Muna Naash - US grants

Mutations in the gene encoding the disk rim specific protein, peripherin/rds, have been implicated in the pathogenesis of both autosomal dominant retinitis pigmentosa (ADRP) and various forms of macular dystrophy (MD). Peripherin/rds plays an important role in the morphogenesis and maintenance of the disk rim structure. This role is supported by interactions with other proteins. The primary goals of the proposed research are to determine the molecular basis of action and functional properties of peripherin/rds and these interacting proteins, and to investigate the molecular abnormalities that lead to photoreceptor degeneration in transgenic models of human retinal diseases. Specific Aims 1, 2 and 3 are to use the structural, electrophysiological and biochemical studies to evaluate transgenic retinas expressing different mutations in the peripherin/rds gene. These mutations include: (1) the ARG172TP mutation that causes MD in humans, (2) the CYS214Ser mutation to determine the role of intermolecular disulfide bonds in the assembly of functional peripherin/rds-rom-1 complexes. We will analyze the effects of these mutations on morphogenesis of the disc membrane and on regulation of complex formation by peripherin/rds and rom-1. We can evaluate the different roles played by peripherin/rds in rods versus cones by comparing the effects of the Arg172Trp and Cys214Ser mutations on the structure and function of rod and cone outer segments. Comparisons of the Cys214 and Cys150Ser transgenic mice will provide insight into the role of inter- and intramolecular disulfide bond formation in peripherin/rds function. In Specific im 4, we will use a yeast two-hybrid system to identify the sites of interaction involved in formation of multimeric complexes by peripherin/rds and rom-1. This system provides a powerful genetic mechanism for detecting protein-protein interactions. We will also use the yeast two-hybrid system to screen a retinal cDNA library for other proteins that are involved in the assembly of functional peripherin/rds-rom-1 complexes. We have identified regions of high homology between all known peripherin/rds and rom-1 that are located in the large intradiscal loop. We hypothesize that these regions mediate the interactions between peripherin/rds-rom-1 complexes and are required to hold the rod discs or cone lamellae in their flattened shape. These interactions may be mediated by direct association of the subunit complexes or indirectly through other proteins. Mutagenesis studies will be performed to evaluate these interactions. These studies will provide insight into the functional role of peripherin/rds in normal and diseased retinas.

DESCRIPTION (provided by applicant): Peripherin/rds (P/rds) interacts with other proteins to build and maintain the disc rim structure of rods and cones. Our goal is to determine the functional properties of P/rds and its interacting partners and to investigate the molecular and biochemical abnormalities associated with disease-causing mutations in this gene. Utilizing proteomics, we have identified several proteins which interact with the P/rds complex. In this application, we will test our main hypothesis that P/rds and rom-1 exist in a primary complex that associates with an auxiliary complex linking the disc rim to the plasma membrane (PM). We propose that the primary complex is composed of hetero- and homo-tetramers, octamers and/or oligomers. Through proteomic studies, potential members of the auxiliary protein complex were identified and we propose to study their role in P/rds complex formation and disc rim morphogenesis of both rods and cones. Aim 1 will test the hypothesis that P/rds complex is different in composition between inner (IS) and outer segment (OS) regions, as well as between rods and cones. Experiments in this aim should investigate the differences in primary complex formation in the IS to that in the OS and identify rod/cone-specific interacting partners. Aim 2 evaluates will investigate the association between P/rds complex and the cone cGMP gated channel. Conventional biochemical techniques of protein-protein interactions and colocalization at the ultrastructural level will confirm and map the sites of their association with P/rds and rom-1. Aim 3 will characterize transgenic mice that express the C150S mutation in either rods or in cones to determine the role of C150S mediated inter-molecular disulfide-linked homodimers on rod and cone disc morphogenesis and its effect on P/rds primary and auxiliary complex assembly. Aim 4 investigates modifications in P/rds-primary or -auxiliary complex formation associated with diseases-causing mutations in P/rds. Our previous results indicate that either self-associations of P/rds or association with rom-1 to form tetramers are critical for stability and OS localization of P/rds and rom-1. R172W and C214S transgenic retinas will be used to define the perturbations in complex assembly and disc structure caused by these mutations in P/rds. Studies put forth in this application will provide biochemical and physiological evidence to define the role of P/rds, and its associated proteins, during genesis of rod and cone OSs. Also, the influence of the disc rim region on phototransductory signal transmission to the PM.

