The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.(1) Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.(2) These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.(3-4) Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.(5-8) In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

Retinal pigment epithelium-specific protein 65 kDa (RPE65) is a key enzyme for the visual cycle in the eye. Rpe65(-/-) mice lack 11-cis-retinal, and show early cone degeneration and mislocalization of cone opsins. The present study investigated whether abnormal modification of cone opsins at the protein level is present in Rpe65(-/-) mice. Retina-RPE-choroids of Rpe65(-/-) mice at 3, 5 and 7 weeks old were used. Immunohistochemistry of opsins was performed using cryosections and retinal flatmounts. We evaluated levels of mRNA for cone and rod opsin genes by RT-PCR and levels of proteins by western blotting. To examine modification patterns of N-glycan in Rpe65(-/-) mice, cone opsins were digested with peptide-N-glycosidase (PNGase) F. S-opsin protein was detected at approximately 40-kDa as a major band in wild-type mice, whereas approximately 42-kDa S-opsin protein was detected in Rpe65(-/-) mice. After PNGase F treatment, mobility of S-opsin protein in wild-type and Rpe65(-/-) mice on SDS-PAGE was similar. In addition, approximately 25-kDa S-opsin polypeptide was notably detected in Rpe65(-/-) mice. Conversely, M-opsin proteins were not observed by immunohistochemistry or western blotting in Rpe65(-/-) mice, but expression of M-opsin mRNA in Rpe65(-/-) mice did not differ significantly from that in wild-type mice at 3 and 5 weeks. Mobility of M-opsin protein in Rpe65(-/-) mice was unchanged. Our data suggest that S-opsin protein is incompletely modified during N-glycan processing in Rpe65(-/-) mice, whereas M-opsin protein is severely reduced by posttranslational degradation in the absence of incomplete N-glycan processing in Rpe65(-/-) mice.

RPE65 is the isomerohydrolase essential for regeneration of 11-cis retinal, the chromophore of visual pigments. Here we compared the impacts of two mutations in RPE65, E417Q identified in patients with Leber congenital amaurosis (LCA), and E417D on isomerohydrolase activity. Although both mutations decreased the stability of RPE65 and altered its sub-cellular localization, E417Q abolished isomerohydrolase activity whereas the E417D mutant retained partial enzymatic activity suggesting that the negative charge of E417 is important for RPE65 catalytic activity. Loss of charge at this position may represent a mechanism by which the E417Q mutation causes blindness in LCA patients.

OBJECTIVE

We have been engaged in an ongoing study to screen candidate genes for mutations in small families with various forms of autosomal recessive retinal dystrophy. Here we report the screening of a cohort of 14 families from Sardinia for mutations in the genes encoding the alpha- and beta-subunits of cGMP-phosphodiesterase and RPE65 (PDE6A, PDE6B, and RPE65).

METHODS

Haplotype analysis was performed on each family using simple sequence repeat markers closely flanking or within each of the three gene candidates. For families in which a gene could not be ruled out from segregating with disease, exons of the gene from proband DNAs were screened for mutations by SSCPE (single strand conformation polymorphism electrophoresis). All variants found by SSCPE were sequenced directly.

RESULTS

By haplotype analysis, 6/14, 11/14, and 4/13 families were ruled out for PDE6A, PDE6B, and RPE65, respectively. A few variants were found in the proband DNAs of the remaining families, but only one was significant: a 20 bp deletion in exon 4 of RPE65. The deletion co-segregated with disease in one family and caused a frame shift that produces a stop codon downstream. It was absent from the other Sardinian families that we tested, and from Sardinian and North American controls. Results of studies of phenotype in homozygotes and heterozygotes in this Sardinian family are compared with those from a non-Sardinian family recently reported to have the same RPE65 mutation.

CONCLUSIONS

This RPE65 mutation, which appears to be quite restricted in its occurrence in Sardinia, leads to childhood onset severe retinal dystrophy or Leber congenital amaurosis. Affecteds of the other 13 plus two additional families were diagnosed with arRP. This family lived in an area of Sardinia where none of the others lived suggesting different ancestral origins.

