Search

Mutations in thePRPH2gene encoding the photoreceptor-specific protein PRPH2 (also known as peripherin-2 or rds) cause a broad range of autosomal dominant retinal diseases. Most of these mutations affect the structure of the light-sensitive photoreceptor outer segment, which is composed of a stack of flattened “disc” membranes surrounded by the plasma membrane. The outer segment is renewed on a daily basis in a process whereby new discs are added at the outer segment base and old discs are shed at the outer segment tip. New discs are formed as serial membrane evaginations, which eventually enclose through a complex process of membrane remodeling (completely in rods and partially in cones). As disc enclosure proceeds, PRPH2 localizes to the rims of enclosed discs where it forms oligomers which fortify the highly curved membrane structure of these rims. In this study, we analyzed the outer segment phenotypes of mice of both sexes bearing a single copy of either the C150S or the Y141C PRPH2 mutation known to prevent or increase the degree of PRPH2 oligomerization, respectively. Strikingly, both mutations increased the number of newly forming, not-yet-enclosed discs, indicating that the precision of disc enclosure is regulated by PRPH2 oligomerization. Without tightly controlled enclosure, discs occasionally over-elongate and form large membranous “whorls” instead of disc stacks. These data show that the defects in outer segment structure arising from abnormal PRPH2 oligomerization are manifested at the stage of disc enclosure.SIGNIFICANCE STATEMENTThe light-sensitive photoreceptor outer segment contains a stack of flattened “disc” membranes that are surrounded, or “enclosed,” by the outer segment membrane. Disc enclosure is an adaptation increasing photoreceptor light sensitivity by facilitating the diffusion of the second messenger along the outer segment axes. However, the molecular mechanisms by which photoreceptor discs enclose within the outer segment membrane remain poorly understood. We now demonstrate that oligomers of the photoreceptor-specific protein peripherin-2, or PRPH2, play an active role in this process. We further propose that defects in disc enclosure because of abnormal PRPH2 oligomerization result in major structural abnormalities of the outer segment, ultimately leading to loss of visual function and cell degeneration in PRPH2 mutant models and human patients.

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3(f/f(iCre75)) and Stx3(f/f(CRX-Cre))] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.

Ariboflavinosis is a pathological condition occurring as a result of riboflavin deficiency. This condition is treatable if detected early enough, but it lacks timely diagnosis. Critical symptoms of ariboflavinosis include neurological and visual manifestations, yet the effects of flavin deficiency on the retina are not well investigated. Here, using a diet induced mouse model of riboflavin deficiency, we provide the first evidence of how retinal function and metabolism are closely intertwined with riboflavin homeostasis. We find that diet induced riboflavin deficiency causes severe decreases in retinal function accompanied by structural changes in the neural retina and retinal pigment epithelium (RPE). This is preceded by increased signs of cellular oxidative stress and metabolic disorder, in particular dysregulation in lipid metabolism, which is essential for both photoreceptors and the RPE. Though many of these deleterious phenotypes can be ameliorated by riboflavin supplementation, our data suggests that some patients may continue to suffer from multiple pathologies at later ages. These studies provide an essential cellular and mechanistic foundation linking defects in cellular flavin levels with the manifestation of functional deficiencies in the visual system and paves the way for a more in-depth understanding of the cellular consequences of ariboflavinosis.

We previously reported a model of progressive retinal degeneration resulting from the knockout of the retina-specific riboflavin binding protein, retbindin (Rtbdn(−/−)). We also demonstrated a reduction in neural retinal flavins as a result of the elimination of RTBDN. Given the role of flavins in metabolism, herein we investigated the underlying mechanism of this retinal degeneration by performing metabolomic analyses on predegeneration at postnatal day (P) 45 and at the onset of functional degeneration in the P120 retinas. Metabolomics of hydrophilic metabolites revealed that individual glycolytic products accumulated in the P45 Rtbdn(−/−) neural retinas along with the elevation of pentose phosphate pathway, while TCA cycle intermediates remained unchanged. This was confirmed by using (13)C-labeled flux measurements and immunoblotting, revealing that the key regulatory step of phosphoenolpyruvate to pyruvate was inhibited via down-regulation of the tetrameric pyruvate kinase M2 (PKM2). Separate metabolite assessments revealed that almost all intermediates of acylcarnitine fatty acid oxidation, ceramides, sphingomyelins, and multiple toxic metabolites were significantly elevated in the predegeneration Rtbdn(−/−) neural retina. Our data show that lack of RTBDN, and hence reduction in flavins, forced the neural retina into repurposing glucose for free-radical mitigation over ATP production. However, such sustained metabolic reprogramming resulted in an eventual metabolic collapse leading to neurodegeneration.

The large number of inherited retinal disease genes (IRD), including the photopigment rhodopsin and the photoreceptor outer segment (OS) structural component peripherin 2 (PRPH2), has prompted interest in identifying common cellular mechanisms involved in degeneration. Although metabolic dysregulation has been shown to play an important role in the progression of the disease etiology, identifying a common regulator that can preserve the metabolic ecosystem is needed for future development of neuroprotective treatments. Here, we investigated whether retbindin (RTBDN), a rod-specific protein with riboflavin binding capability, and a regulator of riboflavin-derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is protective to the retina in different IRD models, one carrying the P23H mutation in rhodopsin (which causes retinitis pigmentosa) and one carrying the Y141C mutation in Prph2 (which causes a blended cone-rod dystrophy). RTBDN levels are significantly upregulated in both the rhodopsin (Rho)P23H/+ and Prph2Y141C/+ retinas. Rod and cone structural and functional degeneration worsened in models lacking RTBDN. In addition, removing Rtbdn worsened other phenotypes, such as fundus flecking. Retinal flavin levels were reduced in RhoP23H/+/Rtbdn&minus, /&minus, and Prph2Y141C/+/Rtbdn&minus, retinas. Overall, these findings suggest that RTBDN may play a protective role during retinal degenerations that occur at varying rates and due to varying disease mechanisms.

Peripherin 2 (PRPH2) is a retina-specific tetraspanin protein essential for the formation of rod and cone photoreceptor outer segments (OS). Patients with mutations in PRPH2 exhibit severe retinal degeneration characterized by vast inter- and intra-familial phenotypic heterogeneity. To help understand contributors to this within-mutation disease variability, we asked whether the PRPH2 binding partner rod OS membrane protein 1 (ROM1) could serve as a phenotypic modifier. We utilized knockin and transgenic mouse models to evaluate the structural, functional and biochemical effects of eliminating one allele of Rom1 (Rom1+/−) in three different Prph2 models which mimic human disease: C213Y Prph2 (Prph2C/+), K153Del Prph2 (Prph2K/+) and R172W (Prph2R172W). Reducing Rom1 in the absence of Prph2 mutations (Rom1+/−) had no effect on retinal structure or function. However, the effects of reducing Rom1 in the presence of Prph2 mutations were highly variable. Prph2K/+/Rom1+/− mice had improved rod and cone function compared with Prph2K/+ as well as amelioration of K153Del-associated defects in PRPH2/ROM1 oligomerization. In contrast, Prph2R172W/Rom1+/− animals had worsened rod and cone function and exacerbated retinal degeneration compared with Prph2R172W animals. Removing one allele of Rom1 had no effect in Prph2C/+. Combined, our findings support a role for non-pathogenic ROM1 null variants in contributing to phenotypic variability in mutant PRPH2-associated retinal degeneration. Since the effects of Rom1 reduction are variable, our data suggest that this contribution is specific to the type of Prph2 mutation.

