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ABSTRACT: RPGR-interacting protein-1 (RPGRIP1) is localized in the photoreceptor-connecting cilium, where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an adeno-associated virus serotype 8 (AAV8) vector, used a rhodopsin kinase gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. After subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in photoreceptors, localized correctly in the connecting cilia, and restored the normal localization of RPGR. Electroretinogram and histological examinations showed better preservation of rod and cone photoreceptor function and improved photoreceptor survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect.
Human gene therapy 04/2010; 21(8):993-1004. · 4.20 Impact Factor
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Hyeong Gon Yu,
Xiaoqing Liu,
Szilard Kiss,
Edward Connolly,
Evangelos S Gragoudas,
Norman A Michaud, Oleg V Bulgakov,
Michael Adamian,
Margaret M DeAngelis,
Joan W Miller,
Tiansen Li,
Ivana K Kim
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ABSTRACT: Age-related degradation of the elastic lamina in Bruch's membrane may have a permissive effect on the growth of choroidal neovascularization (CNV). This study investigated the influence of defective elastic fiber maintenance in the development of laser-induced CNV.
A mouse lacking lysyl oxidase-like (LOXL)-1, an enzyme essential for elastin polymerization, was studied. The morphologic characteristics of the elastic lamina within Bruch's membrane were examined in mutant and wild-type (WT) eyes. Laser-induced CNV was evaluated by fluorescein angiography and choroidal flat mounts. Immunohistochemistry for elastin was performed on the CNV lesions, and vascular endothelial growth factor (VEGF) levels were determined by ELISA. Soluble elastin and matrix metalloproteinase (MMPs) levels were also analyzed by immunoblotting.
The elastic lamina of Bruch's membrane in the LOXL1-deficient mice was fragmented and less continuous than in the WT controls. The mutant mice showed increased levels of soluble elastin peptides and reduced elastin polymer deposition in neovascular membranes. Significantly larger CNV with greater leakage on fluorescein angiography developed in mutant mice. VEGF levels in the RPE/choroid were higher in the knockout mice on days 7 and 14 after laser (P < 0.05). MT1-MMP (MMP14) was also elevated after laser in the LOXL1 mutant eyes compared to the WT controls.
These results show that a systemic defect in elastic fiber deposition affects Bruch's membrane integrity and leads to more aggressive CNV growth. The latter may be partially mediated by abnormal signaling from the accumulation of soluble elastin peptides.
Investigative Ophthalmology & Visual Science 06/2008; 49(6):2599-605. · 3.60 Impact Factor
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ABSTRACT: Gene therapy for retinal degeneration requires well-defined promoters that drive expression in rod and cone photoreceptors. This study was undertaken to develop short, active derivatives of the human rhodopsin kinase (RK) gene promoter for targeting transgene expression in rods and cones. RK, also known as G protein-coupled receptor kinase 1 (GRK1), is a component of the light adaptation pathway expressed in rods and cones.
Human RK (hRK) promoter and its concatemers or derivatives extending into the conserved 5' untranslated region (5'-UTR) were assayed for promoter activity in WERI retinoblastoma or Crx/Sp1-supplemented HEK-293 cells. The derivative displaying the highest activity was linked to a GFP reporter and packaged in a pseudotyped adenoassociated viral vector (AAV2/5). The AAV vector was tested in vivo by subretinal injections in wild-type mice, in the all-cone Nrl(-/-) mice, and in the cone-rich diurnal Nile grass rat (Arvicanthis niloticus). Control eyes received a similar AAV2/5 vector carrying a mouse rod opsin (mOps) promoter-controlled GFP reporter.
The hRK promoter with the full 5' untranslated sequence (-112 to +180) was the most active in cell culture. Delivered by the AAV2/5 vector, RK promoter drove GFP expression specifically in photoreceptors. In rods, hRK promoter-mediated expression was as efficient as, but appeared more uniform than, mOps promoter-mediated expression. In cones, the hRK promoter drove expression, whereas the mOps promoter did not.
The hRK promoter is active and specific for rod and cone photoreceptors. Because of its small size and proven activity in cones, it is a promoter of choice for somatic gene transfer and gene therapy targeting rods and cones.
