New Gene Therapy for Retinitis Pigmentosa Treats Early and Late Stages of the Disease in Dogs


Collaboration between scientists from the University of Pennsylvania and the University of Florida, Gainesville has hit pay dirt when it comes to treating an inherited eye disease. This study used gene therapy to treat the disease and the results of this research project make a definitive contribution to the development of gene therapies for people with the blinding eye disorders for which there is presently no cure.

The disease in question is called retinitis pigmentosa, which is a group of rare, genetic disorders characterized by the degradation and subsequent loss of photoreceptors in the retina. People who suffer from retinitis pigmentosa have difficulty seeing at night and experience a loss of peripheral vision.

As mentioned, retinitis pigmentosa is an inherited disorder that results from mutations in any one of more than 50 different genes. These genes encode proteins that are required for retinal photoreceptors, and mutations in these genes compromises photoreceptor survival and function.

In human patients, retinitis pigmentosa is the most common inherited disease that results in degeneration of the photoreceptors of the retina. Approximately 1 in 4,000 people are affected with retinitis pigmentosa and 10 to 20 percent have a particularly severe form called X-linked retinitis pigmentosa. This disease predominately affects males, who experience night blindness by age 10 and progressive loss of the visual field by age 45. 70 percent of people with the X-linked retinitis pigmentosa harbor loss-of-function mutations in the retinitis pigmentosa GTPase Regulator (RPGR) gene. RPGR encodes a protein that maintains the health and survival of retinal photoreceptors. There are two types of photoreceptors; rods that give us the ability to see in dim light, and cones that allow us to see fine detail and color in bright light. Loss of the RPGR protein damages both types of photoreceptors.

Because there are no treatments for retinitis pigmentosa, gene therapy might be the best option to treat this disease. Fortunately, some varieties of dogs have a naturally occurring, late-stage retinitis pigmentosa that closely resembles the human disease. In previous experiments, gene therapies were used in diseased dogs, but such studies showed that benefits from gene therapy were only observed when it was used in the earliest stages of the disease.

“The study shows that a corrective gene can stop the loss of photoreceptors in the retina, and provides good proof of concept for gene therapy at the intermediate stage of the disease, thus widening the therapeutic window,” said Neeraj Agarwal, Ph.D., a program director at National Eye Institute, a part of the National Institutes of Health, who funded this research.

The dogs used in this study all suffered from a naturally occurring canine form of RPGR X-linked retinitis pigmentosa that is observed in some mixed breeds. These animals provided an excellent model system for their gene therapy tests, since affected dogs with early to late stages of the disease could be treated with the experimental therapy in one eye while the other untreated eye could be evaluated in parallel as a control.

To treat these blind dogs, the team utilized adeno-associated virus (AAV). They engineered AAV particles that possessed the entire RPGR gene. Then they devised a way to deliver these viruses to the retinal cells so that the viruses could infect the retinal cells and produce normal copies of the RPGR protein.

When the eyes treated with the AAV vectors were subjected to detailed imaging, it was clear that the gene therapy protocol arrested the thinning of the retinal layer. This shows that the treatment halted the degeneration of the photoreceptors in the affected dogs. When the treated eyes were compared with the untreated eye, the structure of the rod and cone photoreceptors was obviously improved and better preserved in the treated eye in comparison to the untreated eye. When the neural physiology of the retinas from the treated and untreated eyes was compared, once again, the retinas from the eyes treated with the gene therapy were more normal than the untreated eyes. In fact, the gene therapy halted the photoreceptor cell death associated with retinitis pigmentosa for two and a half years, which was the length of the study.

The team also treated dogs who suffered from later-stage disease in the hope that the gene therapy could not only improve the condition of dogs in the early stages of the disease, but also those with later stages of the disease. Interestingly, the gene therapy also froze the loss of retinal thickness and preserved the structure of surviving photoreceptors, but the retinas in the untreated eyes continued to thin and their photoreceptor function deteriorated as well. When the dogs were sent through an obstacle course and a maze under dim light, the animals did significantly better when they used their eye that had been treated with the gene therapy compared with their performance when they used the untreated eye. This shows that this gene therapy also works in dogs suffering from the late-stages of retinitis pigmentosa.

Can such a therapy be used in people in human clinical trials? Not yet. More safety testing must be done in order to properly determine if it is safe over long periods of time, according to this study’s co-leaders, Gustavo Aguirre, V.M.D., Ph.D., and William Beltran, D.V.M., Ph.D., of the University of Pennsylvania. Other collaborators, University of Pennsylvania scientists Artur Cideciyan, Ph.D., and Samuel Jacobson, M.D., Ph.D. are presently screening potential patients who have RPGR mutations as a prolegomena for a future clinical trial.