Ryan A Kelley,1 Shannon M Conley,1 Rasha Makkia,1 Jamie N Watson,1 Zongchao Han,1 Mark J Cooper,2 Muna I Naash3 1Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; 2Copernicus Therapeutics, Inc., Cleveland, OH, USA; 3Department of Biomedical Engineering, University of Houston, Houston, TX, USA Introduction: DNA nanoparticles (NPs) comprising polylysine conjugated to polyethylene glycol efficiently target murine photoreceptors and the retinal pigment epithelium (RPE) and lead to long-term phenotypic improvement in models of retinal degeneration. Advancing this technology requires testing in a large animal model, particularly with regard to safety. So, herein we evaluate NPs in non-human primates (baboon). Methods and results: NPs with plasmids carrying GFP and a ubiquitous, RPE-specific, or photoreceptor-specific promoter were delivered by either subretinal or intravitreal injection. We detected GFP message and protein in the retina/RPE from eyes dosed with NPs carrying ubiquitously expressed and RPE-specific vectors, and GFP message in eyes injected with NPs carrying photoreceptor-specific vectors. Importantly, we observed NP DNA in the retina/RPE following intravitreal injection, indicating the inner limiting membrane does not prevent NP diffusion into the outer retina. We did not observe any adverse events in any baboon, and there were no NP-associated changes in retinal function. Furthermore, no systemic or local inflammatory reaction to the vectors/injections was observed, and no NP DNA was found outside the eye. Conclusion: Taken together with the well-established rodent safety and efficacy data, these findings suggest that DNA NPs may be a safe and potentially clinically viable nonviral ocular therapy platform for retinal diseases. Keywords: DNA nanoparticles, non-human primate, nonviral gene transfer, baboon, gene therapy, safety, ocular gene transfer

Mutations in peripherin 2 (PRPH2) have been associated with retinitis pigmentosa (RP) and macular/pattern dystrophies, but the origin of this phenotypic variability is unclear. The majority of Prph2 mutations are located in the large intradiscal loop (D2), a region that contains seven cysteines involved in intra- and intermolecular disulfide bonding and protein folding. A mutation at cysteine 213, which is engaged in an intramolecular disulfide bond, leads to butterfly-shaped pattern dystrophy in humans, in sharp contrast to mutations in the adjacent cysteine at position 214 which result in RP. To help understand this unexpected phenotypic variability, we generated a knockin mouse line carrying the C213Y disease mutation. The mutant Prph2 protein lost the ability to oligomerize with rod outer segment membrane protein 1 (Rom1), but retained the ability to form homotetramers. C213Y heterozygotes had significantly decreased overall Prph2 levels as well as decreased rod and cone function. Critically, supplementation with extra wild-type Prph2 protein elicited improvements in Prph2 protein levels and rod outer segment structure, but not functional rescue in rods or cones. These findings suggest that not all interruptions of D2 loop intramolecular disulfide bonding lead to haploinsufficiency-related RP, but rather that more subtle changes can lead to mutant proteins stable enough to exert gain-of-function defects in rods and cones. This outcome highlights the difficulty in targeting Prph2-associated gain-of-function disease and suggests that elimination of the mutant protein will be a pre-requisite for any curative therapeutic strategy.

Peripherin 2 (Prph2) is a photoreceptor tetraspanin, and deletion of codon 153 (K153Δ) leads to retinitis pigmentosa, pattern dystrophy, and fundus flavimaculatus in the same family. To study this variability, we generated a K153Δ-Prph2 knockin mouse. K153Δ-Prph2 cannot form the complexes required for outer segment formation, and in cones cannot interact with its binding partner rod outer segment membrane protein 1. K153Δ causes dominant defects in rod and cone function; however, rod but not cone ultrastructure is improved by the presence of K153Δ-Prph2. Likewise, supplementation of K153Δ heterozygotes with WT-Prph2 results in structural but not functional improvements. These results support the idea that mutations may differentially affect Prph2's role as a structural component, and its role as a functional protein key for organizing membrane domains for cellular signalling. These roles may be different in rods and cones, thus contributing to the phenotypic heterogeneity that characterizes diseases associated with Prph2 mutations.

Purpose Rod photoreceptor outer segment (OS) morphogenesis, structural integrity, and proper signal transduction rely on critical proteins found in the different OS membrane domains (e.g., plasma, disc, and disc rim membrane). Among these key elements are retinal degeneration slow (RDS, also known as peripherin-2), rhodopsin, and the beta subunit of the cyclic nucleotide gated channel (CNGB1a), which have been found to interact in a complex. The purpose of this study was to evaluate the potential interplay between these three proteins by examining retinal disease phenotypes in animal models expressing varying amounts of CNGB1a, rhodopsin, and RDS. Methods Outer segment trafficking, retinal function, and photoreceptor structure were evaluated using knockout mouse lines. Results Eliminating Cngb1 and reducing RDS leads to additive defects in RDS expression levels and rod electroretinogram (ERG) function, (e.g., Cngb1−/−/rds+/− versus rds+/− or Cngb1−/−) but not to additive defects in rod ultrastructure. These additive effects also manifested in cone function: Photopic ERG responses were significantly lower in the Cngb1−/−/rds+/− versus rds+/− or Cngb1−/−, suggesting that eliminating Cngb1 can accelerate the cone degeneration that usually presents later in the rds+/−. This was not the case with rhodopsin; reducing rhodopsin levels in concert with eliminating CNGB1a did not lead to phenotypes more severe than those observed in the Cngb1 knockout alone. Conclusions These data support a role for RDS as the core component of a multiprotein plasma membrane-rim-disc complex that has both a structural role in photoreceptor OS formation and maintenance and a functional role in orienting proteins for optimal signal transduction.

Purpose Riboflavin and its cofactors are essential for cellular energy generation, responses to oxidative stress, and overall homeostasis. Retbindin is a novel retina-specific riboflavin binding protein essential for the maintenance of retinal flavin levels, but its function remains poorly understood. To further elucidate the function of retbindin in retinal health and disease, we evaluated its role in retinal degeneration in a cone-rod dystrophy model associated with the R172W mutation in the photoreceptor tetraspanin Prph2. Methods We performed structural, functional, and biochemical characterization of R172W-Prph2 mice with and without retbindin (Rtbdn-/-/Prph2R172W). Results Retbindin is significantly upregulated during degeneration in the R172W model, suggesting that retbindin plays a protective role in retinal degenerative diseases. This hypothesis was supported by our findings that R172W mice lacking retbindin (Rtbdn-/-/Prph2R172W) exhibit functional and structural defects in rods and cones that are significantly worse than in controls. Retinal flavin levels were also altered in the Rtbdn-/-/Prph2R172W retina. However, in contrast to the Rtbdn-/- retina which has reduced flavin levels compared to wild-type, Rtbdn-/-/Prph2R172W retinas exhibited elevated levels of riboflavin and the flavin cofactor FMN. Conclusions These results indicate that retbindin plays a protective role during retinal degeneration, but that its function is more complex than previously thought, and suggest a possible role for retbindin in protecting the retina from phototoxicity associated with unbound flavins. This study highlights the essential role of precisely regulated homeostatic mechanisms in photoreceptors, and shows that disruption of this metabolic balance can contribute to the degenerative process associated with other cellular defects.