Musashi-1 (Msi1), an RNA-binding protein (RBP), has been postulated to play important roles in the maintenance of the stem-cell state, differentiation, and tumorigenesis. However, the expression and function of Msi1 in differentiated cells remain obscure. Here we show that Msi1 is expressed in mature photoreceptors and retinal pigment epithelium (RPE) cells, and is indispensable for the survival of photoreceptors. We found in the adult newt eye that Msi1 is expressed in all photoreceptors and RPE cells as well as in the retinal stem/progenitor cells in the ciliary marginal zone (CMZ). We found in the analyses of the newt normal and regenerating retinas that the expression profiles of the Msi1 transcripts and protein isoforms in the photoreceptors are different from those in the retinal stem/progenitor cells. Furthermore, we found that all photoreceptors and RPE cells of the adult mice also express Msi1, and that Msi1 knockout (Msi1-KO) results in degeneration of photoreceptors and a lack of a visual cycle protein RPE65 in the microvilli of RPE cells. Taken together, our current results demonstrate that the expression of Msi1 in mature photoreceptors and RPE cells is evolutionarily conserved, and that Msi1 bears essential functions for vision. Considering such an Msi1-KO phenotype in the retina, it is now reasonable to address whether defects of the Msi1 functions are responsible for inherited retinal diseases. Studying the regulation of Msi1 and the target RNAs of Msi1 in photoreceptors and RPE cells might contribute to fundamental and clinical studies of retinal degeneration.

Lecithin retinol acyl transferase (LRAT) has the essential role of catalyzing the transfer of an acyl group from the sn-1 position of lecithin to vitamin A to generate all-trans-retinyl esters (tREs). In vitro studies had shown previously that LRAT also can exchange palmitoyl groups between RPE65, a tRE binding protein essential for vision, and tREs. This exchange is likely to be of regulatory significance in the operation of the visual cycle. In the current study, the substrate specificity of LRAT is explored with palmitoylated amino acids and dipeptides as RPE65 surrogates. Both O- and S-substituted palmitoylated analogues are excellent substrates for tLRAT, a readily expressed and readily purified form of LRAT. Using vitamin A as the palmitoyl acceptor, tREs are readily formed. The cognate of these reactions occurs in crude retinal pigment epithelial (RPE) membranes as well. RPE membranes containing LRAT transfer palmitoyl groups from radiolabeled [1-(14)C]-l-alpha-dipalmitoyl diphosphatidylcholine (DPPC) to RPE65. Palmitoyl transfer is abolished by preincubation with a specific LRAT antagonist both in membranes and with purified tLRAT. These experiments are consistent with an expanded role for LRAT function as a protein palmitoyl transferase.

Previous studies have demonstrated that the intrastriatal implantation of human retinal pigment epithelial cells attached to gelatin microcarriers (hRPE-GM) ameliorates behavioral deficits in animal models of Parkinson disease. However, there are only sparse data on cell survival in the host. In this study, we characterized a variety of retinal pigment epithelial (RPE)-specific markers in vitro and used these markers to investigate the long-term survival of hRPE-GM implants. Sprague-Dawley rats (n = 22) were unilaterally lesioned with 6-hydroxydopamine (6-OHDA) and implanted with hRPE-GM without immunosuppression. Rats were euthanized at 48 hours, 7 days, 4 weeks, and 5 months postimplant and immunohistochemically processed using the following antibodies: 1) human-specific nuclear mitotic apparatus protein (NuMA-Ab2), 2) epithelial-specific extracellular matrix metalloproteinase inducer (EMMPRIN), 3) RPE cell-specific RPE65, and the inflammation markers 4) glial fibrillary acidic protein and 5) ED1 (rat CD68). Our analysis revealed NuMA-, EMMPRIN-, and RPE65-immunoreactive cells at different times postimplant. The morphologic features of hRPE cell implants (at 48 hours and 5 months) were confirmed by electron microscopy. Furthermore, despite evidence of chronic inflammation at the later time point, there is an appreciable number of surviving hRPE cells. This study suggests that hRPE-GM implants can survive in the absence of immunosuppression and can be potentially used as an alternative for treating Parkinson disease.

OBJECTIVE

To present long-term follow-up on a North American patient with Leber congenital amaurosis (LCA) and novel compound heterozygous mutations in the RPE65 gene.

METHODS

Case report.

