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At least 1,387 records · Page 77Linked to original sources
PubMed2006

Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain.

Adeno-associated virus (AAV) vectors have gained a preeminent position in the field of gene delivery to the normal brain through their ability to achieve extensive transduction of neurons and to mediate long-term gene expression with no apparent toxicity. In adult animals direct infusion of AAV vectors into the brain parenchyma results in highly efficient transduction of target structures. However AAV-mediated global delivery to the adult brain has been an elusive goal. In contrast, widespread global gene delivery has been obtained by i.c.v. injection of AAV1 or AAV2 in neonates. Among the novel AAV serotypes cloned and engineered for production of recombinant vectors, AAV8 has shown a tremendous potential for in vivo gene delivery with nearly complete transduction of many tissues in rodents after intravascular infusion. Here we compare the efficiency of an AAV8 serotyped vector with that of AAV1 and AAV2 serotyped vectors for the extent of gene delivery to the brain after neonatal injection into the lateral ventricles. The vectors all encoded green fluorescent protein (GFP) under control of a hybrid CMV enhancer/chicken beta-actin promoter with AAV2 inverted terminal repeats, but differed from each other with respect to the capsid type. A total of 6.8 x 10(10) genome copies were injected into the lateral ventricles of postnatal day 0 mice. Mice were killed at postnatal day 30 and brains analyzed for distribution of GFP-positive cells. AAV8 proved to be more efficient than AAV1 or AAV2 vectors for gene delivery to all of the structures analyzed, including the cerebral cortex, hippocampus, olfactory bulb, and cerebellum. Moreover the intensity of gene expression, assessed using a microarray reader, was considerably higher for AAV8 in all structures analyzed. In conclusion, the enhanced transduction achieved by AAV8 compared with AAV1 and AAV2 indicates that AAV8 is the superior serotype for gene delivery to the CNS.

Animals↗
PubMed2004

Functional expression and display of an antibody Fab fragment in Escherichia coli: study of vector designs and culture conditions.

Several different vector designs are currently being used to display and express Fab molecules in Escherichia coli, but their relative efficiency in phage display and protein expression cannot be compared from the published data. We systematically investigated which vector design most effectively displays and expresses Fab molecules in E. coli using, as a model system, a human Fab against tetanus toxoid (tt). Three different vectors were used in this study: pFab1 where the VL-CL and VH-CH1 genes were driven by two promoters in two separate expression cassettes, and pFab2 and pFab3 that both contain one dicistronic expression cassette with two translation initiation sites and either VH-CH1 before VL-CL or VL-CL before VH-CH1, respectively. The display of tt-Fab on the surface of phage and the expression of tt-Fab protein in E. coli were compared for the aforementioned vectors. Our results showed that the pFab3 vector was most effective in Fab display. A 10-fold increase in the expression of secreted Fab was observed in pFab3 when compared with vectors pFab1 and pFab2. Further experiments were conducted using pFab3 to optimize expression levels using different strains of E. coli and various culture conditions. The highest expression of tt-Fab was obtained using the pFab3 vector in host strain JM105 with an induction temperature at 37 degrees C and IPTG concentration of 0.1 mM.

Bacteriophages↗
PubMed2005

A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.

A set of ligation-independent expression vectors system has been developed for co-expression of proteins in Escherichia coli. These vectors contain a strong T7 promoter, different drug resistant genes, and an origin of DNA replication from a different incompatibility group, allowing combinations of these plasmids to be stably maintained together. In addition, these plasmids also contain the lacI gene, a transcriptional terminator, and a 3' polyhistidine (6x His) affinity tag (H6) for easy purification of target proteins. All of these vectors contain an identical transportable cassette flanked by suitable restriction enzyme cleavage sites for easy cloning and shuttling among different vectors. This cassette incorporates a ligation-independent cloning (LIC) site for LIC manipulations, an optimal ribosome binding site for efficient protein translation, and a 6x His affinity tag for protein purification Therefore, any E. coli expression vector of choice can be easily converted to LIC type expression vectors by shuttling the cassette using the restriction enzyme cleavage sites at the ends. We have demonstrated the expression capabilities of these vectors by co-expressing three bacterial (dsbA, dsbG, and Trx) and also two other mammalian proteins (KChIP1 and Kv4.3). We further show that co-expressed KChIP1/Kv4.3 forms soluble protein complexes that can be purified for further studies.

Base Sequence↗
PubMedDate not supplied

Augmentation of transgene expression in cold-preserved organs using vascular endothelial growth factor receptor-mediated adenoviral vector combined with hyperbaric oxygen.