The retinal disease gene peripherin 2 (PRPH2) is essential for the formation of photoreceptor outer segments (OSs), where it functions in oligomers with and without its homologue ROM1. However, the precise role of these proteins in OS morphogenesis is not understood. By utilizing a knock-in mouse expressing a chimeric protein comprised of the body of Rom1 and the C-terminus of Prph2 (termed RRCT), we find that the Prph2 C-terminus is necessary and sufficient for the initiation of OSs, while OS maturation requires the body of Prph2 and associated large oligomers. Importantly, dominant-negative physiological and biochemical defects in RRCT heterozygous rods are rescued by removing Rom1, suggesting Rom1 is a regulator for OS formation. Our experiments evaluating Prph2 trafficking show that Rom1 is a key determinant of whether Prph2 complexes utilize conventional versus unconventional (Golgi bypass) secretory pathways to reach the OS. These findings significantly advance our understanding of the molecular underpinnings of OS morphogenesis and particularly the role of Rom1.

Mutations in peripherin 2 (PRPH2, also known as Rds), a tetraspanin protein found in photoreceptor outer segments (OSs), cause retinal degeneration ranging from rod-dominant retinitis pigmentosa (RP) to cone-dominant macular dystrophy (MD). Understanding why some Prph2 mutants affect rods while others affect cones remains a critical unanswered question. Prph2 is essential for OS structure and function and exhibits a very specific pattern of oligomerization with its homolog Rom1. Non-covalent Prph2/Rom1 homo- and hetero-tetramers assemble into higher-order covalently linked complexes held together by an intermolecular disulfide bond at Prph2-C150/Rom1-C153. Here we disrupt this crucial bond using a C150S-Prph2 knockin mouse line to study the role of Prph2 higher-order complex formation. We find that C150S-Prph2 traffics to the OS, interacts with Rom1 and forms non-covalent tetramers, but alone cannot support normal OS structure and function. However, C150S-Prph2 supports the initiation or elaboration of OS disc structures, and improves rod OS ultrastructure in the presence of wild-type (WT) Prph2 (i.e. Prph2(C150S/+) versus Prph2(+/−)). Prph2(C150S/+) animals exhibit haploinsufficiency in rods, but a dominant-negative phenotype in cones, suggesting cones have a different requirement for large Prph2 complexes than rods. Importantly, cone but not rod function can be improved by the addition of one Prph2(Y141C) allele, a mutation responsible for pattern dystrophy owing to the extra cysteine. Combined these findings show that covalently linked Prph2 complexes are essential for OS formation, but not for Prph2 targeting to the OS, and that cones are especially sensitive to having a broad distribution of Prph2 complex types (i.e. tetramers and large complexes).

Fibulins 2 and 5 are part of a seven-member family of proteins integral to the retinal extracellular matrix. Our study aimed to further explore the roles of both fibulins in retinal function. We obtained knockout mouse models of both fibulins and performed immunohistochemistry, electroretinography, and histology to investigate the outcome of eliminating these proteins. Immunohistochemical analysis showed that both fibulins are localized to the RPE, choroid, and Bruch's membrane. Functional testing showed a significantly reduced scotopic A response at 1 month of age, when compared to their wild-type counterpart. This functional reduction remained constant throughout the age of the animal and only declined as a result of normal aging. The functional decline was associated with reduced number of photoreceptor cells. The results presented clearly demonstrate that fibulins 2 and 5, as extracellular proteins, are necessary for normal retinal development.

Retbindin (Rtbdn) is a novel protein of unknown function found exclusively in the retina. Recently, our group has suggested, from in silico analysis of the peptide sequence and in vitro binding data, that Rtbdn could function to bind riboflavin (RF) and its derivatives flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), collectively known as flavins. Here we confirm that Rtbdn is capable of flavin binding and that this characteristic can protect photoreceptors from flavin-sensitized light damage.

Considerable progress has been made in the design and delivery of non-viral gene therapy vectors, but, like their viral counterparts, therapeutic levels of transgenes have not met the requirements for successful clinical applications so far. The biggest advantage of polymer-based nanoparticle vectors is the ease with which they can be modified to increase their ability to penetrate the cell membrane and target specific cells by simply changing the formulation of the nanoparticle compaction. We took advantage of this characteristic to improve transfection rates of our particles to meet the transgene levels which will be needed for future treatment of patients. For this study, we successfully investigated the possibility of our established pegylated polylysine particles to be administered via intravitreal rather than subretinal route to ease the damage during injection. We also demonstrated that our particles are flexible enough to sustain changes in the formulation to accommodate additional targeting sequences without losing their efficiency in transfecting neuronal cells in the retina. Together, these results give us the opportunity to even further improve our particles.

For patients with hereditary retinal diseases, retinal gene therapy offers significant promise for the prevention of retinal degeneration. While adeno-associated virus (AAV)-based systems remain the most popular gene delivery method due to their high efficiency and successful clinical results, other delivery systems, such as non-viral nanoparticles (NPs) are being developed as additional therapeutic options. NP technologies come in several categories (e.g., polymer, liposomes, peptide compacted DNA), several of which have been tested in mouse models of retinal disease. Here, we discuss the key biochemical features of the different NPs that influence how they are internalized into cells, escape from endosomes, and are delivered into the nucleus. We review the primary mechanism of NP uptake by retinal cells and highlight various NPs that have been successfully used for in vivo gene delivery to the retina and RPE. Finally, we consider the various strategies that can be implemented in the plasmid DNA to generate persistent, high levels of gene expression.

One-fifth of all cases of Leber congenital amaurosis are type 1 (LCA1). LCA1 is a severe form of retinal dystrophy caused by loss-of-function mutations in guanylate cyclase 1 (GC1), a key member of the phototransduction cascade involved in modulating the photocurrents. Although GC1 has been studied for some time, the mechanisms responsible for its regulation and membrane targeting are not fully understood. We reported earlier that retinal degeneration 3 (RD3) protein interacts with GC1 and promotes its targeting to the photoreceptor outer segments (POS). Here, we extend our studies to show a direct association between RD3 and guanylate cyclase activating protein 1 (GCAP1). Furthermore, we demonstrate that this functional interaction is important for GC1 targeting to POS. We also show that most LCA1-causing mutations in GC1 result in lost GC1 interaction with RD3 or GC1 being targeted to the plasma membrane. Our data suggest that GC1, GCAP1, and RD3 form a complex in the endoplasmic reticulum that targets GC1 to POS. Interruption of this assembly is likely the underlying mechanism for a subset of LCA1. This study offers insights for the development of therapeutic strategies to treat this severe form of blindness. Background: Defects in the function of guanylate cyclase 1 (GC1) cause Leber congenital amaurosis (LCA) type 1. Results: GC1, GCAP1, and RD3 form a complex in the endoplasmic reticulum that targets GC1 to outer segments. Conclusion: A subset of LCA1 is caused by impaired formation of the RD3-GC1-GCAP1 complex. Significance: Understanding the molecular interaction of RD3 with GC1 and GCAP1 has potential therapeutic benefits for LCA1.