[unreadable] DESCRIPTION (provided by applicant): Our research objective is to use DNA nanotechnology and helper-independent Sleeping Beauty Trasposon-Transposase motif to develop a novel and effective therapeutic strategy for eye diseases; particularly those associated with loss-of-function mutations in the retina and retinal pigment epithelium (RPE). Successful application of ocular therapy is contingent upon the efficient delivery and targeting of therapeutic agents in a cell-specific manner. We propose to test the hypothesis that compacted DNA nanoparticles is an effective, efficient, and well-tolerated method for therapeutic gene delivery and targeting to the retina and RPE cells to respectively battle diseases of the retina and RPE. Since nanoparticles are small and condensed, they can pass safely through the cell membrane as well as the nuclear membrane and into the nucleus. Our experimental plan requires three components: engineered nanparticles, genetically engineered therapy, and animal models of ocular diseases, all of which are currently available in our laboratory. Therapeutic rescue of retinal disease will be assessed functionally using electroretinography (ERG) and multifocal ERG, structural analysis will be assessed using immunofluorescence, light and electron microscopy, and biochemical analysis will use real time RT-PCR, RNase protection, Western blot, and ELISA analysis. When taken together, these studies will provide valuable insight into non-viral gene therapy as a prospective method for the treatment of inherited blinding diseases. [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY We aim to advance our current DNA nanoparticle (NP) delivery platform and gene expression to develop safe and effective therapies targeting important photoreceptor-associated ocular disorders caused by defects in large genes. We are merging knowledge in molecular bioengineering, nanoformulation, material science, eye biology/physiology and chemistry to accelerate essential steps for the generation of effective non-viral gene delivery platform for eye diseases. The NPs consist of single molecules of DNA compacted with poly-lysine- PEG polycation and have a diameter of 8-11 nm. Their small size coupled with cellular uptake via cell-surface nucleolin, which efficiently traffics the NPs to the nucleus, accounts for their ability to transfect post-mitotic photoreceptors. Using NPs has led to efficient expression of large genes, an essential prerequisite for targeting hard-to-rescue diseases of the retina. Although upper gene size limitation has not been established, the largest size tested in the lung was 20 kb and in the eye was ~14 kb making NPs an ideal alternative to AAVs for delivery of large genes. We have showed that NP treatment led to efficient transfection of ocular cells including photoreceptors and retinal pigment epithelium, exerted no toxic effects on the eye even after multiple injections, distributed throughout the subretinal space, and mediated appreciable structural/functional rescue in mouse models of retinitis pigmentosa, Leber?s congenital amaurosis, Stargardt?s and diabetic retinopathy. Effective gene expression without toxicity has also been achieved in baboon eyes. These proof- of-principle studies confirms the potential clinical significance of this technology and highlights the value of a large capacity, but emphasized the need for prolonged high levels of gene expression. Our main goal is to enhance photoreceptor uptake of NPs to achieve long-term therapeutic levels of expression of large genes for full phenotypic rescue. We propose to accomplish this by targeted vector engineering to boost expression levels along with NP-delivery platform to enhance their delivery from the vitreous to photoreceptors, achieve pan retinal distribution, promote episomal stability in the nucleus and prevent epigenetic silencing. Subsequently, we will test these optimized NPs/delivery platform(s) for their ability to mediate full phenotypic rescue in a large gene disease model; specifically the Abca4-/- model of Stargardt?s associated with the lack of ABCA4 gene. ABCA4 is a large gene which has not been fully rescued by traditional viral vectors and as a result development of targeted therapeutics for Stargardt?s has lagged. We plan in aim1 to engineer vectors that can achieve therapeutic levels of expression in photoreceptors and in aim 2 to develop effective NP- delivery platforms to enhance photoreceptor uptake of NPs from the vitreous. Aim 3 will test the efficacy of the best delivery platform of the most effective NPs for therapeutically-effective levels of expression in photoreceptors of Abca4-/- mice before and after the onset of the disease. In summary, results from this application will facilitate the advancement of DNA NP use for ocular diseases associated with large genes.

