Stem Cell, Gene Therapies for Retinitis Pigmentosa in Mice

Ricki Lewis, PhD

January 07, 2013

Researchers at Columbia University have tested stem cell and gene therapies in mouse models of retinitis pigmentosa (RP) with promising results, according to 2 studies published in 2012.

Whether stem cell or gene therapy is the optimal approach for a particular disease depends on the degree of tissue degradation. "When cells are dead, gene therapy wouldn't work, but stem cells can replace lost cells to regain lost vision," Stephen Tsang, MD, PhD, leader of both studies and associate professor of pathology, cell biology, and ophthalmology at Columbia University Medical Center and an ophthalmologist at New York–Presbyterian Hospital/CUMC in New York City, told Medscape Medical News.

In contrast, gene therapy can be useful earlier in pathology in still-viable cells. "The 2 approaches will be complementary for personalized medicine," Dr. Tsang said.

Using Human iPS Cells

In an article published online August 9, 2012, and in the September 2012 issue of Molecular Medicine, Yao Li, MD, and colleagues discussed their investigation, in which they transplanted human induced pluripotent stem (iPS) cell-derived retinal pigment epithelium (RPE) cells into the subretinal spaces of 34 mice with the Rpe65rd12 /Rpe65rd12 form of RP. RPE supports the photoreceptors.

This use of iPS cells is a model for autologous transplants. "The main drawback to using embryonic stem cells is that they require immunosuppression therapy to prevent rejection, since they are not derived from the host. iPS cells are generated from a patient's skin," explained Dr. Tsang.

The mice had albinism, which provided contrast against the human cells that have perinuclear melanin granules. The mice also had severe combined immune deficiency to prevent graft-vs-host disease. The investigators previously showed that mouse iPS-derived RPE cells restore retinal function.

A healthy adult provided skin fibroblasts that the researchers cultured with lentivirus-delivered genes encoding transcription factors OCT4, SOX2, KLF4, and MYC. Within 3 weeks, pigmented cells appeared.

Antibody staining of markers (TRA-1-60, SSEA4, NANOG, and SOX2) and a teratoma assay demonstrated pluripotency of the hiPS cells. Then, culturing in differentiation medium guided their fate to RPE. By 12 weeks, from 30% to 50% of the surfaces of 12-well dishes were coated with pigmented epithelium emerging in telltale fluid domes, with characteristic hexagonal shapes, perinuclear melanin granules, and microvilli.

The researchers injected 1000 hiPS-derived RPE cells subretinally into the right eyes of 34 mice at 2 days after birth. Uninjected left eyes controlled for surgical trauma. The mice were killed at 6 months, shortly before they would have died from severe combined immune deficiency.

Several techniques monitored the fate of the injected cells. Light microscopic examination revealed pigmented hiPS-derived RPE integrated into the native, albino RPE, "suggesting successful incorporation of iPS-derived RPE," the researchers write. No tumors formed.

Quantitative polymerase chain reaction detected markers of human fetal RPE and iPS-derived RPE. Undifferentiated iPS cells and donor fibroblasts did not exhibit these markers.

Staining for rhodopsin demonstrated that the hiPS-derived RPE cells phagocytosed photoreceptors. In RP, loss of this phagocytosis impairs vision.

The researchers used the electroretinogram (ERG) response to measure neuronal function, but not all animals could be assessed: 10 had retinal detachment or no ERG response in the treated right eyes, and another 17 right eyes had surgical trauma. Of the remaining 7 mice, the average ERG β-wave peak difference between treated and control eyes was significant at 13.7 μV (P = 0.0246). The improvement was small, the researchers write, because of the small size of the transplanted area.

"It's very exciting that iPS cells are effective, and to see no tumor formation is encouraging," Jeffrey Stern, MD, PhD, director of translational research at the Neural Stem Cell Institute in Rensselaer, New York, told Medscape Medical News.

Gene Therapy Approach

In a second series of experiments, published online October 29, 2012, in Human Molecular Genetics, Katherine J. Wert, MS, and colleagues at Columbia University tested gene therapy in mice with mutations in the α subunit of rod-specific cyclic guanosine monophosphate phosphodiesterase (PDE6). Correction of the β subunit, which causes less severe photoreceptor loss, is effective. Mutations in PDE6 cause 36,000 cases of RP in humans worldwide.

