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The BMP/TGFß-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Morfogenética Ósea 6/metabolismo , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 2/metabolismo , Hipoxia de la Célula , Núcleo Celular/metabolismo , Vía de Señalización Hippo , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Transporte de Proteínas , Porcinos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Gene therapy has been expected to become a novel treatment method since the structure of DNA was discovered in 1953. The morbidity from cardiovascular diseases remains remarkable despite the improvement of percutaneous interventions and pharmacological treatment, underlining the need for novel therapeutics. Gene therapy-mediated therapeutic angiogenesis could help those who have not gained sufficient symptom relief with traditional treatment methods. Especially patients with severe coronary artery disease and heart failure could benefit from gene therapy. Some clinical trials have reported improved myocardial perfusion and symptom relief in CAD patients, but few trials have come up with disappointing negative results. Translating preclinical success into clinical applications has encountered difficulties in successful transduction, study design, endpoint selection, and patient selection and recruitment. However, promising new methods for transducing the cells, such as retrograde delivery and cardiac-specific AAV vectors, hold great promise for myocardial gene therapy. This review introduces gene therapy for ischaemic heart disease and heart failure and discusses the current status and future developments in this field.
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Enfermedad de la Arteria Coronaria , Terapia Genética , Insuficiencia Cardíaca , Isquemia Miocárdica , Insuficiencia Cardíaca/terapia , Humanos , Isquemia Miocárdica/terapia , MiocardioRESUMEN
BACKGROUND: High grade serous ovarian carcinoma (HGSOC) is the most common subtype of epithelial ovarian cancers (EOC) with poor prognosis. In most cases EOC is widely disseminated at the time of diagnosis. Despite the optimal cytoreductive surgery and chemotherapy most patients develop chemoresistance, and the 5-year overall survival being only 25-35%. METHODS: Here we analyzed the gene expression profiles of 10 primary HGSOC tumors and 10 related omental metastases using RNA sequencing and identified 100 differentially expressed genes. RESULTS: The differentially expressed genes were associated with decreased embryogenesis and vasculogenesis and increased cellular proliferation and organismal death. Top upstream regulators responsible for this gene signature were NR5A1, GATA4, FOXL2, TP53 and BMP7. A subset of these genes were highly expressed in the ovarian cancer among the cancer transcriptomes of The Cancer Genome Atlas. Importantly, the metastatic gene signature was suggestive of poor survival in TCGA data based on gene enrichment analysis. CONCLUSION: By comparing the gene expression profiles of primary HGSOC tumors and their matched metastasis, we provide evidence that a signature of 100 genes is able to separate these two sample types and potentially predict patient survival. Our study identifies functional categories of genes and transcription factors that could play important roles in promoting metastases and serve as markers for cancer prognosis.
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Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/patología , Perfilación de la Expresión Génica , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Transcriptoma , Carcinoma Epitelial de Ovario/mortalidad , Línea Celular Tumoral , Biología Computacional/métodos , Femenino , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias Ováricas/mortalidadRESUMEN
The insertion of a stent in diseased arteries is a common endovascular procedure that can be compromised by the development of short- and long-term inflammatory responses leading to restenosis and thrombosis, respectively. While treatment with drugs, either systemic or localized, has decreased the incidence of restenosis and thrombosis these complications persist and are associated with a high mortality in those that present with stent thrombosis. We reasoned that if stents could be made to undergo accelerated endothelialization in the deployed region, then such an approach would further decrease the occurrence of stent thrombosis and restenosis thereby improving clinical outcomes. Toward that objective, the first step necessitated efficient capture of progenitor stem cells, which eventually would become the new endothelium. To achieve this objective, we engineered intrinsic ferromagnetism within nonmagnetizable, biodegradable magnesium (Mg) bare metal stents. Mg stents were coated with biodegradable polylactide (PLA) polymer embedding magnetizable iron-platinum (FePt) alloy nanoparticles, nanomagnetic particles, nMags, which increased the surface area and hence magnetization of the stent. nMags uniformly distributed on stents enabled capture, under flow, up to 50 mL/min, of systemically injected iron-oxide-labeled (IO-labeled) progenitor stem cells. Critical parameters enhancing capture efficiency were optimized, and we demonstrated the generality of the approach by showing that nMag-coated stents can capture different cell types. Our work is a potential paradigm shift in engineering stents because implants are rendered as tissue in the body, and this "natural stealthiness" reduces or eliminates issues associated with pro-inflammatory immune responses postimplantation.