Their results are published in Proceedings of the National Academy of Sciences.

Patient-Specific Stem Cells Plus Personalized Gene Therapy for Blindness


Researchers from Columbia University Medical Center (CUMC) have devised protocols to develop personalized gene therapies for patients with an eye known as retinitis pigmentosa (RP), which is a leading cause of vision loss. While RP can begin during infancy, the first symptoms typically emerge during early adulthood. Typically the disease begins with night blindness, and RP eventually progresses to rob the patients of their peripheral vision. In its later stages, RP destroys photoreceptors in the macula, that region of the retina that provides the best vision under lighted conditions. RP is estimated to affect at least 75,000 people in the United States and 1.5 million worldwide.

The approach utilized by this Columbia team utilizes induced pluripotent stem (iPS) cell technology to transform patient’s skin cells into retinal cells, which are then used as a patient-specific model for disease study and preclinical testing.

The leader of this research group, Stephen H. Tsang, MD, PhD, showed that a form of RP caused by mutations to the MFRP gene compromised the structural integrity of the retinal cells. The MFRP gene encodes a protein called the Membrane Frizzled-Related Protein, which plays an important role in eye development. Mutations in the MFRP gene are associated with small eye conditions such as nanophthalmos, posterior microphthalmia, or retinal issues such as retinitis pigmentosa, foveoschisis, or even optic disc drusen. Tsang and his group, however, showed that the effects of these MFRP mutations could be reversed with gene therapy. Thus this new approach could potentially be used to create personalized therapies for other forms of RP, or even other genetic diseases.

“The use of patient-specific cell lines for testing the efficacy of gene therapy to precisely correct a patient’s genetic deficiency provides yet another tool for advancing the field of personalized medicine,” said Dr. Tsang, the Laszlo Z. Bito Associate Professor of Ophthalmology and associate professor of pathology and cell biology. This work was recently published in the online edition of Molecular Therapy, the official journal of the American Society for Gene & Cell Therapy.

Mutations in more than 60 different genes have been linked to RP. Such a genetic disease is known as a heterogeneous trait and genetic diseases like RP or deafness or other such conditions are very difficult to develop models to study. Animal models, though useful, have significant limitations because of interspecies differences. Eye researchers have also used human retinal cells from eye banks to study RP. This eye tissue comes from the eyes of patients who suffered from the disease and donated their eye tissue to research after death. Unfortunately, despite their usefulness, donated eye tissues typically illustrate the end stage of the disease process. Despite their usefulness, they reveal little about how RP develops. Also, there are no human tissue culture models of RP, since it is dangerous to harvest retinal cells from patients. Finally, human embryonic stem cells could be useful in RP research, but they are fraught with ethical, legal, and technical issues.

However, the Tsang group used iPS technology to transform skin cells from RP patients, each of whom harbored a different MFRP mutation, into retinal cells. Thus they created patient-specific models for studying the disease and testing potential therapies. Because they used iPS technology, Tsang found a way around the limitations and concerns and dog embryonic stem cells. Thus researchers can induce the patient’s own skin cells and de-differentiated them to a more basic, embryonic stem cell–like state. Such cells are “pluripotent,” which means that they can be transformed into specialized cells of various types.

When Tsang and others analyzed these patient-specific cells, they discovered that the primary effect of MFRP mutations is to disrupt the regulation of a cytoskeletal protein called actin, the scaffolding that gives the cell its structural integrity. “Normally, the cytoskeleton looks like a series of connected hexagons,” said Dr. Tsang. “If a cell loses this structure, it loses its ability to function.” They also found that MFRP works in tandem with another gene, CTRP5, and that a balance between the two genes is required for normal actin regulation.

In the next phase of the study, the CUMC team used adeno-associated viruses (AAVs) to introduce normal copies of MFRP into the iPS-derived retinal cells. This successfully restored the cells’ function. Tsang and others used gene therapy to “rescue” mice with RP due to MFRP mutations. According to Dr. Tsang, the mice showed long-term improvement in visual function and restoration of photoreceptor numbers.

“This study provides both in vitro and in vivo evidence that vision loss caused by MFRP mutations could potentially be treated through AAV gene therapy,” said coauthor Dieter Egli, PhD, an assistant professor of developmental cell biology (in pediatrics) at CUMC, who is also affiliated with the New York Stem Cell Foundation.

