共查询到20条相似文献,搜索用时 31 毫秒
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Anliker B Abel T Kneissl S Hlavaty J Caputi A Brynza J Schneider IC Münch RC Petznek H Kontermann RE Koehl U Johnston IC Keinänen K Müller UC Hohenadl C Monyer H Cichutek K Buchholz CJ 《Nature methods》2010,7(11):929-935
We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105(+) endothelial cells, human CD133(+) hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133(+) cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity. 相似文献
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《Journal of receptor and signal transduction research》2013,33(1-3):279-291
AbstractSeveral approaches have been developed for genetic modulations of receptor expression. These initiated with gene cloning and heterologous expression in microinjected Xenopus oocytes, and proceeded through transgenic expression and genomic disruption of receptor genes in mice. In addition, antisense treatments have reduced receptor levels in a transient, reversible manner. Integration of foreign DNA with host genomic sequences yields both cis- and trans-acting responses. These may depend on the DNA integration site, host cells condition and, most importantly, the affected signal transduction circuit. For example, acetylcholinesterase (AChE) overexpression in microinjected Xenopus tadpoles has been shown to upregulate α-bungarotoxin binding levels, indicating trans-acting control conferring overproduction of muscle nicotinic acetylcholine receptors. In transgenic mice expressing human AChE, the hypothermic response to oxotremorine was suppressed, reflecting modified levels of brain muscarinic receptors. To dissociate the feedback processes occurring in transfected cells from responses related to DNA integration, we examined the endogenous expression of the α7 neuronal nicotinic acetylcholine receptor in PC 12 cells transfected with DNA vectors carrying alternative splicing variants of human AChE mRNA. Our findings demonstrate suppression of α7 receptor levels associated with the accumulation of foreign DNA in the transfected cells. Acetylcholine receptor levels thus depend on multiple elements, each of which should be considered when genetic interventions are employed. 相似文献
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Neumann J Stitz J König R Seibold E Norley S Flory E Cichutek K 《Journal of biotechnology》2006,124(3):615-625
Retroviral vectors have yet not been tested for their potential as vaccines despite their frequent utilization in gene therapy allowing for highly efficient gene transfer into a number of cell types and their suitability for large-scale production in biotechnology. To investigate MLV-based vectors suitability for inducing immune response against HIV-1-antigens, we generated a MLV(HIV-1) pseudotype vector enabling CD4-specific transduction of HIV-1 genes env, vpu, tat and rev originating from the pathogenic SHIV-89.6P. Functional expression of the lentiviral genes in packaging cells, human and rhesus CD4+ target cells was demonstrated by various assays. Following highly efficient ex vivo transduction, up to 3.4x10(7) autologous, transfer vector-positive rhesus peripheral blood mononuclear cells (rhPBMCs) were re-inoculated into a rhesus macaque. Five weeks after the initial inoculation HIV-1 Env-specific antibodies were detected using ELISA. ELIspot-assay revealed the induction of a HIV-1 Rev and Env-specific CTL-response 7.5 weeks after immunization. Thus, these novel MLV(HIV-1) vectors facilitate efficient transduction and subsequent expression of HIV-1-genes in CD4-positive host cells. Induction of both humoral and cellular HIV-1-specific immune responses in vivo confirmed their potential as an effective HIV-1 vaccine to be further studied in SHIV/rhesus macaque model of lentivirus infection. 相似文献
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Adenovirus binding to the coxsackievirus and adenovirus receptor or integrins is not required to elicit brain inflammation but is necessary to transduce specific neural cell types
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Intracranial administration of adenovirus vectors elicits rapid, capsid-mediated dose-dependent brain inflammation. The mechanisms through which adenovirus capsids trigger inflammation in the brain remain unknown. We determined whether adenovirus interaction with the primary and secondary cell surface receptors for infection (CAR and alphav integrins) was necessary to trigger acute adenovirus-mediated brain inflammation, and, furthermore, whether capsid mutations that abrogated CAR and integrin binding altered vector tropism in the brain. Vectors ablated for CAR binding, but retaining integrin binding function, transduced equivalent areas of brain compared to vectors with wild-type capsids; however, vector