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Materiales Biocompatibles Revestidos/química , Reestenosis Coronaria/prevención & control , Magnesio/química , Imanes/química , Poliésteres/química , Stents , Trombosis/prevención & control , Aleaciones/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hierro/química , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Platino (Metal)/química , Células Madre/citología , Stents/efectos adversosRESUMEN
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.
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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
Gene therapy is a promising new treatment option for cardiac diseases. For finding the most suitable and safe vector for cardiac gene transfer, we delivered adenovirus (AdV), adeno-associated virus (AAV) and lentivirus (LeV) vectors into the mouse heart with sophisticated closed-chest echocardiography-guided intramyocardial injection method for comparing them with regards to transduction efficiency, myocardial damage, effects on the left ventricular function and electrocardiography (ECG). AdV had the highest transduction efficiency in cardiomyocytes followed by AAV2 and AAV9, and the lowest efficiency was seen with LeV. The local myocardial inflammation and fibrosis in the left ventricle (LV) was proportional to transduction efficiency. AdV caused LV dilatation and systolic dysfunction. Neither of the locally injected AAV serotypes impaired the LV systolic function, but AAV9 caused diastolic dysfunction to some extent. LeV did not affect the cardiac function. We also studied systemic delivery of AAV9, which led to transduction of cardiomyocytes throughout the myocardium. However, also diffuse fibrosis was present leading to significantly impaired LV systolic and diastolic function and pathological ECG changes. Compared with widely used AdV vector, AAV2, AAV9 and LeV were less effective in transducing cardiomyocytes but also less harmful. Local administration of AAV9 was safer and more efficient compared with systemic administration.
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Adenoviridae/genética , Dependovirus/genética , Vectores Genéticos/efectos adversos , Cardiopatías/genética , Cardiopatías/terapia , Lentivirus/genética , Animales , Ecocardiografía Tridimensional , Terapia Genética , RatonesRESUMEN
In our earlier studies, Semliki Forest virus vector VA7 completely eliminated type I interferon (IFN-I)-unresponsive human U87-luc glioma xenografts, whereas interferon-responsive mouse gliomas proved refractory. Here, we describe in two clones of CT26 murine colon carcinoma, opposed patterns of IFN-I responsiveness and sensitivity to VA7. Both CT26WT and CT26LacZ clones secreted biologically active interferon in vitro upon virus infection but only CT26WT cells were protected. Focal infection of CT26WT cultures was self-limiting but could be rescued using IFN-I pathway inhibitor Ruxolitinib or antibody against IFNß. Whole transcriptome sequencing (RNA-Seq) and protein expression analysis revealed that CT26WT cells constitutively expressed 56 different genes associated with pattern recognition and IFN-I signaling pathways, spanning two reported anti-RNA virus gene signatures and 22 genes with reported anti-alphaviral activity. Whereas CT26WT tumors were strictly virus-resistant in vivo, infection of CT26LacZ tumors resulted in complete tumor eradication in both immunocompetent and severe combined immune deficient mice. In double-flank transplantation experiments, CT26WT tumors grew despite successful eradication of CT26LacZ tumors from the contralateral flank. Tumor growth progressed uninhibited also when CT26LacZ inoculums contained only a small fraction of CT26WT cells, demonstrating dominance of IFN responsiveness when heterogeneous tumors are targeted with interferon-sensitive oncolytic viruses.