Adeno-associated virus serotypes 2/8 injected subretinally on postnatal day 5 delivered the DNA encoding the PDE6 α subunit into Pde6anmf363 mice. A capsid mutation sustained the effect of the payload, and a rhodopsin promoter targeted the construct to the rod outer segments.

The researchers treated right eyes and monitored photoreceptor survival and visual function for up to 6 months. As in the stem cell study, anatomical and physiological evidence pointed to efficacy.

The treated eyes produced PDE6 and rhodopsin, but not as much as control eyes. Untreated left eyes had undetectable enzyme levels and low levels of rhodopsin.

Infrared imaging revealed that RPE atrophy starts at 5 months in the untreated eyes and is extensive by 11 months, but the treated eyes, imaged at 5, 7, 9, and 11 months, had minimal atrophy.

Histological sectioning of treated eyes at 2 months showed preservation of photoreceptor cell bodies and outer segments in half of each retina, corresponding to the injection sites. By 6 months, the researchers identified "significantly more photoreceptor nuclei in the rescued portion of the treated eye (3.79 ± 4.21) when compared with the untreated fellow eye (1.44 ± 5.46)."

ERGs revealed activities of both rods and cones in the treated eyes, but at about 20% of normal responses.

The observed effects of single injections in enhancing survival of photoreceptors and improving retinal function, the researchers write, may be sufficient to arrest the disease. They conclude that gene therapy for RP is feasible in humans targeting either the α or β subunit of PDE6. Future studies should be longer, alter the timing, and attempt multiple and re-injections, using the natural Welsh Corgi model and then clinical trials, they suggest.

In an interview with Medscape Medical News, Sally Temple, PhD, scientific director of the Neural Stem Cell Institute, applauds the 2 articles and points out that millions of people suffer from retinal diseases that might be amenable to either stem cell or gene therapies. "With such a big patient population, we should be trying more than one approach." Dr. Temple was not involved with either study.

The Li study was supported by the Joan and Michael Schneeweiss Research Fund. Dr. Li's research is supported by a New York State Stem Cell Science grant. The Bernard and Shirlee Brown Glaucoma Laboratory is supported by the National Institutes of Health, Research to Prevent Blindness, and the Foundation Fighting Blindness. One coauthor is a fellow of the Burroughs-Wellcome Program in Biomedical Sciences and has been supported by the Bernard Becker Association of University Professors in Ophthalmology, Research to Prevent Blindness, the Foundation Fighting Blindness, the Dennis W. Jahnigen Award of the American Geriatrics Society, Joel Hoffman Fund, Gale and Richard Siegel Stem Cell Fund, Charles Culpeper Scholarship, Irma T. Hirschl Charitable Trust, Bernard and Anne Spitzer Stem Cell Fund, Barbara and Donald Jonas Family Fund and Professor Gertrude Rothschild Stem Cell Foundation. For the Wert study, imaging and animal facilities are supported by the National Institutes of Health and unrestricted funds from Research to Prevent Blindness. The Bernard and Shirlee Brown Glaucoma Laboratory is supported by the Department of Defense, the Foundation Fighting Blindness, Schneeweiss Stem Cell Fund, and Tistou and Charlotte Kerstan Foundation. Wert and one coauthor are supported by the National Institutes of Health. One coauthor is a member of the RD-CURE Consortium, a Fellow of the Burroughs-Wellcome Program in Biomedical Sciences, and has been supported by the Bernard Becker Association of University Professors in Ophthalmology Research to Prevent Blindness Award, Dennis W. Jahnigen Award of the American Geriatrics Society, Joel Hoffman Scholarship, Schneeweiss Stem Cell Fund, Irma T. Hirschl Charitable Trust, Crowley Family Fund, Barbara and Donald Jonas Family Fund and Professor Gertrude Rothschild Stem Cell Foundation. The other authors and the commentators have disclosed no relevant financial relationships.

Mol Med. 2012;18:1312-1319. Full text

Hum Mol Genet. Published online October 29, 2012. Abstract