Adenovirus-mediated gene transfer has been widely used in gene therapy for congenital metabolic, cardiovascular, and malignant diseases. It has been reported that a gene transfer technique into transplanted organs may suppress rejection reactions and inhibit preservation injury. However, the magnitude of transgene expression in organs preserved at a cold temperature remains to be determined. In this study, we compared the transgene expression using vascular endothelial growth factor receptor (VEGFR)-mediated adenoviral vector at cold versus warm temperatures alone and combined with hyperbaric oxygen in cold-preserved organs. The transgene expression by porcine endothelial cells transduced with adenoviral vector was significantly higher after a 24 hour-incubation at warm temperature than after a 1 hour-incubation with warm or cold temperature. Moreover, the transgene expression of after a 1-hour incubation at cold temperature was significantly lower than a 1-hour incubation at warm temperature. The VEGFR-mediated adenoviral vector augmented transgene expression during a 1-hour incubation at cold temperature compared to the control vector. A/J skin graft survival in C3H mice was significantly prolonged compared to control or standard vector with CTLA4Ig cDNA using VEGFR-mediated adenoviral vector with CTLA4Ig cDNA in a 1-hour cold preservation. Furthermore, combined use of VEGFR-mediated adenoviral vector with CTLA4Ig cDNA plus FK506 showed an augmented effect on graft prolongation. It is concluded that adenovirus-mediated gene transfer in 1-hour cold-preserved organ is difficult compared to that in the warm condition. However, VEGFR-mediated gene transfer can augment the transgene expression in 1-hour cold-preserved organs, followed by the effective suppression of rejection reactions in allogeneic transplantation.

Abatacept↗
PubMedDate not supplied

Studies on the most efficient vector systems for gene transduction into dendritic cells.

Specialized antigen-presenting cells (APC), known as dendritic cells (DC), play a pivotal role in initiating primary immune responses. Several vector systems, including adenoviral vectors, retroviral vectors, hemagglutinating virus of Japan-related vectors, and the electroporation, have been shown to transduce genes into mouse and human but not rat DC. However, there is no direct evidence to support the view that the currently used vector systems are able to transduce genes into mature DC. Inasmuch as most, if not all, gene transfer studies investigating DC or DC-related cell populations are performed employing heterogeneous-groups of cells, it is therefore important to determine the extent to which gene transduction occurs in bona fide DC. In this study, we provide evidence that none of these vector systems are able to transfer genes into mature rat DC, which are derived from bone marrow cells (BMC), driven by Flt3/Flk2 ligand and IL-6, and purified with CD161a. Nevertheless, the most efficient gene transduction was observed with developing DC progenitor cells during long-term culture of rat BMC. Successful gene transfer was achieved after 2-week culture with an HIV-based lentiviral vector system.

Animals↗
PubMed2004

An adenoviral type 5 vector carrying a type 35 fiber as a vaccine vehicle: DC targeting, cross neutralization, and immunogenicity.

Substituting the coat proteins of adenoviral vector serotype 5 (Ad5) can alter vector tropism and circumvent vector neutralization. Here we report that an Ad5 vector carrying a part of the fiber molecule of human subgroup B adenovirus serotype 35 (Ad5.Fib35) transduces cultured human dendritic cells (DC) and circulating myeloid derived DC with approximately 10-fold greater efficiency than Ad5 in vitro. The improved DC transduction results in increased T-cell activation ex vivo. In vivo however, immunogenicity of the vectors in mice and non-human primates did not correlate with in vitro DC tropism. Ad5.Fib35 was less immunogenic in monkeys than Ad5, despite the improved primate DC tropism of Ad5.Fib35. In mice with high Ad5 vector-specific immunity, Ad5.Fib35 showed no significant difference in anti-insert immunity over Ad5 indicating that fiber exchange alone does not evade pre-existing Ad5 immunity. We thus conclude that, for ex vivo vaccination, Ad5.Fib35 shows promise as vector for loading of DC but is unable to circumvent anti-Ad5 immunity limiting its in vivo utility.

Adenoviridae↗
PubMed2004

Effects of sequences of prokaryotic origin on titer and transgene expression in retroviral vectors.