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Neoplasias del Colon/terapia , Viroterapia Oncolítica , Virus Oncolíticos/genética , Virus de los Bosques Semliki/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Efecto Espectador , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Vectores Genéticos , Proteínas Fluorescentes Verdes/biosíntesis , Interferón Tipo I/farmacología , Interferón Tipo I/uso terapéutico , Interferón beta/metabolismo , Ratones Endogámicos BALB C , Necrosis , Trasplante de Neoplasias , Factor de Transcripción STAT1/metabolismo , Transfección , Resultado del TratamientoRESUMEN
Malignant gliomas (MGs) are cancers with poor prognosis and limited therapeutic options. Herpes Simplex virus-1 thymidine kinase expressed from adenoviruses with prodrug ganciclovir (TK/GCV) is the best-characterized suicide gene therapy, whereas temozolomide (TMZ) is the first-line chemotherapy for MG. However, the potential of their combination has not been studied thoroughly. The aim of this study was to evaluate the therapeutic response of this combination and to study whether addition of valproic acid (VPA) could benefit the treatment outcome. Efficacies of different treatments were first studied in vitro in BT4C rat MG cells. Therapeutic assessment in vivo was done in an immunocompetent rat MG model for treatment efficacy and toxicity. In vitro, VPA was able to significantly enhance cytotoxicity and increase adenovirus-mediated transduction efficiency up to sevenfold. In vivo, rats receiving TK/GCV+TMZ had notably smaller tumors and enhanced survival (P<0.001) in comparison with control rats. However, VPA was not able to further enhance the treatment response in vivo. Leukocytopenia and thrombocytopenia were the major side effects. We conclude that careful optimization of the treatment schedules and doses of individual therapies are necessary to achieve an optimal therapeutic effect with TK/GCV+TMZ combination. No further in vivo benefit with VPA was observed.
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Adenovirus Humanos/genética , Antineoplásicos Alquilantes/uso terapéutico , Dacarbazina/análogos & derivados , Genes Transgénicos Suicidas , Terapia Genética , Glioma/terapia , Adenovirus Humanos/metabolismo , Animales , Antivirales/uso terapéutico , Terapia Combinada , Dacarbazina/uso terapéutico , Ganciclovir/uso terapéutico , Genes Virales , Vectores Genéticos , Glioma/tratamiento farmacológico , Glioma/patología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Masculino , Neoplasias Experimentales , Ratas , Temozolomida , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Resultado del Tratamiento , Células Tumorales Cultivadas , Ácido Valproico/uso terapéuticoRESUMEN
Three-dimensional image analyses are required to improve the understanding of the regulation of blood vessel formation and heterogeneity. Currently, quantitation of 3D endothelial structures or vessel branches is often based on 2D projections of the images losing their volumetric information. Here, we developed SproutAngio, a Python-based open-source tool, for fully automated 3D segmentation and analysis of endothelial lumen space and sprout morphology. To test the SproutAngio, we produced a publicly available in vitro fibrin bead assay dataset with a gradually increasing VEGF-A concentration ( https://doi.org/10.5281/zenodo.7240927 ). We demonstrate that our automated segmentation and sprout morphology analysis, including sprout number, length, and nuclei number, outperform the widely used ImageJ plugin. We also show that SproutAngio allows a more detailed and automated analysis of the mouse retinal vasculature in comparison to the commonly used radial expansion measurement. In addition, we provide two novel methods for automated analysis of endothelial lumen space: (1) width measurement from tip, stalk and root segments of the sprouts and (2) paired nuclei distance analysis. We show that these automated methods provided important additional information on the endothelial cell organization in the sprouts. The pipelines and source code of SproutAngio are publicly available ( https://doi.org/10.5281/zenodo.7381732 ).
