RESUMEN
Lentiviral vectors (LVs) are promising tools for gene therapy. However, scaling up the production methods of LVs in order to produce high-quality vectors for clinical purposes has proven to be difficult. In this article, we present a scalable and efficient method to produce LVs with transient transfection of adherent 293T cells in a fixed-bed bioreactor. The disposable iCELLis bioreactors are scalable with a large three-dimensional (3D) growth area range between 0.53 and 500 m2, an integrated perfusion system, and a controllable environment for production. In this study, iCELLis Nano (2.67-4 m2) was used for optimizing production parameters for scale-up. Transfections were first done using traditional calcium phosphate method, but in later runs polyethylenimine was found to be more reliable and easier to use. For scalable LV production, perfusion rate control by measuring cell metabolite concentrations in the bioreactor leads to higher productivity and reduced costs. Optimization of cell seeding density for targeted cell concentration during transfection, use of low compaction fixed-bed and lowering the culture pH have a positive effect on LV productivity. These results show for the first time that iCELLis bioreactor is scalable from bench level to clinical scale LV production.
Asunto(s)
Reactores Biológicos , Vectores Genéticos , Lentivirus/crecimiento & desarrollo , Cultivo de Virus/métodos , Fosfatos de Calcio/química , Control de Costos , Medios de Cultivo , Glucosa/metabolismo , Células HEK293 , Humanos , Lactatos/metabolismo , Polietileneimina/química , TransfecciónRESUMEN
AIMS: The aim of the study was to evaluate the histopathology of neovascular tufts and vitreous samples collected from patients with diabetes. METHODS: Vitreous samples and neovascular tufts were collected from patients with type 1 (n = 13) and (n = 17) type 2 diabetes with proliferative retinopathy, and from controls with a macular hole (n = 5). Neovessels were analysed using immunohistochemistry and vitreous samples with an enzyme-linked immunosorbent assay (ELISA). The main outcome measure was to examine differences in the levels of growth factors in patients with type 1 and type 2 diabetes with proliferative retinopathy. RESULTS: Vascular endothelial growth factor (VEGF)-A was most strongly present in the samples from patients with type 1 diabetes. In type 2 diabetes, VEGF-D was more abundantly present than in type 1 diabetes. Angiopoietin (ANG)-2 was also abundantly present. Macrophages and nuclear factor kappa B (NFkappaB) were found, indicating the presence of an inflammatory process in the neovascular tissues. CONCLUSIONS: VEGF-A and ANG-2 are equally important in the neovascular process in both type 1 and type 2 diabetes. VEGF-D is abundantly present in type 2 diabetes. In order to achieve better control of diabetic retinopathy, it might be beneficial to develop treatments that prevent the actions of ANG-2 and VEGF-D.
Asunto(s)
Inductores de la Angiogénesis/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Retinopatía Diabética/metabolismo , Neovascularización Retiniana/metabolismo , Adulto , Anciano , Angiopoyetina 1/metabolismo , Angiopoyetina 2/metabolismo , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/patología , Retinopatía Diabética/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor D de Crecimiento Endotelial Vascular/metabolismo , Cuerpo Vítreo/metabolismoRESUMEN
AIM: The aim of this study was to determine dose-response effects of vascular endothelial growth factor A as delivered using an adenoviral vector on vascular growth and pathological changes in the rabbit eye. Moreover, we wanted to develop a large animal model for angioproliferative diseases in the eye. METHODS: Seventeen New Zealand White rabbits were injected with adenoviral vascular endothelial growth factor-A (AdVEGF-A) intravitreally with different doses (10(9)-10(11) vp). Controls were injected with an empty virus (AdCMV). Some animals had a combination of AdVEGF-A and AdsKDR (a soluble form of the VEGF receptor-2). Animals were killed 6 days after the gene transfer. On the basis of these results, 14 rabbits were injected intravitreally with AdVEGF-A or adenoviral LacZ (AdLacZ) with 10(10) vp in a volume of 0.1 mL. Animals were killed 3, 6, 14 and 28 days after the gene transfer, eyes were removed and analysed histologically. RESULTS: In enzyme-linked immunosorbent assay (ELISA) analysis, human VEGF-A was present in vitreous humour in all VEGF-A transduced eyes. The amount of VEGF-A showed a dose-dependent increase with the AdVEGF-A dose and was the highest 6 days after the gene transfer. Histologic analyses revealed an increased capillary area and density in the AdVEGF-A eyes when compared with the AdLacZ eyes (P < 0.05). In the AdVEGF-A/AdsKDR eyes the average capillary area was not increased compared with AdLacZ eyes. CONCLUSION: This model could be useful for large animal studies regarding the pathogenesis of neoangiogenesis and for the development of new therapeutic strategies for angioproliferative diseases of the eye. Our results establish the key role of VEGF-A in the induction of neovascularization and pathological changes in the rabbit eye.