Transcriptionally targeted MLV-based ProCon vectors allow expression of the transduced gene in a promoter-specific manner by replacement of the viral U3 region with a heterologous promoter. In order to evaluate the effects of sequence elements present in ProCon vectors on transgene expression (enhanced green fluorescence protein, EGFP), a series of deletion constructs mimicking the situation in proviral DNA following promoter conversion, where expression of the EGFP gene is driven by three different constitutive promoters (MLV U3, mCMV, and hCMV) in the context of a 5'LTR, respectively, were generated and tested in transient transfection experiments. We discovered that modifications in the 3'LTR have only marginal effects on the EGFP expression and the sequence between the promoter and the transgene did not influence EGFP expression at all. On the other hand, EGFP expression was reduced by up to 17-fold in cells transfected with constructs containing SV40neo and/or pBR322ori sequences. To study this effect in transduced cells, we generated a series of retroviral vectors in which these elements were deleted in various combinations and found that an increase in EGFP expression and viral titer was also consistently obtained using vectors lacking these elements, although this was much smaller than that observed using the expression constructs. A vector containing the gene for puromycin resistance (pac) in place of the neomycin resistance gene (neo) was also tested, and found to result in improved vector titers and transgene expression. We conclude that, where possible, the inclusion of neo and ori sequences in retroviral vectors should be avoided, and that, if selection of infected cells is necessary, the pac, rather than neo gene should be used.

Animals↗
PubMed2005

A nuclear localization signal in the matrix of spleen necrosis virus (SNV) does not allow efficient gene transfer into quiescent cells with SNV-derived vectors.

A major limitation in gene therapy for vectors derived from Moloney murine leukemia virus (MLV) is that they only deliver genes into dividing cells. In this study, a careful comparison of spleen necrosis virus (SNV)-derived vectors with MLV and human immunodeficiency virus (HIV)-1 retroviral vectors indicated that SNV vectors can deliver genes 4-fold more efficiently than MLV vectors into aphidicolin-arrested cells, although at a 25-fold lower efficiency than HIV-1-derived vectors. Furthermore, the addition of a NLS in the SNV matrix (MA) that mimics the one located in HIV-1 MA did not increase the ability of SNV vectors to transfer genes into arrested cells. Also, we found that the RD114 envelope was able to pseudotype SNV viral particles in a very efficient manner.

Animals↗
PubMed2003

Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung.

A human immunodeficiency virus (HIV)-based vector pseudotyped with the Ebola Zaire (EboZ) viral envelope glycoprotein (GP) was recently shown to transduce murine airway epithelia cells in vivo. In this study, the vector was further redesigned to improve gene transfer and also to increase safety. We used mutant EboZ envelopes for pseudotyping, which resulted in higher titers and increased transduction of airway cells in vivo compared to vectors pseudotyped with wild-type EboZ GP. As these envelopes lack regions associated with toxicity of the wild-type EboZ GP, they should also be safer to use for pseudotyping of lentiviral vectors. In addition, lentiviral vectors were created based on feline immunodeficiency virus and shown to have similar efficiency of transduction compared to HIV-based vectors. The creation of lentiviral vectors with highly engineered EboZ envelopes improved the performance of the system and should also increase its safety since only minimal regions of the EboZ envelope, which lack the toxic domain, are used.

Amino Acid Sequence↗
PubMed2004

A new method for recombinant adeno-associated virus vector delivery to murine diaphragm.

Genetically modified mice are important models for evaluation of potential gene therapies for human diseases. However, their small size often precludes the use of clinically feasible methods for vector delivery, therefore, alternative methods must be used. We have developed a gel-based method for delivery of recombinant adeno-associated virus vectors to the mouse diaphragm, an important target organ for many myopathic diseases. We hypothesized that delivery of vectors in a viscous solution would increase transduction by providing a longer exposure time to target cells. We demonstrate that gel-mediated delivery of rAAV serotypes 1, 2, and 5 results in higher transduction efficiencies than free vectors alone when administered in vivo to mouse diaphragms. We further establish greater tropism of rAAV1 vectors for the diaphragm compared to serotypes 2 and 5. This report describes a novel method for efficient delivery of rAAV vectors to the mouse diaphragm and is the first demonstration of gene transfer to the diaphragm using recombinant adeno-associated virus vectors.

Animals↗
PubMed2004

Lentiviral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients leads to functional correction.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with a median survival below the age of 20 due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous hematopoietic stem cells or T cells could represent an alternative treatment applicable to all patients. We investigated whether WAS gene transfer with MMLV-based oncoretroviral and HIV-based lentiviral vectors could restore normal functions of patients' T cells. T cells transduced either with lentiviral vectors expressing the WAS protein (WASP) from the ubiquitous PGK promoter or the tissue-specific WASP promoter or with an oncoretroviral vector expressing WASP from the LTR, reached normal levels of WASP with correction of functional defects, including proliferation, IL-2 production, and lipid raft upregulation. Lentiviral vectors transduced T cells from WAS patients at higher rates, compared to oncoretroviral vectors, and efficiently transduced both activated and naive WAS T cells. Furthermore, a selective growth advantage of T cells corrected with the lentiviral vectors was demonstrated. The observation that lentiviral vector-mediated gene transfer results in correction of T cell defects in vitro supports their application for gene therapy in WAS patients.

