OBJECTIVE

To describe the ocular phenotype of patients with RPE65 mutations in infancy and young childhood.

METHODS

Four children from three families with severe early-onset visual impairment related to electrophysiologically detectable retinal dystrophy were screened for mutations in the RPE65 gene. Visual function from infancy to the age of 10 years was assessed with age-adapted methods. Clinical examinations and electroretinograms (ERGs) were also performed on the six parents.

RESULTS

In all three families, patients were compound heterozygous for mutations of the RPE65 gene (ins144T/IVS1+5G->>A, R91W/Y368H, 1114delA+T457N/IVS1+5G->>A). Visual acuity was measurable in all patients at the age of 6 to 10 years, despite severe visual impairment noted during infancy and congenital nystagmus in three of the four patients. Photophobia was not a feature. Funduscopic changes were discrete, the most prominent finding being increased granularity in the macula and the periphery. Peripheral vision was well preserved, measured by Goldmann perimetry. Rod ERGs were not recordable, whereas cone ERGs were detectable in early childhood. All features taken together suggest a specific form of Leber congenital amaurosis (LCA) distinguishable on clinical grounds. ERGs were normal in five of the six parents. One father had an ERG compatible with congenital stationary night blindness unrelated to his heterozygous state for the RPE65 mutation.

CONCLUSIONS

RPE65 mutations on both alleles may be associated with early-onset severe rod-cone dystrophy. Visual functions of the four patients were better than is usually seen in LCA, in particular in cases associated with retGC1 mutations. RPE65 mutations should be suspected in infants who appear to be blind in dim surroundings but react to objects in bright illumination and have nonrecordable rod ERGs and residual cone ERGs.

Leber congenital amaurosis (LCA) is a molecularly heterogeneous disease group that leads to blindness. LCA caused by RPE65 mutations has been studied in animal models and vision has been restored by subretinal delivery of AAV-RPE65 vector. Human ocular gene transfer trials are being considered. Our safety studies of subretinal AAV-2/2.RPE65 in RPE65-mutant dogs showed evidence of modest photoreceptor loss in the injection region in some animals at higher vector doses. We now test the hypothesis that there can be vectorrelated toxicity to the normal monkey, with its human-like retina. Good Laboratory Practice safety studies following single intraocular injections of AAV-2/2.RPE65 in normal cynomolgus monkeys were performed for 1-week and 3-month durations. Systemic toxicity was not identified. Ocular-specific studies included clinical examinations, electroretinography, and retinal histopathology. Signs of ocular inflammation postinjection had almost disappeared by 1 week. At 3 months, electroretinography in vector-injected eyes was no different than in vehicle-injected control eyes or compared with presurgical recordings. Healed sites of retinal perforation from subretinal injections were noted clinically and by histopathology. Foveal architecture in subretinally injected eyes, vector or vehicle, could be abnormal. Morphometry of central retina showed no photoreceptor layer thickness abnormalities occurring in a dose-dependent manner. Vector sequences were present in the injected retina, vitreous, and optic nerve at 1 week but not consistently in the brain. At 3 months, there were no vector sequences in optic nerve and brain. The results allow for consideration of an upper range for no observed adverse effect level in future human trials of subretinal AAV-2/2.RPE65. The potential value of foveal treatment for LCA and other retinal degenerations warrants further research into how to achieve gene transfer without retinal injury from surgical detachment of the retina.

OBJECTIVE

To evaluate cell survival and tumorigenicity of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM).

METHODS

Sixty-nine rats (38 male, 31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1, 6, and 12 months after surgery. Both ocular tissues and systemic organs (brain, liver, kidneys, spleen, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker), anti-TRA-1-85 (human cell marker), anti-Ki67 (proliferation marker), anti-CD68 (macrophage), and anti-cytokeratin (epithelial marker).

RESULTS

The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P < 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group.

CONCLUSIONS

hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects.

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.

