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At least 1,369 records · Page 76Linked to original sources
PubMed2000

Glucose-responsive gene delivery in pancreatic Islet cells via recombinant adeno-associated viral vectors.

PURPOSE: Recent progress in genetic engineering presents the possibility of providing physiologically regulated glucose metabolism in individuals with diabetes. The objective of this study is to explore the feasibility of obtaining glucose dependent gene expression in the pancreatic beta-cell lines via recombinant adeno-associated virus type 2 (rAAV) mediated gene transfer. METHODS: Two transcription cassettes containing the luciferase gene under the control of the rat insulin I gene promoter and the enhanced green fluorescent protein (EGFP) open reading frame under the control of the immediate early gene promoter of human cytomegalovirus (CMV) were placed in series between the inverted terminal repeats (ITRs) of AAV. The rAAV vectors produced were used to transduce pancreatic beta-cell line grown in the absence or presence of various concentrations of glucose. Luciferase activity assays were performed at 72 hr post-transduction. RESULTS: Glucose-responsive reporter gene expression was obtained in both calcium phosphate transfected HIT-T15 and betaHC-9 cells, demonstrating regulated luciferase gene expression under control of the insulin gene promoter. At MOI of 100, rAAV-transduced betaHC-9 cells exhibited glucose-dependent luciferase activities, which were approximately 4.3 fold higher than those transfected by the calcium phosphate coprecipitation method at 20 mM glucose. CONCLUSIONS: Delivery of the insulin gene promoter via rAAV was shown in this study to result in glucose-dependent control of the reporter gene expression. The results suggest that rAAV is an efficient viral vector for gene transfer into the pancreatic islet cells.

Animals↗
PubMed2000

Spleen necrosis virus-derived C-type retroviral vectors for gene transfer to quiescent cells.

Gene therapy applications of retroviral vectors derived from C-type retroviruses have been limited to introducing genes into dividing target cells. Here, we report genetically engineered C-type retroviral vectors derived from spleen necrosis virus (SNV), which are capable of infecting nondividing cells. This has been achieved by introducing a nuclear localization signal (NLS) sequence into the matrix protein (MA) of SNV by site-directed mutagenesis. This increased the efficiency of infecting nondividing cells and was sufficient to endow the virus with the capability to efficiently infect growth-arrested human T lymphocytes and quiescent primary monocyte-derived macrophages. We demonstrate that this vector actively penetrates the nucleus of a target cell, and has potential use as a gene therapy vector to transfer genes into nondividing cells.

Amino Acid Sequence↗
PubMed2005

A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants.

Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.

Amino Acid Sequence↗
PubMed2003

In vivo high-efficiency transcoronary gene delivery and Cre-LoxP gene switching in the adult mouse heart.

High-efficiency somatic gene transfer in adult mouse heart has not yet been achieved in vivo. Here, we demonstrate high-efficiency in vivo transcoronary gene delivery to the adult murine myocardium using a catheter-based technique with recombinant adenovirus (AdV) and adeno-associated virus (AAV) vectors in normal and genetically engineered mice. The method involves immersion hypothermia followed by transient aortic and pulmonary artery occlusion with proximal intra-aortic segmental injection of cardioplegic solution containing substance P and viral vectors. Gene expression measured using a LacZ marker gene was observed throughout both ventricles. The expression efficiency of a cytoplasmic LacZ marker gene in the left ventricular myocardium was 56.4+/-14.5% (mean+/-s.d.) at 4 days with an AdV vector, and with an AAV vector it was 81.0+/-5.9% at 4 weeks. Following AAV gene transfer, no gene expression was found in kidney, brain, lung, and spleen, but there was slight expression in liver. In addition, we demonstrate temporally controlled genetic manipulation in the heart with an efficiency of 54.6+/-5.2%, by transferring an AdV vector carrying Cre recombinase in ROSA26 flox-LacZ reporter mice. Procedure-related mortality was 16% for AdV and zero for AAV transfer. Thus, this method provides efficient, relatively homogeneous gene expression in both ventricles of the adult mouse heart, and offers a novel approach for conditional gene rescue or ablation in genetically engineered mouse models.

Adenoviridae↗
PubMed1986

A general method for retrieving the components of a genetically engineered fusion protein.

Escherichia coli expression vectors encoding an acid-labile aspartyl-proline (Asp-Pro) dipeptide bridging two protein sequences were constructed and used to synthesize two different bovine growth hormone (bGH) fusion proteins. The codons GAT-CCX coding for Asp-Pro are provided by the recognition site for Bam HI (GGATCC). Treatment of the bGH fusion proteins at low pH in the presence of guanidine hydrochloride releases the bGH moiety from the fusion protein. The release of the bGH from the fusion protein specifically requires the Asp-Pro dipeptide linking the bGH sequence to the fusion protein. The bGH released from the fusion protein retains anti-bGH immunoreactivity as well as the ability to bind to growth hormone receptor in vitro.

Acids↗
PubMed1990

Development of expression vectors for transgenic fish.

Genetic alteration of fish is important for aquatic biotechnology as well as for investigating molecular interactions that occur during vertebrate development. The numerous, large, transparent, and externally fertilized eggs of many fish species make them ideally suitable for genetic manipulation, especially for production of transgenic animals. Genetic engineering of fish requires suitable expression vectors. Accordingly, we developed two fish expression vectors, FV-1 and FV-2, which contain the proximal promoter and enhancer regulatory elements of the carp beta-actin gene and the polyadenylation signal from the salmon growth hormone gene. The two fish expression vectors were tested in microinjected fish eggs and in tissue cultured fish and mammalian cells. These two "all-fish" expression vectors should be useful for genetic engineering of fish and have been used with growth-enhancing genes in transgenic fish.