Rhodopsin, a G-protein coupled receptor, most abundant protein in retinal rod photoreceptors, is glycosylated at asparagines-2 and 15 on its N-terminus. To understand the role of rhodopsin's glycosylation in vivo, we generated and characterized a transgenic mouse model that expresses a non-glycosylated form of rhodopsin. We show that lack of glycosylation triggers a dominant form of progressive retinal degeneration. Electron microscopic examination of retinas at postnatal day 17 revealed the presence of vacuolar structures that distorted rod photoreceptor outer segments and became more prominent with age. Expression of non-glycosylated rhodopsin alone showed that it is unstable and is regulated via ubiquitin-mediated proteasomal degradation at the base of outer segments. We observed similar vacuolization in outer segments of transgenic mice expressing human rhodopsin with a T17M mutation (hT17M), suggesting that the mechanism responsible for the degenerative process in mice expressing the non-glycosylated rhodopsin and the RHO(hT17M) mice is likely the cause of phenotype observed in retinitis pigmentosa patients carrying T17M mutation.

Long-chain PUFAs (LC-PUFAs; C20–C22; e.g., DHA and arachidonic acid) are highly enriched in vertebrate retina, where they are elongated to very-long-chain PUFAs (VLC-PUFAs; C ≥28) by the elongation of very-long-chain fatty acids-4 (ELOVL4) enzyme. These fatty acids play essential roles in modulating neuronal function and health. The relevance of different lipid requirements in rods and cones to disease processes, such as age-related macular degeneration, however, remains unclear. To better understand the role of LC-PUFAs and VLC-PUFAs in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes in rodents with rod- or cone-dominant retinas. We analyzed fatty acid methyl esters and the molecular species of glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) by GC-MS/GC-flame ionization detection and ESI-MS/MS, respectively. We found that whole retinas and OS membranes in rod-dominant animals compared with cone-dominant animals had higher amounts of LC-PUFAs and VLC-PUFAs. Compared with those of rod-dominant animals, retinas and OS membranes from cone-dominant animals also had about 2-fold lower levels of di-DHA (22:6/22:6) molecular species of glycerophospholipids. Because PUFAs are necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods.

Retinal detachment is the physical separation of the retina from the retinal pigment epithelium. It occurs during aging, trauma, or during a variety of retinal disorders such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or as a complication following cataract surgery. This report investigates the role of fibulin 2, an extracellular component, in retinal detachment. A major mechanism for detachment resolution is enhancement of cellular adhesion between the retina and the retinal pigment epithelium and prevention of its cellular migration. This report shows that fibulin 2 is mainly present in the retinal pigment epithelium, Bruch membrane, choriocapillary, and to a lesser degree in the retina. In vitro studies revealed the presence of two isoforms for fibulin 2. The small isoform is located inside the cell, and the large isoform is present inside and outside the cells. Furthermore, fibulin 2 is post-translationally modified by tyrosine sulfation, and the sulfated isoform is present outside the cell, whereas the unsulfated pool is internally located. Interestingly, sulfated fibulin 2 significantly reduced the rate of cellular growth and migration. Finally, levels of fibulin 2 dramatically increased in the retinal pigment epithelium following retinal detachment, suggesting a direct role for fibulin 2 in the re-attachment of the retina to the retinal pigment epithelium. Understanding the role of fibulin 2 in enhancing retinal attachment is likely to help improve the current therapies or allow the development of new strategies for the treatment of this sight-threatening condition.

Mutations in the photoreceptor tetraspanin gene peripherin-2/retinal degeneration slow (PRPH2/RDS) cause both rod- and cone-dominant diseases. While rod-dominant diseases, such as autosomal dominant retinitis pigmentosa, are thought to arise due to haploinsufficiency caused by loss-of-function mutations, the mechanisms underlying PRPH2-associated cone-dominant diseases are unclear. Here we took advantage of a transgenic mouse line expressing an RDS mutant (R172W) known to cause macular degeneration (MD) in humans. To facilitate the study of cones in the heavily rod-dominant mouse retina, R172W mice were bred onto an Nrl(-/-) background (in which developing rods adopt a cone-like fate). In this model the R172W protein and the key RDS-binding partner, rod outer segment (OS) membrane protein 1 (ROM-1), were properly expressed and trafficked to cone OSs. However, the expression of R172W led to dominant defects in cone structure and function with equal effects on S- and M-cones. Furthermore, the expression of R172W in cones induced subtle alterations in RDS/ROM-1 complex assembly, specifically resulting in the formation of abnormal, large molecular weight ROM-1 complexes. Fundus imaging demonstrated that R172W mice developed severe clinical signs of disease nearly identical to those seen in human MD patients, including retinal degeneration, retinal pigment epithlium (RPE) defects and loss of the choriocapillaris. Collectively, these data identify a primary disease-causing molecular defect in cone cells and suggest that RDS-associated disease in patients may be a result of this defect coupled with secondary sequellae involving RPE and choriocapillaris cell loss.

Given the number of monogenic ocular diseases and the number of non-monogenic degenerative ocular diseases for which gene therapy is considered as a treatment, the development of effective therapeutic delivery strategies for DNA is a critical research goal. In this work, nonviral nanoparticles (NPs) composed of glycol chitosan (GCS) and plasmid DNA (pDNA) were generated, characterized, and evaluated. These particles are stable, do not aggregate in saline, are resistant to DNases, and have a hydrodynamic diameter of approximately 250 nm. Furthermore, the plasmid in these NPs was shown to maintain its proper conformation and can be released and expressed inside the cell. To determine whether these NPs would be suitable for intraocular use, pDNA carrying the ubiquitously expressed CBA-eGFP expression cassette was compacted and subretinally injected into adult wild-type albino mice. At day 14 post-injection (PI), substantial green fluorescent protein (GFP) expression was observed exclusively in the retinal pigment epithelium (RPE) in eyes treated with GCS NPs but not in those treated with uncompacted pDNA or vehicle (saline). No signs of gross retinal toxicity were observed, and at 30 days PI, there was no difference in electroretinogram function between GCS NP-, pDNA-, or vehicle-treated eyes. These results suggest that with further development, GCS NPs could be a useful addition to the available repertoire of genetic therapies for the treatment of RPE-associated diseases.

Vertebrate photoreceptors contain a unique tetraspanin protein known as 'retinal degeneration slow' (RDS). Mutations in the RDS gene have been identified in a variety of human retinal degenerative diseases, and more than 70% of these mutations are located in the second intra-discal (D2) loop, highlighting the importance of this region. Here we examined the conformational and thermal stability properties of the D2 loop of RDS, as well as interactions with ROM-1, a non-glycosylated homolog of RDS. The RDS D2 loop was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP). The fusion protein, referred to as MBP-D2, was purified to homogeneity. Circular dichroism spectroscopy showed that the wild-type (WT) D2 loop consists of approximately 21% α-helix, approximately 20% β-sheet and approximately 59% random coil. D2 loop fusion proteins carrying disease-causing mutations in RDS (e.g. R172W, C214S, N244H/K) were also examined, and conformational changes were observed (compared to wild-type D2). In particular, the C150S, C214S and N244H proteins showed significant reductions in α-helicity. However, the thermal stability of the mutants was unchanged compared to wild-type, and all the mutants were capable of interacting with ROM-1, indicating that this functional aspect of the isolated D2 loop remained intact in the mutants despite the observed conformational changes. An I-TASSER model of the RDS D2 loop predicted a structure consistent with the circular dichroism experiments and the structure of the conserved region of the D2 loop of other tetraspanin family members. These results provide significant insight into the mechanism of RDS complex formation and the disease process underlying RDS-associated retinal degeneration.

