RESUMEN
Efficient manufacture of recombinant adeno-associated virus (rAAV) vectors for gene therapy remains challenging. Packaging cell lines containing stable integration of the AAV rep/cap genes have been explored, however rAAV production needs to be induced using wild-type adenoviruses to promote episomal amplification of the integrated rep/cap genes by mobilizing a cis-acting replication element (CARE). The adenovirus proteins responsible are not fully defined, and using adenovirus during rAAV manufacture leads to contamination of the rAAV preparation. 'TESSA' is a helper adenovirus with a self-repressing Major Late Promoter (MLP). Its helper functions enable efficient rAAV manufacture when the rep and cap genes are provided in trans but is unable to support rAAV production from stable packaging cells. Using rAAV-packaging cell line HeLaRC32, we show that expression of the adenovirus L4 22/33K unit is essential for rep/cap amplification but the proteins are titrated away by binding to replicating adenovirus genomes. siRNA-knockdown of the adenovirus DNA polymerase or the use of a thermosensitive TESSA mutant decreased adenovirus genome replication whilst maintaining MLP repression, thereby recovering rep/cap amplification and efficient rAAV manufacture. Our findings have direct implications for engineering more efficient adenovirus helpers and superior rAAV packaging/producer cells.
Asunto(s)
Adenoviridae , Proteínas Virales , Humanos , Transfección , Células HeLa , Plásmidos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Vectores Genéticos/genética , Replicación Viral/genéticaRESUMEN
Recombinant adeno-associated virus (rAAV) shows great promise for gene therapy, however scalability, yield and quality remain significant issues. Here we describe an rAAV manufacturing strategy using a 'helper' adenovirus that self-inhibits its major late promoter (MLP) to truncate its own replication. Inserting a tetracycline repressor (TetR) binding site into the MLP and encoding the TetR under its transcriptional control allowed normal adenovirus replication in the presence of doxycycline but only genome amplification and early gene expression (the 'helper' functions) in its absence. Using this self-inhibiting adenovirus we demonstrate delivery of adenoviral helper functions, AAV rep and cap genes, and the rAAV genome to yield up to 30-fold more rAAV vectors compared to the helper-free plasmid approach and significant improvements in particle infectivity for a range of serotypes. This system allows significant improvements in the production of serotypes rAAV2, rAAV6, rAAV8 and rAAV9, and enables propagation of existing rAAV without transfection, a process that improves batch quality by depleting reverse packaged DNA contaminants. We propose this as a high-yielding, contaminant-free system suitable for scalable rAAV manufacture.
Asunto(s)
Adenoviridae , Dependovirus , Adenoviridae/genética , Dependovirus/genética , Vectores Genéticos/genética , Transfección , Replicación ViralRESUMEN
Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD ) as low as 0.8 nm We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule-positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Molécula de Adhesión Celular Epitelial/inmunología , Proteínas de la Membrana/inmunología , Receptores Quiméricos de Antígenos/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Cricetinae , Cricetulus , Biblioteca de Genes , Humanos , Células Jurkat , Unión ProteicaRESUMEN
Oncolytic viruses represent an emerging approach to cancer therapy. However, better understanding of their interaction with the host cancer cell and approaches to enhance their efficacy are needed. Here, we investigate the effect of chemically induced endoplasmic reticulum (ER) stress on the activity of the chimeric group B adenovirus Enadenotucirev, its closely related parental virus Ad11p, and the archetypal group C oncolytic adenovirus Ad5. We show that treatment of colorectal and ovarian cancer cell lines with thapsigargin or ionomycin caused an influx of Ca2+, leading to an upregulation in E1A transcript and protein levels. Increased E1A protein levels, in turn, increased levels of expression of the E2B viral DNA polymerase, genome replication, late viral protein expression, infectious virus particle production, and cell killing during Enadenotucirev and Ad11p, but not Ad5, infection. This effect was not due to the induction of ER stress, but rather the influx of extracellular Ca2+ and consequent increase in protein kinase C activity. These results underscore the importance of Ca2+ homeostasis during adenoviral infection, indicate a signaling pathway between protein kinase C and E1A, and raise the possibility of using Ca2+ flux-modulating agents in the manufacture and potentiation of oncolytic virotherapies.