RPE65 is essential for the biosynthesis of 11-cis-retinal, the chromophore of rhodopsin. Here, we show that the membrane-associated form (mRPE65) is triply palmitoylated and is a chaperone for all-trans-retinyl esters, allowing their entry into the visual cycle for processing into 11-cis-retinal. The soluble form of RPE65 (sRPE65) is not palmitoylated and is a chaperone for vitamin A, rather than all-trans-retinyl esters. Thus, the palmitoylation of RPE65 controls its ligand binding selectivity. The two chaperones are interconverted by lecithin retinol acyl transferase (LRAT) acting as a molecular switch. Here mRPE65 is a palmitoyl donor, revealing a new acyl carrier protein role for palmitoylated proteins. When chromophore synthesis is not required, mRPE65 is converted into sRPE65 by LRAT, and further chromophore synthesis is blocked. The studies reveal new roles for palmitoylated proteins as molecular switches and LRAT as a palmitoyl transferase whose role is to catalyze the mRPE65 to sRPE65 conversion.

Linkage testing using Affymetrix 6.0 SNP Arrays mapped the disease locus in TCD-G, an Irish family with autosomal dominant retinitis pigmentosa (adRP), to an 8.8 Mb region on 1p31. Of 50 known genes in the region, 11 candidates, including RPE65 and PDE4B, were sequenced using di-deoxy capillary electrophoresis. Simultaneously, a subset of family members was analyzed using Agilent SureSelect All Exome capture, followed by sequencing on an Illumina GAIIx platform. Candidate gene and exome sequencing resulted in the identification of an Asp477Gly mutation in exon 13 of the RPE65 gene tracking with the disease in TCD-G. All coding exons of genes not sequenced to sufficient depth by next generation sequencing were sequenced by di-deoxy sequencing. No other potential disease-causing variants were found to segregate with disease in TCD-G. The Asp477Gly mutation was not present in Irish controls, but was found in a second Irish family provisionally diagnosed with choroideremia, bringing the combined maximum two-point LOD score to 5.3. Mutations in RPE65 are a known cause of recessive Leber congenital amaurosis (LCA) and recessive RP, but no dominant mutations have been reported. Protein modeling suggests that the Asp477Gly mutation may destabilize protein folding, and mutant RPE65 protein migrates marginally faster on SDS-PAGE, compared with wild type. Gene therapy for LCA patients with RPE65 mutations has shown great promise, raising the possibility of related therapies for dominant-acting mutations in this gene.

In mammals, intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate non-image-forming visual functions such as pupillary light reflex (PLR) and circadian photoentrainment. This photosensitivity requires melanopsin, an invertebrate opsin-like protein expressed by the ipRGCs. The precise role of melanopsin remains uncertain. One suggestion has been that melanopsin may be a photoisomerase, serving to regenerate an unidentified pigment in ipRGCs. This possibility was echoed by a recent report that melanopsin is expressed also in the mouse retinal pigment epithelium (RPE), a key center for regeneration of rod and cone pigments. To address this question, we studied mice lacking RPE65, a protein essential for the regeneration of rod and cone pigments. Rpe65-/- ipRGCs were approximately 20- to 40-fold less photosensitive than normal at both single-cell and behavioral (PLR) levels but were rescued by exogenous 9-cis-retinal (an 11-cis-retinal analog), indicating the requirement of a vitamin A-based chromophore for ipRGC photosensitivity. In contrast, 9-cis-retinal was unable to restore intrinsic photosensitivity to melanopsin-ablated ipRGCs, arguing against melanopsin functioning merely in photopigment regeneration. Interestingly, exogenous all-trans-retinal was also able to rescue the low sensitivity of rpe65-/- ipRGCs, suggesting that melanopsin could be a bistable pigment. Finally, we detected no melanopsin in the RPE and no changes in rod and cone sensitivities due to melanopsin ablation. Together, these results strongly suggest that melanopsin is the photopigment in the ipRGCs.