Aim: To evaluate the safety of compacted DNA nanoparticles (NPs) in retinal pigment epithelial (RPE) cells. Materials & methods: Enhanced GFP expression cassettes controlled by the RPE-specific vitelloform macular dystrophy promoter were constructed with and without a bacterial backbone and compacted into NPs formulated with polyethylene glycol-substituted lysine 30-mers. Single or double subretinal injections were administered in adult BALB/c mice. Expression levels of enhanced GFP, proinflammatory cytokines and neutrophil/macrophage mediators, and retinal function by electroretinogram were evaluated at different time-points postinjection. Results: Immunohistochemistry and real-time PCR demonstrated that NPs specifically transfect RPE cells at a higher efficiency than naked DNA and similar results were observed after the second injection. At 6 h postinjections, a transient inflammatory response was observed in all cohorts, including saline, indicating an adverse effect to the injection procedure. Subsequently, no inflammation was detected in all experimental groups. Conclusion: This study demonstrates the safety and efficacy of NP-mediated RPE gene transfer therapy following multiple subretinal administrations.

Mutations in the protein product of the retinal degeneration slow (RDS) gene cause both rod-dominant retinitis pigmentosa and different forms of cone-dominant macular dystrophies. In particular, mutations in codon 244 can cause either of these types of disease. In this study, we examine the biochemical effects of N244H and N244K in an effort to understand the mechanism underlying rod- and cone-dominant defects, respectively. COS-1 cells were cotransfected with either wild-type (WT) RDS or RDS containing an N244H or N244K mutation along with its binding partner, ROM-1 (rod outer segment membrane protein 1). Cell extracts were analyzed for mutant protein stability by Western blot, and localization was examined by immunocytochemistry. Interactions between transfected proteins were assessed by reciprocal co-immunoprecipitation, and nonreducing velocity sedimentation was used to identify the pattern of RDS complex assembly. Interactions were confirmed using GST fusion constructs of WT and mutant RDS in GST pull-down assays from WT mouse retinal extract. In COS-1 cells, recombinant N244H RDS had a weakened ability to assemble into higher-order complexes but retained the ability to co-immunoprecipitate with ROM-1 as well as localize properly throughout the cells. In contrast, recombinant N244K protein did not associate with ROM-1, showed signs of protein aggregation, and colocalized with an ER marker. These experiments support the hypothesis that RDS mutations that interrupt higher-order oligomer formation but still interact with ROM-1 and fold properly in membranes may cause dominant, gain-of-function disease phenotypes while mutations that cause RDS misfolding (and thus incorrect trafficking and assembly) may be associated with a loss-of-function haploinsufficiency phenotype.

The purpose of the present study was to test the therapeutic efficiency and safety of compacted-DNA nanoparticle-mediated gene delivery into the subretinal space of a juvenile mouse model of retinitis pigmentosa. Nanoparticles containing the mouse opsin promoter and wild-type mouse Rds gene were injected subretinally into mice carrying a haploinsufficiency mutation in the retinal degeneration slow (rds(+ or -)) gene at postnatal day (P)5 and 22. Control mice were either injected with saline, injected with uncompacted naked plasmid DNA carrying the Rds gene, or remained untreated. Rds mRNA levels peaked at postinjection day 2 to 7 (PI-2 to PI-7) for P5 injections, stabilized at levels 2-fold higher than in uninjected controls for both P5 and P22 injections, and remained elevated at the latest time point examined (PI-120). Rod function (measured by electroretinography) showed modest but statistically significant improvement compared with controls after both P5 and P22 injections. Cone function in nanoparticle-injected eyes reached wild-type levels for both ages of injections, indicating full prevention of cone degeneration. Ultrastructural examination at PI-120 revealed significant improvement in outer segment structures in P5 nanoparticle-injected eyes, while P22 injection had a modest structural improvement. There was no evidence of macrophage activation or induction of IL-6 or TNF-alpha mRNA in P5 or P22 nanoparticle-dosed eyes at either PI-2 or PI-30. Thus, compacted-DNA nanoparticles can efficiently and safely drive gene expression in both mitotic and postmitotic photoreceptors and retard degeneration in this model. These findings, using a clinically relevant treatment paradigm, illustrate the potential for application of nanoparticle-based gene replacement therapy for treatment of human retinal degenerations.-Cai, X., Conley, S. M., Nash, Z., Fliesler, S. J., Cooper, M. J., Naash, M. I. Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa.

It is commonly assumed that the ultrastructural organization of the rim region of outer segment (OS) discs in rods and lamellae in cones requires functional retinal degeneration slow/rod outer segment membrane protein 1 (Rds/Rom-1) complexes. Cysteine-150 (C150) in Rds has been implicated in intermolecular disulfide bonding essential for functional Rds complexes. Transgenic mice containing the Rds C150S mutation (C150S-Rds) failed to form higher-order Rds oligomers, although interactions between C150S-Rds and Rom-1 occurred in rods, but not in cones. C150S-Rds mice exhibited marked early-onset reductions in cone function and abnormal OS structure. In contrast, C150S-Rds expression in rods partly rescued the rds(+/-) phenotype. Although C150S-Rds was detected in the OSs in rods and cones, a substantial percentage of C150S-Rds and cone opsins were mislocalized to different cellular compartments in cones. The results of this study provide novel insights into the importance of C150 in Rds oligomerization and the differences in Rds requirements in rods versus cones. The apparent OS structural differences between rods and cones may cause cones to be more susceptible to the elimination of higher-order Rds/Rom-1 oligomers (e.g. as mediated by mutation of the Rds C150 residue).

The photoreceptor-specific glycoprotein retinal degeneration slow (RDS, also called PRPH2) is necessary for the formation of rod and cone outer segments. Mutations in RDS cause rod and cone-dominant retinal disease, and it is well established that both cell types have different requirements for RDS. However, the molecular mechanisms for this difference remain unclear. Although RDS glycosylation is highly conserved, previous studies have revealed no apparent function for the glycan in rods. In light of the highly conserved nature of RDS glycosylation, we hypothesized that it is important for RDS function in cones and could underlie part of the differential requirement for RDS in the two photoreceptor subtypes. We generated a knockin mouse expressing RDS without the N-glycosylation site (N229S). Normal levels of RDS and the unglycosylated RDS binding partner rod outer segment membrane protein 1 (ROM-1) were found in N229S retinas. However, cone electroretinogram responses were decreased by 40% at 6 months of age. Because cones make up only 3-5% of photoreceptors in the wild-type background, N229S mice were crossed into the nrl(-/-) background (in which all rods are converted to cone-like cells) for biochemical analysis. In N229S/nrl(-/-) retinas, RDS and ROM-1 levels were decreased by ~60% each. These data suggest that glycosylation of RDS is required for RDS function or stability in cones, a difference that may be due to extracellular versus intradiscal localization of the RDS glycan in cones versus rods.

It is commonly assumed that photoreceptor (PR) outer segment (OS) morphogenesis is reliant upon the presence of peripherin/rds, hereafter termed Rds. In this study, we demonstrate a differential requirement of Rds during rod and cone OS morphogenesis. In the absence of this PR-specific protein, rods do not form OSs and enter apoptosis, whereas cone PRs develop atypical OSs and are viable. Such OSs consist of dysmorphic membranous structures devoid of lamellae. These tubular OSs lack any stacked lamellae and have reduced phototransduction efficiency. The loss of Rds only appears to affect the shape of the OS, as the inner segment and connecting cilium remain intact. Furthermore, these structures fail to associate with the specialized extracellular matrix that surrounds cones, suggesting that Rds itself or normal OS formation is required for this interaction. This study provides novel insight into the distinct role of Rds in the OS development of rods and cones.

