RESUMO
X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a putative secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h-/- mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, although photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.
Assuntos
Proteínas do Olho/genética , Terapia Genética/métodos , Retina/citologia , Envelhecimento , Animais , Moléculas de Adesão Celular/genética , Modelos Animais de Doenças , Eletrorretinografia , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Técnicas de Cultura de Órgãos , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/fisiologia , Retinosquise/genética , Retinosquise/terapiaRESUMO
Delivery of therapeutic genes to a large region of the retina with minimal damage from intraocular surgery is a central goal of treatment for retinal degenerations. Recent studies have shown that AAV9 can reach the central nervous system (CNS) and retina when administered systemically to neonates, which is a promising strategy for some retinal diseases. We investigated whether the retinal transduction efficiency of systemically delivered AAV9 could be improved by mutating capsid surface tyrosines, previously shown to increase the infectivity of several AAV vectors. Specifically, we evaluated retinal transduction following neonatal intravascular administration of AAV9 vectors containing tyrosine to phenylalanine mutations at two highly conserved sites. Our results show that a novel, double tyrosine mutant of AAV9 significantly enhanced gene delivery to the CNS and retina, and that gene expression can be restricted to rod photoreceptor cells by incorporating a rhodopsin promoter. This approach provides a new methodology for the development of retinal gene therapies or creation of animal models of neurodegenerative disease.
Assuntos
Sistema Nervoso Central , Dependovirus/genética , Terapia Genética , Retina/patologia , Degeneração Retiniana/terapia , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Regiões Promotoras Genéticas , Retina/citologia , Retina/crescimento & desenvolvimento , Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Rodopsina/genéticaRESUMO
Neuropeptide Y (NPY)-expressing neurones in the arcuate nucleus densely innervate many hypothalamic nuclei. To determine the neurochemical phenotype of target neurones for NPY, we studied the immunohistochemical localization of the NPY Y1 receptor (Y1R) in discrete subpopulations of neurones in the rat hypothalamus. Among several tested populations, including hypocretin/orexin-, melanin-concentrating hormone (MCH)- and nitric oxide synthase (NOS)-positive neurones, only the latter were found to coexpress the Y1R. Numerous Y1R/NOS-positive neurones were found as a densely packaged group of cells located ventrolateral to the ventromedial nucleus, forming a band ascending towards the fornix. Lower numbers of Y1R/NOS-positive neurones were found in the perifornical area and in the peri- and paraventricular nuclei. Expression of the Y1R gene was found in the same locations in the mouse by colocalizing beta-galactosidase, a Y1R gene reporter, with NOS in a Y1R knockout mouse. To explore possible downstream targets of NO in the rat hypothalamus, the NO-regulated molecule cGMP was analysed immunohistochemically after incubation of brain slices with sodium nitroprusside, an NO donor. We observed several cGMP-positive cell bodies in the arcuate nucleus, cGMP-positive blood vessels and a cGMP-positive network of thin fibres, some of which colocalized with choline acetyltransferase.
