Purpose: To report combined viewpoints on ocular gene therapy from a select group of clinician scientists and a patient advocacy group.

Methods: With the support of Randy Wheelock and Dr. Chris Moen from the Choroideremia Research Foundation (CRF), a special interest group at the 2019 Annual meeting of the Association for Research in Vision and Ophthalmology in Vancouver, Canada, shared their knowledge, experience, concepts, and ideas and provided a forum to discuss therapeutic strategies for the treatment of inherited retinal disorders, using experience in choroideremia (CHM) as a model.

Results: A member of the CRF presented the patient perspective and role in clinical trials. Five clinician scientists presented reasons for limited long-term visual improvement in many gene therapy trials, including challenges with dose, incomplete understanding of photoreceptor metabolism, vector delivery, inflammation, and identification of patients likely to benefit from treatment.

Conclusions: The shared experience of the five clinician scientists indicates that the results of ocular gene therapy for choroideremia have been less successful than for RPE65-related Leber congenital amaurosis. Improvement in vector delivery and developing a better understanding of gene expression in target tissues, treatment dose and side effects, and inflammation, as well as identifying patients who are most likely to benefit without suffering excessive risk, are necessary to advance the development of effective therapies for inherited retinal degenerations.

Translational relevance: Additional long-term data are required to determine if ocular gene therapy will be sufficient to alter natural progression in choroideremia. Combination therapies may have to be considered, as well as alternative vectors that minimize risk.

Keywords: choroideremia; gene therapy; retinal degeneration.

BACKGROUND

Nuclear hormone receptor gene, NR2E3, plays a critical role in retinogenesis and determination of the rod photoreceptor phenotype. Mutations in NR2E3 typically lead to recessive enhanced S-cone syndrome (ESCS), where affected individuals show higher sensitivity to short wavelength light and early onset rod dysfunction. Patients with ESCS present in early childhood with nyctalopia, enhanced sensitivity to blue light and display a very heterogeneic retinal phenotype with varying degrees of clumped pigmentation and occasional retinoschisis.

OBJECTIVE

To confirm the pathogenicity of a novel mutation in NR2E3 using electrophysiological studies.

METHODS

Patient underwent detailed clinical evaluation and ophthalmic imaging followed by next generation sequencing analysis and electrophysiological studies.

RESULTS

We describe a case of a young man of Greek descent with a family history of retinal degeneration. His fundal features at presentation were atypical of ESCS, with striking macular involvement in both eyes, including fibrotic subretinal material overlying the pigment epithelial detachment in one eye and schisis in the other. Genetic testing revealed a novel homozygous variant in NR2E3 gene of uncertain pathogenicity. Instead of performing further genetic analyses, electrophysiological studies showed pathognomonic changes in the S-cone response.

CONCLUSIONS

With the recent clinical endorsement of a gene therapy for RPE65 related-inherited retinal degeneration it is of paramount importance to correctly identify the pathogenic genetic mutation. In this particular syndrome, we highlight the value of electrophysiology to confirm the pathogenicity of a novel mutation in NR2E3 and aid the diagnosis of ESCS, with potential for gene therapy in the future.

Previously it was shown that the circulating rhodopsin mRNA level was higher in diabetic retinopathy (DR). Recent evidence suggests that hypoxia may also be associated with DR. The aim of this study was to investigate the effect of oxygen desaturation on circulating retina-specific mRNA in type 2 diabetic patients. Thirty-five type 2 diabetic patients underwent overnight oximetry. Two parameters from oximetry were used to measure oxygen desaturation: the number of times per hour the oxygen saturation decreased by 4% or greater (number of dips/hr) and percentage of sleep time with oxygen saturation (SpO(2)) <90%. Blood samples were collected into PAXgene Blood RNA tubes. Total RNA was extracted from the samples and reverse-transcribed into cDNA, and retina-specific markers were measured by quantitative real time PCR. In patients with>>/=5 dips/hr, mRNA values for rhodopsin (P= 0.05) and RPE65 (P= 0.044) were significantly higher than in patients with <5 dips/hr. No change was seen in retinoschisin mRNA expression. In patients with preproliferative or proliferative DR, median levels for rhodopsin mRNA and RPE65 mRNA were 30% and 80% higher and retinoschisin mRNA was lower in patients with>>/=5 dips/hr when compared to patients with <5 dips/hr. These results indicate that hypoxia may modulate expression of genes in the retina.

