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At least 1,621 records · Page 90Linked to original sources
PubMed2006

Establishment of a novel foreign gene delivery system combining an HSV amplicon with an attenuated replication-competent virus, HSV-1 HF10.

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have been used widely in genetic engineering with many advantages for gene delivery, being easily constructed. An attenuated and replication-competent HSV-1 HF10 clone demonstrating an oncolytic effect on cancer cells in vitro and in vivo has been applied recently for clinical virotherapy of breast cancers and the present studies were conducted to test its efficacy in combination with an HSV-1 amplicon. For this purpose, a new system was developed to produce high titers of the HSV-1 amplicon vector and the results showed that its package efficiency and the titer ratio to HF10 were improved by passage through two cell lines. A high ratio of amplicon/helper virus HF10 (A/H) (>1) was required to express the foreign gene efficiently. Furthermore, in order to express the foreign gene conditionally, an HSV-1 ICP8 promoter was introduced in place of the human cytomegalovirus MIE promoter, this driving expression of the transgene when replication of HF10 progressed. The methodology for simple preparation of mixtures of viruses containing the amplicon with the oncolytic virus is documented. This system should find application for studies of cancer therapy.

Animals↗
PubMed1993

V79 Chinese hamster cells genetically engineered for cytochrome P450 and their use in mutagenicity and metabolism studies.

V79 Chinese hamster cells are being genetically engineered for stable expression of cytochromes P450 using a SV40 early promoter containing plasmid as the eukaryotic expression vector. V79 cells lack endogenous cytochrome P450 activity. Therefore, genetically engineered V79 cell lines are defined for the cytochrome P450 isoform acquired upon cDNA mediated gene transfer. V79 cells have a longstanding tradition as indicator cells for various biological endpoints, e.g. mutation, chromosomal aberration, cytotoxicity. The genetically engineered V79 cells combine these useful biological endpoints with metabolic competence. In this sense, V79 cell lines genetically engineered for stable expression of cytochromes P450 present newly developed tools for studying and understanding metabolism related problems in toxicology and pharmacology.

Animals↗
PubMed2006

Perspectives of gene therapy in stem cell tissue engineering.

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve tissue function. It is hoped that forming tissue de novo will overcome many problems in plastic surgery associated with such areas as wound healing and the immunogenicity of transplanted tissue that lead to dysfunctional repair. Gene therapy is the science of the transfer of genetic material into individuals for therapeutic purposes by altering cellular function or structure at the molecular level. Recently, tissue engineering has been used in conjunction with gene therapy as a hybrid approach. This combination of stem-cell-based tissue engineering with gene therapy has the potential to provide regenerative tissue cells within an environment of optimal regulatory protein expression and would have many benefits in various areas such as the transplantation of skin, cartilage or bone. The aim of this review is to outline tissue engineering and possible applications of gene therapy in the field of biomedical engineering as well as basic principles of gene therapy, vectors and gene delivery.

Animals↗
PubMed1997

Genetic engineering of baker's and wine yeasts using formaldehyde hyperresistance-mediating plasmids.

Yeast multi-copy vectors carrying the formaldehyde-resistance marker gene SFA have proved to be a valuable tool for research on industrially used strains of Saccharomyces cerevisiae. The genetics of these strains is often poorly understood, and for various reasons it is not possible to simply subject these strains to protocols of genetic engineering that have been established for laboratory strains of S. cerevisiae. We tested our vectors and protocols using 10 randomly picked baker's and wine yeasts all of which could be transformed by a simple protocol with vectors conferring hyperresistance to formaldehyde. The application of formaldehyde as a selecting agent also offers the advantage of its biodegradation to CO2 during fermentation, i.e., the selecting agent will be consumed and therefore its removal during down-stream processing is not necessary. Thus, this vector provides an expression system which is simple to apply and inexpensive to use.

Drug Resistance, Microbial↗
PubMed2003

A comparison of targeting performance of oncoretroviral versus lentiviral vectors on human keratinocytes.

