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At least 1,405 records · Page 78Linked to original sources
PubMed2006

Gene therapy for BCR-ABL+ human CML with dual phosphorylation resistant p27Kip1 and stable RNA interference using an EBV vector.

BACKGROUND: BCR-ABL-mediated chronic myelogenous leukemia (CML) CD34(+) cell proliferation mostly depends on the nucleo-cytoplasmic ratio of the cyclin-dependent kinase inhibitor p27. The ubiquitin-ligase SCF(Skp2) promotes degradation of phosphorylated p27 at T187 in the nucleus, resulting in G1/S progression of the cells. On the other hand, phosphatidylinositol-3-kinase (PI3K)-directed T157 nuclear localization signal (NLS) phosphorylation results in cytoplasmic sequestration of p27, leading to abnormal integrin-mediated proliferation of CD34(+) CML cells. METHODS: We demonstrate the generation of an engineered Epstein-Barr virus (EBV) vector with a BAC backbone that has the unique capacity to carry doubly modified (DM) p27 (i.e. T187A, T157A p27) along with the BCR-ABL siRNA expression construct. The HSV-tk suicide gene has also been incorporated in the same vector, which promotes apoptosis in a BCR-ABL-independent pathway. RESULTS: Expression of DM p27 markedly inhibits proliferation of BCR-ABL(+) primary human CML cells. Moreover, DM p27 strongly inhibits the growth of imatinib-resistant CML cells, compared to the T157A p27 (SM p27). The CML growth inhibition is found to be the result of significant G1/S arrest with concomitant increase in hypophosphorylated retinoblastoma (Rb). Moreover, the EBV vector mediated stable RNA interference induces apoptosis in K562 cells and reduces myeloid colony forming units. CONCLUSIONS: We therefore propose a multi-gene delivery strategy for BCR-ABL(+) CML cells by targeting not only the fusion transcript, but also the downstream signaling, to overcome drug resistance in the acute phase of CML.

Active Transport, Cell Nucleus↗
PubMed1991

Analyses of the requirements for the synthesis of virus-like particles by feline immunodeficiency virus gag using baculovirus vectors.

Feline immunodeficiency virus (FIV) gag gene was expressed in baculovirus vectors to investigate its potential for the assembly of virus-like particles. The unprocessed 50-kDa FIV gag precursor made in infected insect cells by recombinant AcFIVGAG-1 was myristoylated, assembled at the cell surface into virus-like particles (with diameters of approximately 100 nm), and efficiently released into the culture supernatant fluids. The presence of the complete viral-coded protease component of the FIV pol gene engineered into a second expression vector (AcFIVGAG-P5) resulted in the efficient processing of the gag precursor to its component proteins and abolished particle formation and secretion. Insertion of a stop codon in this vector upstream of the putative gag-pol frameshift site (GGGAAAC) resulted in the derivation of an expression vector (AcFIVGAG-R) that made a truncated, unprocessed 46-kDa FIV gag precursor lacking some 34 amino acids in the p10 carboxy-proximal coding region of gag. This vector synthesized tubular structures in the cytoplasm of infected cells and released them into the cell supernatant. The results demonstrate that the FIV gag precursor can spontaneously assemble into virus-like particles without any other virus proteins and that the carboxy-terminal part of the precursor gag protein is essential for such assembly.

Animals↗
PubMed2000

Production of viral vectors for gene therapy applications.

Advances in cell culture engineering, cell metabolism, bioreactor design and operation, and downstream processing will all positively impact the bioprocessing of viral vectors. Design of appropriate vectors and tailoring of packaging cells to support more productive infections will be of paramount importance for production of high-titer and high-quality vectors. Furthermore, quantitative analysis of the infection parameters during virus propagation, such as time of infection, multiplicity of infection, the length of replication cycle, virus half-life, and burst size, will also be important to the process optimization. Finally, procedures for separation, purification and formulation of vector preparations have to be further developed.