In vertebrate retinal photoreceptors, the absorption of light by rhodopsin leads to photoisomerization of 11-cis-retinal to its all-trans isomer. To sustain vision, a metabolic system evolved that recycles all-trans-retinal back to 11-cis-retinal. The importance of this visual (retinoid) cycle is underscored by the fact that mutations in genes encoding visual cycle components induce a wide spectrum of diseases characterized by abnormal levels of specific retinoid cycle intermediates. In addition, intense illumination can produce retinoid cycle by-products that are toxic to the retina. Thus, inhibition of the retinoid cycle has therapeutic potential in physiological and pathological states. Four classes of inhibitors that include retinoid and nonretinoid compounds have been identified. We investigated the modes of action of these inhibitors by using purified visual cycle components and in vivo systems. We report that retinylamine was the most potent and specific inhibitor of the retinoid cycle among the tested compounds and that it targets the retinoid isomerase, RPE65. Hydrophobic primary amines like farnesylamine also showed inhibitory potency but a short duration of action, probably due to rapid metabolism. These compounds also are reactive nucleophiles with potentially high cellular toxicity. We also evaluated the role of a specific protein-mediated mechanism on retinoid cycle inhibitor uptake by the eye. Our results show that retinylamine is transported to and taken up by the eye by retinol-binding protein-independent and retinoic acid-responsive gene product 6-independent mechanisms. Finally, we provide evidence for a crucial role of lecithin: retinol acyltransferase activity in mediating tissue specific absorption and long lasting therapeutic effects of retinoid-based visual cycle inhibitors.

The eye has unique advantages as a target organ for gene therapy of both inherited and acquired ocular disorders and offers a valuable model system for gene therapy. The eye is readily accessible to phenotypic examination and investigation of therapeutic effects in vivo by fundus imaging and electrophysiological techniques. Considerable progress has been made in the development of gene replacement therapies for retinal degenerations resulting from gene defects in photoreceptor cells (rds, RPGRIP, RS-1) and in retinal pigment epithelial cells (MerTK, RPE65, OA1) using recombinant adeno-associated virus and lentivirus-based vectors. Gene therapy also offers a potentially powerful approach to the treatment of complex acquired disorders such as those involving angiogenesis, inflammation and degeneration, by the targeted sustained intraocular delivery of therapeutic proteins. Proposals for clinical trials of gene therapy for early-onset retinal degeneration owing to defects in the gene encoding the visual cycle protein RPE65 have recently received ethical approval.

The accumulation of the lipofuscin fluorophores in retinal pigment epithelial (RPE) cells leads to the blinding degeneration characteristic of Stargardt disease and related forms of macular degeneration. RPE lipofuscin, including the fluorophore A2E, forms in large part as a byproduct of the visual cycle. Inhibiting visual cycle function with small molecules is required to prevent the formation of the retinotoxic lipofuscins. This in turn requires identification of rate-limiting steps in the operation of the visual cycle. Specific, non-retinoid isoprenoid compounds are described here, and shown through in both in vitro and in vivo experiments, to serve as antagonists of RPE65, a protein that is essential for the operation of the visual cycle. These RPE65 antagonists block regeneration of 11-cis-retinal, the chromophore of rhodopsin, thereby demonstrating that RPE65 is at least partly rate-limiting in the visual cycle. Furthermore, chronic treatment of a mouse model of Stargardt disease with the RPE65 antagonists abolishes the formation of A2E. Thus, RPE65 is also on the rate-limiting pathway to A2E formation. These nontoxic isoprenoid RPE65 antagonists are candidates for the treatment of forms of macular degeneration wherein lipofuscin accumulation is an important risk factor. These antagonists will also be used to probe the molecular function of RPE65 in vision.

OBJECTIVE

To determine the impairment of the transient pupillary light reflex (TPLR) due to severe retinal dysfunction and degeneration in a murine model of Leber congenital amaurosis (LCA) and in patients with the disease.

