Stargardt Macular Degeneration Research Articles

Structure and function of ABCA4 and its role in the visual cycle and Stargardt macular degeneration.

ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) transporters that is preferentially localized along the rim region of rod and cone photoreceptor outer segment disc membranes. It uses the energy from ATP binding and hydrolysis to transport N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff base adduct of retinal and phosphatidylethanolamine, from the lumen to the cytoplasmic leaflet of disc membranes. This ensures that all-trans-retinal and excess 11-cis-retinal are efficiently cleared from photoreceptor cells thereby preventing the accumulation of toxic retinoid compounds. Loss-of-function mutations in the gene encoding ABCA4 cause autosomal recessive Stargardt macular degeneration, also known as Stargardt disease (STGD1), and related autosomal recessive retinopathies characterized by impaired central vision and an accumulation of lipofuscin and bis-retinoid compounds. High resolution structures of ABCA4 in its substrate and nucleotide free state and containing bound N-Ret-PE or ATP have been determined by cryo-electron microscopy providing insight into the molecular architecture of ABCA4 and mechanisms underlying substrate recognition and conformational changes induced by ATP binding. The expression and functional characterization of a large number of disease-causing missense ABCA4 variants have been determined. These studies have shed light into the molecular mechanisms underlying Stargardt disease and a classification that reliably predicts the effect of a specific missense mutation on the severity of the disease. They also provide a framework for developing rational therapeutic treatments for ABCA4-associated diseases.

Artificial intelligence for assessment of Stargardt macular atrophy.

Stargardt disease (also known as juvenile macular degeneration or Stargardt macular degeneration) is an inherited disorder of the retina, which can occur in the eyes of children and young adults. It is the most prevalent form of juvenile-onset macular dystrophy, causing progressive (and often severe) vision loss. Images with Stargardt disease are characterized by the appearance of flecks in early and intermediate stages, and the appearance of atrophy, due to cells wasting away and dying, in the advanced stage. The primary measure of late-stage Stargardt disease is the appearance of atrophy. Fundus autofluorescence is a widely available two-dimensional imaging technique, which can aid in the diagnosis of the disease. Spectral-domain optical coherence tomography, in contrast, provides three-dimensional visualization of the retinal microstructure, thereby allowing the status of the individual retinal layers. Stargardt disease may cause various levels of disruption to the photoreceptor segments as well as other outer retinal layers. In recent years, there has been an exponential growth in the number of applications utilizing artificial intelligence for help with processing such diseases, heavily fueled by the amazing successes in image recognition using deep learning. This review regarding artificial intelligence deep learning approaches for the Stargardt atrophy screening and segmentation on fundus autofluorescence images is first provided, followed by a review of the automated retinal layer segmentation with atrophic-appearing lesions and fleck features using artificial intelligence deep learning construct. The paper concludes with a perspective about using artificial intelligence to potentially find early risk factors or biomarkers that can aid in the prediction of Stargardt disease progression.

Mitochondria-dependent phase separation of disease-relevant proteins drives pathological features of age-related macular degeneration.

Age-related macular degeneration (AMD) damages the retinal pigment epithelium (RPE), the tissue that safeguards photoreceptor health, leading to irreversible vision loss. Polymorphisms in cholesterol and complement genes are implicated in AMD, yet mechanisms linking risk variants to RPE injury remain unclear. We sought to determine how allelic variants in the apolipoprotein E cholesterol transporter modulate RPE homeostasis and function. Using live-cell imaging, we show that inefficient cholesterol transport by the AMD risk-associated ApoE2 increases RPE ceramide, leading to autophagic defects and complement-mediated mitochondrial damage. Mitochondrial injury drives redox state–sensitive cysteine-mediated phase separation of ApoE2, forming biomolecular condensates that could nucleate drusen. The protective ApoE4 isoform lacks these cysteines and is resistant to phase separation and condensate formation. In Abca–/– Stargardt macular degeneration mice, mitochondrial dysfunction induces liquid-liquid phase separation of p62/SQSTM1, a multifunctional protein that regulates autophagy. Drugs that decrease RPE cholesterol or ceramide prevent mitochondrial injury and phase separation in vitro and in vivo. In AMD donor RPE, mitochondrial fragmentation correlates with ApoE and p62 condensates. Our studies demonstrate that major AMD genetic and biological risk pathways converge upon RPE mitochondria, and identify mitochondrial stress-mediated protein phase separation as an important pathogenic mechanism and promising therapeutic target in AMD.