METHODS

RPE65 mutation screening and search for sequence changes using Single Strand Conformation Polymorphism and direct DNA sequencing. Ophthalmic examination and electrophysiologic testing.

RESULTS

A 35-year-old female carried two RPE65 mutations: a maternal 961A>T (K303X) nonsense mutation and a paternal 1346A>G (Y431C) missense mutation. She had severe visual deficits and an absence of rod and cone Electroretinogram responses. Visual acuity of 20/60 both eyes and normal color recognition during early childhood declined to 2/200 in the right eye and 1/200 in the left eye at the age of 35.

CONCLUSIONS

The RPE65 mutations K303X and Y431C in compound heterozygous form cause progressive visual compromise that starts in childhood and advances to severe visual loss by the fourth decade of life.

Young Rpe65-deficient dogs have been used as a model for human RPE65 Leber congenital amaurosis (RPE65-LCA) in proof-of-concept trials of recombinant adeno-associated virus (rAAV) gene therapy. However, there are relatively few reports of the outcome of rAAV gene therapy in Rpe65-deficient dogs older than 2 years of age. The purpose of this study was to investigate the success of this therapy in older Rpe65-deficient dogs. Thirteen eyes were treated in dogs between 2 and 6 years old. An rAAV2 vector expressing the human RPE65 cDNA driven by the human RPE65 promoter was delivered by subretinal injection. Twelve of the 13 eyes had improved retinal function as assessed by electroretinography, and all showed improvement in vision at low lighting intensities. Histologic examination of five of the eyes was performed but found no correlation between electroretinogram (ERG) rescue and numbers of remaining photoreceptors. We conclude that functional rescue is still possible in older dogs and that the use of older Rpe65-deficient dogs, rather than young Rpe65-deficient dogs that have very little loss of photoreceptors, more accurately models the situation when treating human RPE65-LCA patients.

A method is described for cultivating retinal pigment epithelial cells from choroidal neovascular membrane (CNV) specimens that were surgically removed in patients with age-related macular degeneration (AMD). CNV specimens of 43 patients were available for cultivation. They were incubated in supplemented DMEM/Ham's F12 cell culture medium on microporous semipermeable filter membranes. Thirty-four specimens gave rise to cell cultures, 28 of which could be subcultivated for up to 15 passages. The membrane type as classified by fluorescence angiography was compared with cellular growth in vitro. Immunocytochemistry revealed a uniform expression of cytokeratin 18 and vimentin, while factor 8, glial fibrillary acidic protein and alpha smooth muscle actin were absent in all 21 cultures stained. The expression of RPE markers cellular retinaldehyde binding protein (CRALBP) and RPE65 was detected by RT-PCR in all cultures tested. An epithelial character of the cultures was supported by the presence of apical microvilli as determined by electron microscopical studies. Therefore, the cell cultures from CNV in AMD bear characteristics of retinal pigment epithelial cells. For the first time, this cell culture system holds the potential to study human RPE cells in the context of neovascular AMD in vitro.

The ability to deliver a large transgene efficiently to photoreceptors using viral vectors remains problematic and yet is critical for the future therapy of inherited retinal diseases such as Stargardt's and Usher's 1B. Herein, we examine the ocular tropism of a HIV-1-based lentivirus vector pseudotyped with Venezuelan equine encephalitis virus-derived glycoprotein (VEEV-G) after intraocular delivery to the posterior and anterior chambers of C57BL/6 wild-type mice. Reporter gene (EGFP) expression was evaluated using in vivo fluorescence imaging followed by postmortem immunohistochemistry and retinal function assessed by electroretinography. Intracameral administration of VEEV-G and vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped vectors resulted in robust transgene expression in the corneal endothelium and trabecular meshwork. After subretinal administration, onset of transgene expression was observed in the retinal pigment epithelium (RPE) 1 day postinjection with both VEEV-G and control VSV-G pseudotypes, but no significant photoreceptor transduction was apparent. Substantial degeneration of the outer nuclear layer was observed with VEEV-G-pseudotyped vector, which corresponded to ablation of retinal function. Subretinal administration of VSV-G was observed to result in significant suppression of electrophysiological function compared with buffer-injected and uninjected control eyes. Suppression of the c-wave amplitude, in addition to reduced RPE65 expression, indicated potential RPE dysfunction. Ex vivo tropism of VSV-G was assessed using organotypic culture of explanted retina harvested from wild-type mice and human patients undergoing retinal detachment surgery to examine the prevention of transduction by physical barriers and species differences in tropism.