The epidermis, like other rapidly renewing tissues, relies on a stem cell compartment to undergo constant regeneration. In order to develop realistic and long-lasting therapeutic approaches for some skin disorders, gene transfer to these critical cells must be obtained. While efficient retroviral ex vivo targeting and transgene integration in human keratinocytes is tightly dependent on proliferation, transferring genetic information to quiescent cells in culture also presents advantages, including the possibility of targeting putative dormant epidermal stem cells. In the present study we compared the efficiency of transduction achieved with a third-generation of human immunodeficiency virus (HIV)-based lentiviral vector to that obtained with a Moloney murine leukemia oncoretroviral vector (MLV) on proliferating and quiescent human keratinocytes growing in vitro in standard Rheinwald and Green cultures as well as in confluent organotypic cultures. Each viral vector contained the enhanced green fluorescent protein (EGFP) as a reporter gene. The lentiviral vector, but not the MLV vector, led to EGFP expression both in nondividing and proliferating epidermal cell populations in vitro. This feature was clearly evident when direct targeting of human keratinocytes, forming part of the epidermal component of an organotypic skin culture, was attempted. Keratinocytes modified by both MLV and the lentiviral vector allowed long-term regeneration of genetically engineered human skin on the backs of immunodeficient nonobese diabetic/severe combined immunodeficiency disorders (NOD/SCID) mice. However, EGFP transgene expression in the context of the MLV (long-terminal repeat [LTR]-driven) or lentiviral vector (cytomegalovirus [CMV]-driven) demonstrated clear differences both in quantitative terms and in the in vivo localization pattern.

Animals↗
PubMed2006

Recombinant polymer-protein fusion: a promising approach towards efficient and targeted gene delivery.

BACKGROUND: Synthetic vectors such as polymers have the potential to reduce the safety problems associated with viral vectors; however, their low transfection efficiency limits their clinical utility. To study the critical steps involved in an efficient transgene expression, there is a need for creative approaches that allow a systematic correlation between gene carrier structure and properties necessary for successful gene transfer. Using recombinant techniques a prototype vector comprised of tandem repeating units fused to a targeting moiety was biosynthesized to mediate gene transfer in mammalian cell lines. The carrier was designed to have the structure of (KHKHKHKHKK)6-FGF2 where lysine (K) residues would allow complexation with plasmid DNA, basic fibroblast growth factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and histidine (H) residues to facilitate escape from the endosomal compartments. METHODS: The gene carrier was biosynthesized in E. coli, purified using a Ni-NTA column, characterized, complexed with pDNA, and the complexes were used to transfect NIH 3T3, T-47D and COS-1 mammalian cell types known to express FGFR. RESULTS: Results demonstrate the successful cloning and expression of the gene carrier with over 95% purity. The molecular weight of the gene carrier was determined by MALDI-TOF to be 27 402. Amino acid content analysis and Western blot confirmed the expression of the gene carrier in E. coli. The vector was able to condense pDNA, induce cell proliferation in NIH 3T3 fibroblasts, and mediate transgene expression in NIH 3T3, T-47D and COS-1 mammalian cell types. CONCLUSION: Genetic engineering techniques show promise for systematic investigation of structure-activity relationships of non-viral gene delivery vectors.

Amino Acid Sequence↗
PubMed1998

A novel strategy of cell targeting based on tissue-specific expression of the ecotropic retrovirus receptor gene.

Gene transfer into specific tissues or cell types is a key technique in the development of gene therapy. Modification of vector particles such that they selectively bind to the target cells has been attempted, but the limitation of this approach is the low transduction efficiency. Here, we show that a two-step gene transfer system can be used for efficient cell targeting. With this strategy, and using a high-titer adenoviral vector containing a tissue-specific promoter, we have engineered a system in which only target cells become susceptible to retrovirus-mediated transduction. In a model experiment, we constructed an adenoviral vector (Ad.AFPEcoRec) containing the ecotropic retrovirus receptor (EcoRec) gene under the control of the alpha-fetoprotein (AFP) promoter. A binding assay showed that after transduction with AD.AFPEcoRec, EcoRec molecules were efficiently expressed in AFP+HepG2 cells, but not in AFP-HeLa and AFP-HLE cells. The EcoRec-expressing HepG2 cells could be stably transduced with ecotropic retroviral vectors, whereas HeLa and HLE cells remained highly resistant to retrovirus-mediated gene transfer. The apparent titer on HepG2 cells was greater than 2 x 10(5) CFU/ml. Because various tissue-specific promoter/enhancer elements are available, the two-step system could be used as a general strategy for both ex vivo and in vivo targeted gene transfer.

3T3 Cells↗
PubMed2006

Therapeutic potential of chitosan and its derivatives in regenerative medicine.