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Células Endoteliales , Neovascularización Fisiológica , Ratones , Animales , Neovascularización Fisiológica/fisiología , Endotelio , Fenómenos Fisiológicos Cardiovasculares , InformáticaRESUMEN
Pro-angiogenic gene therapy is being developed to treat coronary artery disease (CAD). We recently showed that bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor-A synergistically regulate endothelial cell sprouting in vitro. BMP2 was also shown to induce endocardial angiogenesis in neonatal mice post-myocardial infarction. In this study, we investigated the potential of BMP2 gene transfer to improve cardiomyocyte function and neovessel formation in a pig chronic myocardial infarction model. Ischemia was induced in domestic pigs by placing a bottleneck stent in the proximal part of the left anterior descending artery 14 days before gene transfer. Intramyocardial gene transfers with adenovirus vectors (1 × 1012 viral particles/pig) containing either human BMP2 (AdBMP2) or beta-galactosidase (AdLacZ) control gene were performed using a needle injection catheter. BMP2 transgene expression in the myocardium was detected with immunofluorescence staining in the gene transfer area 6 days after AdBMP2 administration. BMP2 gene transfer did not induce angiogenesis or cardiomyocyte proliferation in the ischemic pig myocardium as determined by the quantitations of CD31 or Ki-67 stainings, respectively. Accordingly, no changes in heart contractility were detected in left ventricular ejection fraction and strain measurements. However, BMP2 gene transfer induced pericardial effusion (AdBMP2: 9.41 ± 3.17â mm; AdLacZ: 3.07 ± 1.33â mm) that was measured by echocardiography. Furthermore, an increase in the number of immune cells and CD3+ T cells was found in the BMP2 gene transfer area. No changes were detected in the clinical chemistry analysis of pig serum or histology of the major organs, implicating that the gene transfer did not induce general toxicity, myocardial injury, or off-target effects. Finally, the levels of fibrosis and cardiomyocyte apoptosis detected by Sirius red or caspase 3 stainings, respectively, remained unaltered between the groups. Our results demonstrate that BMP2 gene transfer causes inflammatory changes and pericardial effusion in the adult ischemic myocardium, which thus does not support its therapeutic use in chronic CAD.
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Vascular endothelial growth factor (VEGF)-mediated gene therapy (GT) has shown promising results as a novel method in the treatment of severe cardiovascular diseases. VEGF GT has proved to be safe and well tolerated in short-term studies, but there is only very limited data available on long-term effects. In this study we examined the effects of VEGF GT on patients having received VEGF-A gene transfer for the treatment of symptomatic (that is, claudication or critical lower limb ischemia) peripheral arterial occlusive disease. Out of 54 patients, 25 (46%) were interviewed for this study and 26 (48%) had died during the follow-up. Interviewed patients were treated with adenoviral (n=8, mean age 76 (62.7-90.6) years) or plasmid/liposome (n=8, mean age 84.2 (71.9-94.7) years) vectors compared with a randomized control group (n=10, mean age 80.5 (70.7-88.1) years) using a local balloon catheter device. The follow-up time was 10 years. Causes of death were clarified from hospital records. There were no statistically significant differences between the study groups in the causes of death or in the incidence of cancer (VEGF-Adv 0/10 vs VEGF-p/l 1/8 vs Control 1/7, P=0.5), diabetes (3/10 vs 3/8 vs 2/7, P=1.00) or diabetic retinopathy (0/10 vs 1/8 vs 0/7, P=0.45). In addition, we found no differences in the number of amputations of the treated leg (0/10 vs 3/8 vs 1/7, P=0.17). We conclude that transient VEGF-A-mediated GT did not increase the incidence of cancer, diabetes, retinopathy or any other diseases during the 10-year follow-up time. No significant differences were detected in the number of amputations or causes of death. This study supports our previous findings that local adenovirus and plasmid/liposome-mediated VEGF-A GT is safe and well-tolerated treatment in elderly patients with cardiovascular diseases.