Adenoviridae↗
PubMed1999

Targeting retroviral vectors to CD34-expressing cells: binding to CD34 does not catalyze virus-cell fusion.

We have attempted to engineer murine leukemia virus (MuLV)-based retroviral vectors to specifically transduce cells expressing human CD34, an antigen present on the surface of undifferentiated hematopoietic stem cells. A number of chimeric ecotropic MuLV envelope (Env) proteins were constructed that contained anti-CD34 single-chain antibody variable fragments (scFvs). The scFv-Env proteins were generated either by replacing the receptor-binding domain of Env with the scFv or by inserting the scFv into the N terminus of the Env protein. Only chimeric Env proteins with scFv insertions between amino acids 6 and 7 were incorporated into viral particles, and coexpression of native MuLV Env did not rescue incorporation-defective proteins. In addition, the efficiency of incorporation varied with the specific anti-CD34 scFv that was used. Retroviral vectors containing the scFv-Env proteins bound to CD34+ cells and transduced NIH 3T3 cells expressing human CD34 (3T3-CD34 cells) at approximately twice the efficiency of the parental NIH 3T3 cells. However, the introduction of the mutation D84K, which prevents binding to the ecotropic MuLV receptor mcat-1, prevented transduction of both NIH 3T3 and 3T3-CD34 cells. Complementation cell-cell fusion assays [Zhao et al. (1997). J. Virol. 71, 6967-6972] in 3T3-CD34 cells revealed that although the scFv-Env proteins could contribute postbinding entry functions when bound to mcat-1, they were unable to do so when bound to CD34. Taken together, these data suggest that although the interaction with CD34 effectively increased the concentration of virus on 3T3-CD34 cells, entry could occur only through an interaction with mcat-1; CD34 alone was not capable of triggering the appropriate postbinding changes that lead to viral entry.

3T3 Cells↗
PubMed2004

Characterization of pC7 from Lactobacillus paraplantarum C7 derived from Kimchi and development of lactic acid bacteria--Escherichia coli shuttle vector.

A cryptic plasmid pC7 was characterized, which was isolated from Lactobacillus paraplantarum C7 derived from Kimchi, a traditional korean vegetable food. The plasmid pC7 is a circular molecule of 2,134 base-pairs in length with a G + C content of 38.5%. The nucleotide sequence analysis revealed the presence of an open reading frame encoding a putative 317 amino acids protein homologous to replication proteins RepA. Furthermore, a putative double and a single-strand origin were identified. Together with features of nucleotide sequences, the detection of single-stranded intermediate DNA in Lb. paraplantarum demonstrated that pC7 replicates via a rolling circle mechanism. A cloning vector for lactic acid bacteria was developed on the basis of the pC7 replicon, into which were inserted an erythromycin resistance gene as a marker, multiple cloning sites, and Escherichia coli ColE1 replication origin. E. coli and several species of Lactobacillus and Leuconostoc can be transformed with the resultant vector. Therefore, pC7 derivatives may be useful LAB-E. coli shuttle vectors, which are essential in engineering important strains in food fermentation.

Amino Acid Sequence↗
PubMed2006

High-throughput, library-based selection of a murine leukemia virus variant to infect nondividing cells.

Gammaretroviruses, such as murine leukemia virus (MLV), are functionally distinguished from lentiviruses, such as human immunodeficiency virus, by their inability to infect nondividing cells. Attempts to engineer this property into MLV have been hindered by an incomplete understanding of early events in the viral life cycle. We utilized a transposon-based method to generate saturated peptide insertion libraries of MLV gag-pol variants with nuclear localization signals randomly incorporated throughout these overlapping genes. High-throughput selection of the libraries via iterative retroviral infection of nondividing cells led to the identification of a novel variant that successfully transduced growth-arrested cells. Vector packaging by cotransfection of the gag-pol.NLS variant with wild-type gag-pol produced high-titer virions capable of infecting neurons in vitro and in vivo. The capacity of mutant virions to transduce nondividing cells could help to elucidate incompletely understood mechanisms of the viral life cycle and greatly broaden the gene therapy applications of retroviral vectors. Furthermore, the ability to engineer key intracellular viral infection steps has potential implications for the understanding, design, and control of other post-entry events. Finally, this method of library generation and selection for a desired phenotype directly in a mammalian system can be readily expanded to address other challenges in protein engineering.