Cone photoreceptors have faster light responses than rods and a higher demand for 11-cis retinal (11cRAL), the chromophore of visual pigments. RPE65 is the isomerohydrolase in the retinal pigment epithelium (RPE) that converts all-trans retinyl ester to 11-cis retinol, a key step in the visual cycle for regenerating 11cRAL. Accumulating evidence suggests that cone-dominant species express an alternative isomerase, likely in retinal Müller cells, to meet the high demand for the chromophore by cones. In the present study, we describe the identification and characterization of a novel isomerohydrolase, RPE65c, from the cone-dominant zebrafish retina. RPE65c shares 78% amino acid sequence identity with RPE-specific zebrafish RPE65a (orthologue of human RPE65) and retains all of the known key residues for the enzymatic activity of RPE65. Similar to the other RPE-specific RPE65, RPE65c was present in both the membrane and cytosolic fractions, used all-trans retinyl ester as its substrate and required iron for its enzymatic activity. However, immunohistochemistry detected RPE65c in the inner retina, including Müller cells, but not in the RPE. Furthermore, double-immunostaining of dissociated retinal cells using antibodies for RPE65c and glutamine synthetase (a Müller cell marker), showed that RPE65c co-localized with the Müller cell marker. These results suggest that RPE65c is the alternative isomerohydrolase in the intra-retinal visual cycle, providing 11cRAL to cone photoreceptors in cone-dominant species. Identification of an alternative visual cycle will contribute to the understanding of the functional differences of rod and cone photoreceptors.

OBJECTIVE

Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason, it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE.

METHODS

iPS-RPE was derived from human iPS cells. Immunocytochemistry, RT-PCR, and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT), RPE65, cellular retinaldehyde-binding protein (CRALBP), and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids.

RESULTS

Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol, iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media.

CONCLUSIONS

iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE.

We previously showed that RPE65 does not specifically produce 11-cis-retinol only but also 13-cis-retinol, supporting a carbocation or radical cation mechanism of isomerization. The intrinsic properties of conjugated polyene chains result in facile formation of radical cations in oxidative conditions. We hypothesized that such radical intermediates, if involved in the mechanism of RPE65, could be stabilized by spin traps. We tested a variety of hydrophilic and lipophilic spin traps for their ability to inhibit RPE65 isomerohydrolase activity. We found that the aromatic lipophilic spin traps such as N-tert-butyl-α-phenylnitrone (PBN), 2,2-dimethyl-4-phenyl-2H-imidazole-1-oxide (DMPIO), and nitrosobenzene (NB) strongly inhibit RPE65 isomerohydrolase activity in vitro.

Previously, we showed that mutating RPE65 residue Phe-103 preferentially produces 13-cis-retinol instead of 11-cis-retinol, supporting a carbocation/radical cation mechanism of retinol isomerization. We asked whether this modulation of specificity can occur with residues other than Phe-103 and what role it plays in substrate binding and isomerization. We modeled the substrate-binding cleft of RPE65 to identify residues lining its surface. Many are phenylalanines and tyrosines, including three Phe residues (Phe-61, Phe-312, and Phe-526) forming an arch-like arrangement astride the cleft and Tyr-338. Also, Phe-418 sits at the neck of the cleft, lending a bend to the volume enclosed by the cleft. All mutations of Phe-61, Phe-312, and Phe-418 result in severely impaired or inactive enzyme. However, mutation of Phe-526 and Tyr-338, like Phe-103, decreases 11-cis-retinol formation, whereas increasing the 13-cis isomer. Significantly, 2 of these 3 residues, Phe-103 and Tyr-338, are located on putatively mobile interstrand loops. We propose that residual densities located in the binding cleft of the RPE65 structure represents a post-cleavage snapshot consistent not only with a fatty acid product, as originally modeled, but also an 11-cis-retinol product. Substrate docking simulations permit 11-cis- or 13-cis-retinyl ester binding in this relatively closed cleft, with the latter favored in F103L, F526A, and Y338A mutant structures, but prohibit binding of all-trans-retinyl ester, suggesting that isomerization occurs early in the temporal sequence, with O-alkyl ester cleavage occurring later. These findings provide insight into the mechanism of isomerization central to the visual cycle.

