RESUMO
Loss of cone function in the central retina is a pivotal event in the development of severe vision impairment for many prevalent blinding diseases. Complete achromatopsia is a genetic defect resulting in cone vision loss in 1 in 30,000 individuals. Using adeno-associated virus (AAV) gene therapy, we show that it is possible to target cones and rescue both the cone-mediated electroretinogram response and visual acuity in the Gnat2 ( cpfl3 ) mouse model of achromatopsia.
Assuntos
Defeitos da Visão Cromática/terapia , Modelos Animais de Doenças , Terapia Genética , Células Fotorreceptoras Retinianas Cones/fisiologia , Animais , Defeitos da Visão Cromática/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Both subretinal dosing and intravitreal (IVT) dosing of adeno-associated virus (AAV) in higher species induce mild and transient inflammatory responses that increase with dose. Foreign protein and foreign DNA are known inducers of inflammation, which is also true in the immune-privileged ocular environment. We explored which component(s) of AAV vectors, viral capsid, or viral DNA drive inflammatory responses. Recombinant AAV with three tyrosine to phenylalanine substitutions in the capsid of AAV serotype 2 (rAAV2tYF), and with a generic ubiquitous promoter (cytomegalovirus [CMV]) controlling the expression of humanized green fluorescent protein (hGFP), was processed to enrich for AAV capsids containing genome (full capsids), capsids without genome (empty capsids), and residual material. Nonhuman primate eyes were injected by IVT in both eyes. During in-life, ocular inflammation and development of neutralizing antibodies (NAb) were measured. Following termination, lymph node immunophenotyping was performed, vitreous was processed for cytokine and RNAseq analyses, and ocular sections were assessed for transgene expression (by in situ hybridization) and histopathology. IVT dosing of AAV vectors transiently raised cellular inflammation in the aqueous and induced a more sustained inflammation in the vitreous. Lowering the total capsid dose by removing empty AAV capsids reduced inflammation and improved viral transduction. IVT dosing of AAV induced systemic NAb to AAV irrespective of the vector preparation. Similarly, lymph node immunophenotyping revealed identical profiles irrespective of viral preparation used for dosing. Immune cells in the vitreous were identified based on RNAseq analysis. Three months postdose, cytokine levels were low, indicative of minimal levels of inflammation in agreement with histopathological assessment of the retina.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Animais , Biomarcadores , Proteínas do Capsídeo/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Endoftalmite/diagnóstico , Endoftalmite/genética , Endoftalmite/terapia , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Genoma Viral , Humanos , Imuno-Histoquímica , Camundongos , Transdução Genética , TransgenesRESUMO
Recombinant adeno-associated viral (rAAV) vector-mediated gene therapy is being developed to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. In preparation for a clinical gene therapy trial, we conducted dose range finding (DRF) studies with an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF) vector administered by subretinal injection in a naturally occurring RPGR-mutant canine model (XLPRA2) to compare two different human RPGR (hRPGR) transgenes and to establish a reasonable starting dose for a clinical trial. Different dose levels of two candidate vectors (0.15 mL at 1.2 × 1010-3.0 × 1012 vg/mL of rAAV2tYF-GRK1-hRPGRco or 4 × 1010-3.0 × 1012 vg/mL of rAAV2tYF-GRK1-hRPGRstb), 6.0 × 1011 vg/mL rAAV5-GRK1-hRPGRco reference vector or Vehicle were subretinally administered, and the dogs were followed for 8 weeks postdose. Ophthalmic examinations, analyses of retinal structure by in vivo imaging using confocal scanning laser ophthalmoscopy (cSLO)/optical coherence tomography (OCT) in the Lower (4.0 × 1010 vg/mL) and Lowest (1.2 × 1010 vg/mL) Doses, immunological responses by cell based assays or enzyme-linked immunosorbent assay, RPGR transgene expression, and reversal of opsin mislocalization by immunohistochemistry were performed. No sustained signs of ocular discomfort or ophthalmic complications were noted in any of the injected eyes except some in the High Dose group (3.0 × 1012 vg/mL), which showed signs of retinal detachment and inflammation. A change in fundus reflectivity suggestive of a rescue effect was seen in the High, Mid (6.0 × 1011 vg/mL), and Low (1.2 × 1011 vg/mL) Dose groups. cSLO/OCT demonstrated qualitative and quantitative evidence of rescue effect in eyes treated with the Lower Dose. Anti-hRPGR antibodies were absent, but neutralizing antibody titers against AAV2 were detected in all animals dosed with rAAV2tYF in an apparent dose-related pattern. RPGR expression was stronger for rAAV2tYF-GRK1-hRPGRco compared to rAAV2tYF-GRK1-hRPGRstb at all dose levels. Subretinal administration of rAAV2tYF-GRK1-hRPGRco and rAAV2tYF-GRK1-hRPGRstb both corrected rod and cone opsin mislocalization, two early markers of disease in the XLPRA2 canine model of RPGR-XLRP. These results support the selection and use of rAAV2tYF-GRK1-hRPGRco (AGTC-501) and guided the initial doses in clinical studies in patients with XLRP caused by RPGR mutations.