RESUMEN
The need for CD4+ T cell responses to arise de novo following vaccination can limit the speed of B cell responses. Populations of pre-existing vaccine-induced or anti-viral CD4+ T cells recognising distinct antigens could be exploited to overcome this limitation. We hypothesise that liposomal vaccine particles encapsulating epitopes that are recognised, after processing and B cell MHCII presentation, by pre-existing CD4+ T cells will exploit this pre-existing T cell help and result in improved antibody responses to distinct target antigens displayed on the particle surface. Liposomal vaccine particles were engineered to display the malaria circumsporozoite (CSP) antigen on their surface, with helper CD4+ epitopes from distinct vaccine or viral antigens contained within the particle core, ensuring the B cell response is raised but focused against CSP. In vivo vaccination studies were then conducted in C57Bl/6 mice as models of either vaccine-induced pre-existing CD4+ T cell immunity (using ovalbumin-OVA) or virus-induced pre-existing CD4+ T cell immunity (murine cytomegalovirus-MCMV). Following the establishment of pre-existing by vaccination (OVA in the adjuvant TiterMax® Gold) or infection with MCMV, mice were administered CSP-coated liposomal vaccines containing the relevant OVA or MCMV core CD4+ T cell epitopes. In mice with pre-existing anti-OVA CD4+ T cell immunity, these vaccine particles elicited rapid, high-titre, isotype-switched CSP-specific antibody responses-consistent with the involvement of anti-OVA T helper cells in confirming activation of anti-CSP B cells. Responses were further improved by entrapping TLR9 agonists, combining humoral vaccination signals 'one', 'two' and 'three' within one particle. Herpes viruses can establish chronic infection and elicit significant, persistent cellular immune responses. We then demonstrate that this principle can be extended to re-purpose pre-existing anti-MCMV immunity to enhance anti-CSP vaccine responses-the first description of a strategy to specifically exploit anti-cytomegalovirus immunity to augment vaccination against a target antigen.
Asunto(s)
Linfocitos B/inmunología , Interacciones Huésped-Patógeno/inmunología , Memoria Inmunológica , Activación de Linfocitos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas/inmunología , Virus/inmunología , Animales , Afinidad de Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , Antígenos/inmunología , Linfocitos B/metabolismo , Epítopos/inmunología , Infecciones por Herpesviridae/inmunología , Humanos , Cambio de Clase de Inmunoglobulina/inmunología , Ratones , Muromegalovirus/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Receptor Toll-Like 9/agonistasRESUMEN
The efficacy of vaccine adjuvants such as Toll-like receptor agonists (TLRa) can be improved through formulation and delivery approaches. Here, we attached small molecule TLR-7/8a to polymer scaffolds (polymer-TLR-7/8a) and evaluated how different physicochemical properties of the TLR-7/8a and polymer carrier influenced the location, magnitude and duration of innate immune activation in vivo. Particle formation by polymer-TLR-7/8a was the most important factor for restricting adjuvant distribution and prolonging activity in draining lymph nodes. The improved pharmacokinetic profile by particulate polymer-TLR-7/8a was also associated with reduced morbidity and enhanced vaccine immunogenicity for inducing antibodies and T cell immunity. We extended these findings to the development of a modular approach in which protein antigens are site-specifically linked to temperature-responsive polymer-TLR-7/8a adjuvants that self-assemble into immunogenic particles at physiologic temperatures in vivo. Our findings provide a chemical and structural basis for optimizing adjuvant design to elicit broad-based antibody and T cell responses with protein antigens.