Active Transport, Cell Nucleus↗
PubMed1990

Expression of cauliflower mosaic virus gene I using a baculovirus vector based upon the p10 gene and a novel selection method.

A new baculovirus expression vector based upon the p10 gene of Autographa californica nuclear polyhedrosis virus (AcNPV) and a novel system for the screening of p10 recombinants have been developed. The insertion of a cassette containing the lacZ gene under the control of a heat-shock promoter of Drosophila melanogaster downstream from the cloning site in p10 transfer vectors allows the convenient identification of putative recombinants by virtue of their expression of beta-galactosidase. Using this p10 transfer vector an AcNPV recombinant was engineered with a cDNA copy of gene I of cauliflower mosaic virus (CaMV) in place of the p10 coding sequence. This p10 recombinant expressed CaMV gene I at levels equivalent to those of p10 and polyhedrin, and was shown to be as effective in producing this protein as recombinants exploiting the polyhedrin promoter. CaMV gene I protein formed large numbers of hollow fiber-like structures in the cytoplasm of infected cells. Because the polyhedrin gene remains intact, these p10 expression vectors may be exploited for the expression of heterologous proteins in insects infected per os and for the enhancement of baculovirus pathogenicity for insect control.

Animals↗
PubMed1997

Construction of recombinant adeno-associated virus vector containing the rat preproinsulin II gene.

We have investigated a possible delivery system for the rat preproinsulin II gene (rI2) utilising a recombinant adeno-associated virus (rAAV) vector system, with the long-term goal of engineering stably infected insulin-producing cell lines. The rAAV vector was chosen because it is a safe and nonpathogenic method for gene transfer. The plasmid pBC12BI (ATCC) was purified and digested with restriction enzymes SepI and StuI to release a fragment containing the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter-driven rat preproinsulin II gene (rI2). Subsequently, the RSV-rI2 gene fragment was cloned into the BamHI site of rAAV vector plasmid pWP-19 to produce the rI2 recombinant plasmid designated pLP-1. The pWP-19 also encodes the AAV inverted terminal repeats for integration and replication and the herpes virus thymidine kinase promoter-driven gene for neomycin resistance (neoR). The cell line 293 (ATCC) was then cotransfected with pLP-1 and helper plasmid pAAV/AD, which is required for viral replication. The rAAV genome, now containing rI2, was rescued using adenovirus and packaged into mature AAV virions termed vLP-1. Finally, human pancreatic adenocarcinoma cells (HPAC; ATCC) were exposed to vLP-1, selected for G418 resistance, and screened for insulin production. Successful rescue was confirmed by Southern blot analysis using the rI2 gene probe derived from the original plasmid. The final titer of 1.25 x 10(9) particles/ ml was determined by DNA slot blots using pLP-1 as the standard, HPAC cells were infected with vLP-1 (termed HPAC/rI2). Integration of the rI2 genome in G418-resistant clones was confirmed by Southern blot analysis and again after 6 months in culture by amplification of the rI2 gene by PCR. Insulin gene transcription was confirmed by RT-PCR. We have developed a rAAV-mediated gene transfer system for the rat preproinsulin II gene. Successful transduction and stable integration of rI2 into HPAC was achieved. Production of insulin by HPAC/rI2 was confirmed by RIA and RT-PCR, validating this system as an effective approach to experimental gene therapy.

Adenocarcinoma↗
PubMed2005

HSV trafficking and development of gene therapy vectors with applications in the nervous system.