Assuntos
Dependovirus/genética , Proteínas do Olho/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Mutação , Retinose Pigmentar/terapia , Animais , Cães , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Vetores Genéticos/genética , Masculino , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , TransgenesRESUMO
PURPOSE: Anterior chamber-associated immune deviation (ACAID) is a form of peripheral tolerance achieved via intracameral antigen inoculation. It is well known that ACAID effectively down-regulates potentially destructive immunities such as delayed-type hypersensitivity (DTH) at extraorbital sites. However, what has not been specifically addressed is whether local intraocular tissues are negatively affected from intracamerally placed antigen. Thus, the current study was undertaken to detect and characterize potential pathological effects on intraocular tissues following intracameral inoculation with alloantigen. METHODS: ACAID induced in C57BL/6 hosts via intracameral inoculation with allogeneic (BALB/c) splenocytes was confirmed by the absence of DTH reactivity in the periphery. Injuries to the anterior segment and neuroretina following intracameral inoculation were evaluated pathologically via histological evaluation, molecularly via upregulation of glial fibrillary acidic protein (GFAP), and functionally via assessment of photoreceptor degeneration and electroretinogram (ERG) out to 24 days. In all experiments, intracamerally inoculated mice were compared to sham-operated, and controlled lens-punctured mice--a procedure that elicits intracameral inflammation for positive identification of immunopathological changes. RESULTS: Inflammation of anterior segment tissues persisted out to eight days, despite evidence that significant clearance of allogeneic cells took place within 6 h. In the neuroretina, a transient loss in ERG B-wave amplitudes was detected, but photoreceptor degeneration and GFAP upregulation were not. CONCLUSIONS: Intracameral inoculation with alloantigen leads to anterior segment inflammation and ERG dysfunction; however, this was markedly reduced and transient when compared to strong anterior segment inflammation induced by a more serious lens-puncture wound.
Assuntos
Câmara Anterior/imunologia , Olho/imunologia , Olho/patologia , Tolerância Imunológica , Isoantígenos/imunologia , Animais , Segmento Anterior do Olho/patologia , Eletrorretinografia , Gliose/imunologia , Gliose/patologia , Inflamação/imunologia , Inflamação/patologia , Injeções , Isoantígenos/administração & dosagem , Cristalino/lesões , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Fotorreceptoras/patologia , Retina/imunologia , Retina/fisiopatologia , Baço/imunologia , Fatores de Tempo , Ferimentos Penetrantes/patologiaRESUMO
PURPOSE: To determine the feasibility of targeting gene expression specifically to cone photoreceptors using recombinant adeno-associated virus (rAAV) as the vector. METHODS: An rAAV vector was constructed that contains a 2.1kb upstream sequence of the human red opsin gene to direct green fluorescent protein (GFP) expression. A control construct containing a 472bp mouse rod opsin promoter, previously shown to drive photoreceptor-specific expression, was also used. Each recombinant virus was injected into the subretinal space of rat, ferret or guinea pig eyes. GFP expression was analyzed 4-6 weeks after injection microscopically. RESULT: The human 2.1kb cone opsin gene upstream sequence targeted GFP expression only to a subset of photoreceptors. Cone-specific expression was shown by co-localization of GFP fluorescence and cone-specific opsin antibody staining. Additionally, in rats, expression was specific for L/M-cones whereas no S-cones exhibited GFP fluorescence. The efficiency of rAAV mediated cone transduction surrounding the injection site was high since every L/M-cone antibody-staining cone was also positive for GFP expression. CONCLUSION: The human red/green opsin gene promoter used in this study is sufficient to direct efficient cone-specific gene expression in several mammalian species, suggesting that key cell-type specific regulatory elements must be broadly conserved in mammals. These observations have significance in devising gene therapy strategies for retinal dystrophies that primarily affect cones and point toward a way to functionally dissect the cone opsin promoter in vivo.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas de Bastonetes/genética , Animais , Furões , Marcação de Genes/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Cobaias , Humanos , Mamíferos/metabolismo , Camundongos , Regiões Promotoras Genéticas , Ratos , Opsinas de Bastonetes/metabolismo , Especificidade da EspécieRESUMO
