RESUMEN
BACKGROUND: Photoreceptor (PR) and retinal pigment epithelium (RPE) are the principal cell targets in retinal gene therapy. Recombinant adeno-associated virus (rAAV) has emerged as a very promising vector for gene therapy in hereditary retinal diseases. Gene transfer at different stages of the disease is a practical consideration for future clinical application. METHODS: A rAAV carrying the enhanced green fluorescent protein gene driven by a cytomegalovirus promoter was produced by either co-infecting the 293 cell line with E1-defective adenovirus and purified by CsCl(2) density gradient (CsCl(2)-rAAV), or by transfecting with an adenoviral helper plasmid and purified by iodixanol density gradient followed by heparin column chromatography (heparin-rAAV). The impact of different virus preparations on the patterns of transgene expression was investigated after subretinal injection. Furthermore, rAAV-mediated gene transfer was evaluated at both early and advanced stages of retinal degeneration in four disease models including the RCS rat, rd, RPE(65) (-)/(-) and cathepsin D mutant mice that are associated with PR- or RPE-related gene defects. RESULTS: CsCl(2)-rAAV predominantly transduced RPE and with less efficiency in PR. In contrast, heparin-rAAV predominantly transduced PR but with much less efficiency in RPE. Subretinal injection of either rAAV preparation induced no changes to retinal morphology and retinal-choroidal vasculature. The product of transgene, however, could be observed in multiple tracts in the brain. In the four disease models, target cells were efficiently transduced not only at the early stage, but also at the late stage of disease as long as the target cells were present. CONCLUSIONS: Different preparations of rAAV have an impact on the patterns of transgene expression after subretinal injection. Patients at advanced stages of retinal degeneration may still benefit from rAAV-mediated gene therapy. The possible side effects of transgenic products on the central nervous system should be carefully monitored once therapeutic genes are employed.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Terapia Genética , Degeneración Retiniana/terapia , Animales , Encéfalo/metabolismo , Línea Celular , Virus Defectuosos/genética , Vectores Genéticos , Proteínas Fluorescentes Verdes , Virus Helper/genética , Heparina/genética , Humanos , Proteínas Luminiscentes , Ratones , Ratones Mutantes , Células Fotorreceptoras/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , TransgenesRESUMEN
Diabetic retinopathy, one of the most serious complications of long-term diabetes, could clinically be divided into two stages: 1) background retinopathy that does not cause visual impairment and 2) proliferative retinopathy, which is a potentially blinding condition. This study aims to investigate the correlation between enhancement of vascular endothelial growth factor (VEGF) expression and neovascular changes. A binary recombinant adeno-associated virus construct producing green fluorescent protein (GFP) and VEGF under the control of the human cytomegalovirus promoter, recombinant adeno-associated virus (rAAV).VEGF.GFP, was produced and injected into the subretinal space of C57BL mice. GFP expression was tracked by fluorescence fundus photography, and VEGF expression was confirmed by immunohistochemistry and enzyme-linked immunoassay. Neovascular changes were monitored by fluorescein angiography and histology and by quantifying the number of inner retinal vessels. GFP expression was found in 100% of injected eyes, and vascular changes were detected in 9 of 10 rAAV.VEGF.GFP-injected eyes. Of these, four demonstrated microaneurysms and five showed moderate to severe leakage. There was a statistically significant increase in blood vessel number in the inner nuclear layer (P < 0.03) and dilatation of retinal veins (P < or = 0.05). This work has demonstrated that the development of different stages of diabetic retinopathy is closely correlated with an increased VEGF level in the retina.
Asunto(s)
Dependovirus/genética , Retinopatía Diabética , Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Linfocinas/genética , Retina/metabolismo , Animales , Citomegalovirus/genética , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Factores de Crecimiento Endotelial/análisis , Ensayo de Inmunoadsorción Enzimática , Angiografía con Fluoresceína , Expresión Génica , Vectores Genéticos , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/análisis , Proteínas Luminiscentes/genética , Linfocinas/análisis , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión , Retina/química , Vasos Retinianos/patología , Vasos Retinianos/fisiopatología , Transfección , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , VasodilataciónRESUMEN
The expression of vascular endothelial growth factor has been strongly implicated in the pathogenesis of conditions leading to inappropriate blood vessel growth in the eye. As such, vascular endothelial growth factor is an attractive target for anti-angiogenic therapies designed to treat neovascular eye diseases. One such therapy, antisense gene therapy, is a technique based on the ability of single-stranded DNA or RNA sequences to alter the expression of targeted genes. Recombinant adenoviruses have demonstrated efficient ocular cell transduction with a high level of transgene production. Cauterization of the normally avascular rat cornea results in a strong neovascular response, making it an ideal animal model for the testing of anti-angiogenic therapies. In this study, a recombinant adenovirus system was assessed for the ability to express biologically relevant antisense RNA to reduce vascular endothelial growth factor expression in a rat model of corneal neovascularization. Recombinant adenovirus constructs expressing short and long antisense and sense vascular endothelial growth factor cDNA, under the control of cytomegalovirus major immediate early promoter or the RNA polymerase III promoter, VA1, were constructed. The expression of short and long antisense RNAs was demonstrated by Northern blot hybridization. All constructs were capable of producing RNA, and the highest level of antisense RNA production was detected in retinal pigment epithelial cells which had been transduced with the longer antisense cDNA construct under the control of the VA1 promoter. This construct was also the most efficient in reducing in vitro vascular endothelial growth factor production (P<0.05) and human endothelial cell proliferation. This construct was subsequently injected into rat eyes 24hr prior to cauterization of the cornea and antisense vascular endothelial growth factor expression was demonstrated by in situ hybridization. The resulting neovascular response was clearly inhibited at 4, 7 and 14 days post-cautery, compared to the control injections which demonstrated an intense neovascular response. Only one out of six eyes injected with the long antisense cDNA construct under the control of the VA1 promoter demonstrated any vascular response to cautery. The reduction in the neovascular response was correlated, with significantly lower amounts of vascular endothelial growth factor protein in the corneas (P=0.006). These observations suggest that the specific down-regulation of vascular endothelial growth factor production is sufficient to reduce the corneal neovascular response and that recombinant adenovirus might be a useful vehicle to produce antisense RNA in situ to down-regulate ocular gene expression.