Light accelerates progression of retinal degeneration in many animal models of retinitis pigmentosa (RP). A sequence variant in the Rpe65 gene (Rpe65(450Leu) or Rpe65(450Met)) can act as a modulator of light-damage susceptibility in mice by influencing the kinetics of rhodopsin regeneration and thus by modulating the photon absorption. Depending on exposure duration and light intensity applied, white fluorescent light induces photoreceptor apoptosis and retinal degeneration in wild-type mice by the activation of one of two known molecular pathways. These pathways depend, respectively, on activation of the transcription factor c-Fos/AP-1 and on phototransduction activity. Here we tested Rpe65 as a genetic modifier for inherited retinal degeneration and analysed which degenerative pathway is activated in a transgenic mouse model of autosomal dominant RP. We show that retinal degeneration was reduced in mice expressing the Rpe65(450Met) variant and that these mice retained more visual pigment rhodopsin than did transgenic mice expressing the Rpe65(450Leu) variant. In addition, lack of phototransduction slowed retinal degeneration whereas ablation of c-Fos had no effect. We conclude that sequence variations in the Rpe65 gene can act as genetic modifiers in inherited retinal degeneration, presumably by regulating the daily rate of photon absorption through the modulation of rhodopsin regeneration kinetics. Increased absorption of photons and/or light sensitivity appear to accelerate retinal degeneration via an apoptotic cascade which involves phototransduction but not c-Fos.

Peripherin/rds (P/rds) is a disk rim protein that assembles into homo and hetero complexes with its nonglycosylated homologue, Rom-1, to maintain the integrity of the photoreceptor outer segment. Mutations in the rds gene have been identified in a variety of human retinal degenerative diseases. More than 70% of these mutations are located in the second intradiscal (D2) loop, highlighting the functional importance of this region. This study examines the involvement of different regions of the D2 loop in protein associations using a GST pull-down assay and a heterologous coexpression system. The pull-down assay suggests an association of the N-terminal portion (Phe(120)-Phe(187)) of the D2 loop with Rom-1 as well as with other P/rds molecules. Through peptide competition experiments, the region between Cys(165) and Asn(182) of the D2 loop has been identified as the domain for these associations. In a COS-1 cell heterologous expression system, coexpression of the D2 loop along with the intact P/rds and Rom-1 hindered the association of the two full-length proteins. In contrast to the homo association of P/rds molecules, it seems that the hetero association of P/rds with Rom-1 has a more stringent structural requirement. This work defines the crucial domain of the D2 loop, which mediates homo and hetero associations, specifically the regions that lay between Cys(165) and Asn(182). Elucidation of the molecular mechanisms behind the protein-protein associations of P/rds and its partners may reveal the pathogenic defects arising from the most common mutations in this gene.

Elevation of erythropoietin (Epo) concentrations by hypoxic preconditioning or application of recombinant human Epo (huEpo) protects the mouse retina against light-induced degeneration by inhibiting photoreceptor cell apoptosis. Because photoreceptor apoptosis is also the common path to cell loss in retinal dystrophies such as retinitis pigmentosa (RP), we tested whether high levels of huEpo would reduce apoptotic cell death in two mouse models of human RP. We combined the two respective mutant mouse lines with a transgenic line (tg6) that constitutively overexpresses huEpo mainly in neural tissues. Transgenic expression of huEpo caused constitutively high levels of Epo in the retina and protected photoreceptors against light-induced degeneration; however, the presence of high levels of huEpo did not affect the course or the extent of retinal degeneration in a light-independent (rd1) and a light-accelerated (VPP) mouse model of RP. Similarly, repetitive intraperitoneal injections of recombinant huEpo did not protect the retina in the rd1 and the VPP mouse. Lack of neuroprotection by Epo in the two models of inherited retinal degeneration was not caused by adaptational downregulation of Epo receptor. Our results suggest that apoptotic mechanisms during acute, light-induced photoreceptor cell death differ from those in genetically based retinal degeneration. Therapeutic intervention with cell death in inherited retinal degeneration may therefore require different drugs and treatments.

Retbindin is a novel retina-specific protein of unknown function. In this study, we have used various approaches to evaluate protein expression, localization, biochemical properties, and function. We find that retbindin is secreted by the rod photoreceptors into the inter-photoreceptor matrix where it is maintained via electrostatic forces. Retbindin is predominantly localized at the interface between photoreceptors and retinal pigment epithelium microvilli, a region critical for retinal function and homeostasis. Interestingly, although it is associated with photoreceptor outer segments, retbindin's expression is not dependent on their presence. In vitro, retbindin is capable of binding riboflavin, thus implicating the protein as a metabolite carrier between the retina and the retinal pigment epithelium. Altogether, our data show that retbindin is a novel photoreceptor-specific protein with a unique localization and function. We hypothesize that retbindin is an excellent candidate for binding retinal flavins and possibly participating in their transport from the extracellular space to the photoreceptors. Further investigations are warranted to determine the exact function of retbindin in retinal homeostasis and disease.

Retinal degeneration slow (RDS/PRPH2) is critical for the formation of the disc/lamella rim in photoreceptor outer segments (OSs), but plays a different role in rods vs. cones. Without RDS, rods fail to form OSs, however, cones lacking RDS (in the rds−/−/Nrl−/−) exhibit balloon-like OSs devoid of lamellae. We show that distribution of most proteins in the lamella and PM domains is preserved even in the absence of RDS, rim, and lamella structures. However, the rim protein prominin-1 exhibits altered trafficking and OS localization, suggesting that proper targeting and distribution of rim proteins may require RDS. Our ultrastructural studies show that in cones, OS formation is initiated by the growth of opsin-containing membrane with RDS-mediated rim formation as a secondary step. This is directly opposite to rods and significantly advances our understanding of the role of the rim in cone OS morphogenesis. Furthermore, our results suggest that the unique folded lamella architecture of the cone OS may maximize density or proximity of phototransduction proteins, but is not required for OS function or for protein distribution and retention in different membrane domains.—Conley, S. M., Al-Ubaidi, M. R., Han, Z., Naash, M. I. Rim formation is not a prerequisite for distribution of cone photoreceptor outer segment proteins.

Photoreceptor (PR) cells are prone to accumulation of reactive oxygen species (ROS) and oxidative stress. An imbalance between the production of ROS and cellular antioxidant defenses contributes to PR degeneration and blindness in many different ocular disease states. Yttrium oxide (Y2O3) nanoparticles (NPs) are excellent free radical scavengers due to their non-stoichiometric crystal defects. Here we utilize a murine light stress model to test the efficacy of Y2O3 NPs (~ 10–14 nm in diameter) in ameliorating retinal oxidative stress-associated degeneration. Our studies demonstrate that intravitreal injections of these NPs at doses ranging from 0.1 μM to 5.0 μM two weeks prior to acute light stress protects PRs from degeneration. This protection is reflected both structurally (i.e. decreased light-associated thinning of the outer nuclear layer) and functionally, i.e. in preservation of scotopic and photopic electroretinogram amplitudes. We also observe preservation of structure and function when NPs are delivered immediately after acute light stress, although the magnitude of the preservation is smaller, and only doses ranging from 1.0 μM to 5.0 μM were effective. We show that the Y2O3 NPs are non-toxic and well-tolerated after intravitreal delivery. Our results suggest that Y2O3 NPs have astonishing antioxidant benefits, and with further exploration, may be an excellent strategy for the treatment of oxidative stress associated with multiple forms of retinal degeneration.

