Gene Therapy, Stem Cells: Prime for Vision Restoration

Editor's Note: The second installment of this five-part series, on hearing, will appear in the Oct. 1 issue. Using gene therapy, scientists earlier this year reversed blindness in three dogs afflicted with Leber congenital amaurosis (LCA). The news excited the scientific world and popular press. LCA is a rare, inherited disease characterized by a severe loss of vision at birth. Researchers at the University of Pennsylvania, Cornell University, and University of Florida showed that injecting a good copy of RPE65 protein into the right eye of LCA-afflicted dogs restored sight in that eye.1 But human clinical trials are more than a year away. Graphic: Lisa Damiani This research characterizes the current state of scientific investigation into the world of blindness: virtually all human trials are far down the road, and any findings, so far, pertain only to the particular disease itself. A bright side does exist: vision restoration is a prime candidate for both gene therapy and stem cell transplants. And other researchers are using different approaches in which electrodes implanted on the retina allow blind people to see edges and shapes thanks to electrode stimulation from a camera mounted on a pair of glasses. 2 These researchers, from Johns Hopkins University, are awaiting Food and Drug Administration approval for a Phase I trial, says project director Mark Humayun. "Even restoring some vision in blind patients can have a significant impact on their lives by restoring their independent mobility," Humayun says. The American Foundation for the Blind estimates that about 10 million people in the United States are blind or visually impaired. Retina pigmentosa (RP), a group of inherited diseases that cause progressive photoreceptor degeneration, affects about one in 2,500. Comparatively, about 2,000 individuals have LCA, and only about 15 percent of those cases are caused by the RPE65 mutation, according to researchers. Other causes of blindness concern scientists. With the growth of an aging population, age-related macular degeneration (AMD) will become more prevalent than diabetic retinopathy and glaucoma combined, affecting as many as one in six over age 60, according to the National Eye Institute. Of any place in the body, the retina could be the best candidate for gene therapy and stem cell transplants. Gene therapy complications are attributable, at least in part, to the need for a large quantity of vector because the patient develops an immune reaction to the virus, not the gene, says retinal gene therapy researcher John Flannery, University of California, Berkeley. But the amount of virus injected into the retina is about 1/1000 of what is used for systemic diseases, he says. Also, the blood-ocular barrier within the eye separates it from the rest of the body, acting to protect the retina and preventing the escape of large molecules into the blood stream. Therefore, a virus delivered to the eye is unlikely to have any systemic effect. And, notes Flannery, because the retina is really an extension of the brain into the eye, actually part of the central nervous system, it is particularly amenable to stem cell transplants. Where the Action Is Most research currently focuses on RP, which affects young people and eventually leads to blindness. This disease affects the photoreceptors, located in the rods and cones, which are the retina's specialized cells. RP can be inherited in dominant, recessive, or X-linked fashion. Although RP is the best understood retinal disease, no treatment exists for it or for any other inherited retinal degenerative disease. Ideally, treatments developed for RP will also be applicable to AMD, which affects the macula. Courtesy of Michael YoungImage of mouse retinal stem cells (RSCs, green), which differentiate into cells of retinal lineage when grafted to the diseased or injured adult mouse retina [recoverin (red) and GFP (green) co-expression (yellow) in mechanically injured B6 mouse photoreceptor layer]. Researchers are testing different approaches to slow RP's progression. Gene replacement therapy is being used to treat recessive or X-linked diseases. Ribozymes, which act as molecular scissors to reduce the mutant gene product, or neurotrophins, which inhibit apoptotic cell death, are used for dominant diseases. Flannery and collaborator Matthew LaVail, director of the Center for the Study of Retinal Degeneration, University of California, San Francisco, have created transgenic rats that carry gene mutations for dominant forms of RP. Collaborating with researchers elsewhere, they use these rats to develop gene therapy techniques that slow the photoreceptor cells' degeneration. A few years ago, ribozyme gene therapy dramatically reduced vision loss in studies involving these rats; photoreceptor cell function was as much as 93 percent greater in the treated eyes than in the untreated eyes.3 University of Florida researchers inserted ribozymes into AAV vectors, which the Flannery and LaVail laboratories then injected into the model's subretinal space. The ribozymes located and then bound to the mutated mRNA of the defective protoreceptor, cutting it in half. Thus decreasing the amount of the injury-causing protein before it could damage the photoreceptor. For autosomal dominant RP, the Florida team created a ribozyme that recognizes the mRNA that encodes the mutant P23H rhodopsin, and destroys it before the rhodopsin makes the photoreceptor-degenerating P23H protein. The photoreceptors survive with the single normal copy of the rhodopsin gene in their other allele. Photoreceptor loss occurred whether the disease was treated early or later, Flannery says.4 Another approach involved injecting the rats with vectors carrying neurotrophins, which are naturally occurring, protein growth factors that obstruct apoptosis. Previous rat work in LaVail's lab showed that the therapy slowed disease progress for about 10 days. Building on these findings, Flannery added genes that encode the neurotrophins into the virus vector. The genes prompted the retina to secrete the neurotrophins in perpetuity and slowed the disease progression by 30-40 percent, Flannery says. 5-7 Courtesy of Mark Humayun, Wilmer Eye InstituteResearchers from the Wilmer