Functional Characterization of ABCA4 Missense Variants Linked to Stargardt Macular Degeneration

ABCA4 is an ATP-binding cassette (ABC) transporter expressed in photoreceptors, where it transports its substrate, N-retinylidene-phosphatidylethanolamine (N-Ret-PE), across outer segment membranes to facilitate the clearance of retinal from photoreceptors. Mutations in ABCA4 cause Stargardt macular degeneration (STGD1), an autosomal recessive disorder characterized by a loss of central vision and the accumulation of bisretinoid compounds. The purpose of this study was to determine the molecular properties of ABCA4 variants harboring disease-causing missense mutations in the transmembrane domains. Thirty-eight variants expressed in culture cells were analyzed for expression, ATPase activities, and substrate binding. On the basis of these properties, the variants were divided into three classes: Class 1 (severe variants) exhibited significantly reduced ABCA4 expression and basal ATPase activity that was not stimulated by its substrate N-Ret-PE; Class 2 (moderate variants) showed a partial reduction in expression and basal ATPase activity that was modestly stimulated by N-Ret-PE; and Class 3 (mild variants) displayed expression and functional properties comparable to normal ABCA4. The p.R653C variant displayed normal expression and basal ATPase activity, but lacked substrate binding and ATPase activation, suggesting that arginine 653 contributes to N-Ret-PE binding. Our classification provides a basis for better understanding genotype–phenotype correlations and evaluating therapeutic treatments for STGD1.

Cell-Type-Specific Complement Profiling in the ABCA4-/- Mouse Model of Stargardt Disease.

Stargardt macular degeneration is an inherited retinal disease caused by mutations in the ATP-binding cassette subfamily A member 4 (ABCA4) gene. Here, we characterized the complement expression profile in ABCA4−/− retinae and aligned these findings with morphological markers of retinal degeneration. We found an enhanced retinal pigment epithelium (RPE) autofluorescence, cell loss in the inner retina of ABCA4−/− mice and demonstrated age-related differences in complement expression in various retinal cell types irrespective of the genotype. However, 24-week-old ABCA4−/− mice expressed more c3 in the RPE and fewer cfi transcripts in the microglia compared to controls. At the protein level, the decrease of complement inhibitors (complement factor I, CFI) in retinae, as well as an increased C3b/C3 ratio in the RPE/choroid and retinae of ABCA4−/−, mice was confirmed. We showed a corresponding increase of the C3d/C3 ratio in the serum of ABCA4−/− mice, while no changes were observed for CFI. Our findings suggest an overactive complement cascade in the ABCA4−/− retinae that possibly contributes to pathological alterations, including microglial activation and neurodegeneration. Overall, this underpins the importance of well-balanced complement homeostasis to maintain retinal integrity.

Evidence of complement dysregulation in outer retina of Stargardt disease donor eyes

Stargardt macular degeneration (STGD) is a central blinding disease caused by loss of or dysfunctional ABCA4 transporter in both photoreceptors and retinal pigment epithelial (RPE) cells. Toxic bisretinoid-lipofuscin buildup in the RPE cells is a pathological hallmark of STGD patients and its mouse model, the Abca4−/−. These vitamin A-derived fluorophores have been shown to induce oxidative stress, stimulate complement activity, and cause chronic inflammation of the RPE. In vivo modulation of complement regulatory pathway in the STGD mouse model has partially rescued the STGD phenotype suggesting that complement attack on the RPE is an important etiologic factor in disease pathogenesis. While bisretinoid-dependent complement activation was further evidenced in cultured RPE cells, this pathway has never been investigated directly in the context of RPE from STGD donor eyes. In the current study, we evaluate the complement reactivity in postmortem donor eyes of clinically diagnosed STGD patients. All three STGD donor eyes RPE displayed strong immunoreactivity for an antibody specific to 4-Hydroxynonenal, a lipid peroxidation byproduct. Also, unlike the control eyes, all three STGD donor eyes showed significantly increased membrane attack complex deposition on the RPE cells. In STGD eyes, increased MAC accumulation was mirrored by elevated C3 fragments internalized by the RPE and inversely correlated with the levels of complement factor H, a major complement regulatory protein. Here, we report the first direct evidence of RPE complement dysregulation as a causative factor in developing Stargardt phenotype.