BACKGROUND

The positive results of pioneering clinical trials using gene therapy as treatment for patients with Leber congenital amaurosis (LCA) have ushered in a new era of molecular retinal therapeutics for LCA, other blinding retinal disorders and gene therapy applications.

METHODS

This review describes the role of retinal pigment epithelium-specific 65 kDa protein (RPE65) in the visual cycle and how RPE65 deficiency results in LCA; the extensive preclinical studies with recombinant adeno-associated virus (rAAV)-RPE65 gene vectors; and the human rAAV-RPE65 and related gene therapy clinical trials and studies. The literature search included a review of primary sources (e.g., journal articles) that reported study data results and key secondary sources such as meta-reviews available through PubMed, as well as reviews of clinical trial descriptions and results as reported in clinicaltrials.gov, conference publications and news releases.

CONCLUSIONS

LCA-RPE65 gene therapy is an example of successful, innovative, translational research. Further research is needed regarding how retinal gene therapy can be improved.

OBJECTIVE

To determine the clinical phenotypes in carriers with probable disease-causing sequence variations in 1 of 6 genes established to cause Leber congenital amaurosis (LCA).

METHODS

Observational prospective comparative study.

METHODS

Thirty carriers with various probable disease-causing sequence variations in 1 of 6 genes known to cause LCA.

METHODS

After the establishment of various disease-causing sequence variations in 37 (33.6%) of 110 patients with LCA, we examined a number of carriers who were either parents or offspring and who were willing to participate in our study. Evaluations included assessment of visual acuity, slit-lamp biomicroscopy, dilated fundus examination, and full-field electroretinogram (ERG) measurements.

METHODS

Dilated fundus examination and full-field ERGs.

RESULTS

Of the 30 carriers with probable disease-causing sequence variations for LCA, 5 (16.7%) carriers had an AIPL1 variation, 4 (13.3%) CRB1, 0 (0%) CRX, 5 (16.7%) GUCY2D, 9 (30%) RPE65, and 7 (23.3%) carriers had a RPGRIP1 variation. Twenty-nine (96.7%) carriers had 20/20 or better visual acuity in their better seeing eye with correction. Drusenlike deposits were more selectively observed in carriers with mutations in the AIPL1, CRB1, RPE65, and RPGRIP1 genes, whereas mild peripheral chorioretinal atrophy was only observed in AIPL1 and RPE65 carriers. A reduced dark-adapted isolated rod ERG response and/or maximal combined cone and rod response was recorded in carriers with mutations in the AIPL1, GUCY2D, and RPGRIP1 genes. A reduced light-adapted ERG response to a single-flash and/or 32-Hz flicker was recorded in carriers with mutations in the AIPL1, CRB1, GUCY2D, and RPGRIP1 genes. Overall, our cohort of LCA carriers did not describe significant subjective visual difficulties, including nyctalopia and/or photosensitivity.

CONCLUSIONS

The variation of phenotypic expression in carriers among 5 LCA genotypes indicates that there is considerable phenotypic overlap. However, phenotypic trends were noted in carriers' fundus findings and ERG responses for each genetic subtype. Observations of phenotypic associations with specific disease-causing sequence variations in carriers have potential practical value for molecular screening strategies of patients with LCA.

The visual cycle, the biochemical process by which the light-sensitive isomer of vitamin A is continually recycled, is crucial to vision in a healthy eye. More than 150 years of research into this remarkable biochemical process has given invaluable understanding in debilitating visual diseases that impact thousands of individuals worldwide, many of them children. The visual cycle spans photoreceptor cells in the retina and the underlying retinal pigment epithelium (RPE) and requires a protein called RPE65 for its function. In many ways, RPE65 is the capstone to the cyclical processing of vitamin A in the eye, and the discovery of this retinol isomerase helped fill a critical gap in the understanding of retinoid processing in vision. This chapter will focus on the history of visual cycle research, from the first experiments well over a century ago to the discovery of RPE65. Because of the undeniable importance of RPE65 in the visual cycle, this chapter will also focus on the protein structure and mechanism by which it converts light-insensitive all-trans-vitamin A to light-sensitive 11-cis-vitamin A for continued visual function. Finally, this chapter will briefly discuss RPE65 and its known disease associations in the clinical setting. Thanks to the efforts of researchers for well over a century in studying the visual cycle, the medical community is now poised to make significant gains in the treatment of blindness.