PURPOSE: To develop a gene therapy system that specifically targets transgene expression to S-cones of the mammalian retina, the authors coupled recombinant AAV-mediated delivery with the use of a human blue-opsin (HB) promoter to drive expression. METHODS: Two regions of the HB promoter sequence, HB569 and HB996, were amplified from human DNA, cloned into an AAV vector cassette upstream of the green fluorescent protein (GFP) gene, and packaged into AAV2 and AAV5 capsids. Eyes of postnatal day (P) 40 to P48 Sprague-Dawley rats were subretinally injected with 2 muL vector. Animals were humanely killed 2 to 3 weeks or 20 months after injection, and the pattern and persistence of GFP expression were analyzed in the treated retinas by immunohistochemistry, Western blotting, and RT-PCR. RESULTS: AAV5.HB.GFP vectors targeted photoreceptor transduction with an efficiency 20-fold higher than analogous serotype 2 vector. Both AAV5.HB.GFP vectors exhibited similar transduction efficiencies with patterns of GFP expression that did not vary depending on the size of the HB promoter used. Transgene expression was exclusively localized to photoreceptors of retinas treated with either vector. Furthermore, GFP expression was observed for at least 20 months. Dual GFP immunostaining with S- or M-opsin antibodies and GFP/PNA labeling revealed that cones coexpressing S-opsin/GFP or M-opsin/GFP constituted 37.5% +/- 8% and 13.5% +/- 3% of the GFP-positive photoreceptors, respectively, whereas rods constituted 49% +/- 5% of the GFP-positive photoreceptors. Because cones constitute approximately 1% of adult rat retinal photoreceptors, it was estimated that the relative transduction efficiency of AAV5.HB.GFP vectors was approximately 100:1 for cones versus rods. CONCLUSIONS: AAV5.HB.GFP vector injected into the subretinal space of Sprague-Dawley rats targeted gene expression to photoreceptor cells with an efficiency approximately 20-fold higher than that for AAV2.HB.GFP. Transgene expression regulated by the human blue cone-promoter persisted at least for 20 months. Cones coexpressing S-opsin and the GFP transgene appeared to prevail, confirming that in addition to having properties of the AAV serotype, the promoter choice is key to fine-tuning transgene delivery and expression in specific retinal cells. The system described here may be effective in a therapeutic setting in which strong S-cone transgene expression is required.
Assuntos
Regulação da Expressão Gênica/fisiologia , Genes Reporter/genética , Regiões Promotoras Genéticas , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , Animais , Western Blotting , Dependovirus/genética , Marcação de Genes , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Imuno-Histoquímica , Masculino , Microscopia de Fluorescência , Ratos , Ratos Sprague-Dawley , Células Fotorreceptoras Retinianas Cones/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução Genética , TransgenesRESUMO
PURPOSE: We have previously found that the -385 to +86 portion of the mouse rod opsin promoter (mOP500) can limit recombinant adeno-associated virus (rAAV)-mediated transgene expression to photoreceptor cells when delivered subretinally. However, the photoreceptor (PR) subtype-specificity of expression remains unclear. Here, we evaluated whether the presence of certain cis-elements in this proximal promoter, such as the rod-specific, neural retina leucine zipper protein (NRL) response element (NRE), can render it a driver of rod-specific expression. METHODS: Subretinal injections of a serotype 5 rAAV vector carrying the green fluorescent protein (GFP) cDNA, driven by mOP500, were administered to male Sprague-Dawley rats at postnatal day (P) 40-48. Two weeks to eight months later, the distribution of GFP-expressing cells in the retina was characterized by GFP-, cone-specific alpha-transducin-immuno-, and peanut agglutinin-lectin histochemistry and by morphological criteria. The same viral suspension was also injected sub-retinally into rhodopsin-knockout rho (-/-) mice either at P18 or P78, and retinas were analyzed by immunohistochemistry and PNA lectin histochemistry two weeks later. RESULTS: GFP reactivity was found exclusively in the outer nuclear layer (ONL) of rat retinas two weeks after treatment, with abundant reporter gene expression observed in both rods and cones. GFP-positive cones, defined by their typical morphology and the co-linearity of PNA-lectin labeling with GFP-immunoreactivity, were found in all regions of the transduced retinas. GFP-positive cones constituted up to 6% of the total GFP-positive photoreceptors. By eight months post-injection, a low level of GFP-reactivity was additionally observed in the inner nuclear layer (INL) and ganglion cell layer. Photoreceptor-specific GFP expression was also seen in the rho (-/-) mice at both ages tested. In pups injected at P18, costaining with PNA-lectin revealed that up to 15% of the GFP-positive photoreceptors were cones. Despite only a single row of photoreceptors remaining in these knockout mice by P90, numerous GFP-positive cones were still present. CONCLUSIONS: Subretinal delivery of rAAV5 harboring a reporter gene driven by mOP500 results in passenger gene expression in both rod and cones, indicating that this promoter is photoreceptor-specific but not rod-specific. The lack of photoreceptor subtype-specificity suggests that although cones do not express the NRL and NR2E3 trans-factors considered necessary for activation of mOP500, other general transcription factors in cones may compensate.