The efficacy of non-viral genetic therapies has historically been limited by transient gene expression and vector loss. Scaffold matrix attachment regions (S/MARs) have been shown to augment transcription, promote episomal maintenance, and provide insulator-like function to DNA in in vitro and in vivo systems. Here we explore the ability of S/MAR elements to mediate these effects in retinal pigment epithelial (RPE) cells with the eventual goal of improving the persistence of expression of our non-viral gene delivery tools. We engineered an RPE-specific reporter vector with or without an S/MAR immediately downstream of the eGFP expression cassette. We show that the S/MAR vector is maintained as an episome for up to 1 year. Experiments in which rhodamine-labeled DNA was delivered to the subretinal space of mice show better persistence of the S/MAR-containing vector in the RPE than the non-S/MAR vector. These results suggest that inclusion of the S/MAR region promotes episomal maintenance of plasmid DNA in the RPE after subretinal delivery and that inclusion of this DNA element may be beneficial for non-viral ocular gene transfer.

Mutations in the photoreceptor-specific flippase ABCA4 lead to accumulation of the toxic bisretinoid A2E, resulting in atrophy of the retinal pigment epithelium (RPE) and death of the photoreceptor cells. Many blinding diseases are associated with these mutations including Stargardt's disease (STGD1), cone-rod dystrophy, retinitis pigmentosa (RP), and increased susceptibility to age-related macular degeneration. There are no curative treatments for any of these dsystrophies. While the monogenic nature of many of these conditions makes them amenable to treatment with gene therapy, the ABCA4 cDNA is 6.8 kb and is thus too large for the AAV vectors which have been most successful for other ocular genes. Here we review approaches to ABCA4 gene therapy including treatment with novel AAV vectors, lentiviral vectors, and non-viral compacted DNA nanoparticles. Lentiviral and compacted DNA nanoparticles in particular have a large capacity and have been successful in improving disease phenotypes in the Abca4 (-/-) murine model. Excitingly, two Phase I/IIa clinical trials are underway to treat patients with ABCA4-associated Startgardt's disease (STGD1). As a result of the development of these novel technologies, effective therapies for ABCA4-associated diseases may finally be within reach.

To define the physiological and structural changes that may accompany aging in the normal mouse retina.C57BL/6 mice were maintained under cyclic light for either 2, 6, or 12 months. After rod- and cone-mediated corneal electroretinograms (ERG's) were recorded from anesthetized animals, the retinal structure was quantitatively examined. Photoreceptor cell density was measured within 100-microm regions of the central superior and inferior retina. Cone photoreceptor subtypes were identified by immunocytochemistry.The amplitudes of rod- and cone-mediated ERG's were reduced in older mice, although the overall ERG wave-form did not change appreciably and implicit times were not changed in an age-dependent fashion. In comparison, there was no significant age-related decline in rod or cone photoreceptor density.The amplitude of the mouse ERG declines with age. This change does not appear to reflect a change in the structural integrity of the photoreceptor cells. In functional studies of murine models of late-onset retinal disorders, it will be important to take these changes into consideration.

We have recently noted that the inner nuclear layer (INL) and the inner plexiform layer (IPL) were significantly thinner in mice homozygous for the nervous defect (nr / nr) than in control (nr /+ or +/+) littermates. Here, we have carried out a series of anatomical studies to further understand these inner retinal changes. At postnatal day (P) 13, there was no difference in the inner retina between nervous and control mice, while a significant difference was observed at P30. Similar changes were not seen in other mouse models of photoreceptor degeneration. There was a significant reduction in the density of cells in the INL that were stained by antibodies against the inhibitory neurotransmitters GABA and glycine. These results indicate that the nervous defect causes a degeneration of one or more sub-types of amacrine cells, in addition to the loss of cerebellar Purkinje cells and retinal photoreceptors that is known to occur in these mutant animals. Finally, evidence is provided that photoreceptors die by an apoptotic pathway in nervous mice.

The VPP mouse is a transgenic strain carrying three mutations (P23H, V20G, P27L) near the N-terminus of opsin, the apoprotein of rhodopsin, the rod photopigment. These animals exhibit a slowly progressive degeneration of the rod photoreceptors, and concomitant changes in retinal function that mimic those seen in humans with autosomal dominant retinitis pigmentosa resulting from a point mutation (P23H) in opsin. In the present study we attempted to determine whether the disease process prevents the translocation of mutant opsin to the rod outer segments of transgenic mice, and whether it affects the photochemical properties of the rhodopsin present within their rod outer segments. Immunocytochemistry with a monoclonal antibody against a region of the C-terminus that recognizes epitopes common to both normal and mutant opsin (monoclonal antibody-1D4), and a polyclonal antibody that reacts preferentially with the mutant opsin (anti-VPP), were used to identify the opsin present in the rods of three-week-old VPP mice and normal littermates. Absorbance spectra, photosensitivity, and regeneration kinetics of rhodopsin in rod outer segment disc membranes were analysed by spectrophotometry. Western blot analysis with anti-VPP antibody indicated the specific binding of this antibody to the mutant opsin. Immunolocalization with monoclonal antibody-1D4 and anti-VPP antibodies suggested a normal translocation of the mutant protein to the outer segments. Aside from a small disparity in the absorbance spectra of rhodopsin obtained from normal and VPP retinas, there were no significant differences in either the ability of opsin to bind 11- cis retinal chromophore, or in the photic sensitivity of rhodopsin. The results indicate that mutant opsin is translated and incorporated into the rod outer segment disc membranes of VPP mice, and that the photochemical properties of rhodopsin in the rods of VPP retinas are similar to those of rhodopsin in normal retinas.

Retinitis pigmentosa is a heterogeneous group of inherited retinal disorders in which the photoreceptor cells degenerate. A line of transgenic mice expresses a mutant opsin gene that encodes three missense mutations near the amino terminus, including P23H, which is the basis for a common form of dominant retinitis pigmentosa. By studying the photoreceptor cells of these mice and their normal littermates, we found that: (1) opsin was routed correctly, (2) the concentration of opsin in the disk membranes appeared normal by freeze fracture analysis, (3) the amount of disk membrane shedding was normal, but (4) the basal disks of the outer segments were disorganized, indicating defective disk membrane morphogenesis. Defective disk membrane morphogenesis appears to result in the formation of fewer mature disks, thus accounting for observed gradual shortening of the photoreceptor outer segments with age. We suggest that abnormal disk membrane morphogenesis is the primary cellular defect that leads to blindness, and that it arises from the inability of nascent disk membranes, containing normal and mutant opsin, to interact normally with each other.

Mutations in the peripherin/rds gene have been reported to be associated with different forms of human autosomal dominant retinitis pigmentosa (ADRP) and macular degeneration (MD). To better understand the disruptive role of these mutations, knowledge of the structure-function relationship of the peripherin/rds gene is needed. To facilitate that, genomic clones encoding the mouse gene were isolated using bovine cDNA sequences as probes. Sequence analysis of clone lambda 6-1-1, that contained the entire coding sequence for the mouse peripherin/rds, yielded the exon-intron organization of the gene. The gene is composed of three exons (581, 247, and 213 bp) and two introns with the first and second introns 8.6 kb and 3.7 kb in size, respectively. Two major (1.6 and 2.7 kb) and three minor (4.0, 5.5, 6.5 kb) transcripts were detected on RNA blots. The major transcripts first appeared in the brain at embryonic day 13 and in the retina at postnatal day 1. Transcripts were missing in brain and eye of mice at embryonic day 15. Several transcription start sites were mapped within 26 nucleotides approximately 200 bp upstream from the translation initiation site. However, transcripts varied in the lengths of their 3' untranslated portion as a result of the utilization of different polyadenylation signals.