Antibodies↗
PubMed2005

Lentiviral vectors pseudotyped with glycoproteins from Ross River and vesicular stomatitis viruses: variable transduction related to cell type and culture conditions.

HIV-1-derived lentiviral vectors have been pseudotyped with various envelope glycoproteins to alter their host range. Previously, we found that envelope glycoproteins derived from the alphavirus Ross River virus (RRV) can pseudotype lentiviral vectors and mediate efficient transduction of a variety of epithelial and fibroblast-derived cell lines. In this study, we have investigated transduction of hematopoietic cells using RRV-pseudotyped vectors encoding the enhanced green fluorescent protein (EGFP). RRV-mediated transduction of human CD34+ cord blood cells and progenitors was very inefficient, even at multiplicities of infection of 100 (0.4% EGFP-positive progenitor colonies). Inefficient transduction was also observed in a variety of hematopoietic cell lines. However, two erythroleukemia-derived cell lines and monocytic cells that were driven to macrophage-like differentiation were moderately transduced. Transduction of hematopoietic cells with a control VSV-G-pseudotyped lentiviral vector was generally efficient, but unexpectedly decreased up to threefold upon stimulation of lymphocytic cell lines or primary murine bone marrow cells. Also, the tested hematopoietic cell lines were essentially nonpermissive for adeno-associated type 2 (AAV) vectors, and this was not affected by lineage, activity, or differentiation. Treatment of permissive 293 cells with proteases revealed that transduction with both the RRV- and the VSV-G-pseudotyped vectors in part depends on the presence of cell surface proteins. These results show a severely restricted ability of RRV glycoproteins to mediate transduction in hematopoietic cells that is likely due to specific receptor requirements that differ from those of VSV-G and AAV. Conversely, transduction with the VSV glycoprotein is affected by cellular activation more than widely believed. Our findings suggest that the envelope glycoproteins and culture conditions employed need to be carefully evaluated for each application. Furthermore, the uniquely restricted host range of RRV-pseudotyped vectors may aid in the design of novel cell-selective transduction strategies.

Antigens, CD34↗
PubMed2005

Gene transfer to repopulating human CD34+ cells using amphotropic-, GALV-, or RD114-pseudotyped HIV-1-based vectors from stable producer cells.

A novel, stable human immunodeficiency virus type 1 vector packaging system, STAR, was tested for its ability to transduce human cord blood CD34+ progenitor cells assayed both in vitro and after transplantation into NOD/SCID mice. Vectors pseudotyped with three different gammaretrovirus envelopes were used: the amphotropic MLV envelope (MLV-A), a modified gibbon ape leukemia virus envelope (GALV+), and a modified feline endogenous virus RD114 envelope (RDpro). Gene transfer to freshly thawed CD34+ cells in the absence of cytokines was very low. Addition of cytokines increased gene transfer efficiency significantly and this was further augmented if the cells were prestimulated for 24 h. Concentration of the vectors (15-fold) by low-speed centrifugation increased gene transfer to CD34+ cells in vitro even further. More than 90% of cells were transduced with a single exposure to the RDpro vector as determined by GFP expression using flow cytometry. The two other pseudotypes transduced approximately 65-70% of the cells under the same conditions. Transplantation of CD34+ cells prestimulated for 24 h and then transduced with a single exposure to concentrated vector revealed that the RDpro vector transduced 55.1% of NOD/SCID repopulating human cells, which was significantly higher than the MLV-A (12.6%)- or GALV+ (25.1%)-pseudotyped vectors.

Animals↗
PubMed2005

Semliki forest virus vectors engineered to express higher IL-12 levels induce efficient elimination of murine colon adenocarcinomas.

To evaluate the use of alphavirus vectors for tumor treatment we have constructed and compared two Semliki Forest virus (SFV) vectors expressing different levels of IL-12. SFV-IL-12 expresses both IL-12 subunits from a single subgenomic promoter, while in SFV-enhIL-12 each IL-12 subunit is expressed from an independent subgenomic promoter fused to the SFV capsid translation enhancer. This latter strategy provided an eightfold increase of IL-12 expression. We chose the poorly immunogenic MC38 colon adenocarcinoma model to evaluate the therapeutic potential of SFV vectors. A single intratumoral injection of 10(8) viral particles of SFV-IL-12 or SFV-enh-IL-12 induced>or=80% complete tumor regressions with long-term tumor-free survival. However, lower doses of SFV-enhIL-12 were more efficient than SFV-IL-12 in inducing antitumoral responses, indicating a positive correlation between the IL-12 expression level and the therapeutic effect. Moreover, repeated intratumoral injections of suboptimal doses of SFV-enhIL-12 increased the antitumoral response. In all cases SFV vectors were more efficient at eliminating tumors than a first-generation adenovirus vector expressing IL-12. In addition, the antitumoral effect of SFV vectors was only moderately affected by preimmunization of animals with high doses of SFV vectors. This antitumoral effect was produced, at least partially, by a potent CTL-mediated immune response.