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Terapia Genética/efectos adversos , Enfermedades Vasculares Periféricas/terapia , Factor A de Crecimiento Endotelial Vascular/genética , Adenoviridae/genética , Anciano , Anciano de 80 o más Años , Amputación Quirúrgica , Catéteres , Causas de Muerte , Ensayos Clínicos Fase III como Asunto , Femenino , Estudios de Seguimiento , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Isquemia/terapia , Pierna/irrigación sanguínea , Masculino , Persona de Mediana Edad , Enfermedades Vasculares Periféricas/mortalidadRESUMEN
BACKGROUND: Very recently, cognitively normal, middle-aged adults with severe obstructive sleep apnea (OSA) were shown to have regional cortical amyloid-ß deposits. In the normal brain, amyloid tracer (e.g., [11C]-PiB) uptake is observed in white matter (WM) but not in cortical gray matter (GM), resulting in clear GM-WM contrast. There are no reports on possible changes in this contrast in severe OSA. OBJECTIVES: Evaluate changes in the global [11C]-PiB GM-WM contrast and study if factors reflecting clinical and imaging characteristics are associated with them. DESIGN AND SETTING: Cross-sectional imaging study. PARTICIPANTS: 19 cognitively intact middle-aged (mean 44 years) patients with severe OSA (Apnea-Hypopnea Index >30/h), carefully selected to exclude any other possible factors that could alter brain health. MEASUREMENTS: Detailed neuroimaging (amyloid PET, MRI). Signs of possible alterations in amyloid tracer GM-WM contrast and kinetics were studied with static and dynamic [11C]-PiB PET and WM structures with detailed 3.0T MRI. RESULTS: Static [11C]-PiB PET uptake showed significantly decreased GM-WM contrast in 5 out of 19 patients. This was already clearly seen in visual evaluation and also detected quantitatively using retention indexes. Dynamic imaging revealed decreased contrast due to alterations in trace accumulation in the late phase of [11C]-PiB kinetics. Decreased GM-WM contrast in the late phase was global in nature. MRI revealed no corresponding alterations in WM structures. Importantly, decreased GM-WM contrast was associated with smoking (p = 0.007) and higher Apnea-Hypopnea Index (p = 0.001). CONCLUSIONS: Severe OSA was associated with decreased GM-WM contrast in amyloid tracer uptake, with significant correlation with clinical parameters of smoking and AHI. The results support and further extend the current understanding of the deleterious effect of severe OSA on proper amyloid clearance, possibly reflecting dysfunction of the brain glymphatic system.
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Apnea Obstructiva del Sueño , Sustancia Blanca , Adulto , Amiloide/metabolismo , Compuestos de Anilina , Radioisótopos de Carbono , Humanos , Persona de Mediana Edad , Tomografía de Emisión de Positrones/métodos , Apnea Obstructiva del Sueño/diagnóstico por imagen , Tiazoles , Sustancia Blanca/diagnóstico por imagenRESUMEN
Several gene therapy approaches have been designed for the treatment of cardiovascular diseases. A positive finding is that the safety of cardiovascular gene therapy has been excellent even in long-term follow-up. However, several hurdles to this field are still present. A major disappointing feature of the trials is that while preclinical and uncontrolled phase-I gene therapy trials have been positive, none of the randomized controlled phase-II/III cardiovascular gene therapy trials have shown clinically relevant positive effects. Low gene transfer efficiency seems to be associated with several trials. A sophisticated efficient delivery method for cardiovascular applications is still lacking and only low concentrations of the gene product are produced in the target tissues. Only a few gene therapy vectors can be produced in large scale. In addition, inflammatory reactions against vectors and inability to regulate gene expression are still present. Furthermore, a strong placebo effect is affecting the results in gene therapy trials, and long-term trials have become more difficult to conduct because of the multiplicity of therapies applied simultaneously on the patients. This review summarizes advances and obstacles of current cardiovascular clinical gene therapy trials.