METHODS

Direct TPLR was elicited in anesthetized, dark-adapted Rpe65(-/-) and control mice with full-field light stimuli (0.1 second duration) of increasing intensities (-6.6 to +2.3 log scot-cd. m(-2)). 9-cis-Retinal was administered orally to a subset of Rpe65(-/-) mice, and TPLR was recorded 48 hours after the treatment. TPLR was also measured in a group of patients with LCA.

RESULTS

Baseline pupillary diameters in Rpe65(-/-) and control mice were similar. TPLR thresholds of Rpe65(-/-) mice were elevated by 5 log units compared with those of control animals. The waveform of the TPLR in Rpe65(-/-) mice was similar to that evoked by 4.8-log-unit dimmer stimuli in control mice. Treatment of Rpe65(-/-) mice with 9-cis-retinal lowered the TPLR threshold by 2.1 log units. Patients with LCA had baseline pupillary diameters similar to normal, but the TPLR was abnormal, with thresholds elevated by 3 to more than 6 log units. When adjusted to the elevation of TPLR threshold, pupillary constriction kinetics in most patients were similar to those in normal subjects.

CONCLUSIONS

Pupillometry was used to quantify visual impairment and to probe transmission of retinal signals to higher nervous centers in a murine model of LCA and in patients with LCA. Mouse results were consistent with a dominant role of image-forming photoreceptors driving the early phase of the TPLR when elicited by short-duration stimuli. The objective and noninvasive nature of the TPLR measurement, and the observed post-treatment change toward normal in the animal model supports the notion that this may be a useful outcome measure in future therapeutic trials of LCA.

The RPE65 gene encodes a 65-kDa microsomal protein expressed exclusively in retinal pigment epithelium (RPE). Mutations in the human RPE65 gene have recently been identified in patients with autosomal recessive, severe, childhood-onset retinal dystrophy. Here we report the characterization of a 2.4-kb canine Rpe65 cDNA. The longest open reading frame predicts a 533-amino-acid protein with a calculated molecular mass of about 61 kDa prior to protein modification. Sequence comparison shows that RPE65 is highly conserved throughout mammalian evolution. We have identified a homozygous 4-bp deletion (485delAAGA) in putative exon 5 of the canine Rpe65 gene in affected animals of a highly inbred kinship of Swedish briard/briard-beagle dogs, in which an autosomal recessive, early-onset, and progressive retinal dystrophy segregates. The deletion results in a frameshift and leads to a premature stop codon after inclusion of 52 canine RPE65-unrelated amino acids from residue 153 onward. More than two-thirds of the wildtype polypeptide chain will be missing, and the mutant protein is most likely nonfunctional (null allele). Clinical features of the canine disease are quite similar to those described in human. Therefore this form of canine retinal dystrophy provides an attractive animal model of the corresponding human disorder with immediate significance for various therapeutic approaches, including RPE transplantation.

OBJECTIVE

Acute white-light damage to rods depends on the amount of rhodopsin available for bleaching during light exposure. Bleached rhodopsin is metabolically regenerated through the visual cycle involving the pigment epithelium, or photochemically by deep blue light through photoreversal of bleaching. Because photoreversal is faster than metabolic regeneration of rhodopsin by several orders of magnitude, the photon catch capacity of the retina is significantly augmented during blue-light illumination, which may explain the greater susceptibility of the retina to blue light than to green light. However, blue light can also affect function of several blue-light-absorbing enzymes that may lead to the induction of retinal damage. Therefore, this study was conducted to test whether rhodopsin and its bleaching intermediates play a role in blue-light-induced retinal degeneration.