Eye Institute have devised a pair of glasses that incorporates a camera to capture images and transmit the image signal to implanted electronics on the retinal surface, thus enabling some degree of sight. Flannery is looking to combine the two complementary approaches. "In a perfect world, [there] would be a 30 percent slowing as we've seen in the ribozyme, and a similar amount of slowing with the growth factor, combining for 50 to 60 percent slowing of degeneration." Flannery is developing a technique to inject both vectors by mixing the two in a syringe. To date, this vision-saving gene therapy has only been tested on transgenic rats, which develop RP in a similar fashion to humans, making them a good predictive model. But the lack of appropriate larger animal models has slowed progress to human clinical trials. This sort of gene therapy can't be used in cases where live photoreceptors no longer exist: gene therapy requires a live cell. So in severe macular degeneration or late stages of RP, or any other disorder in which the photoreceptors are dead and irreparable, gene therapy will not work. Stem cell transplantation might. Progress in Stem Cell Transplantation Ongoing studies by Henry Klassen, at Children's Hospital, Orange County, Calif., and Michael Young at the Schepens Eye Research Institute at Harvard Medical School have shown encouraging results in transplanting neural stem cells into the adult host retina. Young and Klassen met 15 years ago in the lab of Ray Lund, University of Pittsburgh. Since then, they have continued collaborating to achieve photoreceptor replacement through stem cell transplantation. In their most significant finding to date, they discovered that stem cells respond better in the diseased eye than in the normal eye, indicating a response to some sort of injury cue, Klassen says. "When we put stem cells into the adult central nervous system of the degenerating rat eye, we saw this profound migration and integration of cells," says Klassen. "That was startling."8 They also found that neural stem cells integrated with the retina and in some cases looked like retinal cells. But the transplanted cells fell short of differentiation--the ultimate quest--and failed to become functional retinal cells. "They were not really becoming photoreceptors, and that is what we need to replace in retinal degenerations," Young says. Courtesy of John G. FlanneryTransverse section of a rat retina two months after a subretinal injection of AAV-CMV-gfp vector. In pursuit of differentiation, Young changed his focus from neural to retinal stem cells. "I think we're pretty close to our goal of transplanting retinal stem cells and having them become true functional photoreceptors in the host," he says. "But we're not there yet." Once differentiation is achieved, then further research will follow to show that this approach restores vision. A better understanding of the molecules present on the stem cells' surface might give researchers a better idea of how the molecules will behave in the eye. Klassen's work involves growing stem cell lines and examining their surface molecules. Although the key to controlling stem cell differentiation and proliferation is by controlling growth factors, managing them requires knowing what receptors are on the cells' surface. Courtesy of Michael YoungImages of adult hippocampal progenitor cells (AHPCs, green) grafted onto normal adult Fischer 344 rats that received a mechanical lesion. Widespread migration and integration of grafted cells was seen (top), with confocal imaging (bottom) demonstrating that cells grafted in this setting can take up residence in the nuclear layers of the retina and adopt morphological phenotypes strikingly similar to that of normal host retinal neurons. Recently, Klassen says he discovered that neural stem cells are partly immunoprivileged because they do not express a molecule called MHC class II, normally found in passenger leukocytes. Immunoprivileged cells can survive transplantation even when transferred to sites where tissue would otherwise be rejected because they have no passenger leukocytes. Most tissue transplants, even embryonic neural tissue, carry microglia and other cells that can function as passenger leukocytes. Both the eye and the brain are immune-privileged sites, making them less prone to reject a cell transplant. "There's hope that these kinds of transplants might not require any sort of immunosuppressants," Klassen says. "That would be a huge step forward at reducing the risk of transplantation." The ultimate solution to vision restoration, suggests Flannery, might be blending both gene therapy and stem cell transplantation. "One of the things we're considering is trying to use the viruses we use in gene therapy to transfect the stem cells, giving them genes that force them to become photoreceptors." Jennifer Fisher Wilson (jfwilson@snip.net) is a contributing editor for The Scientist. References 1. G.M. Acland et al., "Gene therapy restores vision in a canine model of childhood blindness," Nature Genetics, 28:92-5, 2001. 2. M.S. Humayun et al., "Electrical Stimulation of the Human Retina," Vision Research, 39:2569-76, 1999. 3. A.S. Lewin et al., "Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa," Nature Medicine, 4:967-71, 1998. 4.M.M. LaVail et al., "Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy," Proceedings of the National Academy of Sciences, 97:11488-93, 2000. 5. L.H. McGee Sanftner et al., "Recombinant AAV-mediated delivery of a tet-inducible reporter gene to the rat retina," Molecular Therapy, 3:688-96, 2001. 6. E.S. Green et al., "Two animal models of retinal degeneration are rescued by recombinant adeno-associated virus-mediated production of FGF-5 and FGF-18," Molecular Therapy, 3:507-15, 2001. 7. D. Lau et al., "Retinal degeneration is slowed in transgenic rats by AAV-mediated delivery of FGF-2," Investigative Ophthalmology and Visual Science, 41:3622-33, 2000. 8. M.J. Young et al., "Neuronal differentiation and morphological integration of hippocampal progenitor cells transplanted to the retina of immature and mature dystrophic rats," Molecular and Cellular Neuroscience, 16:197-205, 2000.

Gene Therapy, Stem Cells: Prime for Vision Restoration

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