OBJECTIVE

Bisretinoids form in photoreceptor cells and accumulate in retinal pigment epithelium (RPE) as lipofuscin. To examine the role of these fluorophores as mediators of retinal light damage, we studied the propensity for light damage in mutant mice having elevated lipofuscin due to deficiency in the ATP-binding cassette (ABC) transporter Abca4 (Abca4(-/-) mice) and in mice devoid of lipofuscin owing to absence of Rpe65 (Rpe65(rd12)).

METHODS

Abca4(-/-), Rpe65(rd12), and wild-type mice were exposed to 430-nm light to produce a localized lesion in the superior hemisphere of retina. Bisretinoids of RPE lipofuscin were measured by HPLC. In histologic sections, outer nuclear layer (ONL) thickness was measured as an indicator of photoreceptor cell degeneration, and RPE nuclei were counted.

RESULTS

As shown previously, A2E levels were increased in Abca4(-/-) mice. These mice also sustained light damage-associated ONL thinning that was more pronounced than in age-matched wild-type mice; the ONL thinning was also greater in 5-month versus 2-month-old mice. Numbers of RPE nuclei were reduced in light-stressed mice, with the reduction being greater in the Abca4(-/-) than wild-type mice. In Rpe65(rd12) mice bisretinoid compounds of RPE lipofuscin were not detected chromatographically and light damage-associated ONL thinning was not observed.

CONCLUSIONS

Abca4(-/-) mice that accumulate RPE lipofuscin at increased levels were more susceptible to retinal light damage than wild-type mice. This finding, together with results showing that Rpe65(rd12) mice did not accumulate lipofuscin and did not sustain retinal light damage, indicates that the bisretinoids of retinal lipofuscin are contributors to retinal light damage.

Increased exposure to blue or visible light, fluctuations in oxygen tension, and the excessive accumulation of toxic retinoid byproducts places a tremendous amount of stress on the retina. Reduction of visual chromophore biosynthesis may be an effective method to reduce the impact of these stressors and preserve retinal integrity. A class of non-retinoid, small molecule compounds that target key proteins of the visual cycle have been developed. The first candidate in this class of compounds, referred to as visual cycle modulators, is emixustat hydrochloride (emixustat). Here, we describe the effects of emixustat, an inhibitor of the visual cycle isomerase (RPE65), on visual cycle function and preservation of retinal integrity in animal models. Emixustat potently inhibited isomerase activity in vitro (IC50 = 4.4 nM) and was found to reduce the production of visual chromophore (11-cis retinal) in wild-type mice following a single oral dose (ED50 = 0.18 mg/kg). Measure of drug effect on the retina by electroretinography revealed a dose-dependent slowing of rod photoreceptor recovery (ED50 = 0.21 mg/kg) that was consistent with the pattern of visual chromophore reduction. In albino mice, emixustat was shown to be effective in preventing photoreceptor cell death caused by intense light exposure. Pre-treatment with a single dose of emixustat (0.3 mg/kg) provided a ~50% protective effect against light-induced photoreceptor cell loss, while higher doses (1-3 mg/kg) were nearly 100% effective. In Abca4-/- mice, an animal model of excessive lipofuscin and retinoid toxin (A2E) accumulation, chronic (3 month) emixustat treatment markedly reduced lipofuscin autofluorescence and reduced A2E levels by ~60% (ED50 = 0.47 mg/kg). Finally, in the retinopathy of prematurity rodent model, treatment with emixustat during the period of ischemia and reperfusion injury produced a ~30% reduction in retinal neovascularization (ED50 = 0.46mg/kg). These data demonstrate the ability of emixustat to modulate visual cycle activity and reduce pathology associated with various biochemical and environmental stressors in animal models. Other attributes of emixustat, such as oral bioavailability and target specificity make it an attractive candidate for clinical development in the treatment of retinal disease.

Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.

OBJECTIVE

To define the molecular basis of retinal degeneration in consanguineous Pakistani pedigrees with early onset retinal degeneration.

METHODS

A cohort of 277 individuals representing 26 pedigrees from the Punjab province of Pakistan was analyzed. Exomes were captured with commercial kits and sequenced on an Illumina HiSeq 2500. Candidate variants were identified using standard tools and analyzed using exomeSuite to detect all potentially pathogenic changes in genes implicated in retinal degeneration. Segregation analysis was performed by dideoxy sequencing and novel variants were additionally investigated for their presence in ethnicity-matched controls.

RESULTS

We identified a total of nine causal mutations, including six novel variants in RPE65, LCA5, USH2A, CNGB1, FAM161A, CERKL and GUCY2D as the underlying cause of inherited retinal degenerations in 13 of 26 pedigrees. In addition to the causal variants, a total of 200 variants each observed in five or more unrelated pedigrees investigated in this study that were absent from the dbSNP, HapMap, 1000 Genomes, NHLBI ESP6500, and ExAC databases were identified, suggesting that they are common in, and unique to the Pakistani population.

CONCLUSIONS

We identified causal mutations associated with retinal degeneration in nearly half of the pedigrees investigated in this study through next generation whole exome sequencing. All novel variants detected in this study through exome sequencing have been cataloged providing a reference database of variants common in, and unique to the Pakistani population.

Cynops pyrrhogaster (the Japanese common newt) regenerates neural retina from retinal pigmented epithelium (RPE) cells. Otx2 is a transcription factor that is involved in RPE cell differentiation. To understand the role of Otx2 during transdifferentiation of RPE cells, we cloned a Cynops Otx2 cDNA, and explored its expression by RT-PCR, immunohistochemistry and in situ hybridization. The expression of Otx2 was compared with the localization of a proliferating cell marker (PCNA), RPE cell markers (RPE65, CRBP) and an RPE and Muller glial cell marker (CRALBP). At the early stage of regeneration, 2 to 3 cell layered regenerating retina consisting of pigmented cells uniformly expressed Otx2 and other markers. Following this stage, 4-cell layered regenerating retina consisted of two distinct layers, pigmented monolayer (the outer layer) attached to Bruch's membrane and presumptive neural retina (the inner layers). In the outer layer, Otx2 and CRBP expression was maintained and majority of cells lost PCNA expression. Some of cells maintained RPE65. In the inner layers, expression of Otx2, CRBP and RPE65 was downregulated, but a majority of those cells maintained PCNA expression. These results indicate that spatiotemporal regulation of Otx2 expression is consistent with those of RPE markers. Otx2 may play a pivotal role in maintenance and specification of RPE cells during neural retina regeneration. In contrast to RPE cell markers, CRALBP was expressed in both the pigmented and the de-pigmented layers. This observation implicates the appearance of Muller glial cells in an early phase of regenerating retina.

The visual cycle is a sequential enzymatic reaction for vitamin A, all-trans-retinol, occurring in the outer layer of the human retina and is essential for the maintenance of vision. The central source of retinol is derived from dietary intake of both retinol and pro-vitamin A carotenoids. A series of enzymatic reactions, located in both the photoreceptor outer segment and the retinal pigment epithelium, transform retinol into the visual chromophore 11-cis-retinal, regenerating visual pigments. Retina specific proteins carry out the majority of the visual cycle, and any significant interruption in this sequence of reactions is capable of causing varying degrees of blindness. Among these important proteins are Lecithin:retinol acyltransferase (LRAT) and retinal pigment epithelium-specific 65-kDa protein (RPE65) known to be responsible for esterification of retinol to all-trans-retinyl esters and isomerization of these esters to 11-cis-retinal, respectively. Deleterious mutations in these genes are identified in human retinal diseases that cause blindness, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Herein, we discuss the pathology of 11-cis-retinal deficiency caused by these mutations in both animal disease models and human patients. We also review novel therapeutic strategies employing artificial visual chromophore 9-cis-retinoids which have been employed in clinical trials involving LCA patients.