tropsim was dramatically altered. Vectors with wild-type capsids predominantly transduced oligodendrocytes, whereas mutation of the fiber protein to ablate CAR binding resulted in a loss of oligodendrocyte transduction and a consequent redirection of transduction to neurons and other types of glial cells. Combined mutations of fiber and penton base that ablate both CAR and integrin binding almost abolished brain transduction. Thus, doubly-ablated capsids engineered to express new ligands should allow complete vector retargeting in the central nervous system. Although transduction by the doubly-ablated vectors was reduced by greater than 95%, inflammation was not reduced compared to wild-type vectors, demonstrating that brain inflammation occurs independently of adenovirus binding and infection of cells via CAR and integrin receptors. 相似文献
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Efficient and selective gene transfer into primary human brain tumors by using single-chain antibody-targeted adenoviral vectors with native tropism abolished 总被引:7,自引:0,他引:7
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van Beusechem VW Grill J Mastenbroek DC Wickham TJ Roelvink PW Haisma HJ Lamfers ML Dirven CM Pinedo HM Gerritsen WR 《Journal of virology》2002,76(6):2753-2762
The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeting adenoviral vectors toward tumor cells may improve cancer gene therapy procedures by providing augmented tumor transduction and decreased toxicity to normal tissues. Targeting requires both the complete abolition of native tropism and the addition of a new specific binding ligand onto the viral capsid. Here we accomplished this by using doubly ablated adenoviral vectors, lacking coxsackievirus-adenovirus receptor and alpha(v) integrin binding capacities, together with bispecific single-chain antibodies targeted toward human epidermal growth factor receptor (EGFR) or the epithelial cell adhesion molecule. These vectors efficiently and selectively targeted both alternative receptors on the surface of human cancer cells. Targeted doubly ablated adenoviral vectors were also very efficient and specific with primary human tumor specimens. With primary glioma cell cultures, EGFR targeting augmented the median gene transfer efficiency of doubly ablated adenoviral vectors 123-fold. Moreover, EGFR-targeted doubly ablated vectors were selective for human brain tumors versus the surrounding normal brain tissue. They transduced organotypic glioma and meningioma spheroids with efficiencies similar to those of native adenoviral vectors, while exhibiting greater-than-10-fold-reduced background levels on normal brain explants from the same patients. As a result, EGFR-targeted doubly ablated adenoviral vectors had a 5- to 38-fold-improved tumor-to-normal brain targeting index compared to native vectors. Hence, single-chain targeted doubly ablated adenoviral vectors are promising tools for cancer gene therapy. They should provide an improved therapeutic index with efficient tumor transduction and effective protection of normal tissue. 相似文献
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In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors 总被引:9,自引:0,他引:9
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Ulrich Kuhn Atsushi Terunuma Wolfgang Pfutzner Ruth Ann Foster Jonathan C. Vogel 《Journal of virology》2002,76(3):1496-1504
For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture. 相似文献
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Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system.In brain research,a series of powerful and novel gene editing technologies are based on these systems.Although many viral vectors are used in rodents,their full application has been limited in non-human primates.To identify viral vectors that can stably and effectively express exogenous genes within nonhuman primates,eleven commonly used recombinant adeno-associated viral and lentiviral vectors,each carrying a gene to express green or red fluorescence,were injected into the parietal cortex of four rhesus monkeys.The expression of fluorescent cells was used to quantify transfection efficiency.Histological results revealed that recombinant adeno-associated viral vectors,especially the serotype 2/9 coupled with the cytomegalovirus,human synapsin Ⅰ,or Ca2+/calmodulin-dependent protein kinase Ⅱ promoters,and lentiviral vector coupled with the human ubiquitin C promoter,induced higher expression of fluorescent cells,representing high transfection efficiency.This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates (NHPs).These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates. 相似文献
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Recombinant human adenovirus: targeting to the human transferrin receptor improves gene transfer to brain microcapillary endothelium 总被引:6,自引:0,他引:6