Asunto(s)
Adyuvantes Inmunológicos/química , Receptores Toll-Like/agonistas , Vacunas/inmunología , Animales , Portadores de Fármacos/química , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunologíaRESUMEN
Nanomedicines have provided fresh impetus in the fight against cancer due to their selectivity and power. However, these agents are limited when delivered intravenously due to their rapid clearance from the bloodstream and poor passage from the bloodstream into target tumours. Here we describe a novel stealthing strategy which addresses both these limitations and thereby demonstrate that both the passive and mechanically-mediated tumour accumulation of the model nanomedicine adenovirus (Ad) can be substantially enhanced. In our strategy gold nanoparticles were thoroughly modified with 2kDa polyethyleneglycol (PEG) and then linked to Ad via a single reduction-cleavable 5kDa PEG. The resulting Ad-gold-PEG construct was compared to non-modified Ad or conventionally stealthed Ad-poly[N-(2-hydroxypropyl)methacrylamide] (Ad-PHPMA). Notably, although Ad-gold-PEG was of similar size and surface charge to Ad-PHPMA the increase in density, resulting from the inclusion of the gold nanoparticles, provided a substantial enhancement of ultrasound-mediated transport. In an in vitro tumour mimicking phantom, the level and distance of Ad-gold-PEG transport was shown to be substantially greater than achieved with Ad-PHPMA. In in vivo studies 0.1% of an unmodified Ad dose was shown to accumulate in tumours, whereas over 12% of the injected dose was recovered from the tumours of mice treated with Ad-gold-PEG and ultrasound. Ultimately, a significant increase in anti-tumour efficacy resulted from this strategy. This stealthing and density-increasing technology could ultimately enhance clinical utility of intravenously delivered nanoscale medicines including viruses, liposomes and antibodies.
Asunto(s)
Adenoviridae/genética , Oro , Nanopartículas del Metal , Polietilenglicoles , Animales , Línea Celular Tumoral , Femenino , Oro/administración & dosificación , Oro/química , Proteínas Fluorescentes Verdes/genética , Humanos , Hígado/metabolismo , Nanopartículas del Metal/administración & dosificación , Nanopartículas del Metal/química , Ratones Endogámicos BALB C , Ratones Desnudos , Nanomedicina , Neoplasias/metabolismo , Neoplasias/terapia , Viroterapia Oncolítica , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Ácidos Polimetacrílicos/administración & dosificación , Ácidos Polimetacrílicos/química , UltrasonidoRESUMEN
Wild-type Sindbis virus (SV) shows promise as an oncolytic agent, although potential off-target replication is a safety concern. To remove possible pathology reflecting virus replication in liver, muscle, and/or hematopoietic cells, microRNA (miR)-response elements (MREs) to liver-specific miR122, muscle-specific miR133a and miR206, or hematopoietic-specific miR142-3p were inserted into the Sindbis viral genome. We compared the effectiveness of MREs in two distinct genomic locations and found better tissue-specific attenuation when they were inserted into the structural polyprotein coding region (up to 6000-fold selectivity with miR142-3p) rather than into the 3' untranslated region (up to 850-fold with miR142-3p). While this degree of tissue-specific attenuation may be effective for relieving pathology in vivo, genetic instability of RNA viruses raises concerns over the mutation or loss of MREs conferring safety. Genetically modified SVs containing a reporter transgene, used as a surrogate for virus replication, mutated quickly in vitro, losing 50% transgene sequence within 6.2 passages. Using a shorter insert containing MREs but no transgene, complete genetic stability was observed over at least 10 passages. We conclude that SV may be genetically modified to improve clinical properties, but attention must be paid to ensure that genetic stability is sufficient for intended applications.