The purpose of this study was to investigate if the enhanced bioaccumulation of lutein in retina and brain of breastfed, compared to formula-fed, infant monkeys was associated with higher levels of serum total and HDL cholesterol, apolipoproteins, or mRNA/protein expression of carotenoid-related genes. Newborn rhesus macaques were either breastfed, fed a carotenoid-supplemented formula, or fed an unsupplemented formula for 6 months (n = 8, 8, 7). Real-time qPCR and western blotting were performed in two brain regions (occipital cortex and cerebellum) and two retina regions (macular and peripheral retina). Breastfed infants had higher serum total cholesterol, HDL cholesterol, apoA-I, and apoB-100 levels than the combined formula-fed groups (P < 0.05). Breast milk or infant formulas did not alter expression of the nine genes (CD36, SCARB1, SCARB2, LDLR, STARD3, GSTP1, BCO1, BCO2, RPE65) examined except for SCARB2 in the retina and brain regions. In conclusion, dietary regimen did not impact the expression of carotenoid-related genes except for SCARB2. However, carotenoid-related genes were differentially expressed across brain and retina regions. Breastfed infants had higher serum total and HDL cholesterol, and apolipoproteins, suggesting that lipoprotein levels might be important for delivering lutein to tissues, especially the macular retina, during infancy.

A great deal of evidence has confirmed that electromagnetic fields (EMFs) can affect the central nervous system. In this study, cultured neonatal human retinal pigment epithelial (hRPE) cells were exposed to pulsed EMF of 1 mT intensity and 50 Hz frequency 8 h daily for 3 days. In addition to cell proliferation and cell death assays, immunocytochemistry for RPE65, PAX6, nestin, and cytokeratin 8/18 proteins were performed. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed for NES, PAX6, RPE65, and ACTA2 gene expression. Exposed hRPE cells did not demonstrate significant change in terms of cytomorphology, cell proliferation, or cell death. Protein expression of PAX6 was decreased in treated cells compared to controls and remained unchanged for RPE65, cytokeratin 8/18, and nestin. Gene expressions of NES, RPE65, and PAX6 were decreased in treated cells as compared to controls. Gene expression of ACTA2 did not significantly change. In conclusion, viability of cultivated neonatal hRPE cells did not change after short exposure to a safe dose of pulsed EMF albeit that both gene and protein expressions of retinal progenitor cell markers were reduced. Whether longer exposure durations that are being constantly produced by widely-used electronic devices may induce significant changes in these cells, needs further investigation. Bioelectromagnetics. 39:585-594, 2018. © 2018 Wiley Periodicals, Inc.

OBJECTIVE

To examine the expression and promoter activation of the retinal pigment epithelium (RPE)-preferentially expressed Rpe65 gene in the commonly available RPE cell lines.

METHODS

Reverse transcription coupled to polymerase chain reaction (RT-PCR) was performed after total RNA extraction from different RPE (ARPE-19, monkey, hTERT-RP1 and D407) and non-RPE (COS-7, HeLa, HepG2 and HS27) cell lines. Promoter activity was assayed by transient transfection of luciferase reporter constructs containing nested deletions of the 5' flanking region of the mouse Rpe65 gene. The involvement of a putative TATA box in the basal promoter expression was studied by site-directed mutagenesis in D407 cells and binding of TATA box-related transcription factors to that region was demonstrated by Electrophoretic Mobility Shift Assays (EMSA).

RESULTS

Expression of the human RPE65 cDNA was observed in all the RPE cell lines tested, and in COS-7 cells (monkey RPE65 cDNA). Transient transfections of the mouse Rpe65 promoter/luciferase transgene containing nested deletions of the Rpe65 5' flanking region showed that fragments containing bases -655 to +48 and -1240 to +48 generated specific promoter activity only in the D407 cell line. A promoter fragment from -49 to +48 directed basal promoter activity in all the cell lines tested. Part of this basal activity was due to a putative TATA box that specifically binds transcription factors contained in a D407 nuclear extract.

CONCLUSIONS

Although transcription of the Rpe65 gene occurs in all the tested cell lines, we find that the D407 cell line is the only one capable of directing specific mouse Rpe65 promoter activity. This limits the study of the transcriptional regulation of the mouse Rpe65 gene in vitro to this particular cell line.