METHODS

Eyes of anesthetized rats and mice that did or did not contain rhodopsin were exposed to green (550 +/- 10 nm) or deep blue (403 +/- 10 nm) light for up to 2 hours. Rats with nearly rhodopsinless retinas were obtained by bleaching rhodopsin in animals with inhibited metabolic rhodopsin regeneration-that is, under halothane anesthesia. In addition, Rpe65(-/-) mice that are completely without rhodopsin were used to test the susceptibility to blue-light damage of a rodent retina completely devoid of the visual pigment. Effects of illumination on photoreceptor morphology were assessed 24 hours or 10 days thereafter by morphologic and biochemical methods.

RESULTS

Exposure to blue light resulted in severe retinal damage and activation of the transcription factor AP-1 in rats. In contrast, green light had no effect. When rhodopsin was almost completely bleached by short-term green-light exposure while metabolic regeneration (but not photoreversal) was prevented by halothane anesthesia, blue-light exposure induced distinct lesions in rat retinas. When both metabolic rhodopsin regeneration and photoreversal of bleaching were almost completely inhibited, blue-light exposure caused only very moderate lesions. When mice without rhodopsin were exposed to blue light, no damage occurred, in contrast to wild-type control mice.

CONCLUSIONS

Short time exposure to blue light has deleterious effects on retinal morphology. Because damage was observed only in the presence of the visual pigment, blue-light-induced retinal degeneration is rhodopsin mediated. Absorption of blue light by other proteins is not sufficient to induce light damage. Photoreversal of bleaching, which occurs only in blue but not in green light, increases the photon-catch capacity of the retina and may thus account for the difference in the damage potential between blue and green light.

The U.S. Food and Drug Administration recently approved phase I/II clinical trials for embryonic stem (ES) cell-based retinal pigmented epithelium (RPE) transplantation, but this allograft transplantation requires lifelong immunosuppressive therapy. Autografts from patient-specific induced pluripotent stem (iPS) cells offer an alternative solution to this problem. However, more data are required to establish the safety and efficacy of iPS transplantation in animal models before moving iPS therapy into clinical trials. This study examines the efficacy of iPS transplantation in restoring functional vision in Rpe65(rd12)/Rpe65(rd12) mice, a clinically relevant model of retinitis pigmentosa (RP). Human iPS cells were differentiated into morphologically and functionally RPE-like tissue. Quantitative real-time polymerase chain reaction (RT-PCR) and immunoblots confirmed RPE fate. The iPS-derived RPE cells were injected into the subretinal space of Rpe65(rd12)/Rpe65(rd12) mice at 2 d postnatally. After transplantation, the long-term surviving iPS-derived RPE graft colocalized with the host native RPE cells and assimilated into the host retina without disruption. None of the mice receiving transplants developed tumors over their lifetimes. Furthermore, electroretinogram, a standard method for measuring efficacy in human trials, demonstrated improved visual function in recipients over the lifetime of this RP mouse model. Our study provides the first direct evidence of functional recovery in a clinically relevant model of retinal degeneration using iPS transplantation and supports the feasibility of autologous iPS cell transplantation for retinal and macular degenerations featuring significant RPE loss.

OBJECTIVE

The Rpe65-/- mouse, used as a model for Leber congenital amaurosis, has slow rod degeneration and rapid cone loss, presumably because of the mistrafficking of cone opsins. This animal does not generate 11-cis retinal, and both cone loss and rod response are restored by 11-cis retinal administration. Similarly, the Lrat-/- mouse does not produce 11-cis retinal. The authors sought to determine whether the same effects on rod and cone opsins in the Rpe65-/- mouse are also present in the Lrat-/- mouse, thereby establishing that these changes can be attributed to the lack of 11-cis retinal rather than to some unknown function of RPE65.

METHODS

Rod and cone opsins were localized by immunohistochemical methods. Functional opsin levels were determined by regeneration with 11-cis retinal. Isorhodopsin levels were determined from pigment extraction. Opsin phosphorylation was determined by mass spectrometry.