Assuntos
Dependovirus , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Regiões Promotoras Genéticas , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Opsinas de Bastonetes/metabolismo , Animais , Proteínas de Fluorescência Verde/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas de Bastonetes/genéticaRESUMO
PURPOSE: To generate an animal model of retinal degeneration by using AAV-mediated ribozyme knockdown of the gamma-subunit of the rod cGMP phosphodiesterase (PDEgamma) mRNA in the retina of wild-type mice. METHODS: Two hammerhead ribozymes, HRz35 and HRz42, were designed to target the PDEgamma gene in wild-type C57BL/6 mice. The efficiency and specificity of the ribozyme cleavage was tested in vitro against three different types of target: short synthetic RNA oligomers, longer targets transcribed from clones, and full-length mRNA from total retinal RNA extracts. After in vitro validation, the ribozymes were cloned and packaged in a recombinant adenoassociated virus (rAAV) containing a proximal 472-bp murine rod opsin promoter (MOPS) to drive ribozyme synthesis. Three-week-old wild-type C57BL/6 mice were injected subretinally with the vectors. For treated versus partner control retinas, responses to light were measured by full-field electroretinography (ERG), and retinal tissues were examined by light microscopy. Messenger RNA and protein levels of PDEgamma gene were monitored by reverse transcription-polymerase chain reaction (RT-PCR) and Western immunoblot assay. RESULTS: The ribozymes had comparable in vitro kinetic properties in multiple turnover kinetic analyses. Ribozyme HRz35 exhibited a K(cat) of 0.48 minute(-1) and a K(m) of 980 nM, and HRz42 showed a K(cat) of 0.17 minute(-1) and a K(m) of 971 nM. Both ribozymes cleaved at accessible sites in the RNA, as they digested long transcripts transcribed from clones and full-length mRNA from total retinal RNA extracts in vitro. At increasing intervals after subretinal injection with either AAV ribozyme, a 30% to 90% reduction in a- and b-wave amplitudes was observed compared with those in contralateral control eyes that were not injected. Retinal tissue analysis showed that loss of the photoreceptor cells and PDEgamma mRNA and protein paralleled the ERG results. CONCLUSIONS: Ribozyme-mediated somatic knockdown of wild-type PDEgamma mRNA in vivo can efficiently reduce the target RNA leading to a loss in rod photoreceptors and in rod-mediated ERG amplitudes, thus generating an animal model of retinal degeneration resembling human RP in an essentially normal adult retina. This vector ribozyme technique should be applicable to other genes associated with RP and perhaps also to mRNAs of phototransduction genes not yet associated with RP. Application of this approach may be age and species independent.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , RNA Catalítico/farmacologia , RNA Mensageiro/metabolismo , Degeneração Retiniana/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/fisiopatologia , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Animais , Western Blotting , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Dependovirus/genética , Modelos Animais de Doenças , Eletrorretinografia , Vetores Genéticos , Injeções , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmídeos , RNA Catalítico/genética , Degeneração Retiniana/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