BACKGROUND: Cell-based transplantation, tissue engineering and gene therapy are important therapeutic strategies for present and future regenerative medicine. One challenge is to present the target cells in a suitable matrix to allow the cells to survive the wound contraction, tissue repair, and remodeling in certain tissues. Recently, functional biomaterial research has been directed towards the development of improved scaffolds and new drug delivery systems for regenerative medicine. MATERIALS AND METHODS: A literature survey was performed in basic and clinic publications relevant to the therapeutic potential of chitosan and its derivatives in regenerative medicine. In this review the functional properties and potential applications of chitosan and its derivatives in regenerative medicine are presented and discussed. RESULTS: Chitosan can be obtained by alkaline deacetylation of chitin and is found to be a natural-based nontoxic, biocompatible, and biodegradable polymer with anti-microbial activity. Chitosan and its derivatives could accelerate wound healing by enhancing the functions of inflammatory cells and repairing cells. Recent studies further indicated that chitosan and its derivatives also are novel scaffold materials for tissue engineering and are-promising non-viral vectors for gene delivery. CONCLUSIONS: Regenerative medicine has entered a new era with the development of modern science and technology. The novel properties of chitosan make it a versatile biomaterial for cell therapy, tissue engineering and gene therapy. It is hoped that these diverse approaches for regenerative medicine will translate from "bench to bedside" in the future.

Animals↗
PubMed1998

Recombinant herpes simplex virus type 1 engineered for targeted binding to erythropoietin receptor-bearing cells.

The utility of recombinant herpes simplex virus type 1 (HSV-1) vectors may be expanded by manipulation of the virus envelope to achieve cell-specific gene delivery. To this end, an HSV-1 mutant virus deleted for glycoprotein C (gC) and the heparan sulfate binding domain of gB (KgBpK-gC-) was engineered to encode different chimeric proteins composed of N-terminally truncated forms of gC and the full-length erythropoietin hormone (EPO). Biochemical analyses demonstrated that one gC-EPO chimeric molecule (gCEPO2) was posttranslationally processed, incorporated into recombinant HSV-1 virus (KgBpK-gCEPO2), and neutralized with antibodies directed against gC or EPO in a complement-dependent manner. Moreover, KgBpK-gCEPO2 recombinant virus was specifically retained on a soluble EPO receptor column, was neutralized by soluble EPO receptor, and stimulated proliferation of FD-EPO cells, an EPO growth-dependent cell line. FD-EPO cells were nevertheless refractory to productive infection by both wild-type HSV-1 and recombinant KgBpK-gCEPO2 virus. Transmission electron microscopy of FD-EPO cells infected with KgBpK-gCEPO2 showed virus endocytosis leading to aborted infection. Despite the lack of productive infection, these data provide the first evidence of targeted HSV-1 binding to a non-HSV-1 cell surface receptor.

Animals↗
PubMed2004

Engineering ex vivo-expanded marrow stromal cells to secrete calcitonin gene-related peptide using adenoviral vector.

Calcitonin gene-related peptide (CGRP) is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs) hold promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated CGRP gene transfer into ex vivo-expanded MSCs, rat MSCs were isolated, ex vivo expanded, and transduced with adenoviruses. Adprepro-CGRP and AdntlacZ, adenoviral vectors containing prepro-CGRP or nuclear-targeted beta-galactosidase reporter gene ntlacZ under the control of Rous sarcoma virus promoter, were used. In this study, it can be shown that transduction efficiency of adenoviral-mediated gene transfer into ex vivo-expanded MSCs is dose dependent, transgene expression persists for more than 21 days in culture, and adenoviral transduction does not alter the proliferation or viability of MSCs. Transduced MSCs retain multipotentiality and transgene expression after cell differentiation. The expression and secretion of CGRP by Adprepro- CGRP-transduced MSCs was confirmed by Western blot analysis and enzyme immunoassay. The secretion of CGRP by Adprepro-CGRP-transduced MSCs is dose dependent, and the transduced cells release as much as 9.5 +/- 0.4 pmol CGRP/1 x 10(6) cells/48 hours (mean +/- standard error of mean, n = 3) into culture medium at a multiplicity of infection of 300. Furthermore, culture supernatant from Adprepro-CGRP-transduced MSCs increases intracellular cyclic AMP levels in pulmonary artery smooth muscle cells in culture. These findings suggest that replication-deficient recombinant adenovirus can be used to gene engineer ex vivo-expanded MSCs and that high-level secretion of biologically active CGRP can be achieved, underscoring the clinical potential of using this novel adult stem cell-based cell and gene therapy strategy for the treatment of cardiovascular diseases.