Herpes simplex virus type 1 (HSV-1) is a neurotropic double-stranded DNA virus that causes cold sores, keratitis, and rarely encephalitis in humans. Nonpathogenic HSV-1 gene transfer vectors have been generated by elimination of viral functions necessary for replication. The life cycle of the native virus includes replication in epithelial cells at the site of initial inoculation followed by retrograde axonal transport to the nuclei of sensory neurons innervating the area of cutaneous primary infection. In this review, we summarize the current understanding of the molecular basis for HSV cell entry, nuclear transport of the genome, virion egress following replication, and retrograde and anterograde axonal transport in neurons. We discuss how each of these properties has been exploited or modified to allow the generation of gene transfer vectors with particular utility for neurological applications. Recent advances in engineering virus entry have provided proof of principle that vector targeting is possible. Furthermore, significant and potentially therapeutic modifications to the pathological responses to various noxious insults have been demonstrated in models of peripheral nerve disease. These applications exploit the natural axonal transport mechanism of HSV, allowing transgene expression in the cell nucleus within the inaccessible trigeminal ganglion or dorsal root ganglion, following the noninvasive procedure of subcutaneous vector inoculation. These findings demonstrate the importance of understanding basic virology in the design of vector systems and the powerful approach of exploiting favorable properties of the parent virus in the generation of gene transfer vectors.

Active Transport, Cell Nucleus↗
PubMed2004

Expression and purification of immunologically reactive DPPD, a recombinant Mycobacterium tuberculosis skin test antigen, using Mycobacterium smegmatis and Escherichia coli host cells.

DPPD is a Mycobacterium tuberculosis recombinant antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both guinea pigs and humans infected with M. tuberculosis. In addition, earlier clinical studies with DPPD suggested that this molecule could improve the specificity of the tuberculin skin test, which is used as an important aid for the diagnosis of tuberculosis. However, these studies could only be performed with DPPD engineered as a fusion molecule with another Mycobacterium spp. protein because no expression of DPPD could be achieved as a single molecule or as a conventional fusion protein in any commercial system. Although recombinant fusion proteins are in general suitable for several biological studies, they are by definition not ideal for studies involving highly purified and defined polypeptide sequences. Here, we report two alternative approaches for the expression of immunologically reactive recombinant genuine DPPD. The first approach used the rapidly growing, nonpathogenic Mycobacterium smegmatis as host cells transformed with the pSMT3 plasmid vector containing the full-length DPPD gene. The second approach used Escherichia coli transformed with the pET-17b plasmid vector containing the DPPD gene engineered in a three-copy fusion manner in tandem with itself. Though at low levels, expression and purification of immunologically reactive DPPD in M. smegmatis could be achieved. More abundant expression and purification of DPPD as a homo-trimer molecule was achieved in E. coli (> or =2 mg/L of bacterial broth cultures). Interestingly, expression could only be achieved in host cells transformed with the DPPD gene containing its leader peptide. However, the expressed proteins lacked the leader sequence, which indicates that processing of the M. tuberculosis DPPD gene was accurately achieved and necessary in both M. smegmatis and E. coli. More importantly, the delayed type hypersensitivity reactions elicited by purified molecules in guinea pigs infected with M. tuberculosis were indistinguishable from that elicited by purified protein derivative. Because the DPPD gene is present only in the tuberculosis-complex organisms of the Mycobacterium genus, these highly purified molecules should be helpful in identifying individuals sensitized with tubercle bacilli.

Animals↗
PubMed2004

In vitro and in vivo characterisation of endothelial cell selective adenoviral vectors.

