimed at understanding several types of inherited muscle disease, which lead to skeletal, and heart muscle dysfunction. The discovery that AAV vectors can lead to sustained expression in muscle tissue was made by Dr. Byrne and colleagues just before coming to the University of Florida at the time the center was founded. Since that time a number of notable milestones have been achieved. A critical question to establishing safety of AAV vectors in human studies was a thorough understanding of safety studies in animals. For this purpose the Powell Center was awarded a $6 million NIH grant to establish a Center for Toxicology studies. Results of these safety studies were required to achieve FDA approval of several other NIH- and industry-sponsored studies. Two studies in a type of inherited emphysema known as alpha-one antitrypsin deficiency have been conducted and the latest of these studies has been the first to establish a true dose response that would establish the basis for approval of the candidate drug.

A second study done in muscle cells was conducted in collaboration with clinician scientists at Nationwide Children’s Hospital. The study by Dr. Jerry Mendell and UF researchers was recognized last month by the Annals of Neurology prize for an outstanding contribution to clinical neuroscience this year. The paper “Sustained Alpha-Sarcoglycan Gene Expression after Gene Transfer in Limb-Girdle Muscular Dystrophy, Type 2D” is recognized as the first successful human gene transfer for muscular dystrophy.

Finally, building on the UF expertise in neuromedicine, the Powell Gene Therapy Center team led by Dr. Byrne is in the midst of the first gene therapy trial for ventilatory failure due to another form of muscular dystrophy, know as Pompe disease. Children with this fatal condition are now enrolled in a study to test AAV vectors as a way to restore independent breathing by correcting the gene defect in the diaphragm muscle and spinal cord. These efforts are part of the pipeline of research in neuromuscular disease, including an important MRI study in Duchenne muscular dystrophy, led by Dr. Krista Vandenborne, chair of the department of physical therapy at UF’s College of Public Health and Health Professions.

The latest research grant at UF in gene therapy pertains to the osteoarthritis studies of Dr. Ghivizzani. In osteoarthritis, the equilibrium of the breakdown and rebuilding of cartilage surrounding the head of bones is disturbed. Consequently, the protective cartilage begins to break down faster than it can be repaired, causing pain and stiffness in the joints of its sufferers. There is currently no cure for the condition, but its symptoms can be treated with medication. As explained by Dr. Ghivizzani, “our bodies produce many reparative proteins, and it’s possible to use these proteins in drugs. The problem is that our bodies are engineered so that proteins are broken down quite quickly.” Ghivizzani and his team are attempting a variation of gene therapy. Instead of trying to replace a defective gene, they are attempting to maintain production of regenerative proteins by injecting AAV viruses loaded with healthy, protein-producing DNA into the cells of the affected joints. “By putting the therapeutic gene material into cells, they will produce the proteins as long as they’re alive,” Ghivizzani explains. “So we’ve spent a lot of time figuring out how to deliver it efficiently and safely.”

Successful trials in mice have been encouraging, but the key question of how the results in mice can be translated to horses and humans are being studied in this newly funded research, which will examine the capacity of the gene-based treatment to inhibit the onset and progression of an osteoarthritic model in thoroughbred horses. In addition, Dr. Ghivizanni and his team will address the questions of how much virus to deliver, how long they remain functional, whether re-dosing is effective and whether there will be an immune response. Discussions with the FDA about a phase 1 safety trial are scheduled for later this month.

Just as I was completing this issue of On the Same Page, there was an online publication in The New England Journal of Medicine (Nathwani AC, et al. December 10, 2011) from University College London Cancer Institute, reporting successful gene therapy in six men with severe Hemophilia B, an X-linked bleeding disorder that results from a defect in the gene encoding coagulation factor IX. The investigators infused a modified AAV vector with the normal gene, based on previous work suggesting that after a single administration of vector, there can be continuous endogenous production of normal factor IX. Interestingly, only a small rise in circulating factor IX (to about 1% of normal levels) can substantially ameliorate the bleeding phenotype. The investigators concluded that: “Follow-up of larger numbers of patients for longer periods of time is necessary to fully define the benefits and risks and to optimize dosing. However, this gene-therapy approach…has the potential to convert the severe bleeding phenotype into a mild form of the disease or to reverse it entirely.”

In 1988, 35 years after his letter to Nature with Frances Crick describing the DNA helix structure, James Watson said: “We used to think that our fate was in our stars, but now we know that, in large measure, our fate is in our genes.” Now, a short 25 years later, the field of genetics pioneered by Watson and Crick is finding ways to help people whose fate includes diseases that can be ameliorated by gene therapy. UF is at the forefront in blazing this path.

Wishing readers of On the Same Page the very best for the holiday season and New Year ahead.

Forward Together,

David S. Guzick, M.D., Ph.D.