Functional analysis and classification of homozygous and hypomorphic ABCA4 variants associated with Stargardt macular degeneration.

Stargardt macular degeneration (Stargardt disease 1 [STGD1]) is caused by mutations in the gene encoding ABCA4, an ATP-binding cassette protein that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across photoreceptor membranes. Reduced ABCA4 activity results in retinoid accumulation leading to photoreceptor degeneration. The disease onset and severity vary from severe loss in visual acuity in the first decade to mild visual impairment late in life. We determined the effect of 22 disease-causing missense mutations on the expression and ATPase activity of ABCA4 in the absence and presence of N-Ret-PE. Three classes were identified that correlated with the disease onset in homozygous STGD1 individuals: Class 1 exhibited reduced ABCA4 expression and ATPase activity that was not stimulated by N-Ret-PE; individuals homozygous for these variants had an early disease onset (≤13years); Class 2 showed reduced ATPase activity with limited stimulation by N-Ret-PE; these correlated with moderate disease onset (14-40years); and Class 3 displayed high expression and ATPase activity that was strongly activated by N-Ret-PE; these were associated with late disease onset (>40years). On the basis of our results, we introduce a functionality index for gauging the effect of missense mutations on STGD1 severity. Our studies support the mild phenotype exhibited by the p.Gly863Ala, p.Asn1868Ile, and p.Gly863Ala/p.Asn1868Ile variants.

The CFTR Corrector, VX-809 (Lumacaftor), Rescues ABCA4 Trafficking Mutants: a Potential Treatment for Stargardt Disease.

Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.

Recombinant Manganese Peroxidase Reduces A2E Burden in Age-Related and Stargardt's Macular Degeneration Models.

Macular degeneration is hallmarked by retinal accumulation of toxic retinoid species (e.g., A2E) for which there is no endogenous mechanism to eliminate it. This ultimately results in progressive dysfunction and loss of vision either in advanced age for genetically normal patients (age-related macular degeneration) or in adolescence for those with inherited genetic mutations (Stargardt's disease). In this article, we present a proof-of-concept study for an enzyme-based therapy to remove these retinoids, modeled on traditional enzyme replacement therapy. Recombinant manganese peroxidase (rMnP) is produced in Pichia pastoris. In vitro, we demonstrate that rMnP breaks down A2E and other lipofuscin fluorophores with limited cellular toxicity, and as this enzyme is mannosylated, it can be taken up into cells through mannose receptor-dependent endocytosis. In vivo, we demonstrate that rMnP can significantly reduce the A2E burden when administered by intravitreal injections. Together, these data provide encouraging results toward the development of an enzyme-based therapy for macular degeneration and indicate the need for additional work to characterize the molecular mechanism of A2E breakdown and to improve the pharmacological parameters of the enzyme.

Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4-/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk-/- but not Abca4-/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4-/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4-/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.

ATP-binding cassette subfamily A, member 4 intronic variants c.4773+3A>G and c.5461-10T>C cause Stargardt disease due to defective splicing.

