RESUMO
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.
Assuntos
Avidina/metabolismo , Perfilação da Expressão Gênica/métodos , Vetores Genéticos/genética , Lentivirus/genética , Estreptavidina/metabolismo , Animais , Baculoviridae/genética , Biotinilação , Encéfalo/metabolismo , Linhagem Celular Tumoral , Meios de Cultura , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Técnicas de Transferência de Genes , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligantes , Imageamento por Ressonância Magnética/métodos , Masculino , Proteína Cofatora de Membrana/metabolismo , Glicoproteínas de Membrana/metabolismo , Plasmídeos , Ratos , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Técnicas Estereotáxicas , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Transdução Genética/métodos , Transdução Genética/normas , Transgenes , Proteínas do Envelope Viral/metabolismo , Tropismo Viral/genéticaRESUMO
As gene therapy has matured from clinical trials to the first commercial products, understanding of the mechanisms of gene delivery has increased tremendously. This has also been reflected in viral vector development, creating a number of new approaches to tackle issues in transduction efficiency, biodistribution and viral safety. This review will highlight the most important issues and advancements in vector development, administration, surface modification, integration to host genome and safety. The gene therapy products currently available or near market approval, based on p53 expression (Gendicine and Advexin), conditionally replicative adenoviruses (Oncorine) and thymidine kinase + ganciclovir therapy (Cerepro), are introduced with emphasis on the molecular mechanisms of action.
Assuntos
Terapia Genética , Adenoviridae/genética , Vetores Genéticos/imunologia , Humanos , Vírus Oncolíticos/genética , Timidina Quinase/genética , Timidina Quinase/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/farmacologiaRESUMO
Non-invasive imaging provides essential information regarding the biodistribution of gene therapy vectors and it can also be used for the development of targeted vectors. In this study, we have utilized micro Single-photon emission computed tomography to visualize biodistribution of a (99m)Tc-polylys-ser-DTPA-biotin-labelled avidin-displaying baculovirus, Baavi, after intrafemoral (i.f.), intraperitoneal (i.p.), intramuscular (i.m.) and intracerebroventricular (i.c.v.) administration. The imaging results suggest that the virus can spread via the lymphatic network after different administration routes, also showing accumulation in the nasal area after systemic administration. Extensive expression in the kidneys and spleen was seen after i.p. administration, which was confirmed by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Additionally, transduction of kidneys was seen with i.m. and i.f. administrations. We conclude that baculovirus may be beneficial for the treatment of kidney diseases after i.p. administration route.
Assuntos
Baculoviridae/fisiologia , Vetores Genéticos , Rim/virologia , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , Animais , Biotina , Terapia Genética , Imuno-Histoquímica , Injeções Intramusculares , Injeções Intraperitoneais , Injeções Intraventriculares , Rim/diagnóstico por imagem , Nefropatias/terapia , Masculino , Ácido Pentético , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pertecnetato Tc 99m de Sódio , Baço/virologiaRESUMO
We describe here a technique for the visualization of viral vector delivery by magnetic resonance imaging (MRI) in vivo. By conjugating avidin-coated baculoviral vectors (Baavi) with biotinylated ultra-small superparamagnetic iron oxide particles (USPIO), we are able to produce vector-related MRI contrast in the choroid plexus cells of rat brain in vivo over a period of 14 days. Ten microlitres of 2.5 x 10(10) PFU/ml nuclear-targeted LacZ-encoding Baavi with bUSPIO coating was injected into rat brain ventricles and visualized by MRI at 4.7 T. As baculoviruses exhibit restricted cell-type specificity in the rat brain, altered MRI contrast was detected in the choroid plexus of the injected ventricles. No specific signal loss was detected when wild-type baculoviruses or intact biotinylated USPIO particles were injected into the lateral ventricles. Cryosectioned brains were stained for nuclear-targeted beta-galactosidase gene expression, which was found to colocalize with MRI contrast. This study provides the first proof of principle for robust and non-invasive viral vector MRI by using avidin-displaying viruses in vivo. Considering the widespread use of MRI in current medical imaging, the approach is likely to provide numerous future applications in imaging of therapeutic gene transfer.
Assuntos
Baculoviridae/ultraestrutura , Encéfalo/virologia , Terapia Genética/métodos , Imageamento por Ressonância Magnética , Animais , Baculoviridae/genética , Biomarcadores , Compostos Férricos , Vetores Genéticos/administração & dosagem , Nanopartículas , Ratos , Distribuição Tecidual , Transdução Genética , beta-Galactosidase/genéticaRESUMO
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.