PURPOSE: The primary cause of vision loss in people more than 50 years of age in developed nations is age-related macular degeneration (ARMD). The wet form of ARMD is characterized by choroidal neovascularization (CNV). A prior study has shown that adult hematopoietic stem cells (HSCs) contribute to approximately 50% of newly formed vasculature in CNV. Stromal-derived factor (SDF)-1 is involved with homing of HSCs from bone marrow to target tissue. Vascular endothelial cadherin (VE-cadherin, or CD144) is involved in endothelial cell adhesion. Preventing homing and/or adhesion of progenitor cells to damaged choroid could reduce CNV. METHODS: Adult C57BL/6J mice were lethally irradiated, and then received a transplant of purified c-kit+Sca-1+ HSCs from the bone marrow of green fluorescent protein (gfp) homozygous donor mice. Bruch's membrane rupture by laser photocoagulation was used to induce CNV. Animals were injected subretinally with anti-SDF-1, anti-CD144, or control, before or after laser photocoagulation. The eyes were enucleated, and the neural retinas were separated from the RPE/choroid/sclera complex. All tissues were flatmounted and qualitatively and quantitatively assessed by fluorescence microscopy. RESULTS: CNV lesions from eyes treated with anti-CD144 showed significantly less incorporation of gfp+ cells compared with those treated with anti-SDF-1. Antibody treatment generally reduced the degree of gfp+ stem cell recruitment and incorporation into the CNV lesions, compared with the control. Treatment with either antibody also significantly reduced the size of the CNV lesions. CONCLUSIONS: These results indicate that homing and adhesion of progenitor cells to CNV may be targeted differentially or in combination to prevent CNV.
Assuntos
Neovascularização de Coroide/metabolismo , Endotélio Vascular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Anticorpos/uso terapêutico , Antígenos CD , Caderinas/imunologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12 , Quimiocinas CXC/imunologia , Quimera , Neovascularização de Coroide/patologia , Proteínas de Fluorescência Verde , Transplante de Células-Tronco Hematopoéticas , Fotocoagulação a Laser , Proteínas Luminescentes , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de FluorescênciaRESUMO
PURPOSE: Insulin-like growth factor (IGF)-1 and its receptor (IGF-1R) are associated with abnormal retinal neovascularization. Ribozymes were designed that selectively decreased the expression of the IGF-1R and these ribozymes were tested in angiogenesis models in vitro and in vivo. METHODS: Two hammerhead ribozymes were designed that cleave the human IGF-1R mRNA. The ribozymes were cloned into recombinant adeno-associated viral vectors (rAAV). The rAAV constructs were transfected into human retinal endothelial cells (HRECs). IGF-1R mRNA and protein levels were examined and the modified Boyden chamber assay used to examine ribozyme effects on cell migration. These constructs were injected intravitreally into mice to determine the effect of the ribozymes on retinal neovascularization in a mouse model of oxygen-induced retinopathy. RESULTS: Relative quantitative RT-PCR analysis showed that IGF-1R Rz1 reduced IGF-1R mRNA levels by 40% +/- 10% (P = 0.003), and Western blot analysis showed a 41% +/- 5% (P = 4.6 x 10(-5)) reduction of IGF-1R protein, confirming that this ribozyme reduces IGF-1R expression. IGF-1R Rz1 also reduced IGF-1-induced cell migration by 90% +/- 5% (P = 2.9 x 10(-9)) showing that IGF-1R Rz1 reduces IGF-1R function in HRECs. IGF-1R Rz1 also reduced the amount of preretinal neovascularization by 65% +/- 6% (P = 2.7 x 10(-5)), as measured by the average number of endothelial preretinal nuclei per section. CONCLUSIONS: These studies demonstrate that the IGF-1R ribozymes are effective at reducing the expression and function of the IGF-1R in vitro and in vivo. Therefore, the IGF-1R ribozymes are an effective method for studying the process of angiogenesis and may ultimately be effective as gene therapy tools for the reduction of pathologic retinal angiogenesis.