RESULTS

Rods in both models degenerated slowly. Regenerable rod opsin levels were similar over the 6-month time course investigated, rod opsin was phosphorylated at a low level (approximately 10%), and minimal 9-cis retinal was generated by a nonphotic process, giving a trace light response. In both models, S-opsin and M/L-opsin failed to traffic to the cone outer segments appropriately, and rapid cone degeneration occurred. Cone opsin mistrafficking in both models was arrested on 11-cis retinal administration.

CONCLUSIONS

These data show that the Lrat-/- and Rpe65-/- mice are comparable models for studies of Leber congenital amaurosis and that the destructive cone opsin mistrafficking is caused by the lack of 11-cis retinal.

BACKGROUND

RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice.

RESULTS

Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease.

CONCLUSIONS

By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65(-/-) mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.

Leber's congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies responsible for congenital blindness. Genetic heterogeneity of LCA has been suspected since the report by Waardenburg of normal children born to affected parents. In 1995, we localized the first disease causing gene, LCA1, to chromosome 17p13 and confirmed the genetic heterogeneity. In 1996, we ascribed LCA1 to mutations in the photoreceptor-specific guanylate cyclase gene (retGC1). RetGC1 is an essential protein implicated in the phototransduction cascade, especially in the recovery of the dark state after the excitation process of photoreceptor cells by light stimulation. In 1997, mutations in a second gene were reported in LCA, the RPE65 gene, which is the first specific retinal pigment epithelium gene. The protein RPE65 is implicated in the metabolism of vitamin A, the precursor of the photoexcitable retinal pigment (rhodopsin). Finally, a third gene, CRX, implicated in photoreceptor development, has been suspected of causing a few cases of LCA. Taken together, these three genes account for only 27% of LCA cases in our series. The three genes encode proteins that are involved in completely different physiopathologic pathways. Based on these striking differences of physiopathologic processes, we reexamined all clinical physiopathological discrepancies and the results strongly suggested that retGC1 gene mutations are responsible for congenital stationary severe cone-rod dystrophy, while RPE65 gene mutations are responsible for congenital severe but progressive rod-cone dystrophy. It is of tremendous importance to confirm and to refine these genotype-phenotype correlations on a large scale in order to anticipate the final outcome in a blind infant, on the one hand, and to further guide genetic studies in older patients on the other hand.

BACKGROUND

RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA).

RESULTS

RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean +/- standard deviation [SD] = 0.07% +/- 0.06% and volume = 1.3 +/- 0.6 cm(3)). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% +/- 0.06%) and volume (8.2 +/- 0.8 cm(3)) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1-4 y of age. Human RPE65-LCA patients (ages 18-23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 +/- 0.5 mm) was within the normal range (3.2 +/- 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 +/- 1.2 cm(3)) compared to controls (29.7 +/- 8.3 cm(3), p < 0.001) when stimulated with lower intensity light. Unexpectedly, cortical response volume (41.2 +/- 11.1 cm(3)) was comparable to normal (48.8 +/- 3.1 cm(3), p = 0.2) with higher intensity light stimulation.

CONCLUSIONS

Visual cortical responses dramatically improve after retinal gene therapy in the canine model of RPE65-LCA. Human RPE65-LCA patients have preserved visual pathway anatomy and detectable cortical activation despite limited visual experience. Taken together, the results support the potential for human visual benefit from retinal therapies currently being aimed at restoring vision to the congenitally blind with genetic retinal disease.

OBJECTIVE

To assess the frequency of mutations in the CRX, GUCY2D, and RPE65 genes in patients with Leber congenital amaurosis (LCA).

METHODS

One hundred seventy-six probands with a clinical diagnosis of LCA were from 9 countries, with the largest subgroup being 39 probands from India.

METHODS

Samples were screened with single-strand conformation polymorphism analysis followed by DNA sequencing of 3 genes (CRX, GUCY2D, and RPE65) known to be associated with LCA.

RESULTS

Of the 176 probands, 28 (15.9%) harbored possible disease-causing mutations. The relative contribution of each gene to the total number of mutations was as follows: CRX, 2.8%; GUCY2D, 6.3%; and RPE65, 6.8%. No patients who harbored mutations in these genes had associated systemic abnormalities. Molecular diagnosis allowed definitive genetic counseling in a family affected with Best disease and LCA.