Adenocarcinoma↗
PubMed2005

Stable transduction of primary human monocytes by simian lentiviral vector PBj.

Despite the ability to infect nonproliferating cells, current lentiviral vectors are inefficient at mediating gene transfer into quiescent primary human cells such as monocytes. Here, a replication-incompetent vector based on a molecular clone of simian immunodeficiency virus strain PBj (SIVsmmPBj1.9) was generated that, in contrast to lenti- and gamma-retroviral control vectors, enabled transfer of heterologous genes into human diploid fibroblasts and cell lines blocked in the G(0) phase of the cell cycle. Moreover, freshly isolated human monocytes refractory to HIV-1-derived vectors were efficiently transduced by the PBj vector independent of the viral Nef protein. Stable chromosomal integration of PBj-derived viral expression vectors was verified in transduced cells. The capability of the PBj vector to transduce quiescent cells such as unstimulated primary human monocytes is an important extension of human gene therapy perspectives.

Blotting, Southern↗
PubMed2005

Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice.

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases by stable vector insertion into the patients' chromosomes. However, three cases of T cell lymphoproliferative disease have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In two of these cases vector insertion into the LMO2 locus was implicated in leukemogenesis, demonstrating that a more profound understanding is required of the genetic and molecular effects imposed on the host by vector integration or transgene expression. In vivo models to test for retro- and lentiviral vector safety prior to clinical application are therefore needed. Here we present a high incidence of lentiviral vector-associated tumorigenesis following in utero and neonatal gene transfer in mice. This system may provide a highly sensitive model to investigate integrating vector safety prior to clinical application.

Animals↗
PubMed2005

Rapid Kupffer cell death after intravenous injection of adenovirus vectors.

When adenovirus vectors are injected intravenously, they are quickly taken up by Kupffer cells in the liver. We report that this causes rapid necrosis of Kupffer cells in mice at doses of 10(11) particles/kg or higher. By 10 min after intravenous vector injection, Kupffer cells were permeable to propidium iodide and trypan blue. This coincided with a sharp rise in serum lactate dehydrogenase. Ultrastructural examination showed degeneration of Kupffer cells, including complete disappearance of chromatin by 1 h. After an initial intravenous injection of vector, dead Kupffer cells were unable to take up a second dose of vector, and hepatic transgene expression from the second dose was augmented. Death of Kupffer cells did not affect serum levels of IL-6 or IL-12. There was no immediate change in the number of Kupffer cells in the liver, but a significant decline was found by 4 h after injection of vector. Interestingly, substantial numbers of vector-containing Kupffer cells were found in pulmonary capillaries, indicating that they had been swept out of the liver. Together these results show that an intravenous injection of adenovirus vector causes synchronous and surprisingly rapid Kupffer cell death.

Adenoviridae↗
PubMed2005

Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells.

Severe adverse events related to insertional mutagenesis have reinforced interest in self-inactivating (SIN) retroviral vectors lacking enhancer-promoter sequences in the long terminal repeats (LTRs). Here, we have compared the potency of gammaretroviral and lentiviral vectors expressing the P140K mutant of O(6)-methylguanine-DNA methyltransferase (MGMT). MGMT-P140K is a clinically relevant selection marker that mediates a strong survival advantage in hematopoietic cells exposed to alkylating agents. We designed gammaretroviral and lentiviral vectors that contained identical enhancer-promoter sequences located either in the LTR or downstream of the packaging region, for internal initiation of transcription from SIN backbones. Gammaretroviral vectors with intact LTRs containing enhancer-promoter sequences showed both higher titers and higher expression levels than the lentiviral counterparts, likely a result of suboptimal RNA processing of the lentiviral leader region. In the SIN context, gammaretroviral and lentiviral vectors with comparable internal cassettes had similar expression properties. Interestingly, gammaretroviral SIN vectors pseudotyped with RD114/TR had a higher transduction efficiency on proliferating human CD34(+) cells than lentiviral counterparts. These results encourage further investigations into the formation of retroviral hybrid vectors that combine the desired properties of high efficiency and increased biosafety.

Animals↗