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Some inborn errors of metabolism due to deficiencies of soluble lysosomal enzymes cause global neurodegenerative disease. Representative examples include the infantile and late infantile forms of the ceroid lipofuscinoses (CLN1 or CLN2 deficiency, respectively) and mucopolysaccharidoses type VII (MPS VII), a deficiency of beta-glucuronidase. Treatment of the central nervous system component of these disorders will require widespread protein or enzyme replacement, either through dissemination of the protein or through dissemination of a gene encoding it. We hypothesize that transduction of brain microcapillary endothelium (BME) with recombinant viral vectors, with secretion of enzyme product basolaterally, could allow for widespread enzyme dissemination. To achieve this, viruses should be modified to target the BME. This requires (i) identification of a BME-resident target receptor, (ii) identification of motifs targeted to that molecule, (iii) the construction of modified viruses to allow for binding to the target receptor, and (iv) demonstrated transduction of receptor-expressing cells. In proof of principal experiments, we chose the human transferrin receptor (hTfR), a molecule found at high density on human BME. A nonamer phage display library was panned for motifs which could bind hTfR. Forty-three clones were sequenced, most of which contained an AKxxK/R, KxKxPK/R, or KxK motif. Ten peptides representative of the three motifs were cloned into the HI loop of adenovirus type 5 fiber. All motifs tested retained their ability to trimerize and bind transferrin receptor, and seven allowed for recombinant adenovirus production. Importantly, the fiber-modified viruses facilitated increased gene transfer (2- to 34-fold) to hTfR expressing cell lines and human brain microcapillary endothelia expressing high levels of endogenous receptor. Our data indicate that adenoviruses can be modified in the HI loop for expanded tropism to the hTfR. 相似文献
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Adeno-associated virus vectors for gene transfer to the brain 总被引:4,自引:0,他引:4
Okada T Nomoto T Shimazaki K Lijun W Lu Y Matsushita T Mizukami H Urabe M Hanazono Y Kume A Muramatsu S Nakano I Ozawa K 《Methods (San Diego, Calif.)》2002,28(2):237-247
Gene therapy is a novel method under investigation for the treatment of neurological disorders. Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system. Adeno-associated virus (AAV) is a potentially useful gene transfer vehicle for neurologic gene therapies. The advantages of AAV vector include the lack of any associated disease with a wild-type virus, the ability to transduce nondividing cells, the possible integration of the gene into the host genome, and the long-term expression of transgenes. The development of novel therapeutic strategies for neurological disorder by using AAV vector has an increasing impact on gene therapy research. This article describes methods that can be used to generate rodent and nonhuman primate models for testing treatment strategies linked to pathophysiological events in the ischemic brain and neurodegenerative disorders such as Parkinson's disease. 相似文献
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Intraventricular brain injection of adeno-associated virus type 1 (AAV1) in neonatal mice results in complementary patterns of neuronal transduction to AAV2 and total long-term correction of storage lesions in the brains of beta-glucuronidase-deficient mice 总被引:4,自引:0,他引:4
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Passini MA Watson DJ Vite CH Landsburg DJ Feigenbaum AL Wolfe JH 《Journal of virology》2003,77(12):7034-7040
Inherited metabolic disorders that affect the central nervous system typically result in pathology throughout the brain; thus, gene therapy strategies need to achieve widespread delivery. We previously found that although intraventricular injection of the neonatal mouse brain with adeno-associated virus serotype 2 (AAV2) results in dispersed gene delivery, many brain structures were poorly transduced. This limitation may be overcome by using different AAV serotypes because the capsid proteins use different cellular receptors for entry, which may allow enhanced global targeting of the brain. We tested this with AAV1 and AAV5 vectors. AAV5 showed very limited brain transduction after neonatal injection, even though it has different transduction patterns than AAV2 in adult brain injections. In contrast, AAV1 vectors, which have not been tested in the brain, showed robust widespread transduction. Complementary patterns of transduction between AAV1 and AAV2 were established and maintained in the adult brain after neonatal injection. In