Asunto(s)
Inestabilidad Genómica , Virus Oncolíticos/genética , Virus Sindbis/fisiología , Regiones no Traducidas 3' , Animales , Línea Celular , Cricetinae , Genoma Viral , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Hígado/metabolismo , Hígado/virología , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Músculos/metabolismo , Músculos/virología , Elementos de Respuesta/genética , Virus Sindbis/genéticaRESUMEN
Cancer arises from 'self' in a series of steps that are all subject to immunoediting. Therefore, therapeutic cancer vaccines must stimulate an immune response against tumour antigens that have already evaded the body's immune defences. Vaccines presenting a tumour antigen in the context of obvious danger signals seem more likely to stimulate a response. This approach can be facilitated by genetic engineering using recombinant viral vectors expressing tumour antigens, cytokines, or both, from an immunogenic virus particle. We overview clinical attempts to use these agents for systemic immunisation and contrast the results with strategies employing direct intratumoural administration. We focus on the challenge of producing an effective response within the immune-suppressive tumour microenvironment, and discuss how the technology can overcome these obstacles.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Neoplasias/inmunología , Neoplasias/terapia , Vacunas Virales/uso terapéutico , Animales , Vacunas contra el Cáncer/inmunología , Virus ADN/inmunología , Humanos , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéutico , Vacunas Virales/inmunologíaRESUMEN
Intravenous delivery of therapeutic virus particles remains a major goal for virotherapy of metastatic cancer. Avoiding phagocytic capture and unwanted infection of nontarget cells is essential for extended plasma particle kinetics, and simply ablating one or the other does not give extended plasma circulation. Here we show that polymer coating of adenovirus type 5 (Ad5) can combine with predosing strategies or Kupffer cell ablation to achieve systemic kinetics with a half-life >60 min, allowing ready access to peripheral tumors. Accumulation of virus particles within tumor nodules is proportional to the area under the plasma concentration/time curve. Polymer coating wild-type Ad5 in this way is known to decrease hepatic toxicity, increasing the dose of virus particles that can be safely administered. Using polymer-coating technology to deliver a replicating Ad5 systemically, virus replication and transgene expression was almost totally confined to tumor tissues, giving a much improved therapeutic index compared with uncoated virus, and complete control of human HepG2 tumor xenografts.
Asunto(s)
Acrilamidas/química , Adenoviridae/química , Adenoviridae/fisiología , Neoplasias Hepáticas/terapia , Viroterapia Oncolítica/métodos , Animales , Femenino , Células HEK293 , Células Hep G2 , Humanos , Hígado/patología , Hígado/virología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Ratones , Ratones Endogámicos BALB CRESUMEN
Building on their success in vaccination, many groups are now exploring the use of viruses as anticancer agents. In general, viral therapeutics provide the possibility to express anticancer proteins directly at the tumour site, decreasing exposure to normal tissue during delivery and maximising therapeutic index. Some viruses are also 'oncolytic', either naturally or by design, and these agents function to kill cancer cells selectively before spreading to infect adjacent cells and repeat the process. This whole field of cancer 'virotherapy' is moving forward rapidly at the moment, with notable clinical successes demonstrated with a range of oncolytic agents developed as directly oncolytic and also as oncolytic cancer vaccines. Given the versatility of oncolytic viruses to express therapeutic proteins we anticipate this approach will provide the platform for useful application of a broad range of innovative biological therapies.
Asunto(s)
Vacunas contra el Cáncer/farmacología , Neoplasias/terapia , Neoplasias/virología , Viroterapia Oncolítica/métodos , Virus Oncolíticos/fisiología , Animales , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Humanos , Neoplasias/genética , Neoplasias/inmunología , Virus Oncolíticos/genética , Virus Oncolíticos/inmunologíaRESUMEN
Tumour Necrosis Factor alpha (TNF) is a pleiotropic pro-inflammatory cytokine with known vascular permeabilising activity. It is employed during isolated limb perfusion to enhance delivery of chemotherapeutic drugs into tumour tissue. The use of conditionally-replicating lytic viruses, so called 'oncolytic virotherapy', provides a new approach to cancer treatment that is currently limited by the low efficiency of extravasation of viral particles into tumours. We report here evidence that TNF significantly enhances the delivery of virus particles through the endothelial layer to allow access to tumour cells both in vitro and in vivo. Intravenous administration of TNF resulted in a 3- to 6-fold increase in EL4 tumour uptake of Evans Blue/Albumin, adenovirus and long-circulating polymer coated adenovirus. Interestingly, endothelial permeabilisation could be suppressed in vitro and in vivo by Y-27632, a Rho kinase inhibitor, without inhibiting viral infection. These data indicate that TNF can enhance the delivery of virus particles into tumours through a Rho A/Rho kinase dependent mechanism and may be a valuable strategy for increasing the delivery of oncolytic viruses and other therapeutic agents.