DESCRIPTION (provided by applicant): Human blinding disorders are often initiated by hereditary mutations that impact rod and/or cone photoreceptors (PRs) and cause subsequent cellular death. Generally, the disease phenotype can be predicted from the specific mutation since many PR genes are specific to rods or cones;however certain genes, such as Retinal Degeneration Slow (Rds), are expressed in both cell types and cause different forms of retinal disease affecting rods, cones, or both. Rds is a member of the tetraspanin family known to interact with other proteins to build a membrane domain responsible for the morphogenesis of the rod and cone PR disc rim. Without this protein the outer segments (OSs) fail to develop, and over 80 different Rds mutations have been shown to cause several inherited retinal diseases. The goals of this program are: 1) to uncover the mechanisms underlying the differential behavior of Rds in rods and cones;2) to identify Rds'interacting partners from both PRs types;and 3) to determine how Rds complexes traffic to OSs and assemble the disc rim. Our ultimate goal is to determine how the structure of Rds, Rds/Rom-1 complexes, and Rds interactions with secondary unknown binding partners affect the development of the OS and the formation of the rim microdomain. Using biochemical, structural and functional approaches along with different genetically modified mouse models, we have shown that Rds functions differently in rods vs. cones, Rds inter-molecular disulfide oligomerization occurs in the OS, and that Rds traffics in the inner segment as homo- and hetero-tetramers with its partner Rom-1. In this application, we will test the hypothesis that the differential roles of Rds in rods and cones are a result of the presence of distinct Rds-associated partners in the rod OS vs the cone OS. Four aims are proposed to test our hypotheses. Aim 1 will identify Rds/Rom-1 associated partners involved in Rds complex trafficking and OS assembly in rods &cones. In this aim, we will use proteomic studies on affinity purified Rds complexes from various retinal samples isolated from different genetically modified models to identify and study interacting proteins. Aim 2 will determine the functional role of the Rds C-terminal region in rod and cone OS morphogenesis. Transgenic mice expressing a chimeric protein made of the body of Rom-1 but with the Rds C-terminal region have been generated and will be evaluated structurally, functionally, and biochemically. Aim 3 will evaluate the role of Rds in cone OS lamellae formation. We have shown that in the absence of Rds, rods do not form OSs and enter apoptosis, whereas cone PRs develop viable but dysmorphic OS structures devoid of lamellae that are able to photo-transduce. Aim 4 will study the differential role of certain Rds residues in rod vs. cone OSs morphogenesis and maintenance. Experiments put forth in this application will provide biochemical and physiological evidence for the role of Rds and its associated proteins during genesis of rod and cone OSs. PUBLIC HEALTH RELEVANCE: This proposal is designed to further our understanding of the role of Rds in outer segment morphogenesis and maintenance. Mutations in the Rds gene lead to a variety of debilitating retinal disorders. Our studies will extend our fundamental knowledge of Rds and other molecules involved in normal disc membrane morphogenesis and turnover processes, both steps are essential for normal vision but poorly understood. Also, our work will provide a basis for understanding how different mutations in the Rds gene can result in diverse retinal diseases. We will employ different genetically modified mouse models to test our main hypothesis that in rods, Rds plays an important role in rod rim outer segment morphogenesis and closure of the disc rim while in cones, Rds is only necessary for the evagination step of the lamellae formation. We also hypothesize that the differential behavior of Rds in rods vs. cones is a function of differences in Rds-associated protein partners and in this application we propose to identify these partners and test their functional role in normal and diseased retinas.