More than 100 different mutations in the RPE65 gene are associated with inherited retinal degeneration. Although some missense mutations have been shown to abolish isomerase activity of RPE65, the molecular bases leading to loss of function and retinal degeneration remain incompletely understood. Here we show that several missense mutations resulted in significant decrease in expression level of RPE65 in the human retinal pigment epithelium cells. The 26S proteasome non-ATPase regulatory subunit 13, a newly identified negative regulator of RPE65, mediated degradation of mutant RPE65s, which were misfolded and formed aggregates in the cells. Many mutations, including L22P, T101I, and L408P, were mapped on nonactive sites of RPE65. Enzyme activities of these mutant RPE65s were significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. 4-phenylbutyrate (PBA) displayed a significant synergistic effect on the low temperature-mediated rescue of the mutant RPE65s. Our results suggest that a low temperature eye mask and PBA, a FDA-approved oral medicine, may provide a promising "protein repair therapy" that can enhance the efficacy of gene therapy for delaying retinal degeneration caused by RPE65 mutations.

Using an in vivo radiolabeling technique, we investigated the movement of retinoid into the retinal pigment epithelium (RPE) of the abcr-/- mouse, which lacks the photoreceptor ABCR protein and is a model for Stargardt disease. Eye tissues and serum obtained from dark-adapted, 5- to 8-month-old abcr-/- and control mice following the intraperitoneal injection of all-trans ((3)H)retinol were analyzed to determine the inferred influx of retinoid from the serum into the RPE. At 4.5 hr post-injection, the inferred all-trans retinol influx in abcr-/- mice, which possess the leucine 450 variant of RPE65 protein, was 0.011 +/- 0.004 nmol (n = 3). This value did not differ significantly from that determined in age-matched controls possessing the methionine 450 variant of RPE65 (0.015 +/- 0.003 nmol; n = 3) or from 3-month-old wildtype mice that possess the leucine 450 RPE65 variant (0.020 +/- 0.007; n = 4). Thus, the absence of ABCR does not significantly compromise the passage of retinoid from the serum into the RPE under dark-adapted conditions.

To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 10(8) viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes.

BACKGROUND

In 2007, clinical trials began for gene-replacement therapy for RPE65-associated Leber congenital amaurosis. To enroll, subjects must have both disease-causing RPE65 alleles identified. Determining which patients have true disease-causing mutations requires a multistep approach.

METHODS

This study is a retrospective case series using the estimate of pathogenic probability (EPP) algorithm and genotyping of family members to establish phase.

RESULTS

Five probands and their families were studied. Patient 1 had genetic testing elsewhere and was reported to have 2 disease-causing AIPL1 mutations. The family received incorrect prenatal counseling based on this result. We found both variations to be benign ethnic polymorphisms (EPP = 0). Case 2 had possible disease-causing mutations in RPE65, RPGRIP1, and CRB1; however, screening of family members revealed that only CRB1 variations were disease causing and the RPE65 change was a polymorphism found in 11% of African Americans. Case 3 had a diagnosis of CRB1-associated Leber congenital amaurosis, but this mutation had an EPP = 0; a true homozygous disease-causing mutation was later found in RDH12. Patient 4 had 3 mutations found in RPE65, but only 2 were disease causing. Patient 5 had a homozygous mutation in RPE65. Only Patients 4 and 5 would be eligible for clinical trials of RPE65 gene replacement.

CONCLUSIONS

To be eligible for participation in current RPE65 gene therapy trials, patients' DNA must contain 2 correctly segregating alleles with an EPP = 2 or 3. Interpretation of DNA variants is complex; genetic misdiagnosis may lead to ineffective treatment in some patients and lack of treatment in others.

In retinal pigment epithelium (RPE), RPE65 catalyzes the isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol in the visual cycle and controls the rhodopsin regeneration rate. However, the mechanisms by which these processes are regulated are still unclear. Fatty Acid Transport Protein 1 (FATP1) is involved in fatty acid uptake and lipid metabolism in a variety of cell types. FATP1 co-localizes with RPE65 in RPE and inhibits its isomerase activity in vitro. Here, we further investigated the role of FATP1 in the visual cycle using transgenic mice that overexpress human FATP1 specifically in the RPE (hFATP1TG mice). The mice displayed no delay in the kinetics of regeneration of the visual chromophore 11-cis-retinal after photobleaching and had no defects in light sensitivity. However, the total retinoid content was higher in the hFATP1TG mice than in wild type mice, and the transgenic mice also displayed an age-related accumulation (up to 40%) of all-trans-retinal and retinyl esters that was not observed in control mice. Consistent with these results, hFATP1TG mice were more susceptible to light-induced photoreceptor degeneration. hFATP1 overexpression also induced an ~3.5-fold increase in retinosome autofluorescence, as measured by two-photon microscopy. Interestingly, hFATP1TG retina contained ~25% more photoreceptor cells and ~35% longer outer segments than wild type mice, revealing a non-cell-autonomous effect of hFATP1 expressed in the RPE. These data are the first to show that FATP1-mediated fatty acid uptake in the RPE controls both retinoid metabolism in the outer retina and photoreceptor development.