Asunto(s)
Adenoviridae/fisiología , Permeabilidad Capilar/efectos de los fármacos , Viroterapia Oncolítica/métodos , Virus Oncolíticos/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Virión/fisiología , Quinasas Asociadas a rho/metabolismo , Animales , Línea Celular , Endotelio Vascular/efectos de los fármacos , Humanos , Ratones , Neoplasias/terapia , Transducción de Señal/efectos de los fármacosRESUMEN
MicroRNAs are small non-coding RNA molecules that regulate mRNA translation and stability by binding to complementary sequences usually within the 3' un-translated region (UTR). We have previously shown that the hepatic toxicity caused by wild-type Adenovirus 5 (Ad5WT) in mice can be prevented by incorporating 4 binding sites for the liver-specific microRNA, mir122, into the 3' UTR of E1A mRNA. This virus, termed Ad5mir122, is a promising virotherapy candidate and causes no obvious liver pathology. Herein we show that Ad5mir122 maintains wild-type lytic activity in cancer cells not expressing mir122 and assess any effects of possible mir122 depletion in host cells. Repeat administration of 2×10(10) viral particles of Admir122 to HepG2 tumour bearing mice showed significant anti-cancer efficacy. RT-QPCR showed that E1A mRNA was down-regulated 29-fold in liver when compared to Ad5WT. Western blot for E1A confirmed that all protein variants were knocked down. RT-QPCR for mature mir122 in infected livers showed that quantity of mir122 remained unaffected. Genome wide mRNA microarray profiling of infected livers showed that although the transcript level of >3900 different mRNAs changed more than 2-fold following Ad5WT infection, less than 600 were changed by Ad5mir122. These were then filtered to select mRNAs that were only altered by Ad5mir122 and the remaining 21 mRNAs were compared to predicted mir122 targets. No mir122 target mRNAs were affected by Ad5 mir122. These results demonstrate that the exploitation of microRNA regulation to control virus replication does not necessarily affect the level of the microRNA or the endogenous mRNA targets.
Asunto(s)
Adenoviridae/genética , MicroARNs/uso terapéutico , Neoplasias Experimentales/terapia , Animales , Células Hep G2 , Humanos , Hígado/química , Ratones , MicroARNs/análisis , Trasplante Heterólogo , Resultado del Tratamiento , ViriónRESUMEN
The endothelium imposes a structural barrier to the extravasation of systemically delivered oncolytic adenovirus (Ad). Here, we introduced a transendothelial route of delivery in order to increase tumor accumulation of virus particles (vp) beyond that resulting from convection-dependent extravasation alone. This was achieved by engineering an Ad encoding a syncytium-forming protein, gibbon ape leukemia virus (GALV) fusogenic membrane glycoprotein (FMG). The expression of GALV was regulated by a hybrid viral enhancer-human promoter construct comprising the human cytomegalovirus (CMV) immediate-early enhancer and the minimal human endothelial receptor tyrosine kinase promoter ("eTie1"). Endothelial cell-selectivity of the resulting Ad-eTie1-GALV vector was demonstrated by measuring GALV mRNA transcript levels. Furthermore, Ad-eTie1-GALV selectively induced fusion between infected endothelial cells and uninfected epithelial cells in vitro and in vivo, allowing transendothelial virus penetration. Heterofusion of infected endothelium to human embryonic kidney 293 (HEK 293) cells, in mixed in vitro cultures or in murine xenograft models, permitted fusion-dependent transactivation of the replication-deficient Ad-eTie1-GALV, due to enabled access to viral E1 proteins derived from the HEK 293 cytoplasm. These data provide evidence to support our proposed use of GALV to promote Ad penetration through tumor-associated vasculature, an approach that may substantially improve the efficiency of systemic delivery of oncolytic viruses to disseminated tumors.
