RESUMEN
Substantial advances in our understanding of lentivirus lifecycles and their various constituent proteins have permitted the bioengineering of lentiviral vectors now considered safe enough for clinical trials for both lethal and non-lethal diseases. They possess distinct properties that make them particularly suitable for gene delivery in ophthalmic diseases, including high expression, consistent targeting of various post-mitotic ocular cells in vivo and a paucity of associated intraocular inflammation, all contributing to their ability to mediate efficient and stable intraocular gene transfer. In this review, the intraocular tropisms and therapeutic applications of both primate and non-primate lentiviral vectors, and how the unique features of the eye influence these, are discussed. The feasibility of therapeutic targeting using these vectors in animal models of both anterior and posterior ophthalmic disorders has been established, and has, in combination with substantial progress in enhancing lentiviral vector bio-safety over the past two decades, paved the way for the first human ophthalmic clinical trials using lentivirus-based gene transfer vectors.
Asunto(s)
Neovascularización Coroidal/genética , Técnicas de Transferencia de Gen , Glaucoma/genética , Lentivirus , Degeneración Retiniana/genética , Animales , Neovascularización Coroidal/terapia , Modelos Animales de Enfermedad , Expresión Génica , Terapia Genética , Vectores Genéticos/administración & dosificación , Glaucoma/terapia , Humanos , Degeneración Retiniana/terapiaRESUMEN
Insertional mutagenesis following gene therapy with gammaretroviral vectors can cause the development of lymphoproliferation in children with X-linked severe combined immunodeficiency. In experimental studies, recombinant adeno-associated virus (rAAV) vectors have also been reported to increase susceptibility to carcinogenesis. The possibility of vector-induced transformation in quiescent ocular cells is probably significantly lower than in mitotically active cells, but given the increasing number of clinical applications of rAAV and lentiviral vectors for ocular disease, a specific assessment of their oncogenic potential in the eye is important. In this study, we investigated the effect of rAAV2/2 and integrating HIV-1 vectors upon the incidence of ocular neoplasia in p53 tumour-suppressor gene-knockout (p53(-/-)) mice, which are highly susceptible to intraocular malignant transformation. Subretinal injections of high titre rAAV2/2 or integrating HIV-1 vectors induced no tumours in p53(-/-) or p53(+/-) animals, nor significantly affected their natural longevity. We conclude that any insertional events arising from subretinal delivery of these vectors appear insufficient to cause intraocular malignancy, even in highly susceptible animals. These findings support the continued development of these vectors for ocular applications.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen/efectos adversos , Vectores Genéticos/efectos adversos , Lentivirus/genética , Proteína p53 Supresora de Tumor/genética , Animales , Transformación Celular Neoplásica/genética , Electrorretinografía , Neoplasias del Ojo/genética , Técnicas de Inactivación de Genes , Terapia Genética , Vectores Genéticos/administración & dosificación , Proteínas Fluorescentes Verdes , Ratones , Retina , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
We evaluated the efficacy of equine infectious anaemia virus (EIAV)-based lentiviral vectors encoding endostatin (EIAV.endostatin) or angiostatin (EIAV.angiostatin) in inhibiting angiogenesis and vascular hyperpermeability in the laser-induced model of choroidal neovascularisation (CNV). Equine infectious anaemia virus.endostatin, EIAV.angiostatin or control (EIAV.null) vectors were administered into the subretinal space of C57Bl/6J mice. Two weeks after laser injury CNV areas and the degree of vascular hyperpermeability were measured by image analysis of in vivo fluorescein angiograms. Compared with EIAV.null-injected eyes, EIAV.endostatin resulted in a 59.5% (P<0.001) reduction in CNV area and a reduction in hyperpermeability of 25.6% (P<0.05). Equine infectious anaemia virus.angiostatin resulted in a 50.0% (P<0.05) reduction in CNV area and a 23.9% (P<0.05) reduction in hyperpermeability. Equine infectious anaemia virus.endostatin, but not EIAV.angiostatin significantly augmented the frequency of apoptosis within the induced CNV as compared with injected controls. TdT-dUTP terminal nick end labeling analysis 5 weeks post-injection, and histological and retinal flatmount analysis 12 months post-injection revealed no evidence of vector- or transgene expression-related deleterious effects on neurosensory retinal cells, or mature retinal vasculature in non-lasered eyes. Highly expressing EIAV-based vectors encoding endostatin or angiostatin effectively control angiogenesis and hyperpermeability in experimental CNV without long-term deleterious effects, supporting the use of such a strategy in the management of patients with exudative age-related macular degeneration.
Asunto(s)
Angiostatinas/genética , Neovascularización Coroidal/terapia , Endostatinas/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Virus de la Anemia Infecciosa Equina/genética , Inhibidores de la Angiogénesis/genética , Animales , Apoptosis , Permeabilidad Capilar , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/fisiopatología , Angiografía con Fluoresceína , Vectores Genéticos/genética , Etiquetado Corte-Fin in Situ , Rayos Láser , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Neovascularización Patológica , Transducción Genética/métodos , Regulación hacia ArribaRESUMEN
BACKGROUND: We have developed minimal non-primate lentiviral vectors based on the equine infectious anaemia virus (EIAV). We evaluated the in vivo expression profiles of these vectors delivered regionally to ocular tissues to define their potential utility in ocular gene therapy. METHODS: EIAV vectors pseudotyped with VSV-G or rabies-G envelope proteins were delivered subretinally, intravitreally or into the anterior chambers (intracameral administration) in mice. Reporter gene (eGFP) expression was analysed using in vivo retinal imaging or histological examination of eyes and brains at intervals between 3 days and 16 months. We investigated the effects of vector titre, pseudotype, genome configuration, site of intraocular administration, intentional retinal trauma and the degree of retinal maturation on the spatial and temporal expression profiles of these vectors. RESULTS: Subretinal vector delivery resulted in efficient and stable transduction of retinal pigment epithelial (RPE) cells and variable transduction of photoreceptors up to 16 months post-injection. Retinal trauma facilitated the local transduction of neurosensory retinal cells. Intracameral administration of VSV-G- but not rabies-G-pseudotyped vectors produced stable eGFP expression in corneal endothelial cells and trabecular meshwork. CONCLUSIONS: The cellular tropism and expression kinetics of optimised EIAV vectors after intraocular administration make them attractive vehicles for delivering therapeutic genes in the management of inherited and acquired retinal and anterior segment disorders.
Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética/métodos , Virus de la Anemia Infecciosa Equina/genética , Enfermedades de la Retina/terapia , Animales , Femenino , Perfilación de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Genoma , Proteínas Fluorescentes Verdes/biosíntesis , Cinética , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras , Epitelio Pigmentado Ocular/fisiología , Retina/lesiones , Enfermedades de la Retina/genética , Transducción Genética , TropismoRESUMEN
The Royal College of Surgeons (RCS) rat is a well-characterized model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding , a receptor tyrosine kinase found in RPE cells, that is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.