Inherited retinal dystrophies (IRDs) represent a group of progressive conditions affecting the retina. There is a great genetic heterogeneity causing IRDs, and to date, more than 260 genes are associated with IRDs. Stargardt disease, type 1 (STGD1) or macular degeneration with flecks, STGD1 represents a disease with early onset, central visual impairment, frequent appearance of yellowish flecks and mutations in the ATP-binding cassette subfamily A, member 4 (ABCA4) gene. A large number of intronic sequence variants in ABCA4 have been considered pathogenic although their functional effect was seldom demonstrated. In this study, we aimed to reveal how intronic variants present in patients with Stargardt from the same Swedish family affect splicing. The splicing of the ABCA4 gene was studied in human embryonic kidney cells, HEK293T, and in human retinal pigment epithelium cells, ARPE-19, using a minigene system containing variants c.4773+3A>G and c.5461-10T>C. We showed that both ABCA4 variants, c.4773+3A>G and c.5461-10T>C, cause aberrant splicing of the ABCA4 minigene resulting in exon skipping. We also demonstrated that splicing of ABCA4 has different outcomes depending on transfected cell type. Two intronic variants c.4773+3A>G and c.5461-10T>C, both predicted to affect splicing, are indeed disease-causing mutations due to skipping of exons 33, 34, 39 and 40 of ABCA4 gene. The experimental proof that ABCA4 mutations in STGD patients affect protein function is crucial for their inclusiontofuture clinical trials; therefore, functional testing of all ABCA4 intronic variants associated with Stargardt disease by minigene technology is desirable.

Localization and functional characterization of the p.Asn965Ser (N965S) ABCA4 variant in mice reveal pathogenic mechanisms underlying Stargardt macular degeneration.

ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) proteins that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across outer segment disc membranes thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptor cells. Mutations in the gene encoding ABCA4 are responsible for Stargardt disease (STGD1), an autosomal recessive retinal degenerative disease that causes severe vision loss. To define the molecular basis for STGD1 associated with the p.Asn965Ser (N965S) mutation in the Walker A motif of nucleotide binding domain 1 (NBD1), we generated a p.Asn965Ser knockin mouse and compared the subcellular localization and molecular properties of the disease variant with wild-type (WT) ABCA4. Here, we show that the p.Asn965Ser ABCA4 variant expresses at half the level of WT ABCA4, partially mislocalizes to the endoplasmic reticulum (ER) of photoreceptors, is devoid of N-Ret-PE activated ATPase activity, and causes an increase in autofluorescence and the bisretinoid A2E associated with lipofuscin deposits in retinal pigment epithelial cells as found in Stargardt patients and Abca4 knockout mice. We also show for the first time that a significant fraction of WT ABCA4 is retained in the inner segment of photoreceptors. On the basis of these studies we conclude that loss in substrate-dependent ATPase activity and protein misfolding are mechanisms underlying STGD1 associated with the p.Asn965Ser mutation in ABCA4. Functional and molecular modeling studies further suggest that similar pathogenic mechanisms are responsible for Tangiers disease associated with the p.Asn935Ser (N935S) mutation in the NBD1 Walker A motif of ABCA1.

Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease

Recessive Stargardt macular degeneration (STGD1) is caused by mutations in the gene for the ABCA4 transporter in photoreceptor outer segments. STGD1 patients and Abca4-/- (STGD1) mice exhibit buildup of bisretinoid-containing lipofuscin pigments in the retinal pigment epithelium (RPE), increased oxidative stress, augmented complement activation and slow degeneration of photoreceptors. A reduction in complement negative regulatory proteins (CRPs), possibly owing to bisretinoid accumulation, may be responsible for the increased complement activation seen on the RPE of STGD1 mice. CRPs prevent attack on host cells by the complement system, and complement receptor 1-like protein y (CRRY) is an important CRP in mice. Here we attempted to rescue the phenotype in STGD1 mice by increasing expression of CRRY in the RPE using a gene therapy approach. We injected recombinant adeno-associated virus containing the CRRY coding sequence (AAV-CRRY) into the subretinal space of 4-wk-old Abca4-/- mice. This resulted in sustained, several-fold increased expression of CRRY in the RPE, which significantly reduced the complement factors C3/C3b in the RPE. Unexpectedly, AAV-CRRY-treated STGD1 mice also showed reduced accumulation of bisretinoids compared with sham-injected STGD1 control mice. Furthermore, we observed slower photoreceptor degeneration and increased visual chromophore in 1-y-old AAV-CRRY-treated STGD1 mice. Rescue of the STGD1 phenotype by AAV-CRRY gene therapy suggests that complement attack on the RPE is an important etiologic factor in STGD1. Modulation of the complement system by locally increasing CRP expression using targeted gene therapy represents a potential treatment strategy for STGD1 and other retinopathies associated with complement dysregulation.