Asunto(s)
Modelos Animales de Enfermedad , Ojo/irrigación sanguínea , Neovascularización Patológica/etiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adenoviridae/genética , Animales , Barrera Hematorretinal , Capilares/patología , Neovascularización Coroidal/etiología , Retinopatía Diabética/etiología , Relación Dosis-Respuesta a Droga , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Neovascularización Patológica/metabolismo , Conejos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/fisiología , Cuerpo Vítreo/metabolismoRESUMEN
Pseudotyping of viral vectors has been widely used to enhance viral transduction efficiency. One of the most popular pseudotyping proteins has been the G-protein of the vesicular stomatitis virus, VSV-G. In the present study, we show that the 21-amino-acid ectodomain with transmembrane and cytoplasmic tail domains of VSV-G (VSV-GED) augments baculovirus-mediated gene delivery in vertebrate cells by aiding viral entry. The VSV-GED pseudotyped virus replicated efficiently in insect cells yielding high titers. Five out of six studied cell lines showed improved transduction, as measured by a number of transduced cells or transgene expression level. Nearly 15-fold increase in the transduction efficiency was detected in rat malignant glioma cells as compared to the control virus. In the rat brain, transgene expression could be detected in the walls of lateral ventricles and in subarachnoid membranes. Increased transduction efficiency was also observed in the rabbit muscle. Our results suggest that VSV-GED enhances baculoviral gene transfer by augmenting gp64-mediated endosomal release. Moreover, no cytotoxicity was associated with improved gene transfer efficiency. Thus, VSV-GED pseudotyping provides a simple means to enhance baculovirus-mediated gene transfer in vitro and in vivo.
Asunto(s)
Baculoviridae/genética , Terapia Genética/métodos , Glioma/terapia , Glicoproteínas de Membrana/genética , Neoplasias de Tejido Nervioso/terapia , Transducción Genética/métodos , Proteínas del Envoltorio Viral/genética , Animales , Encéfalo/metabolismo , Células Cultivadas , Endosomas/metabolismo , Vectores Genéticos/administración & dosificación , Immunoblotting , Glicoproteínas de Membrana/efectos adversos , Glicoproteínas de Membrana/metabolismo , Modelos Animales , Músculo Esquelético/metabolismo , Estructura Terciaria de Proteína , Conejos , Ratas , Ratas Endogámicas , Proteínas del Envoltorio Viral/efectos adversos , Proteínas del Envoltorio Viral/metabolismoRESUMEN
BACKGROUND: The role of vascular endothelial growth factors (VEGFs) in intimal hyperplasia and atherogenesis remains unknown. Several studies have suggested that some members of the VEGF family reduce intimal hyperplasia, but others have proposed that VEGFs accelerate restenosis and atherosclerosis. This investigation conducted a comparative study with adenoviruses encoding different VEGFs in a rabbit carotid artery collar model of intimal hyperplasia in order to analyze the role of VEGFs in the formation of intimal hyperplasia. MATERIALS AND METHODS: Intimal hyperplasia was induced in the carotid arteries of cholesterol fed New Zealand White rabbits using a silastic collar. Adenoviral vectors encoding VEGF-A, VEGF-B, VEGF-C, VEGF-C(DeltaNDeltaC), VEGF-D and VEGF-D(DeltaNDeltaC) were delivered to the adventitia using the collar as a gene delivery device. Adeno-LacZ was used as a control. RESULTS: A significant (P < 0.01) increase in the intima/media ratio was observed in the arteries transduced with VEGF-A, VEGF-D and VEGF-D(DeltaNDeltaC). There was a significant increase in the number of proliferating cells in the adventitia, media and intima of the VEGF-A, VEGF-D and the VEGF-D(DeltaNDeltaC) transduced arteries. The majority of medial smooth muscle cells in these arteries had a synthetic phenotype. The presence of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the VEGF-A, VEGF-D and the VEGF-D(DeltaNDeltaC) transduced arteries was significantly increased. A significant positive correlation was observed between adventitial angiogenesis and intimal hyperplasia. CONCLUSIONS: Adventitial delivery of adenoviruses encoding VEGF-A, VEGF-D and VEGF-D(DeltaNDeltaC) increased intimal hyperplasia in the rabbit collar model. Adventitial angiogenesis correlated positively with the intimal hyperplasia. These results indicated that efficient adventitial production of VEGF-A, VEGF-D and VEGF-D(DeltaNDeltaC) can cause thickening of the inner layer of the artery in rabbits.