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Enfermedades Cardiovasculares/terapia , Ensayos Clínicos como Asunto , Terapia Genética , Técnicas de Transferencia de Gen , Vectores Genéticos/efectos adversos , Humanos , Selección de Paciente , Efecto PlaceboRESUMEN
Vascular endothelial growth factor A (VEGF-A) induces strong angiogenesis and it has been widely used in proangiogenic gene therapy studies. However, little is known about long-term effects of VEGF-A expression in skeletal muscle. Here the long- term effects of adeno-associated virus (AAV) encoding human VEGF-A(165) (AAV-VEGF-A) gene transfer in normal and ischemic rabbit hindlimb skeletal muscles were studied. AAV-LacZ was used as a control. In one-year follow-up, a remarkable increase in skeletal muscle perfusion compared with AAV-LacZ was observed measured with Doppler and contrast pulse sequence ultrasound. Angiogenesis was also seen in histology as enlarged and sprouting capillaries. In addition to favorable angiogenic effects, aberrant vascular structures with CD31 positive cell layers were seen inside muscle fibers after AAV-VEGF-A gene transfer. Importantly, we found increased amounts of extracellular matrix with a high number of macrophages and fibrosis in AAV-VEGF-A transduced muscles. No changes in skeletal muscle morphology were detected in AAV-LacZ transduced muscles. Our results indicate that local AAV-VEGF-A gene transfer efficiently promotes long-term angiogenesis in large animal model. However, non-regulated expression of VEGF-A causes unfavorable changes in muscle morphology, which suggests the need for regulation of the transgene expression in long-term AAV-mediated VEGF-A gene transfer applications.
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Dependovirus , Fibrosis/etiología , Terapia Genética/métodos , Músculo Esquelético/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Estudios de Seguimiento , Terapia Genética/efectos adversos , Humanos , Isquemia/terapia , Músculo Esquelético/patología , Neovascularización Fisiológica/genética , Conejos , Factor A de Crecimiento Endotelial Vascular/efectos adversosRESUMEN
Lentivirus can be engineered to be a highly potent vector for gene therapy applications. However, generation of clinical grade vectors in enough quantities for therapeutic use is still troublesome and limits the preclinical and clinical experiments. As a first step to solve this unmet need we recently introduced a baculovirus-based production system for lentiviral vector (LV) production using adherent cells. Herein, we have adapted and optimized the production of these vectors to a suspension cell culture system using recombinant baculoviruses delivering all elements required for a safe latest generation LV preparation. High-titer LV stocks were achieved in 293T cells grown in suspension. Produced viruses were accurately characterized and the functionality was also tested in vivo. Produced viruses were compared with viruses produced by calcium phosphate transfection method in adherent cells and polyethylenimine transfection method in suspension cells. Furthermore, a scalable and cost-effective capture purification step was developed based on a diethylaminoethyl monolithic column capable of removing most of the baculoviruses from the LV pool with 65% recovery.
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Baculoviridae/genética , Técnicas de Cultivo de Célula , Vectores Genéticos , Lentivirus/genética , Lentivirus/aislamiento & purificación , Animales , Línea Celular , Etanolaminas , Organismos Modificados Genéticamente , Ratas , Transducción Genética , TransfecciónRESUMEN
The lymphatic vasculature transports extravasated tissue fluid, macromolecules and cells back into the blood circulation. Recent reports have focused on the molecular mechanisms regulating the lymphatic vessels. Vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to stimulate lymphangiogenesis and their receptor, VEGFR-3, has been linked to human hereditary lymphedema. Here we show that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal. Transgenic mice develop a lymphedema-like phenotype characterized by swelling of feet, edema and dermal fibrosis. They survive the neonatal period in spite of a virtually complete lack of lymphatic vessels in several tissues, and later show regeneration of the lymphatic vasculature, indicating that induction of lymphatic regeneration may also be possible in humans.
