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National Eye Institute Science Advances

Canine Study Puts Gene Therapy for X-linked Retinitis Pigmentosa in Sight

A team of National Eye Institute (NEI)-funded scientists used gene therapy to treat canines with a form of X-linked retinitis pigmentosa (XLRP), an inherited eye disease that damages light sensing cells in the retina called photoreceptors and causes gradual vision loss that can lead to blindness. The study provides strong evidence a similar approach might work in humans.

"These are very promising results that demonstrate for the first time in a large animal model of retinitis pigmentosa that corrective gene therapy can be used both to prevent retinal degeneration and rescue photoreceptor cells if treatment is given after disease onset," said William Beltran, D.V.M, Ph.D., an assistant professor of ophthalmology at the University of Pennsylvania School of Veterinary Medicine and lead author of the study report, which appeared February 7, 2012, in Proceedings of the National Academy of Sciences.

RPGR gene therapy preserves retinal thickness.
RPGR gene therapy preserves retinal thickness. Retinal maps reconstructed from multiple OCT scans. Color indicates retinal thickness. (A) Dotted line indicates injection area in treated retina. (B) Cross-sectional retinal OCT scan located at the treated/ nontreated transition zone (black arrow in A). (C) Retinal thickness map of control eye.

The most common form of XLRP is caused by a mutation in the RPGR gene located on the X chromosome. Expression of the gene supports rod and cone photoreceptors. These cells convert light into nerve signals the brain perceives as images. Photoreceptors are located in the layer of tissue in the back of the eye called the retina. Without normal RPGR function, photoreceptors and other retinal cells slowly deteriorate, along with vision.

XLRP, as with all X-linked traits, almost exclusively affects males. An estimated 1 in 30,000 boys are born with XLRP. In most cases, night blindness occurs at around age 10. By age 45, most men with XLRP are legally blind. In canines, the disease is called X-linked progressive retinal atrophy (XLPRA) and also stems from an RPGR gene mutation.

To get normal RPGR gene inside the photoreceptors, the scientists injected one of each dog's eyes with adeno-associated virus (AAV) particles, each packed with a normal copy of the human RPGR gene. A solution containing the AAV vector was injected into the space between the photoreceptor cell layer and the adjacent retinal pigment epithelial layer. AAV is an ideal gene therapy vector because it infects cells but doesn't cause illness. The other eye served as a control and was injected with a solution lacking AAV vector.

Once the RPGR gene was introduced, photoreceptors began expressing normal RPGR protein, preventing degeneration in treated areas of the retina. Photoreceptor health was evaluated by measuring retinal thickness using optical coherence tomography (OCT), a procedure similar to ultrasound that can create high-resolution images of subsurface tissues, or in this case, the back of the eye. In contrast to ultrasound, OCT employs light waves instead of sound waves. Treated areas retained near-normal retinal thickness while untreated areas of the retina became thinner (see image).

Staining of retinal tissues showed that RPGR gene therapy preserved not only the photoreceptors but other retinal cell types, too, and maintained normal retinal morphology. XLRP, XLPRA, and other diseases that affect photoreceptors have secondary effects on inner retinal cells, such as bipolar cells, which are essential for conducting visual signals to the brain.

"Rescue of photoreceptor cells accompanied a dramatic improvement of the connections between the photoreceptors and inner retinal cells, which suggests that image transfer from the retina to the brain will be improved," said Gustavo Aguirre, V.M.D., Ph.D., Professor of Medical Genetics and Ophthalmology, University of Pennsylvania School of Veterinary Medicine, and a co-author of the report.

Electroretinography-a test that measures electrical activity of retinal cells-exhibited stronger signals in treated eyes.

The scientists also demonstrated RPGR gene therapy could reverse XLPRA, a finding with enormous implications for human therapy because it suggests treatment is still possible after disease onset.

Effectiveness of the technique in canines is a promising sign it will work in humans. Several of the report's authors were involved in pre-clinical dog studies that led to the development of human gene therapy for a different type of inherited retinal degeneration called Leber congenital amaurosis (LCA). The subsequent breakthrough LCA clinical trials, which began in 2007, demonstrated gene therapy is safe and effective at restoring some vision and set the stage for future gene therapies for eye diseases.

Gene therapy for XLRP must clear several obstacles before moving into clinical trials. First, the scientists must identify critical periods during the course of the disease when treatment will be most effective. Determining how late in the course of retinal degeneration a therapeutic effect can still be achieved will be essential when selecting patients for potential clinical trials. More pre-clinical work will help identify critical treatment windows and correlate them to retinal thickness or other biomarkers that can be measured noninvasively in human patients.

Second, the scientists must make sure transgenic expression through gene therapy approximates a normal level of RPGR gene activity. Compared to the LCA gene, relatively little is known about RPGR function. In the current study, the scientists studied RPGR vectors with two different gene promoters. Gene promoters are stretches of DNA at the beginning of gene sequences that control gene expression. One of the promoters led to much higher gene activity than the other.

The AAV vector concentration is another factor that influences gene expression. More vector leads to greater gene expression. Additional studies will identify the ideal combination of promoter and AAV vector concentration that can restore vision without causing unintentional side effects due to RPGR overexpression.

And third, the scientists must ensure that the treatment is durable and safe. Patients in the LCA gene therapy trials retained vision gained and experienced very few adverse events. Beltran is cautiously optimistic the same will be true for XLRP gene therapy. "You can extrapolate from what has been learnt from the LCA gene therapy trials, but every new combination of a viral vector, promoter, and transgene should be considered as a new drug and its safety and efficacy thoroughly evaluated," he said.

The study involved close collaboration with scientists at University of Pennsylvania's Scheie Eye Institute, including Samuel Jacobsen and Arthur Cideciyan; University of Florida scientists William Hauswirth and Alfred Lewin; and former University of Michigan scientists Anand Swaroop, now at the National Institutes of Health, and Hermant Khanna, now at University of Massachusetts Medical School.

- By Dustin C. Hays

Publication Citation:

  • Beltran WA, Cideciyan AV, Lewin AS, et al. Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa. Proceedings of the National Academy of Sciences USA. Feb 7 2012;109(6):2132-2137. PubMed

Support for this research was provided in part by NIH grants EY-06855, EY-17549, EY-007961, EY-021721, P30 EY-001583, and 2PNEY018241.

Last Reviewed: July 2012

Department of Health and Human Services NIH, the National Institutes of Health