Studies of antigen presentation in retina using mice that expressed green fluorescent protein (GFP) from a transgenic CD11c promoter found that retinal GFPhi cells possessed antigen presentation function. Subsequent studies found that these high GFPhi cells preferentially localized to sites of retinal injury, consistent with their APC function. Interest in the roles of macrophages in degenerative CNS diseases led us to study the GFPhi cells in a retinal model of neurodegeneration. We asked if apoptotic cone photoreceptor cell death in Rpe65-/- knockout mice induced the GFPhi cells, explored their relationship to resident microglia (MG), and tested their role in cone survival.

Rpe65-/- mice were bred to CD11cGFP mice on the B6/J background. CD11cGFPRpe65-/- mice were also backcrossed to CX3CR1YFP-creERROSADTA mice so that CX3CR1+ mononuclear cells could be depleted by Tamoxifen. Retinas were analyzed by immunohistochemistry, confocal microscopy, fluorescence fundoscopy and flow cytometry.

Elevated numbers of GFPhi cells were concentrated in photoreceptor cell layers of CD11cGFPRpe65-/- mice coinciding with the peak of cone death at 2 to 4weeks of age, and persisted for at least 14months. After the initial wave of cone loss, a slow progressive loss of cones was found that continued to retain GFPhi cells in the outer retina. Sustained, four-week Tamoxifen depletions of the GFPhi cells and MG in Rpe65-/- mice from day 13 to day 41, and from day 390 to day 420 promoted a small increase in cone survival. We found no evidence that the GFPhi cells were recruited from the circulation; all data pointed to a MG origin. MG and GFPhi cells were well segregated in the dystrophic retina; GFPhi cells were foremost in the photoreceptor cell layer, while MG were concentrated in the inner retina.

The expression of GFP on a subset of retinal mononuclear cells in CD11cGFP mice identified a distinct population of cells performing functions previously attributed to MG. Although GFPhi cells dominated the macrophage response to cone death in the photoreceptor cell layer, their ablation led to only an incremental increase in cone survival. The ability to identify, ablate, and isolate these cells will facilitate analysis of this activated, antigen-presenting subset of MG.

OBJECTIVE

We wish to identify transcriptional factors involved in regulation binding to the proximal promoter region of the RPE65 gene that confers RPE-specific expression.

METHODS

We incubated human D407 RPE cell nuclear extract with double-stranded (sense 5-prime biotinylated) oligonucleotides, based on the RPE65 proximal gene promoter, bound to streptavidin-Dynabeads. Bound nuclear proteins were eluted, separated on SDS-PAGE, and analyzed by mass spectrometry. Peptide sequence was used to identify cDNA clones that were subcloned into pCDNA3.1 for expression and co-transfection into D407 cells to assess transcriptional activation of mouse Rpe65 gene promoter/reporter constructs. SiRNA interference was used to suppress ZNF492 expression.

RESULTS

We identified a D407 nuclear protein binding to biotinylated-DNA/streptavidin beads as the product of clone KIAA1473 encoding a protein named ZNF492. ZNF492 has an open reading frame of 531 amino acids with a truncated N-terminus and lacks the usual Krüppel-associated box-A (KRAB-A) while KRAB-B remains intact and has 12 C2H2 zinc-fingers in tandem arrangement. Co-expression in D407 cells of ZNF492 protein did not activate TR1, a mouse Rpe65 gene promoter/reporter construct with 49-bp 5-prime flanking sequence, but did activate construct TR2, containing 188-bp 5-prime flanking sequence, by 2.5-fold, and the longer constructs TR4, containing 655-bp 5-prime flanking sequence, and TR5, containing 1240-bp 5-prime flanking sequence, by about 2-fold. SiRNA-mediated suppression of ZNF492 in D407 resulted in decreased Rpe65 promoter activity.