Adenoviridae↗
PubMed1999

Identification of side-chain clusters in protein structures by a graph spectral method.

This paper presents a novel method to detect side-chain clusters in protein three-dimensional structures using a graph spectral approach. Protein side-chain interactions are represented by a labeled graph in which the nodes of the graph represent the Cbeta atoms and the edges represent the distance between the Cbeta atoms. The distance information and the non-bonded connectivity of the residues are represented in the form of a matrix called the Laplacian matrix. The constructed matrix is diagonalized and clustering information is obtained from the vector components associated with the second lowest eigenvalue and cluster centers are obtained from the vector components associated with the top eigenvalues. The method uses global information for clustering and a single numeric computation is required to detect clusters of interest. The approach has been adopted here to detect a variety of side-chain clusters and identify the residue which makes the largest number of interactions among the residues forming the cluster (cluster centers). Detecting such clusters and cluster centers are important from a protein structure and folding point of view. The crucial residues which are important in the folding pathway as determined by PhiF values (which is a measure of the effect of a mutation on the stability of the transition state of folding) as obtained from protein engineering methods, can be identified from the vector components corresponding to the top eigenvalues. Expanded clusters are detected near the active and binding site of the protein, supporting the nucleation condensation hypothesis for folding. The method is also shown to detect domains in protein structures and conserved side-chain clusters in topologically similar proteins.

Algorithms↗
PubMed2006

Transposon-free insertions for insect genetic engineering.

Methods involving the release of transgenic insects in the field hold great promise for controlling vector-borne diseases and agricultural pests. Insect transformation depends on nonautonomous transposable elements as gene vectors. The resulting insertions are stable in the absence of suitable transposase, however, such absence cannot always be guaranteed. We describe a method for post-integration elimination of all transposon sequences in the pest insect Medfly, Ceratitis capitata. The resulting insertions lack transposon sequences and are therefore impervious to transposase activity.

Animals↗
PubMed2002

Rapid induction of specific cytotoxic T lymphocytes against melanoma-associated antigens by a recombinant vaccinia virus vector expressing multiple immunodominant epitopes and costimulatory molecules in vivo.

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8(+) T cells could be observed. Tetramer staining also revealed significant increases in specific CD8(+) T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.

Antigens, Neoplasm↗
PubMed1970

Evaluation of ultra-low-volume insecticide dispensing systems for use in single-engined aircraft and their effectiveness against Aedes aegypti populations in South-East Asia.

An evaluation study of ultra-low-volume (ULV) spraying of insecticide from aircraft was carried out in Thailand, to determine if this technique could be used for the emergency control of Aedes aegypti, the major vector of haemorrhagic fever. A small, single-engined aircraft, a Cessna-180, was used in the trials and 2 types of spraying equipment were tested; both were found to be equally effective. The aircraft was fitted with 6 spraying nozzles and flew at an altitude of 150 feet (46 m) at a speed of 100 miles/h (161 km/h). The insecticide used was 95% technical grade malathion and swaths 75 feet wide (22.8 m) were laid down; the rate of application was 3 US fl oz/acre (219 ml/ha).Trials were made in 3 villages near Bangkok and it became apparent that a small aircraft could not produce the required even distribution of insecticide; the rate of application was therefore increased to 6 US fl oz/acre (438 ml/ha). This increased rate appeared to compensate for the narrow width of the swath and produced very satisfactory mortalities in caged mosquitos as well as in natural populations.The size and distribution of droplets was monitored by the use of oil-sensitive red dye cards which showed that there was a good penetration of insecticide into dwellings, etc. Trial results were evaluated by biting counts, bioassays of Aedes and Culex adults and larvae, Culex dips and ovitraps. Biossays indicated that the 6 US fl oz/acre rate of application was almost 100% effective in the open and produced satisfactory mortalities inside markets and dwellings.It was concluded that larger aircraft would be required to treat areas of more than 1000 acres (405 ha) and congested city areas but that the rate of application of insecticide could be considerably lower. Nevertheless, small aircraft can be useful in smaller and less congested areas.

Aedes↗
PubMed1994

Replicating vectors for gene therapy of cancer: risks, limitations and prospects.