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells

Neutrophil myeloperoxidase (MPO) catalyzes the H2O2-dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N-retinylidene-N-retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N-retinylidene-N-retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death.

Insights into the Molecular Properties of ABCA4 and Its Role in the Visual Cycle and Stargardt Disease.

ABCA4 is a member of the A-subfamily of ATP-binding cassette (ABC) transporters localized in rod and cone outer segment disc membranes. Over 800 mutations in ABCA4 are now known to cause Stargardt macular degeneration and related retinal degenerative diseases. Biochemical studies have shown that ABCA4 transports or flips the 11-cis and all-trans isomers of N-retinylidene-phosphatidylethanolamine across disc membranes, thereby facilitating the removal of retinal from disc membranes through the visual cycle and preventing the accumulation of potentially toxic bisretinoid compounds in photoreceptor and retinal pigment epithelial cells. This chapter summarizes studies which have led to our understanding of the role of ABCA4 in the visual cycle and Stargardt disease.

ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11- cis -retinal

The visual cycle is a series of enzyme-catalyzed reactions which converts all-trans-retinal to 11-cis-retinal for the regeneration of visual pigments in rod and cone photoreceptor cells. Although essential for vision, 11-cis-retinal like all-trans-retinal is highly toxic due to its highly reactive aldehyde group and has to be detoxified by either reduction to retinol or sequestration within retinal-binding proteins. Previous studies have focused on the role of the ATP-binding cassette transporter ABCA4 associated with Stargardt macular degeneration and retinol dehydrogenases (RDH) in the clearance of all-trans-retinal from photoreceptors following photoexcitation. How rod and cone cells prevent the accumulation of 11-cis-retinal in photoreceptor disk membranes in excess of what is required for visual pigment regeneration is not known. Here we show that ABCA4 can transport N-11-cis-retinylidene-phosphatidylethanolamine (PE), the Schiff-base conjugate of 11-cis-retinal and PE, from the lumen to the cytoplasmic leaflet of disk membranes. This transport function together with chemical isomerization to its all-trans isomer and reduction to all-trans-retinol by RDH can prevent the accumulation of excess 11-cis-retinal and its Schiff-base conjugate and the formation of toxic bisretinoid compounds as found in ABCA4-deficient mice and individuals with Stargardt macular degeneration. This segment of the visual cycle in which excess 11-cis-retinal is converted to all-trans-retinol provides a rationale for the unusually high content of PE and its long-chain unsaturated docosahexaenoyl group in photoreceptor membranes and adds insight into the molecular mechanisms responsible for Stargardt macular degeneration.

Visual exploration of objects and scenes in people with Stargardt disease and macular degeneration

Visual exploration of objects and scenes in people with Stargardt disease and macular degeneration

Evaluation of the ELOVL4, PRPH2 and ABCA4 genes in patients with Stargardt macular degeneration

Mutations in the ATP-binding cassette, subfamilyA, member 4 (ABCA4), elongation of very long chain fatty acids4 (ELOVL4) and peripherin-2 (PRPH2) genes have been identified in patients with Stargardt macular degeneration (STGD). The aim of this study was to investigate which of these genes is responsible for susceptibility in Chinese patients. A total of 41probands with STGD or suspected STGD were enrolled in the study. The coding regions and adjacent intronic sequences of the ELOVL4 and PRPH2 genes and 3 coding exons of the ABCA4 gene were amplified by polymerase chain reaction (PCR). The nucleotide sequences of the amplicons were determined by Sanger sequencing. Three novel heterozygous missense mutations in the ABCA4 gene were identified: c:2633C>A (p:Ser878X), c:5646G>A (p:Met1882Ile) and c:6389T>A (p:Met2130Lys). These mutations were not present in 176 normal individuals and were predicted to be pathogenic. Two benign variations were found: a reported variation, c:5682G>C in ABCA4 and a novel variation, c:699G>A in ELOVL4. In addition, 5single nucleotide polymorphisms (SNPs: rs3812153, rs7764439, rs390659, rs434102 and c:929G>A) were detected in ELOVL4 and PRPH2. The c:929G>A variation has not been previously reported. We conclude that no pathogenic variations in ELOVL4 and PRPH2 were detected in the Chinese STGD patients. Our results imply that ABCA4 is more likely to be significant in Chinese STGD patients.