CONCLUSIONS

We have identified ZNF492, a KRAB-zinc finger protein, by its interaction with immobilized RPE65 promoter DNA sequence. This KRAB-zinc finger protein serves as a moderate transcriptional factor for Rpe65 gene upregulation. In ZNF492, absence of KRAB-A might reduce or prevent co-repressor binding to account for the modest upregulation of Rpe65 gene expression.

Culturing of human retinal pigment epithelial cells (hRPE) is the initial step in cell therapy of some retinal diseases. To transfer these cells into clinical use, it is necessary to guarantee that they are well differentiated and contamination free. Fluorescence microscopy is the easiest method to do this, but it is associated with operator subjectivity, and the results are highly variable. The aim of this study was to demonstrate the practicality of implementing flow cytometry (FC) analysis to determine the purity of human RPE primary cell cultures. An ARPE19 cell line, human skin fibroblasts, hRPE, and human corneal epithelial cells were analysed by FC to determine the percentage of the hRPE population expressing RPE65 and epithelial and fibroblast proteins. The cell viability and DNA content also were determined. FC analysis showed that the hRPE cells were healthy, stable, and expressed RPE65 protein in the study working conditions. The density of RPE65 protein expression decreased during passages 2 to 10, which was confirmed using a Western blot technique. However, the hRPE cells did not express the 112-kDa epithelial and fibroblast proteins in the current working conditions. These findings suggested that FC facilitates a detailed analysis of human RPE primary cell cultures, a necessary step in developing new cell therapies for retinal diseases.

OBJECTIVE

To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis.

METHODS

We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6.

RESULTS

No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice.

CONCLUSIONS

Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

The aim of the present study was to investigate the changes in retinal gene expression at three time points and assess the underlying molecular mechanisms of diabetic retinopathy (DR) in a streptozotocin (STZ)-induced diabetes rat model using bioinformatics analysis. The gene expression profile of GSE28831 was extracted from the Gene Expression Omnibus database and differentially expressed genes (DEGs) were identified at three different time points (1, 4 and 12 weeks) using the limma package in R language. Gene ontology (GO) enrichment analysis of DEGs was performed followed by a principal component and pathway enrichment analysis of the selected DEGs along with protein-protein interaction network construction at the three time points. A total of 402, 105 and 213 DEGs were screened at 1, 4 and 12 weeks, respectively. In addition, the expression of 8 genes was identified to be significantly different at different time points, including cytochrome P450 2B2 (CYP2B2; downregulated gene; P=0.048; at 1 week), mannan binding lectin-associated serine protease-2 (MASP2; downregulated gene; P=0.044), lecithin retinol acyltransferase (LRAT; downregulated gene; P=0.015), retinal pigment epithelium (RPE)-specific protein 65 kDa (RPE65; downregulated gene; P=0.025), 11-cis-retinoldehydrogenase (RDH5; downregulated gene; P=0.04; at 4 weeks), mitogen-activated protein kinase 13 (MAPK13; upregulated gene; P=0.036), LRAT (downregulated gene; P=0.01) and RPE65 (downregulated gene; P=0.009; at 12 weeks). Furthermore, pathway enrichment and GO enrichment analyses revealed that DEGs at 4 weeks were primarily enriched in retinol metabolism and processes associated with visual functions, including 'visual perception' and 'retinol metabolism'. DEGs, including CYP2B2, MASP2, LRAT, RPE65, RDH5 and MAPK13 may be potential targets for the diagnosis and treatment of DR. Thus, the current study demonstrated that abnormal visual functions occur at 4 weeks in STZ-induced diabetic rats. This may provide a scientific basis for the diagnosis and treatment of DR because DEGs may be used to facilitate the development of novel therapeutic strategies to diagnose and treat DR.