There are good theoretical arguments for exploring the use of replicating gene-transfer vectors for human cancer therapy. Such vectors should be derived from weakly pathogenic human viruses with initially broad tissue tropism. Coat protein engineering and promoter engineering might be used successfully to narrow the tropism of the vector, enhancing its ability to target tumour cells. Killing of uninfected 'bystander' tumour cells could be achieved through prodrug activation by a vector-encoded enzyme. Rapid elimination of infused vector particles by circulating antiviral antibody would limit access to tumour deposits after repeated administration, but might be circumvented by the use of infectious nucleic acid which is poorly imunogenic [64]. This putative therapeutic strategy is illustrated in Figure 1.

Gene Transfer Techniques↗
PubMed2005

Simultaneous production of two foreign proteins from a polyvirus-based vector.

With the aim of developing a biotechnological tool for the production of foreign proteins in plants, we first engineered an infectious turnip mosaic virus (TuMV) cDNA that contained the jellyfish green fluorescent protein (GFP) gene or the bacterial beta-glucuronidase (GUS) gene (uidA). Two insertion sites were assessed, either between P1 and HCPro cistrons or Pol and CP cistrons. In each construct, the junctions flanking the inserted gene coded for P1 and/or VPg-Pro cleavage recognition site sequences, to produce free GUS or GFP. After transfection by particle bombardment on Brassica perviridis, characteristic symptoms for TuMV infection appeared and Western blot analyses showed that GFP and GUS had been excised from the viral polyprotein. No significant differences in expression level were noticed between the two insertion sites. By RT-PCR, gfp was found to be stable over 30 days post-transfection (dpt) while uidA was gradually lost at 15 dpt. We also created two constructs containing either gene at each insertion sites on the same molecule. Attenuated systemic symptoms were observed after particle bombardment on B. perviridis and Western blot analyses showed that both foreign proteins were produced. Also, the same stability/instability as for the single-gene constructs were observed. These results indicate that it is possible to produce at least two foreign proteins simultaneously in a TuMV-based vector.

Amino Acid Sequence↗
PubMedDate not supplied

Nonviral vector loaded collagen sponges for sustained gene delivery in vitro and in vivo.

BACKGROUND: Naked DNA and standard vectors have previously been used for gene delivery from implantable carrier matrices with great potential for gene therapeutic assistance of wound healing or tissue engineering. We have previously developed copolymer-protected gene vectors which are inert towards opsonization. Here we examine their potency in carrier-mediated gene delivery in comparison to standard vectors using a vector-loaded collagen sponge model. METHODS: Equine collagen type I sponges were loaded by a lyophilization method with naked DNA, polyethylenimine (PEI)-DNA, DOTAP/cholesterol-DNA and copolymer-protected PEI-DNA. These preparations were characterized in terms of vector-release, cell growth on the matrices and reporter gene expression by cells colonizing the sponges in vitro and in vivo. Subcutaneous implantation of sponges in rats served as an in vivo model. RESULTS: At the chosen low vector dose, the loading efficiency was at least 86%. Naked DNA-loaded collagen matrices lost 77% of the DNA dose in an initial burst in aqueous buffer in vitro. The other preparations examined displayed a sustained vector release. There was no difference in cell growth and invasion of the sponges between vector-loaded and untreated collagen grafts. Reporter gene expression from cells colonizing the sponges in vitro was observed for not more than 7 days with naked DNA, whereas the lipoplex and polyplex preparations yielded long-term expression throughout the experimental period of up to 56 days. The highest expression levels were achieved with the PEI-DNA-PROCOP (protective copolymer) formulation. Upon subcutaneous implantation in rats, no luciferase expression was detected with naked DNA preparations. DOTAP/cholesterol-DNA and PEI-DNA-loaded implants lead to reporter gene expression for at least 3 days, but with poor reproducibility. PEI-DNA-PROCOP collagen matrices yielded consistently the highest reporter gene expression levels for at least 7 days with good reproducibility. CONCLUSIONS: With the preparation method chosen, lipoplex- and polyplex-loaded collagen sponges are superior in mediating sustained gene delivery in vitro and local transfection in vivo as compared to naked DNA-loaded sponges. Protective copolymers are particularly advantageous in promoting the tranfection capacity of polyplex-loaded sponges upon subcutaneous implantation, likely due to their stabilizing and opsonization-inhibiting properties.

3T3 Cells↗