Gene therapy in rare diseases

AbstractPurpose Stargardt disease is caused by a mutation of the ABCR gene leading to photoreceptor degeneration and vision loss. Genetic replacement StarGen™ uses LentiVector® technology to deliver a corrected version of the ABCR gene. A single administration of the product directly to the retina could provide long‐term or potentially permanent correction. The primary objective of StarGen™ study is to assess the safety and tolerability of ascending doses of StarGen™ in adult patients with Stargardt macular degeneration. Up to 28 patients 18 years or older with a differing level of advancement of SMD will be included in this study in which a dose‐escalation phase of StarGen™ is followed by a dose confirmation phase. In Retinitis Pigmentosa, preservation or partial restoration of impaired cone function offers very promising perspectives based on neuroprotection, prosthetics or optogenetics. We demonstrated that cone cell function loss might result from the loss of expression of Rod‐derived Cone Viability Factor (RdCVF) consecutive to degeneration of rod photoreceptors directly affected by causative mutations. Administration of RdCVF, irrespective of the gene defect, induced in relevant animal models a strong preservation of cone cell function related to the maintenance of rod outer segments (1). Recently, work conducted by Botond Roska and our group showed that in advanced cases, cone cell bodies of dormant cones can be reactivated by vectorization of halorhodopsins (2). Over the past years we have followed a cohort of over 3000 patients and studied the morphology and function of cone photoreceptors, using novel high resolution imaging.. We showed that cone outer segment degeneration during the disease course can be documented and that patients suitable for clinical trials testing neuroprotection or optogenetics can be selected on this basis. A comprehensive personalized set of therapeutic strategies can be tailored on this basis. Such advances, unprecedented in the field of currently untreatable retinal diseases, offer hope for visual rehabilitation. To evaluate and document such functional outcomes, we have developed novel tools for assessing reproducibly visual impairment and restoration, as well as palliative aids and associated training protocols.Conclusion StarGen™: A Phase I/IIa Dose Escalation Safety Study of Subretinally Injected StarGen™ Administered to Patients With Stargardt Macular Degeneration Sponsored by Oxford BioMedica UK Ltd. Principal Investigator Prof. Jose‐Alain Sahel Centre Hospitalier National d’Ophtalmologie des Quinze‐Vingts, Paris France – Professor David Wilson ‐Casey Eye Institute, Portland Oregon Léveillard T, Sahel JA. Rod‐derived cone viability factor for treating blinding diseases: from clinic to redox signaling. Sci Transl Med. 2010 Apr 7;2(26):26ps16. Busskamp V et al. Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa. Science. 2010 Jul 23;329(5990):413‐7.

ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer

ATP-binding cassette (ABC) transporters comprise a superfamily of proteins, which actively transport a variety of compounds across cell membranes. Mammalian and most eukaryotic ABC transporters function as exporters, flipping or extruding substrates from the cytoplasmic to the extracellular or lumen side of cell membranes. Prokaryotic ABC transporters function either as exporters or importers. Here we show that ABCA4, an ABC transporter found in retinal photoreceptor cells and associated with Stargardt macular degeneration, is a novel importer that actively flips N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic leaflet of disc membranes, thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptors. ABCA4 also actively transports phosphatidylethanolamine in the same direction. Mutations known to cause Stargardt disease decrease N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine transport activity of ABCA4. These studies provide the first direct evidence for a mammalian ABC transporter that functions as an importer and provide insight into mechanisms underlying substrate transport and the molecular basis of Stargardt disease.