Smoking is one of the most important factors associated with the development of lung cancer. However, the signaling pathways and driver genes in smoking-associated lung adenocarcinoma remain unknown. The present study analyzed 433 samples of smoking-associated lung adenocarcinoma and 75 samples of non-smoking lung adenocarcinoma from the Cancer Genome Atlas database. Gene Ontology (GO) analysis was performed using the Database for Annotation, Visualization and Integrated Discovery and the ggplot2 R/Bioconductor package. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using the R packages RSQLite and org.Hs.eg.db. Multivariate Cox regression analysis was performed to screen factors associated with patient survival. Kaplan-Meier and receiver operating characteristic curves were used to analyze the potential clinical significance of the identified biomarkers as molecular prognostic markers for the five-year overall survival time. A total of 373 differentially expressed genes (DEGs; |log2-fold change|≥2.0 and P<0.01) were identified, of which 71 were downregulated and 302 were upregulated. These DEGs were associated with 28 significant GO functions and 11 significant KEGG pathways (false discovery rate <0.05). Two hundred thirty-eight proteins were associated with the 373 differentially expressed genes, and a protein-protein interaction network was constructed. Multivariate regression analysis revealed that 7 mRNAs, cytochrome P450 family 17 subfamily A member 1, PKHD1 like 1, retinoid isomerohydrolase RPE65, neurotensin receptor 1, fetuin B, insulin-like growth factor binding protein 1 and glucose-6-phosphatase catalytic subunit, significantly distinguished between non-smoking and smoking-associated adenocarcinomas. Kaplan-Meier analysis demonstrated that patients in the 7 mRNAs-high-risk group had a significantly worse prognosis than those of the low-risk group. The data obtained in the current study suggested that these genes may serve as potential novel prognostic biomarkers of smoking-associated lung adenocarcinoma.

Docosahexaenoic acid (DHA, 22 : 6) is an essential omega-3 long-chain polyunsaturated fatty acid that plays a pivotal role in vision. The purpose of this study was to clarify the cellular uptake and binding processes of free and protein-bound unesterified DHA in retinal pigment epithelial cell (RPE) line ARPE-19 as a model of the human outer blood-retinal barrier and isolated porcine RPE cell fractions. Uptake of free [14C]DHA by ARPE-19 cells was saturable with a Michaelis-Menten constant of 283 µM, and was significantly inhibited by eicosapentaenoic acid, arachidonic acid, and linoleic acid, but not by oleic acid. Further, the uptakes of [14C]DHA associated with retinol-binding protein ([14C]DHA-RBP), [14C]DHA associated with low-density lipoprotein ([14C]DHA-LDL) and [14C]DHA associated with bovine serum albumin ([14C]DHA-BSA) in ARPE-19 cells increased time-dependently at 37°C, and were significantly reduced at 4°C, suggesting the involvement of energy-dependent transport processes. [14C]DHA-LDL uptake by ARPE-19 cells was significantly inhibited by excess unlabeled LDL, but not by an inhibitor of scavenger receptor B type I. Fatty acid transport protein (FATP) 2 and 4 mRNAs were expressed in ARPE-19 cells, and [14C]DHA uptake was observed in FATP2- and FATP4-expressing Xenopus oocytes. Photo-reactive crosslinking and mass spectrometry analyses identified 65-kDa retinal pigment epithelium-specific protein (RPE65) as a DHA-binding protein in porcine RPE cell membrane fractions. Thus, RPE cells possess multiple cellular transport/binding processes for unesterified DHA, involving at least partly FATP2, FATP4, LDL, RBP, and RPE65.

Mutations in the RPE65 gene are associated with autosomal recessive early onset severe retinal dystrophy. Morphological and functional studies indicate early and dramatic loss of rod photoreceptors and early loss of S-cone function, while L and M cones remain initially functional. The Swedish Briard dog is a naturally occurring animal model for this disease. Detailed information about rod and cone reaction to RPE65 deficiency in this model with regard to their location within the retina remains limited. The aim of this study was to analyze morphological parameters of cone and rod viability in young adult RPE65 deficient dogs in different parts of the retina in order to shed light on local disparities in this disease. In retinae of affected dogs, sprouting of rod bipolar cell dendrites and horizontal cell processes was dramatically increased in the inferior peripheral part of affected retinae, while central inferior and both superior parts did not display significantly increased sprouting. This observation was correlated with photoreceptor cell layer thickness. Interestingly, while L/M cone opsin expression was uniformly reduced both in the superior and inferior part of the retina, S-cone opsin expression loss was less severe in the inferior part of the retina. In summary, in retinae of young adult RPE65 deficient dogs, the degree of rod bipolar and horizontal cell sprouting as well as of S-cone opsin expression depends on the location. As the human retinal pigment epithelium (RPE) is pigmented similar to the RPE in the inferior part of the canine retina, and the kinetics of photoreceptor degeneration in humans seems to be similar to what has been observed in the inferior peripheral retina in dogs, this area should be studied in future gene therapy experiments in this model.