Assuntos
Endotélio Vascular/efeitos dos fármacos , RNA Catalítico/farmacologia , Receptor IGF Tipo 1/genética , Neovascularização Retiniana/prevenção & controle , Animais , Western Blotting , Movimento Celular , Clonagem Molecular , Dependovirus/genética , Endotélio Vascular/metabolismo , Feminino , Terapia Genética , Vetores Genéticos , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos , Gravidez , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/metabolismo , Neovascularização Retiniana/metabolismo , Vasos Retinianos/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Viable strategies for retinal gene therapy must be designed to cope with the genetic nature of the disease and/or the primary pathologic process responsible for retinal malfunction. For dominant gene defects the aim must be to destroy the presumably toxic gene product, for recessive gene defects the direct approach aims to provide a wild-type copy of the gene to the affected retinal cell type, and for diseases of either complex or unknown genetic origin, more general cell survival strategies that deal with preserving affected retinal cells are often the best and only option. Hence examples of each type of therapy will be briefly discussed in several animal models, including ribozyme therapy for autosomal dominant retinitis pigmentosa in the transgenic P23H opsin rat, beta-PDE gene augmentation therapy for autosomal recessive retinitis pigmentosa in the rd mouse, glial cell-derived neurotrophic factor (GDNF) gene therapy for autosomal dominant RP in the transgenic S334ter opsin rat and pigment epithelial cell-derived neurotrophic factor (PEDF) gene therapy for neovascular retinal disease in rodents. Each employs a recombinant AAV vectored passenger gene controlled by one of several promoters supporting either photoreceptor-specific expression or more general retinal cell expression depending on the therapeutic requirements.
Assuntos
Dependovirus , Proteínas do Olho , Terapia Genética , Vetores Genéticos , Doenças Retinianas/terapia , Animais , Genes Dominantes , Genes Recessivos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Camundongos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Proteínas/genética , Proteínas/metabolismo , RNA Catalítico , Doenças Retinianas/genética , Neovascularização Retiniana/terapia , Serpinas/genética , Serpinas/metabolismoRESUMO
5A11/Basigin is an immunoglobulin-like glycoprotein expressed on the surface of Müller cells, the apical and basal surfaces of the retinal pigmented epithelium, and photoreceptor cell bodies and their inner segments. Disruption of the 5A11/Basigin gene in the mouse results in photoreceptor degeneration and a corresponding decrease in electroretinogram amplitudes in mature mice. The purpose of this study was to examine the electrophysiology of the 5A11/Basigin null mouse retina at earlier ages than previously examined. Although the architecture of the 5A11/Basigin null mouse retina appears normal, the ERG amplitudes are severely depressed at eye opening, indicating failure in retinal maturation.
Assuntos
Antígenos CD , Antígenos de Neoplasias , Antígenos de Superfície , Proteínas Aviárias , Proteínas Sanguíneas , Glicoproteínas de Membrana/genética , Retina/fisiopatologia , Animais , Basigina , Eletrorretinografia , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Retina/química , Rodopsina/análiseAssuntos
Proteínas do Olho/genética , Terapia Genética/métodos , Retinosquise/genética , Retinosquise/terapia , Animais , Discoidinas , Modelos Animais de Doenças , Eletrorretinografia/métodos , Proteínas do Olho/fisiologia , Imuno-Histoquímica , Lectinas/química , Camundongos , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Retina/metabolismo , Retinosquise/patologia , Fatores de TempoRESUMO
X-linked juvenile retinoschisis (RS) is a common cause of juvenile macular degeneration in males. RS is characterized by cystic spoke-wheel-like maculopathy, peripheral schisis, and a negative (b-wave more reduced than a-wave) electroretinogram (ERG). These symptoms are due to mutations in the RS1 gene in Xp22.2 leading to loss of functional protein. No medical treatment is currently available. We show here that in an Rs1h-deficient mouse model of human RS, delivery of the human RS1 cDNA with an AAV vector restored expression of retinoschisin to both photoreceptors and the inner retina essentially identical to that seen in wild-type mice. More importantly, unlike an earlier study with a different AAV vector and promoter, this work shows for the first time that therapeutic gene delivery using a highly specific AAV5-opsin promoter vector leads to progressive and significant improvement in both retinal function (ERG) and morphology, with preservation of photoreceptor cells that, without treatment, progressively degenerate.