DESCRIPTION (provided by applicant): The goal of this program is to advance current DNA nanoparticle (NP) delivery and expression technologies to develop safe and effective therapies for important ocular disorders affecting the photoreceptor (PR) and retinal pigment epithelial (RPE) cells. The program will merge experts with molecular bioengineering, vision science, physics, and chemistry to accelerate essential steps for the generation of effective ocular non-viral gene therapy. The DNA NPs consist of single molecules of DNA compacted with lysine-PEG polycations and have a minimum diameter of 8-11 nm. Their small size, coupled with a specific uptake mechanism that efficiently traffics the NPs to the nucleus and bypasses the lysosomal degradation system, likely accounts for the ability of the NPs to robustly transfect post-mitotic, differentiated cells. The counter-ion used at the time of formulation determines the NP shape and both rod-like and ellipsoidal NPs show robust transfection of ocular cells, including RPE, PRs and retinal ganglion cells (RGCs). Moreover, murine RDS NPs have demonstrated partial phenotypic correction in a retinitis pigmentosa mouse model of RDS haploinsufficiency. Having previously shown proof-of-principle for the effective use of these NPs in the eye, in this application we will take the necessary steps to optimize the particles for clinical ocular use. First, we will optimize the NP formulation (NP shape, size, and chemical composition) for PR- and RPE-specific gene transfer (Aim #1). Second, we will engineer clinically-appropriate DNA vectors (Aim #2) to generate persistent, high levels of transgene expression. Thirds, we will test the ability of these NPs to target the macula in a non-human primate model (baboon) (Aim #3) including an assessment of whether non-invasive intravitreal delivery of NPs can transfect macular/foveal cones. This is a novel and critical development for clinical application of this technology, as many retinal degenerations target the macula. Furthermore, it is a step that cannot be modeled by a mouse system due to the absence of a macula in the rodent retina. We will also conduct toxicology and DNA biodistribution studies in baboons, including a detailed evaluation of brain visual pathways. Building on the documented ability of the current NP formulation to penetrate deep retinal layers, we hypothesize that this efficiency can be improved by the NP formulation optimization program detailed in Aim #1, enabling robust foveal cone gene transfer. In summary, results from this application will facilitate preclinical trial evaluations of DNA NPs for ocular gene delivery. PUBLIC HEALTH RELEVANCE: This program is designed to advance our compacted DNA nanotechnology to facilitate its future use as a clinical gene therapy treatment for ocular diseases. Preclinical studies are planned to lay the foundation for use of this technology as a clinically viable gene delivery system for ocular diseases such as retinitis pigmentosa (RP), various macular dystrophies (MD), and diabetic retinopathy. The minimum diameter (8-11 nm) of our NPs is much smaller than any viral vector, and may permit uniquely effective and safe intravitreal ocular dosing to deliver genes into RPE cells and foveal cones.