Asunto(s)
Adenoviridae/metabolismo , Fusión Celular/métodos , Células Gigantes/metabolismo , Virus de la Leucemia del Gibón/genética , Glicoproteínas de Membrana/genética , Terapia Molecular Dirigida/métodos , Migración Transendotelial y Transepitelial/genética , Adenoviridae/genética , Animales , Antígenos Virales/genética , Antígenos Virales/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/virología , Elementos de Facilitación Genéticos , Células Epiteliales/metabolismo , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células Gigantes/citología , Células HEK293 , Hepatocitos/citología , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones SCID , Neoplasias/irrigación sanguínea , Neoplasias/terapia , Neoplasias/virología , Viroterapia Oncolítica/métodos , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Trasplante Heterólogo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Virales/farmacología , Virión , Replicación Viral/genéticaRESUMEN
A significant limitation to the use of viruses as systemic vectors is the susceptibility of the vector to inactivation and clearance by various blood components. Despite much focus on antibodies as the primary neutralizing molecules in blood, other mechanisms inactivate and clear virus particles from the bloodstream in both naïve and pre-immune hosts. This review provides an overview of the major blood components that interact with enveloped viruses. The mechanisms of action of these blood components by which virus particles are inactivated are also discussed. In addition, important blood components that act as barriers to the systemic delivery of therapeutic viruses are identified, and recent advances in overcoming these barriers are highlighted. Particular attention is given to the field of oncolytic virotherapy in which adequate intravenous virus delivery is critical for therapeutic success.
Asunto(s)
Ensayo de Materiales/métodos , Virus/inmunología , Animales , Proteínas del Sistema Complemento/inmunología , Humanos , Viroterapia Oncolítica/métodos , Viroterapia Oncolítica/tendencias , Virus Oncolíticos/inmunologíaRESUMEN
Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5x10(10) viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.
Asunto(s)
Adenoviridae/fisiología , Hepatocitos/virología , MicroARNs/metabolismo , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Alanina Transaminasa/sangre , Secuencia de Aminoácidos , Animales , Fusión Artificial Génica , Aspartato Aminotransferasas/sangre , Sitios de Unión/genética , Línea Celular Tumoral , Fluorescencia , Regulación Viral de la Expresión Génica , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hígado/metabolismo , Hígado/patología , Hígado/virología , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/virología , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Datos de Secuencia Molecular , Viroterapia Oncolítica , Distribución Tisular , Imagen de Cuerpo EnteroRESUMEN
A report on the symposium 'In vivo barriers to gene delivery', Cold Spring Harbor, USA, 26-29 November 2007.
Asunto(s)
Técnicas de Transferencia de Gen , Expresión Génica , Técnicas de Transferencia de Gen/normas , Técnicas de Transferencia de Gen/tendencias , Vectores Genéticos/inmunología , Humanos , Transgenes/genéticaRESUMEN
The nucleolus is a dynamic sub-nuclear structure which is involved in ribosome subunit biogenesis, modulation of cell growth and response to cell stress. The nucleolar proteome varies particularly with regard to the cell cycle. Viral proteins can localise to the nucleolus and using the coronavirus nucleocapsid (N) protein as a model, the cell cycle dependent trafficking of viral proteins to the nucleolus was investigated. Cell synchronisation studies coupled to live cell confocal microscopy indicated that nucleolar localisation of N protein was greater in the G2/M phase of the cell cycle than at other stages. This result was reinforced when FRAP and FLIP analysis indicated that N protein was more mobile within the nucleoplasm and nucleolus in the G2/M phase of the cell cycle. The data suggested that viral nucleolar proteins can also localise to the nucleolus in a cell cycle dependent manner and this may be related to dynamic trafficking.