Assuntos
Moléculas de Adesão Celular/genética , Proteínas do Olho/genética , Terapia Genética , Retina/fisiopatologia , Degeneração Retiniana/genética , Retinosquise/terapia , Animais , Dependovirus/genética , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho/metabolismo , Ligação Genética , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Knockout , Retina/patologia , Retinosquise/genética , Retinosquise/fisiopatologia , TransfecçãoRESUMO
Autoantibodies against recoverin are found in the sera of patients with cancer-associated retinopathy syndrome, a paraneoplastic disease associated with retinal degeneration. We have previously shown that anti-recoverin autoantibodies induced photoreceptor apoptotic cell death after injection into the vitreous of Lewis rats. Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of a number of neuronal cell types, including photoreceptors. In this study, we examined whether an adeno-associated virus (AAV)-mediated delivery of gene encoding the human CNTF protected photoreceptor cells from anti-recoverin antibody-induced death. One month after subretinal injection of the AAV-CNTF gene into one eye and a control vector into the other eye, an anti-recoverin antibody was injected to induce retinal cell death in Lewis rats. Subretinal administration of the virus led to an efficient transduction of photoreceptors, as indicated by immunostaining of retinas with anti-CNTF. Histological examination of the corresponding retinas showed that photoreceptor cells were significantly protected from apoptotic death in the CNTF-treated eyes. CNTF treatment of the retinas resulted in a time-dependent activation of STAT 3. The present study shows that an AAV-mediated delivery of CNTF may protect photoreceptors from antibody-induced cell death through the activation of STAT3 and the suppression of caspase 3 activity, a key caspase leading to apoptosis. Thus, CNTF may be a useful treatment for human antibody-mediated retinal degeneration.
Assuntos
Apoptose , Autoanticorpos/toxicidade , Proteínas do Olho , Lipoproteínas , Proteínas do Tecido Nervoso , Células Fotorreceptoras de Vertebrados/patologia , Receptor do Fator Neutrófico Ciliar/genética , Degeneração Retiniana/imunologia , Degeneração Retiniana/terapia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Doenças Autoimunes/terapia , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dependovirus/genética , Terapia Genética , Vetores Genéticos , Hipocalcina , Humanos , Modelos Biológicos , Ratos , Ratos Endogâmicos Lew , Receptor do Fator Neutrófico Ciliar/metabolismo , Recoverina , Retina/imunologia , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/patologia , Fator de Transcrição STAT3 , Transativadores/metabolismo , Transdução GenéticaRESUMO
The beta-glucuronidase-deficient mucopolysaccharidosis type VII (MPS VII) mouse accumulates partially degraded glycosaminoglycans in many cell types, including retinal pigmented epithelial (RPE) cells in the eye. This lysosomal storage in RPE cells leads to progressive retinal degeneration and reduced function as measured by flash electroretinography (ERG). The impact of AAV-mediated intraocular gene therapy on pathology and retinal function was examined in normal and MPS VII mice treated at 4 weeks of age, when lysosomal storage is evident but functional impairment is minimal in affected animals. At 16 weeks, an age at which untreated MPS VII mice have advanced histologic lesions and significantly reduced ERG amplitudes, treated eyes had nearly normal levels of beta-glucuronidase activity, preservation of cells in the outer nuclear layer of the retina, and decreased lysosomal storage within the RPE. The AAV-treated MPS VII mice also had significantly increased dark-adapted ERG amplitudes compared to untreated MPS VII mice. Although retinal function was improved, the efficacy of the treatment depended heavily on parameters related to the injection procedure, such as the injection volume, injection site, and vector dose. These data suggest that intraocular AAV-mediated therapy may be efficacious for treating the retinal disease associated with certain lysosomal storage diseases.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Glucuronidase/genética , Mucopolissacaridose VII/terapia , Epitélio Pigmentado Ocular/patologia , Retina/fisiopatologia , Animais , Eletrorretinografia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Glucuronidase/análise , Glucuronidase/metabolismo , Glicosaminoglicanos/metabolismo , Injeções/métodos , Lisossomos/metabolismo , Camundongos , Camundongos Mutantes , Mucopolissacaridose VII/metabolismo , Mucopolissacaridose VII/fisiopatologia , Epitélio Pigmentado Ocular/metabolismo , Retina/química , Retina/patologia , Distribuição Tecidual , Corpo Vítreo/fisiologiaRESUMO
The neuromuscular disease myotonic dystrophy (DM) is caused by microsatellite repeat expansions at two different genomic loci. Mutant DM transcripts are retained in the nucleus together with the muscleblind (Mbnl) proteins, and these abnormal RNAs somehow interfere with pre-mRNA splicing regulation. Here, we show that disruption of the mouse Mbnl1 gene leads to muscle, eye, and RNA splicing abnormalities that are characteristic of DM disease. Our results support the hypothesis that manifestations of DM can result from sequestration of specific RNA binding proteins by a repetitive element expansion in a mutant RNA.