CONCLUSIONS

Molecular diagnosis may be of benefit to patients affected with LCA. The relative paucity of mutations found in this study suggests that more LCA-associated genes remain to be discovered.

CONCLUSIONS

Molecular diagnosis can confirm and clarify the diagnosis of LCA. As genotype data accumulate, clinical phenotypes associated with specific mutations will be established. This will facilitate the counseling of patients on their visual prognosis and the likelihood of associated systemic anomalies.

The isomerization of all-trans-retinyl ester to 11-cis-retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle and is essential for normal vision. Recently, we have established that protein RPE65 is the isomerohydrolase catalyzing this reaction. The present study investigated if metal ions are required for the isomerohydrolase activity of RPE65. The conversion of all-trans-[3H]retinol to 11-cis-[3H]retinol was used as the measure for isomerohydrolase activity. Metal chelators 2,2'-bipyridine and 1,10-phenanthroline both showed dose-dependent inhibitions of the isomerohydrolase activity in bovine RPE microsomes, with IC50 values of 0.5 and 0.2 mm, respectively. In the same reaction systems, however, lecithin-retinol acyltransferase (LRAT) activity was not affected by these metal chelators. The isomerohydrolase activity inhibited by the metal chelators was restored by FeSO4 but not by CuSO4, ZnCl2, or MgCl2. Moreover, addition of Fe(III) citrate or FeCl3 did not restore the activity, indicating that Fe2+ is the metal ion essential for the isomerohydrolase activity. To confirm this result in recombinant RPE65, we expressed RPE65 in a 293A cell line stably expressing LRAT. In vitro activity assay showed that both metal chelators inhibited isomerohydrolase activity of recombinant RPE65. The addition of FeSO4 restored the enzymatic activity of the recombinant RPE65. Further, two specific iron-staining methods showed that purified RPE65 contains endogenous iron. Inductively coupled plasma mass spectrometry measurements showed that bovine RPE65 binds iron ion with a stoichiometry of 0.8 +/- 0.1. These results indicate that RPE65 is an iron-dependent isomerohydrolase in the visual cycle.

C57BL/6J-c(2J) (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J x BALB/c)F(1) x c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c. When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases.

OBJECTIVE

Recent in vitro evidence has shown that RPE65 is the isomerohydrolase that converts all-trans retinyl ester to 11-cis retinal, the chromophore for visual pigments in vertebrates. Homozygous RPE65 knockout (Rpe65-/-) mice lack 11-cis retinoids and have early cone degeneration. The purpose of this study is to determine whether RPE65 gene delivery restores the isomerohydrolase activity and normal profile of endogenous retinoids in Rpe65-/- mice.

METHODS

Adenovirus-expressing RPE65 (Ad-RPE65) was injected into the subretinal space of Rpe65-/- mice. The expression of RPE65 was determined by immunohistochemistry and Western blot analysis. The isomerohydrolase activity was measured in vitro in eyecup homogenates. Endogenous retinoid profile in the eyecups was analyzed by high-performance liquid chromatography (HPLC). Photoreceptor-specific gene expression was determined with real-time RT-PCR. Cone degeneration was determined by cone-specific staining and counting cones in flatmounted retina.

RESULTS

High levels of RPE65 expression from the Ad-RPE65 injection generated robust isomerohydrolase activity in the eyecup of Rpe65-/- mice, at levels comparable to those in wild-type (wt) mice. Consequently, the RPE65 gene delivery resulted in substantial amounts of 11-cis retinal in Rpe65-/- mice. The RPE65 gene delivery prevented the downregulation of cone-specific genes, including both cone opsins and cone tranducin alpha subunit in Rpe65-/- mice. Moreover, the Ad-RPE65 injection also prevented massive cone degeneration at early ages of Rpe65-/- mice.