DESCRIPTION (provided by applicant): Retinal degeneration slow (RDS) is a photoreceptor-specific glycoprotein critical for rod and cone outer segment (OS) rim formation and maintenance. Rim formation is critical for OS structure; in the absence of RDS no rod OSs are formed and no rod function is detected while cones develop dysmorphic balloon-like OSs that lack lamellae but contain cone opsins and retain ~50% of function. Developing a thorough understanding of the role and function of RDS in rods and cones is critical as over 100 different mutations in RDS cause retinal degenerative diseases with widely varying phenotypes including rod-dominant autosomal dominant retinitis pigmentosa (ADRP) and multiple classes of cone-dominant macular dystrophy (MD). Studies have suggested that mutations that cause rod-dominant disease result in null alleles likely due to protein misfolding while mutations that cause cone-dominant disease have a more complex pathology. Assessment of disease in patients has shown that in many cases, RDS-associated macular vision loss is not only associated with primary defects in photoreceptors, but with subsequent secondary toxicity and changes in the retinal pigment epithelium (RPE) and choroidal/retinal vasculature. Nothing is known about the link between these primary defects and the blinding secondary sequellae; however, for the first time we have generated mouse models which facilitate study of this issue. The diversity in RDS-associated phenotypes seen in patients coupled with data from cone and rod- dominant mouse models suggests that rods and cones have different requirements for RDS. It is not clear why these two cell types should be so differently affected by different mutations, primarily because our current knowledge of the precise function of RDS in these two cell types is incomplete. Here we plan to focus our investigation on understanding how RDS functions differently in rods and cones with particular interest in RDS initial complex formation and trafficking, role in cone vs. rod OS structures, and effects of RDS mutations on overall retinal health. We will conduct a careful assessment of the molecular properties of RDS and RDS mutants, with specific regard to complex assembly in the IS and trafficking to the OS in Aim 1; and study the role of RDS in OS structures, disc/rim formation, and disc sizing in Aim 2. Of particular interest will be differences in these processes between rods and cones. Links between RDS-mutation-associated photoreceptor defects and alterations in the RPE and choroid will be assessed in Aim 3. We have a set of unique tools at our disposal and advanced genetically engineered mouse models to study how mutations in RDS result in diverse retinal pathologies as a consequence of abnormal OS formation and the role of RDS in the closed rim structure in rods and the open rim structures in cones. Given the lack of effective treatments for RDS-associated disease these studies are essential.

PROJECT SUMMARY Flavins are essential cofactors for wide-ranging metabolic processes; hence they are particularly critical in highly metabolically active tissues. The retina, in which levels of flavins are several folds higher than in blood, is an important example. The physiological significance of modulating levels of retinal flavins is underscored by the observation that riboflavin deficiency results in photosensitivity and degeneration, a process thought to result from lipid peroxidation. Unbound flavins are toxic so, in practice, flavins are virtually always bound to flavin binding proteins. Although tight regulation of flavin levels is clearly critical for maintaining retinal homeostasis, nothing is known about the mechanisms governing their uptake, regulation, or binding proteins. We have recently begun studying a novel, and highly relevant candidate retinal flavin binding protein called Retbindin (Retb). Retb has homology to chicken riboflavin binding protein (RBP), and we have shown in vitro and in retinal explant that Retb binds flavins. This, coupled with the importance of flavins in the retina, led us to hypothesize that Retb's function is tied to flavin regulation. We recently reported that Retb is exclusively expressed by rods, secreted into the interphotoreceptor matrix (IPM) and maintained via electrostatic forces at the interface between photoreceptors and retinal pigment epithelium (RPE) microvilli, a region critical for retinal function and homeostasis. This localization combined with its ability to bind flavins, implicates Retb as a potential carrier of flavins between the retina and the RPE. To further assess Retb's function in the retina, we generated a knockout mouse (Retb-/-), in which Retb sequence was replaced with eGFP. Electroretinography revealed an age- and dose-dependent decline in rod and cone responses at postnatal days (P) 120 and 240 and a concurrent degeneration of rods and cones. We also show flavin levels significantly reduced in P45 Retb-/- retina, prior to the onset of degeneration. In light of the potential pathological consequences of elimination of Retb, coupled with a complete lack of knowledge of Retb function, we propose to explore the role of flavins in the retina and determine how Retb could regulate their levels. Specifically, we propose three aims. First, we will determine the functional role of Retb in the photoreceptor cells by evaluating Retb-/- and Retb+/- animals at different ages and under various lighting/dietary treatments as well as identify Retb binding partners. Second, we will take a metabolomic approach to identify the metabolic pathways that are affected by the absence of Retb. Third, because we observed levels of Retb are significantly elevated in the retinas of animal models of retinal diseases, we will test the role Retb plays in the development and progression of retinal degenerations such as retinitis pigmentosa. We have also initiated the generation of a Retb over-expresser mouse model to be used to assess the capability of Retb in ameliorating disease progression. These experiments are highly significant, not just to further our knowledge of a poorly understood protein critical for retinal function, but also to explore the role of metabolic dysregulation in retinal homeostasis and diseases.