Investigative Ophthalmology & Visual Science 10/2007; 48(9):3954-61. · 3.60 Impact Factor
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ABSTRACT: Usher syndrome type IIA (USH2A), characterized by progressive photoreceptor degeneration and congenital moderate hearing loss, is the most common subtype of Usher syndrome. In this article, we show that the USH2A protein, also known as usherin, is an exceptionally large ( approximately 600-kDa) matrix protein expressed specifically in retinal photoreceptors and developing cochlear hair cells. In mammalian photoreceptors, usherin is localized to a spatially restricted membrane microdomain at the apical inner segment recess that wraps around the connecting cilia, corresponding to the periciliary ridge complex described for amphibian photoreceptors. In sensory hair cells of the cochlea, it is associated transiently with the hair bundles during postnatal development. Targeted disruption of the Ush2a gene in mice leads to progressive photoreceptor degeneration and a moderate but nonprogressive hearing impairment, mimicking the visual and hearing deficits in USH2A patients. These data suggest that usherin is required for the long-term maintenance of retinal photoreceptors and for the development of cochlear hair cells. We propose a model in which usherin in photoreceptors is tethered via its C terminus to the plasma membrane and its large extracellular domain projecting into the periciliary matrix, where they may interact with the connecting cilium to fulfill important structural or signaling roles.
Proceedings of the National Academy of Sciences 04/2007; 104(11):4413-8. · 9.68 Impact Factor
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ABSTRACT: Sonic hedgehog (Shh) signaling is required for the growth and patterning of many tissues in vertebrate embryos, but important aspects of the Shh signal transduction pathway are poorly understood. For example, the vesicle transport protein Rab23 is a cell autonomous negative regulator of Shh signaling, but the process affected by Rab23 has not been defined. Here, we demonstrate that Rab23 acts upstream of Gli transcription factors in patterning neural cell types in the spinal cord. Double mutant analysis indicates that the primary target of Rab23 is the Gli2 activator and that Rab23 and Gli3 repressor have additive effects on patterning. Analysis of Gli3 protein suggests that Rab23 also has a role in promoting the production of Gli3 repressor. Although the membrane proteins Patched and Smoothened change subcellular localization in response to Shh, double mutant analysis demonstrates that Rab23 does not work through either Patched or Smoothened. Instead, Rab23 appears to regulate subcellular localization of essential components of the Hedgehog pathway that act downstream of Smoothened and upstream of Gli proteins.
Developmental Biology 03/2006; 290(1):1-12. · 4.07 Impact Factor
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ABSTRACT: Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a member of the FK-506-binding protein family expressed specifically in retinal photoreceptors. Mutations in AIPL1 cause Leber congenital amaurosis, a severe early-onset retinopathy that leads to visual impairment in infants. Here we show that knockdown of AIPL1 expression in mice also produces a retinopathy but over a more extended time course. Before any noticeable pathology, there was a reduction in the level of rod cGMP phosphodiesterase (PDE) proportional to the decrease in AIPL1 expression, whereas other photoreceptor proteins were unaffected. Consistent with less PDE in rods, flash responses had a delayed onset, a reduced gain, and a slower recovery of flash responses. We suggest that AIPL1 is a specialized chaperone required for rod PDE biosynthesis. Thus loss of AIPL1 would result in a condition that phenocopies retinal degenerations in the rd mouse and in a subgroup of human patients.
Proceedings of the National Academy of Sciences 10/2004; 101(38):13903-8. · 9.68 Impact Factor
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ABSTRACT: Sonic hedgehog (SHH) is a secreted morphogen that regulates the patterning and growth of many tissues in the developing mouse embryo, including the central nervous system (CNS). We show that a member of the FK506-binding protein family, FKBP8, is an essential antagonist of SHH signaling in CNS development. Loss of FKBP8 causes ectopic and ligand-independent activation of the Shh pathway, leading to expansion of ventral cell fates in the posterior neural tube and suppression of eye development. Although it is expressed broadly, FKBP8 is required to antagonize SHH signaling primarily in neural tissues, suggesting that hedgehog signal transduction is subject to cell-type specific modulation during mammalian development.
Development 06/2004; 131(9):2149-59. · 6.60 Impact Factor