CONCLUSIONS

RPE65 gene delivery generates isomerohydrolase activity and restores retinoid profile in Rpe65-/- mice. Regeneration of 11-cis retinal is essential for survival of cone photoreceptors.

OBJECTIVE

Fundus autofluorescence is due to accumulation of lipofuscin in the retinal pigment epithelium (RPE) resulting from incomplete digestion of N-retinylidene-phosphatidyl-ethanolamine from shed photoreceptor outer segment discs. Alteration in autofluorescence reflects changes in lipofuscin content of the RPE. Mutations on both alleles of RPE65 result in absent or largely decreased formation of rhodopsin, due to a defect in all-trans retinol isomerization in the RPE. Autofluorescence could therefore be altered. This study was conducted to evaluate fundus autofluorescence in patients with early-onset severe retinal dystrophy (EOSRD, or early-onset rod-cone dystrophy) associated with mutations on both alleles of RPE65.

METHODS

Case series.

METHODS

Ten 10- to 55-year-old patients with EOSRD and compound heterozygous or homozygous mutations in RPE65. For comparison, 6 heterozygous parents and 2 patients with other forms of EOSRD were examined.

METHODS

Participants underwent, in addition to standard clinical and electrophysiological examination, autofluorescence imaging using a confocal scanning laser ophthalmoscope. Three of the patients were also examined by optical coherence tomography (OCT) to evaluate the status of retinal degeneration. Mutations in 7 patients have been reported previously; the other patients were investigated by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing for mutations in RPE65 and lecithin retinol acyltransferase (LRAT).

METHODS

Fundus autofluorescence and OCT.

RESULTS

Absent or minimal autofluorescence was found in all patients with compound heterozygous or homozygous RPE65 mutations. Autofluorescence was normal in the heterozygous parents. Autofluorescence was present in 2 children with EOSRD not associated with mutations in RPE65 or LRAT, another gene involved in retinol recycling. Optical coherence tomography in younger patients revealed an intraretinal appearance similar to that of their healthy, heterozygous parents.

CONCLUSIONS

Lack of autofluorescence in patients with EOSRD associated with mutations in RPE65 is in accordance with the biochemical defect and can be used as a clinical marker of this genotype. Optical coherence tomography results in younger patients would indicate still viable photoreceptors despite the absence of autofluorescence.

The first event in light perception is absorption of a photon by the retinaldehyde chromophore of an opsin pigment in a rod or cone photoreceptor cell. This induces isomerization of the chromophore, rendering the bleached pigment insensitive to light. Restoration of light sensitivity requires chemical reisomerization of retinaldehyde via a multistep enzyme pathway, called the visual cycle, in cells of the retinal pigment epithelium (RPE). Interphotoreceptor retinoid-binding protein (IRBP) is present in the extracellular space between photoreceptors and the RPE. IRBP is known to bind visual retinoids. Previous studies on irbp(-/-) mice suggested that IRBP plays an insignificant role in opsin-pigment regeneration. However, the mice in these studies were uncontrolled for a severe mutation in the rpe65 gene. Rpe65 catalyzes the rate-limiting step in the visual cycle. Here, we examined the phenotype in irbp(-/-) mice homozygous for the wild-type (Leu450) rpe65 gene. We show that lack of IRBP causes delayed transfer of newly synthesized chromophore from RPE to photoreceptors. Removal of bleached chromophore from photoreceptors is also delayed in irbp(-/-) retinas after light exposure. It was previously shown that rods degenerate in irbp(-/-) mice. Here, we show that cones and rods degenerate at similar rates. However, cones are more affected functionally and show greater reductions in outer segment length than rods in irbp(-/-) mice. The disproportionate reductions in cone function and outer-segment length appear to result from mistrafficking of cone opsins due to impaired delivery of retinaldehyde chromophore, which functions as a chaperone for cone opsins but not rhodopsin.