BACKGROUND: Both viral and non-viral gene transfer vectors transduce vascular endothelial cells (EC) with low efficiency compared with other cell types such as hepatocytes. Generation of EC-selective vectors would enhance the clinical utility of gene therapy for diverse vascular-targeted applications. METHODS: 12mer peptides derived by in vitro phage display with EC binding specificity [MTPFPTSNEANL (MTP) and MSLTTPPAVARP (MSL)] were inserted at position T542 in the exposed HI loop of the adenovirus (Ad) serotype 5 fiber using overlapping oligonucleotides; in combination with a double point mutation (KO1) to ablate virus : cell binding via the coxsackie-adenovirus receptor (CAR). The resulting modified viruses were tested in vitro and in vivo for their ability to direct endothelial-specific gene transfer. RESULTS: Peptide insertion was not deleterious to fiber trimerisation or virion maturation. In vitro gene transfer studies using a panel of cell types demonstrated that both peptide-targeted Ad vectors mediated efficient CAR-independent gene transfer to vascular EC compared with non-modified Ads. Neither peptide supported gene delivery to non-EC. Upon systemic injection into mice and subsequent evaluation of transgene expression we failed to observe a reduction in hepatic Ad accumulation but observed a significant elevation in beta-galactosidase in blood vessels with the MSLTTPPAVARP-targeted Ad vector. CONCLUSIONS: We have genetically engineered two novel Ads that transduce human EC selectively in vitro, one of which leads to altered Ad biodistribution in vivo. The successful generation of genetically engineered tropism for EC has broad implications for cardiovascular gene therapy. Further modifications to the Ad capsid will be required to improve in vivo biodistribution profiles.

Adenoviridae↗
PubMed1983

Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein.

A complementary (cDNA) molecule encoding the structural proteins of Semliki Forest virus (SFV) has been inserted into a Simian virus 40-derived eucaryotic expression vector lacking introns. Introduction of the recombinant DNA into nuclei of baby hamster kidney cells results in the synthesis of authentic SFV membrane glycoproteins E1 and E2. The glycoproteins are both transported to the cell surface and induce cell-cell fusion after a brief treatment of the cells with low pH medium. The pH dependence of the fusion reaction was the same as that induced by virus particles (White, J., J. Kartenbeck, and A. Helenius, 1980, J. Cell Biol., 89:674-679). Transfection of cells with another recombinant DNA molecule in which the SFV cDNA is engineered into the same expression vector including an intron has been shown before to result in the expression of only the E2 protein on the cell surface, whereas the E1 protein is trapped in the rough endoplasmic reticulum (Kondor-Koch, C., H. Riedel, K. Söderberg, and H. Garoff, 1982, Proc. Natl. Acad. Sci. USA, 79:4525-4529). Such cells do not exhibit pH-dependent polykaryon formation, suggesting that the E1 protein is necessary for fusion activity. Immunoblotting experiments show that the RER-trapped E1 protein expressed from the DNA construction with an intron has a smaller apparent molecular weight than authentic E1, and that is has lost its amphipathic characteristics.

Animals↗
PubMed1999

Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3' copy-back ambisense rabies virus.

Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.

3' Untranslated Regions↗
PubMed2001

Cross-strain protection against clinical and laboratory strains of Pseudomonas aeruginosa mediated by dendritic cells genetically modified to express CD40 ligand and pulsed with specific strains of Pseudomonas aeruginosa.

We have shown that dendritic cells (DCs) genetically engineered with a recombinant adenovirus vector (Ad) to express CD40 ligand (CD40L) elicit specific humoral immunity against the Pseudomonas aeruginosa laboratory strain PAO1, without CD4(+) T cell help. In the present study, using several different strains of P. aeruginosa, we examine whether this strategy is generally applicable to enhancing clinically relevant pathogen-specific immunity. Mice immunized with DCs modified with CD40L and pulsed with heat-killed P. aeruginosa clinical strain PA514, originally isolated from the sputum of an individual with cystic fibrosis, survived lethal respiratory challenge with PA514-impregnated agar beads. Consistent with this effective in vivo protection, the immunized mice generated high levels of serum isotype-switched antibodies directed against PA514 without concomitant nonspecific elevations of total serum immunoglobulin levels. The CD40L genetically engineered DCs pulsed with seven of eight different strains of P. aeruginosa afforded significant, albeit variable, cross-protection against lethal respiratory challenge with a clinical (PA514) or laboratory (PAO1) strain of P. aeruginosa. CD40L genetically modified DCs pulsed with a clinical (PA514) or laboratory (PAO1) strain of P. aeruginosa initiated cross-reacting antibody responses against each other, but not against Escherichia coli and vice versa. These observations may be useful in developing vaccines for infectious diseases, including P. aeruginosa infection.