the majority of structures, AAV1 transduced many more cells than AAV2. Both vectors transduced mostly neurons, indicating that differential expression of receptors on the surfaces of neurons occurs in the developing brain. The number of cells positive for a vector-encoded secreted enzyme (beta-glucuronidase) was notably greater and more widespread in AAV1-injected brains. A comprehensive analysis of AAV1-treated brains from beta-glucuronidase-deficient mice (mucopolysaccharidosis type VII) showed complete reversal of pathology in all areas of the brain for at least 1 year, demonstrating that the combination of this serotype and experimental strategy is therapeutically effective for treating global neurometabolic disorders. 相似文献
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Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+ signal transduction. 总被引:22,自引:0,他引:22
T Abe H Sugihara H Nawa R Shigemoto N Mizuno S Nakanishi 《The Journal of biological chemistry》1992,267(19):13361-13368
A cDNA clone for a new metabotropic glutamate receptor, mGluR5, was isolated through polymerase chain reaction-mediated DNA amplification by using primer sequences conserved among the metabotropic glutamate receptor (mGluR) family and by the subsequent screening of a rat brain cDNA library. The cloned receptor consists of 1171 amino acid residues and exhibits a structural architecture common to the mGluR family, possessing a large extracellular domain preceding the seven putative membrane-spanning segments. mGluR5 shows the highest sequence similarity to mGluR1 among the mGluR members and is coupled to the stimulation of phosphatidylinositol hydrolysis/Ca2+ signal transduction in Chinese hamster ovary cells transfected with the cloned cDNA. This receptor also resembles mGluR1 in its agonist selectivity and antagonist responses; the potency rank order of agonists for mGluR5 was determined to be quisqualate greater than L-glutamate greater than or equal to ibotenate greater than trans-1-aminocyclopentane-1,3-dicarboxylate. Blot and in situ hybridization analyses indicated that mGluR5 mRNA is widely distributed in neuronal cells of the central nervous system and is expressed differently from mGluR1 mRNA in many brain regions. This investigation thus demonstrates that there is an additional mGluR subtype which closely resembles mGluR1 in its signal transduction and pharmacological properties and is expressed in specialized neuronal cells in the central nervous system. 相似文献
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Recombinant adenoviral (rAd) vectors elicit potent cellular and humoral immune responses and show promise as vaccines for HIV-1, Ebola virus, tuberculosis, malaria, and other infections. These vectors are now widely used and have been generally well tolerated in vaccine and gene therapy clinical trials, with many thousands of people exposed. At the same time, dose-limiting adverse responses have been observed, including transient low-grade fevers and a prior human gene therapy fatality, after systemic high-dose recombinant adenovirus serotype 5 (rAd5) vector administration in a human gene therapy trial. The mechanism responsible for these effects is poorly understood. Here, we define the mechanism by which Ad5 targets immune cells that stimulate adaptive immunity. rAd5 tropism for dendritic cells (DCs) was independent of the coxsackievirus and adenovirus receptor (CAR), its primary receptor or the secondary integrin RGD receptor, and was mediated instead by a heparin-sensitive receptor recognized by a distinct segment of the Ad5 fiber, the shaft. rAd vectors with CAR and RGD mutations did not infect a variety of epithelial and fibroblast cell types but retained their ability to transfect several DC types and stimulated adaptive immune responses in mice. Notably, the pyrogenic response to the administration of rAd5 also localized to the shaft region, suggesting that this interaction elicits both protective immunity and vector-induced fevers. The ability of replication-defective rAd5 viruses to elicit potent immune responses is mediated by a heparin-sensitive receptor that interacts with the Ad5 fiber shaft. Mutant CAR and RGD rAd vectors target several DC and mononuclear subsets and induce both adaptive immunity and toxicity. Understanding of these interactions facilitates the development of vectors that target DCs through alternative receptors that can improve safety while retaining the immunogenicity of rAd vaccines. 相似文献
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Lilley CE Groutsi F Han Z Palmer JA Anderson PN Latchman DS Coffin RS 《Journal of virology》2001,75(9):4343-4356
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Abad JL Serrano F San Román AL Delgado R Bernad A González MA 《The journal of gene medicine》2002,4(1):27-37