Retinal dystrophies (RD) are a rare genetic disorder with high genetic heterogeneity. This study aimed at identifying disease-causing variants in fifteen consanguineous Tunisian families. Full ophthalmic examination was performed. Index patients were subjected to IROme analysis or whole exome sequencing followed by homozygosity mapping. All detected variations were confirmed by direct Sanger sequencing. Mutation analysis in our patients revealed two compound heterozygous mutations p.(R91W);(V172D) in RPE65, and five novel homozygous mutations: p.R765C in CNGB1, p.H337R in PDE6B, splice site variant c.1129-2A>> G and c.678_681delGAAG in FAM161A and c.1133 + 3_1133 + 6delAAGT in CERKL. The latter mutation impacts pre-mRNA splicing of CERKL. The other changes detected were six previously reported mutations in CNGB3 (p.R203*), ABCA4 (p.W782*), NR2E3 (p.R311Q), RPE65 (p.H182Y), PROM1 (c.1354dupT) and EYS (c.5928-2A>> G). Segregation analysis in each family showed that all affected individuals were homozygotes and unaffected individuals were either heterozygote carriers or homozygous wild type allele. These results confirm the involvement of a large number of genes in RD in the Tunisian population.

Over the last decade, significant progress has been made in the development of gene therapy strategies for the treatment of neovascular disorders and inherited dystrophies of the retina. Of all tested viral vectors, recombinant adeno-associated virus (rAAV) vectors, have been shown to be optimal vectors for gene transfer to the retina. Broadly speaking, two gene therapy strategies are used to treat retinal diseases; the first being corrective expression in the retina of the mutated gene (i. e., specific gene therapy) and the second being therapeutic expression of, for example, neurotrophic or antiangiogenic factors, in cases of neurodegenerative or neovascular, respectively, disorders (non-specific gene therapy). The naturally occurring RPE65 (-/-) Briard dog model has been successfully treated by specific gene transfer protocols and, based on these studies, the first clinical phase I trials are in preparation or have already begun. To avoid potential negative side effects due to the expression of neurotrophic and/or antiangiogenic factors in the retina, the expression of these transgenes needs to be regulated into a therapeutic window. Several regulatory systems have been tested in the retina of large animal models and may soon be used in clinical applications.

The inherited retinal dystrophies comprise a large number of disorders characterized by a slow and progressive retinal degeneration. They are the result of mutations in genes that express in either the photoreceptor cells or the retinal pigment epithelium. The mode of inheritance can be autosomal dominant, autosomal recessive, X linked recessive, digenic or mitochondrial DNA inherited. At the moment, there is no treatment for these conditions and the patients can expect a progressive loss of vision. Accurate genetic counseling and support for rehabilitation are indicated. Research into the molecular and genetic basis of disease is continually expanding and improving the prospects for rational treatments. In this way, gene therapy, defined as the introduction of exogenous genetic material into human cells for therapeutic purposes, may ultimately offer the greatest treatment for the inherited retinal dystrophies. The eye is an attractive target for gene therapy because of its accessibility, immune privilege and translucent media. A number of retinal diseases affecting the eye have known gene defects. Besides, there is a well characterized animal model for many of these conditions. Proposals for clinical trials of gene therapy for inherited retinal degenerations owing to defects in the gene RPE65, have recently received ethical approval and the obtained preliminary results brought large prospects in the improvement on patient's quality of life.

Rapid progress in knowledge of the organization of the dog genome has facilitated the identification of the mutations responsible for numerous monogenic diseases, which usually present a breed-specific distribution. The majority of these diseases have clinical and molecular counterparts in humans. The affected dogs have thus become valuable models for preclinical studies of gene therapy for problems such as eye diseases, immunodeficiency, lysosomal storage diseases, hemophilia, and muscular dystrophy. Successful gene therapies in dogs have significantly contributed to decisions to run clinical trials for several human diseases, such as Leber's congenital amaurosis 2-LCA2 (caused by a mutation of RPE65), X-linked retinitis pigmentosa-XLRP (caused by mutation RPGR), and achromatopsia (caused by mutation of CNGB3). Promising results were also obtained for canine as follows: hemophilia (A and B), mucopolysaccharidoses (MPS I, MPS IIIB, MPS VII), leukocyte adhesion deficiency (CLAD), and muscular dystrophy (a counterpart of human Duchenne dystrophy). Present knowledge on molecular background of canine monogenic diseases and their successful gene therapies prove that dogs have an important contribution to preclinical studies.