Asunto(s)
Arterias Carótidas/patología , Túnica Íntima/patología , Factores de Crecimiento Endotelial Vascular/administración & dosificación , Animales , Capilares , División Celular/fisiología , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Hiperplasia , Operón Lac , Macrófagos/patología , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/análisis , Microscopía Electrónica/métodos , Neovascularización Patológica/patología , Conejos , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Factor D de Crecimiento Endotelial Vascular/administración & dosificaciónRESUMEN
Gene transfer to the vessel wall using vascular endothelial growth factors (VEGFs) has shown therapeutic potential for the treatment of restenosis. In this study, we evaluated the effect of catheter-mediated adenoviral (Ad) gene transfer of the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) in balloon-denuded cholesterol-fed rabbit aorta. AdLacZ was used as a control. Transduced VEGF-D(DeltaNDeltaC) mRNA was detectable in the arterial wall with RT-PCR at 6, 14 and 28 days. Gene transfer efficiency as detected with X-gal staining 6 days after the AdLacZ transduction was 1.91 +/- 1.32% in intima. AdVEGF-D(DeltaNDeltaC) gene transfer led to 52% reduction in intima/media ratio (I/M) as compared to the AdLacZ controls at 14 days time point. At 6 days there were no differences in I/M, but the number of macrophages in the vessel wall was 85% lower in the AdVEGF-D(DeltaNDeltaC) group as compared to the controls. The therapeutic effect was no longer detectable 28 days after the gene transfer. The therapeutic effect of VEGF-D(DeltaNDeltaC) was nitric oxide (NO)-dependent as the feeding of NO synthase inhibitor, L-NAME, blocked the reduction in intimal thickening. It is concluded that AdVEGF-D(DeltaNDeltaC) gene transfer reduces intimal thickening and macrophage influx into the vessel wall in balloon-denuded rabbit aortas.
Asunto(s)
Adenoviridae/genética , Enfermedades de la Aorta/terapia , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Transducción Genética/métodos , Factor D de Crecimiento Endotelial Vascular/genética , Animales , Aorta , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Cateterismo , Constricción Patológica/terapia , Neovascularización Patológica , Óxido Nítrico/metabolismo , Conejos , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Túnica Íntima/metabolismo , Túnica Íntima/patología , Factor D de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Transfection of oocytes should be avoided in somatic gene therapy. However, several viral vectors including adenoviruses can transfect zona-pellucida-free eggs in vitro. During early stages of development, oocytes of postnatal ovaries lack the zona pellucida. Therefore, they may be susceptible to gene transfer and unintended toxic effects. The purpose of this study was to see whether the injection of adenoviruses (1 x 10(10) PFU) or plasmid (500 microg)/DOTMA:DOPE (1:2) liposomes directly into uterine arteries in pregnant rabbits leads to transfection of oocytes and other types of ovarian cells. LacZ and herpes simplex virus thymidine kinase (HSV-TK) were used as transgenes. It was found that both adenovirus and plasmid vectors transfected oocytes at the primordial and primary follicle stage when they were not protected by the zona pellucida, whereas no transfection was seen in oocytes surrounded by the zona pellucida. Efficient transfection of corpus luteum and granulosa cells was also detected by adenoviral and plasmid vectors. Transfection of oocytes and other ovarian cells was verified by X-gal staining and laser microdissection, followed by PCR analysis. HSV-TK gene transfer, followed by ganciclovir treatment, led to destruction of a significant number of oocytes, whereas HSV-TK gene transfer alone did not lead to toxic effects. It is concluded that the presence of a high concentration of adenovirus or plasmid vectors via the uterine artery may lead to transfection of zona-pellucida-free oocytes and other ovarian cells.