Dr. Tsang thinks this approach could potentially be used to study other forms of RP. “Through genome-sequencing studies, hundreds of novel genetic spelling mistakes have been associated with RP,” he said. “But until now, we’ve had very few ways to find out whether these actually cause the disease. In principle, iPS cells can help us determine whether these genes do, in fact, cause RP, understand their function, and, ultimately, develop personalized treatments.”

A Protein from Fat-Based Stem Cells Prevents Light-Induced Damage to the Retina


Japanese researchers from Gifu Pharmaceutical University and Gifu University have reported that a type of protein found in stem cells taken from adipose (fat) tissue can reverse and prevent age-related, light-induced retinal damage in mice. These results may lead to treatments for patients faced with permanent vision loss.

According to the work done by these two research teams led by Drs. Hideaki Hara and Kazuhiro Tsuruma, a single injection of fat-derived stem cells (ASCs) reduced the retinal damage induced by light exposure in mice. This study also discovered that when fat-derived stem cells were grown in culture with retinal cells, the stem cells prevented the retinal cells from suffering damage after exposure to hydrogen peroxide and visible light both in the culture and in the retinas of live mice.

Additionally, Hara and Tsuruma and their colleagues discovered a protein in fat-derived stem cells called “progranulin.” This protein, progranulin, seems to play a central role in protecting other cells from suffering light-induced eye damage.

In the retina, which lies at the back of the eye, excessive light exposure causes degeneration of the photoreceptor cells that respond to light. Several studies have suggested that a long-term history of exposure to light might be an important factor in the onset of age-related macular degeneration. Photoreceptor loss is the primary cause of blindness in particular eye-specific degenerative diseases such as age-related macular degeneration and retinitis pigmentosa.

“However, there are few effective therapeutic strategies for these diseases,” Hideaki Hara, Ph.D., R.Ph., and Kazuhiro Tsuruma, Ph.D., R.Ph.

“Recent studies have demonstrated that bone marrow-derived stem cells protect against central nervous system degeneration with limited results. Just like the bone marrow stem cells, ASCs also self-renew and have the ability to change, or differentiate, as they grow. But since they come from fat, they can be obtained more easily under local anesthesia and in large quantities.”

The fat tissue used in the study was taken from mice and processed in the laboratory to isolate the fat-based stem cells. Afterwards, those cells were tested with cultured mouse retinal cells, and they show a robust protective effect. These successes suggested to the team to test their theory on a live group of mice that had retinal damage after exposure to intense levels of light.

Five days after receiving injections of the fat-based stem cells, the animals were tested for photoreceptor degeneration and retinal dysfunction. The results showed the degeneration had been significantly inhibited.

“Progranulin was identified as a major secreted protein of ASCs, which showed protective effects against retinal damage in culture and in animal tests using mice,” Drs. Hara and Tsuruma said. “As such, it may be a potential target for the treatment of degenerative diseases of the retina such as age-related macular degeneration and retinitis pigmentosa. The ASCs reduced photoreceptor degeneration without engraftment, which is concordant with the results of previous studies using bone marrow stem cells.”

“This study, suggesting that the protein progranulin may play a pivotal role in protecting against retinal light-induced damage, points to the potential for new therapeutic approaches to degenerative diseases of the retina,” said, Anthony Atala, MD, editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine, where this work was published.

Stem Cell Treatments for Retinitis Pigmentosa Inch Toward Clinical Trials


Retinitis Pigmentosa or RP is the most common form of inherited blindness. There are many different genes involved in the onset of RP. Molecular defects in more than 40 different genes can cause “isolated RP” and defects in more than 50 different genes can cause “syndromic RP.” Not only are there a host of different genes involved in RP, two patients with exactly the same molecular lesion can have a type of RP that differs substantially in its presentation.

The retina at the back of the eye is composed of two thick layers known as the inner neural retina and the outer pigmented retina. The neural retina consists of an outer layer of photoreceptors that are connected to an inner layer of bipolar cells. The bipolar cells connect with ganglion cells that have axons that extend to the optic nerve. The photoreceptor cells have their tips embedded in the pigmented retina, and the pigmented retina maintain and nourish the photoreceptors.