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Linfedema/patología , Neovascularización Patológica , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Transducción de Señal , Animales , Línea Celular , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Humanos , Ganglios Linfáticos/irrigación sanguínea , Ratones , Ratones Transgénicos , Fenotipo , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Solubilidad , Factor C de Crecimiento Endotelial Vascular , Factor D de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial VascularRESUMEN
Vascular endothelial growth factor (VEGF) has been shown to stimulate angiogenesis and myocardial perfusion. The short-term safety of VEGF gene therapy is excellent. However, there are only limited results regarding the long-term effects. The Kuopio Angiogenesis Trial (KAT) studied the efficiency and short-term safety of the local VEGF-A(165) gene transfer in 103 patients with coronary artery disease. Three patient groups received either VEGF as an adenoviral (n=37), or as a plasmid/liposome vector (n=28), or as a placebo (n=38), during coronary angioplasty and stenting (percutaneous coronary intervention, PCI)AQ1. The aim of this study was to examine the long-term effects and safety of VEGF gene therapy. Patients were interviewed by telephone or with a questionnaire on their current status of health, coronary and other cardiovascular events and symptoms, working ability, exercise tolerance, other diseases, such as cancer and diabetes, as well as their personal experience of the treatment. Causes of death were clarified from hospital records. The total follow-up time was 8.1 years (range 6.9-9.7 years). Overall 82% of the patients were reached across the study. Eight (7.5%) of the patients died during the follow-up, but there was no significant difference in mortality between the groups (3/32 vs 2/26 vs 3/31 VEGF-adenovirus vs VEGF-plasmid/liposome vs placebo, respectively; P=0.88). The incidence of major adverse cardiovascular events (MACEs) (10 vs 11 vs 15; P=0.85), cancer (1 vs 4 vs 2; P=0.38) or diabetes (2 vs 2 vs 2; P=0.97) did not differ between the groups. Local intracoronary VEGF gene transfer is safe and does not increase the risk of MACE, arrhythmias, cancer, diabetes or other diseases.
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Enfermedad Coronaria/terapia , Terapia Genética/efectos adversos , Factor A de Crecimiento Endotelial Vascular/genética , Adenoviridae/genética , Adulto , Anciano , Angioplastia Coronaria con Balón , Enfermedades Cardiovasculares/etiología , Terapia Combinada , Método Doble Ciego , Estudios de Seguimiento , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Humanos , Liposomas , Persona de Mediana Edad , Plásmidos , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin-biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.
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Avidina/metabolismo , Perfilación de la Expresión Génica/métodos , Vectores Genéticos/genética , Lentivirus/genética , Estreptavidina/metabolismo , Animales , Baculoviridae/genética , Biotinilación , Encéfalo/metabolismo , Línea Celular Tumoral , Medios de Cultivo , Ensayo de Inmunoadsorción Enzimática , Receptores ErbB/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Técnicas de Transferencia de Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ligandos , Imagen por Resonancia Magnética/métodos , Masculino , Proteína Cofactora de Membrana/metabolismo , Glicoproteínas de Membrana/metabolismo , Plásmidos , Ratas , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Técnicas Estereotáxicas , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único/métodos , Transducción Genética/métodos , Transducción Genética/normas , Transgenes , Proteínas del Envoltorio Viral/metabolismo , Tropismo Viral/genéticaRESUMEN
Despite promising preclinical results, the clinical benefits of cancer gene therapy have been modest heretofore. The main obstacle continues to be the level and persistence of gene delivery to sufficiently large areas of the tumor. One approach for overcoming this might entail extended local virus release. We studied the utility of silica gel monoliths for delivery of adenovirus to advanced orthotopic gastric and pancreatic cancer tumors. Initially, the biochemical properties of the silica-virus matrix were studied and nearly linear release as a function of time was detected. Virus stayed infective for weeks at +37 degrees C and months at +4 degrees C, which may facilitate storage and distribution. In vivo, extended release of functional replication deficient and also replication-competent, capsid-modified oncolytic viruses was seen. Treatment of mice with pancreatic cancer doubled their survival (P<0.001). Also, silica gel-based delivery slowed the development of antiadenovirus antibodies.