Adenoviridae↗
PubMed1990

A latent, nonpathogenic HSV-1-derived vector stably expresses beta-galactosidase in mouse neurons.

A genetically engineered herpes simplex virus variant was constructed for use as a stable gene vector for neurons. To inhibit replication, the agent possessed a deletion in the immediate early gene ICP4, and to minimize reactivation from the latent state, the gene encoding the latency-associated transcript was deleted. The E. coli beta-galactosidase gene under the control of the Maloney murine leukemia virus long terminal repeat promoter was inserted into the ICP4 region. When introduced into the peripheral nervous system, this virus established latent infections and stably expressed beta-galactosidase in primary sensory neurons. Expression of beta-galactosidase over a more limited time period was observed when the latent infection was established in motor neurons of the hypoglossal nucleus. Agents of this general design have considerable potential for use as gene vectors for studies of neuronal function and correction of genetic defects affecting neurons.

Acute Disease↗
PubMed1996

Gene transduction into murine primitive hematopoietic cells with 2-gene retroviral vectors using a Transwell coculture system.

The present study aims at expressing a reporter gene in hematopoietic cells in vivo by introducing it into primitive hematopoietic cells with a 2-gene retroviral vector. Various constructs of retroviral vectors containing the human IL-2 receptor alpha chain gene (TAC) as the reporter and the neomycin phosphotransferase gene (neo) as a selectable marker were engineered, and the effectiveness of these vectors for expression of the reporter gene was evaluated after transfection into the packaging cell line GP + E86. It was found that the highest levels of reporter gene expression were attained with constructs ordered 5' long terminal repeat (LTR)-TAC-internal promoter-neo-3' LTR. In experiments investigating the expression of a reporter gene in hematopoietic cells, we used the Escherichia coli beta-galactosidase gene (lacZ) instead of TAC, because a very sensitive detection method was available for lacZ. For transduction of hematopoietic progenitors, packaging cell lines producing recombinant viruses were cultured in a Transwell hung into a Dexter-type bone marrow (BM) culture. The BM cells were selected with G418, and transferred into irradiated recipient mice. LacZ enzyme activity was detectable in the peripheral blood lymphocytes (PBL) of recipients taken 8 wk after reconstitution.

Animals↗
PubMed2001

Protection of rabbits against rabbit hemorrhagic disease virus by immunization with the VP60 protein expressed in plants with a potyvirus-based vector.

A new plum pox potyvirus (PPV)-based vector has been constructed for the expression of full-length individual foreign proteins. The foreign sequences are cloned between the NIb replicase and capsid protein (CP) cistrons. The heterologous protein is split from the rest of the potyviral polyprotein by cleavage at the site that originally separated the NIb and CP proteins and at an additional NIa protease recognition site engineered at its amino-terminal end. This vector (PPV-NK) has been used to clone different genes, engendering stable chimeras with practical applications. We have constructed a chimera expressing high levels of jellyfish green fluorescent protein, which can be very useful for the study of PPV molecular biology. The VP60 structural protein of rabbit hemorrhagic disease virus (RHDV) was also successfully expressed by making use of the PPV-NK vector. Inoculation of extracts from VP60-expressing plants induced a remarkable immune response against RHDV in rabbits, its natural host. Moreover, these animals were protected against a lethal challenge with RHDV.

Amino Acid Sequence↗