Pigmented Retina

If the pigmented retina does not function properly, then the effects are most profoundly displayed in the photoreceptors. Photoreceptors respond to light and the constant exposure to light causes the photoreceptors to take a beating. The byproducts of all that light-induced damage accumulates at the tips of the photoreceptors cells, and these rubbish-filled tips are taken a gulped down by the cells of the pigmented retina. The pigmented retina cells degrade the damaged byproducts and recycle the precursor molecules. Without properly functioning pigmented retina cells, the photoceptors cells accumulate toxic light damage and then eventually die. Photoreceptor cell death is the end product of RP, and it results in blindness.

There is no cure for RP, and the treatments available are very hit-and-miss. For this reason, cell therapies have been examined in a variety of animal models of RP, which, in many cases, closely mimic the human disease to some degree.

Two different experimental treatments, one with induced pluripotent stem cells (iPSCs) and another with gene therapy have produced long-term improvement in visual function in mice with RP. These studies have been conducted at the Columbia University Medical Center (CUMC).

Stephen Tsang, associate professor of pathology, cell biology and ophthalmology who led both studies commented: “While these therapies still need to be refined, the results are highly encouraging. We’ve never seen this type of improvement in retinal function in mouse models of RP. We hope we may finally have something to offer patients with this form of vision loss.”

In one study, CUMC researchers tested the long-term safety and efficacy of iPSC grafts into the pigmented retina to restore visual function in a mouse model of RP. The mice were injected with undifferentiated iPSCs when they were five years old, and the cells differentiated into retinal pigmented epithelial (RPE) cells and integrated into the retinas. None of the mice that received these transplantations developed tumors over their lifetimes.

To test the effects of the implanted cells on the vision of the mice, Tsang’s group used electrophysiological measurements of the retina. In RP mice, as they become blind, the electrophysiology of the retina becomes rather abnormal, but in these mice implanted with the iPSCs, the electrophysiology of their retinas were not only normal, but stayed normal for a long period of time.

According to Tsang: “This is the first evidence of lifelong neuronal recovery in an animal model using stem cell transplants, with vision improvement persisting throughout the lifespan.”

In 2011, the FDA approved clinical trials of embryonic stem cell (ESC) transplants for the treatment of macular degeneration, but this treatment requires the application of drugs that suppress the immune system. Such drugs have rather nasty side effects.

“Our study focused on patient-specific iPS cells, which offer a compelling alternative,” Tsang said. “The iPS cells can provide a potentially unlimited supply of cells for functional rescue and optimization. Also, since they would come from a patient’s own body, immunosuppression would not be necessary to prevent rejection after transplantation.”

Theoretically, iPSC transplants, could also be used to treat age-related macular degeneration, which is the leading cause of vision loss in older adults.

In a second approach to treating RP, CUMC scientists tested a gene therapy protocol in RP mice. A specific type of RP that results from mutations in a PDE6alpha gene was used as a model system for gene therapy protocol. This particular type of RP is rather common in humans. The CUMC scientists injected a virus into one of the eyes of afflicted mice. This virus was engineered to express the PDE6alpha gene when it entered cells. Because this virus is the AAV or adenovirus-associated virus, it only spreads in the presence of adenovirus. Without a helper adenovirus in the retina, the engineered virus particles will infect the cells they initially contact, but they will not produce a productive infection. However, ferry the genes inside them to the cell they initially infect. This the engineered AAV particles are excellent vehicles for getting genes inside cells without causing an infection.

Examination of the mice six months later, the photoreceptors in the AAV-treated eyes were healthy and these eyes were able to see, but the uninjected eyes were unable to see and their photoreceptors were mostly dead.

Again Tsang commented: “These results provide support that RP due to PDE6alpha deficiency in humans is also likely to be treatable by gene therapy.”

CUMC and its teaching-hospital affiliate, New York-Presbyterian Hospital are part of an international consortium that was recently formed to bring this PDE6A gene therapy to patients. Pending FDA approval, clinical trials could begin within a year.

See  Li, Y., Tsai, Y.T., Hsu, C.W., Erol, D., Yang, J., Wu, W.H., Davis, R.J., Egli, D., and Tsang, S.H. Long-term safety and efficacy of human induced pluripotent stem cell (iPS) grafts in a preclinical model of retinitis pigmentosa. Mol Med. 2012 Aug 9. doi: 10.2119/molmed. 2012.00242. [Epub ahead of print] (2012).

Wert KJ, Davis RJ, Sancho-Pelluz J, Nishina PM, Tsang S.H. Gene therapy provides long-term visual function in a pre-clinical model of retinitis pigmentosa. Hum. Mol. Genet. (2012) doi: 10.1093/hmg/dds46