Mutations in the retinal pigment epithelium (RPE) gene RPE65 are associated with multiple blinding diseases including Leber's Congenital Amaurosis (LCA). Our goal has been to develop persistent, effective non-viral genetic therapies to treat this condition. Using precisely engineered DNA vectors and high capacity compacted DNA nanoparticles (NP), we previously demonstrated that both plasmid and NP forms of VMD2-hRPE65-S/MAR improved the disease phenotypes in an rpe65(-/-) model of LCA up to 6 months post-injection (PI), however the duration of this treatment efficacy was not established. Here, we test the ability of these vectors to sustain gene expression and phenotypic improvement for the life of the animal. NPs or naked DNA were subretinally injected in rpe65(-/-) mice at postnatal day (P) 16 and evaluated at 15 months PI. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were performed at PI-15 months and demonstrated appreciable expression of transferred RPE65 (levels were 32% of wild-type [WT] for NPs and 44% of WT for naked DNA). No reduction in expression at the message level was observed from PI-6 month data. Spectral electroretinography (ERG) demonstrated significant improvement in cone ERG amplitudes in treated versus uninjected animals. Most importantly, we also observed reduced fundus autofluorescence in the eyes injected with NP and naked DNA compared to uninjected counterparts. Consistent with these observations, biochemical studies showed a reduction in the accumulation of toxic retinyl esters in treated mice, suggesting that the transferred hRPE65 was functional. These critical results indicate that both NP and uncompacted plasmid VMD2-hRPE65-S/MAR can mediate persistent, long-term improvement in an RPE-associated disease phenotype, and suggest that DNA NPs, which are non-toxic and have a large payload capacity, expand the treatment repertoire available for ocular gene therapy.

Mutations in peripherin 2 (PRPH2), also known as retinal degeneration slow/RDS, lead to various retinal degenerations including retinitis pigmentosa (RP) and macular/pattern dystrophy (MD/PD). PRPH2-associated disease is often characterized by a phenotypic variability even within families carrying the same mutation, raising interest in potential modifiers. PRPH2 oligomerizes with its homologue rod outer segment (OS) membrane protein 1 (ROM1), and non-pathogenic PRPH2/ROM1 mutations, when present together, lead to digenic RP. We asked whether ROM1 could modify the phenotype of a PRPH2 mutation associated with a high degree of intrafamilial phenotypic heterogeneity: Y141C. In vitro, Y141C-Prph2 showed signs of retention in the endoplasmic reticulum (ER), however co-expression with Rom1 rescued this phenotype. In the heterozygous Y141C knockin mouse model (Prph2Y/+), Y141C-Prph2 and Rom1 formed abnormal complexes but were present at normal levels. Abnormal complexes were eliminated in the absence of Rom1 (Prph2Y/+/Rom1-/-) and total Prph2 levels were reduced to those found in the haploinsufficient Prph2+/- RP model. The biochemical changes had functional and structural consequences; while Prph2Y/+ animals exhibited a cone-rod electroretinogram defect, Prph2Y/+/Rom1-/- animals displayed a rod-dominant phenotype and OSs similar to those seen in the Prph2+/-. These data show that ablation of Rom1 results in the conversion of an MD/PD phenotype characterized by cone functional defects and the formation of abnormal Prph2/Rom1 complexes to an RP phenotype characterized by rod-dominant functional defects and reductions in total Prph2 protein. Thus one method by which ROM1 may act as a disease modifier is by contributing to the large variability in PRPH2-associated disease phenotypes.

The nuclear receptor NR2E3 promotes expression of rod photoreceptor genes while repressing cone genes. Mice lacking NR2E3 (Nr2e3(rd7/rd7) referred to here as rd7) are a model for enhanced S-cone syndrome, a disease associated with increased sensitivity to blue light and night blindness. Rd7 retinas have reduced levels of the outer segment (OS) structural protein retinal degeneration slow (RDS). We test the hypothesis that increasing RDS levels would improve the Rd7 phenotype. Transgenic mice over-expressing normal mouse peripherin/RDS (NMP) in rods and cones were crossed onto the rd7 background. Disease phenotypes were assessed in NMP/rd7 eyes and compared to wild-type (WT) and rd7 eyes at postnatal day 30. NMP/rd7 retinas expressed total RDS (transgenic and endogenous) message at WT levels, and NMP protein was correctly localized to the OS. NMP/rd7 retinas have shorter OSs compared to rd7 and WT and significantly reduced number of rosettes. NMP/rd7 mice also exhibited significant deficits in scotopic ERG amplitudes compared to rd7 while photopic amplitudes remained unaffected. Protein levels of rhodopsin, RDS, and the RDS homologue ROM-1 were significantly reduced in the NMP/rd7 retinas compared to rd7. We show that correcting the levels of RDS gene expression does not improve the phenotype of the rd7 suggesting that RDS deficiency is not responsible for the defect in this model. We suggest that the specific rod defect in the NMP/rd7 is likely associated with ongoing problems in the rd7 that are related to the expression of cone genes in rod cells, a characteristic of the model.

Mutations in the photoreceptor-specific flippase ABCA4 are associated with Stargardt disease and many other forms of retinal degeneration that currently lack curative therapies. Gene replacement is a logical strategy for ABCA4-associated disease, particularly given the current success of traditional viral-mediated gene delivery, such as with adeno-associated viral (AAV) vectors. However, the large size of the ABCA4 cDNA (6.8 kbp) has hampered progress in the development of genetic treatments. Nonviral DNA nanoparticles (NPs) can accommodate large genes, unlike traditional viral vectors, which have capacity limitations. We utilized an optimized DNA NP technology to subretinally deliver ABCA4 to Abca4-deficient mice. We detected persistent ABCA4 transgene expression for up to 8 months after injection and found marked correction of functional and structural Stargardt phenotypes, such as improved recovery of dark adaptation and reduced lipofuscin granules. These data suggest that DNA NPs may be an excellent, clinically relevant gene delivery approach for genes too large for traditional viral vectors.

Previously, we demonstrated that CK30PEG10k-compacted DNA nanoparticles (NPs) efficiently target photoreceptor cells and improve visual function in a retinitis pigmentosa model. Here, we test the ability of these NPs in driving transgene expression in the retinal pigment epithelium (RPE), using an RPE-specific reporter vector (VMD2-eGFP). NPs, uncompacted plasmid, or saline were subretinally delivered to adult BALB/c mice. NP-based expression was specific to RPE cells and caused no deleterious effects on retinal structure and function. eGFP expression levels in NP-injected eyes peaked at post-injection day 2 (PI-2), stabilized at levels ~3-fold higher than in naked DNA-injected eyes, and remained elevated at the latest time-point examined (PI-30). Unlike naked DNA, which only transfected cells at the site of injection, NPs were able to transfect cells throughout the RPE. Subretinal injections of rhodamine labeled NPs and naked DNA showed comparable initial uptake into RPE cells. However, at PI-7 and -30 days significantly more fluorescence was detected inside the RPE of NP-injected eyes compared to naked DNA, suggesting NPs are stable inside the cell which could possibly lead to higher and sustained expression. Overall, our results demonstrate that NPs can efficiently deliver genes to the RPE and hold great potential for the treatment of RPE-associated diseases.