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.2001 Oct 23;98(22):12584-9.
doi: 10.1073/pnas.221364198. Epub 2001 Oct 9.

Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk

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Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk

D Vollrath et al. Proc Natl Acad Sci U S A..

Abstract

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

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Figures

Figure 1
Figure 1
Ad-Mertk expressesMertk mRNA and protein in infected HeLa cells. (A) Cells were infected with different amounts (0, 50, or 500 μl) of cell lysate containing Ad-Mertk for 24 or 48 h, and RNA was isolated. cDNA was synthesized in the presence (+) or absence (−) of reverse transcriptase. PCR was performed to detect a fragment of ratMertk (arrow), and products were resolved by agarose gel electrophoresis. The left-most lane is a 100-bp ladder. (B) An immunoblot was performed on total protein from cells infected with (1) no virus, (2) Ad-GFP, or (3) Ad-Mertk, and probed with a polyclonal antibody directed against rat Mertk.
Figure 2
Figure 2
Light micrographs of a single RCS rat retina injected in the superior hemisphere with Ad-Mertk at P22 and taken at P52. (A) Peripheral retina in the inferior hemisphere shows a thick OS debris zone (D) containing numerous macrophages (M). (B) Posterior retina in the inferior hemisphere. (C) Posterior retina in the superior hemisphere. (D) Peripheral retina in the superior hemisphere in the region of Ad-Mertk injection. (Bar = 20 μm.)
Figure 3
Figure 3
Measurements of the ONL thickness along the vertical meridian of the eye from the optic nerve head (ONH) to the ora serrata of wild-type RCS-rdy+ rats (■;n = 4) and retinal dystrophic RCS rats taken at P52-P57. To illustrate the distribution and magnitude of rescue, representative sections of eyes injected with either stock Ad-Mertk (○;n = 1), diluted Ad-Mertk (●;n = 7) or dialyzed Ad-Mertk (⧫;n = 3) are compared with those of uninjected RCS rats (X;n = 3).
Figure 4
Figure 4
(A andB) Light micrographs of retinal sections of an RCS rat at P52 that received stock Ad-Mertk at P22. (A) Tips of rod OS (ROS) reach the apical surface of the RPE (arrows). (B) Numerous phagosomes are present in the RPE cytoplasm (arrowheads), demonstrating ingestion of shed ROS membranes. (Bar = 15 μm.) (C andD) Electron micrographs of a retinal section comparable to that inB. (C) A large phagosome (P) is found in the cytoplasm and near the nucleus of an RPE cell. Tips of ROS are apposed to the apical surface of the RPE cell. ×10,625. (D) Higher magnification (×28,600) of a serial section to (C) illustrating ROS-like lamellar disk membranes within the phagosome.
Figure 5
Figure 5
Scotopic ERG intensity series from the uninjected eye (Left) and contralateral dialyzed Ad-Mertk-injected eye (Right) of an RCS rat at P60. Vertical arrows indicate application of the stimulus. Slanted arrows indicate STRs recorded at threshold stimulus intensity. The difference in the STR threshold between the control and treated eyes in this animal is greater than two log units.
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