Actins↗
PubMed2004

[Construction of combined site-directed random mutation libraries of recombinant human lymphotoxin].

To construct the combined site-directed random mutation library of recombinant human Lymphotoxin (rhLT) for in vitro molecular evolution study, and to study the structure and function relationship. The random point mutations at the sites of 46,106 and 130 were individually generated by overlap PCR amplification with the random nucleotide primers. The three point mutations were combined and cloned into pMD-18T vector to construct the combined mutation library. DNA sequencing was used to evaluate the diversity and randomness of the mutation sites. The combined mutation library was re-engineered, inserted in prokaryotic expression vector pBV220, transformed and expressed in Escherichia coli strain DH5alpha. The biological activity of some of the mutants was tested in 1929 mouse fibroblast cells. As much as 1.5 x 10(5) clones were obtained, which represents 4.5 times of the complete mutation libraries at 99% confidence. Sequencing 50 clones revealed no obvious bias in the nucleotide and amino acid mutations at the sites. Among the 30 expressed samples underwent for the bioassay, 70% (21 samples) were inactive, 23.3% (7 samples) had lower activity than rhLT, the remaining 6.7% (2 samples) had higher activity than rhLT. The combined site-directed random mutation library of rhLT has been constructed successfully. In combination with phase display, the library is ready for in vitro molecular evolution study.

Amino Acid Sequence↗
PubMed2005

[The construction of pSecTag2/B vector modified by osteoprotegerin].

PURPOSE: In order to treat periodontitis by using tissue engineering and gene engineering technology, we constructed mammalian secreted expression pSecTag2/B-OPG vector, the cDNA sequence encoding osteoprotegerin(OPG) obtaining from 293 cell. METHODS: The primers were designed based on the human OPG cDNA sequence. Total mRNA was isolated from 293 cells and RT-PCR was performed .The fragment was recombined into pSecTag2/B vector and sequenced by automatic sequence analyzer. RESULTS: The sequences of OPG cDNA from 293 cell by RT-PCR were completely identical to the sequences provided by GenBank [gi:33878056]. After polymerase chain reaction and the recombinant plasmid digesting with Hind III,EcoR I and BamH I,1% agarose electrophoresis showed several fragments, which were consistent with predicted size. CONCLUSION: From cultured 293 cells the cDNA has been cloned and pSecTag2/B-OPG vector has been constructed successfully. Supported by Research Fund (No.03043304) from Anhui Provincial Natural Science Foundation.

Animals↗
PubMed1991

'Phytoantibodies': a general vector for the expression of immunoglobulin domains in transgenic plants.

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immunohistochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.

Agrobacterium tumefaciens↗
PubMed2000

Bifidobacterium longum as a delivery system for cancer gene therapy: selective localization and growth in hypoxic tumors.

A fundamental obstacle in gene therapy for cancer is the specific delivery of an anticancer gene product to a solid tumor, and yet no systemic delivery system that specifically targets solid tumors currently exists. A strain of domestic bacteria, Bifidobacterium longum, which is nonpathogenic and anaerobic, selectively localized and proliferated in several types of mouse solid tumors after systemic application. In this report, we further describe a novel approach to cancer gene therapy in which genetically engineered Bifidobacterium is used as a tumor-specific vector. Similarly to wild-type B. longum, genetically engineered B. longum could be detected in tumor tissue only and was not found in a large survey of normal mouse tissues after intravenous injection. This finding strongly suggests that obligate anaerobic bacteria such as Bifidobacterium can be used as highly specific gene delivery vectors for cancer gene therapy.

Animals↗
PubMed2000

Long-term and significant correction of brain lesions in adult mucopolysaccharidosis type VII mice using recombinant AAV vectors.

Most lysosomal storage diseases, including mucopolysaccharidosis, affect the central nervous system (CNS). They often induce severe and progressive mental retardation. Replacement therapy by purified enzyme infusions is a promising approach for the treatment of peripheral organs but without effect on CNS pathology because the enzyme cannot cross the blood-brain barrier. Intracranial injection of recombinant adeno-associated virus (AAV) vectors offers an alternative for sustained local enzyme delivery from genetically engineered cells. We stereotactically injected an AAV vector containing the human beta-glucuronidase cDNA into the striatum of adult mice severely affected by mucopolysaccharidosis type VII at the time of treatment. Six weeks later, beta-glucuronidase activity in the injected hemisphere was comparable to that of heterozygous mice, which have a normal phenotype. Areas staining positive for enzyme activity enlarged with time, representing more than 10% of the hemisphere volume by 16 weeks. A complete reversion of lysosomal storage lesions was evident in these areas, as well as in most neurons located in surrounding negative areas and in the noninjected hemisphere. Thus, a single intracerebral injection of AAV vectors could achieve a broad and sustained lysosomal enzyme delivery, allowing for stable reversion of storage lesions in a significant fraction of the adult brain.

Animals↗
PubMedDate not supplied

Semliki forest virus-based expression for versatile use in receptor research.

Semliki Forest virus (SFV) vectors have been generated for highly efficient studies on gene expression in a variety of mammalian host cells, including immortalized cell lines as well as primary cells in culture. Moreover, SFV expression has been scaled up for mammalian suspension cultures in spinner flasks and bioreactors for production of large quantities of recombinant proteins for drug screening and purification. The strong preference of expression in neuronal cells in primary cell cultures, in organotypic hippocampal slices and in vivo has made SFV vectors attractive for neurobiological studies. Additionally, the engineering of novel, less cytotoxic and temperature-sensitive SFV mutant vectors has further increased their application range.

Animals↗
PubMed1995

Recombinant retroviral vector interferes with the detection of amphotropic replication competent retrovirus in standard culture assays.

Many protocols for gene therapy employ recombinant retroviral vectors, which are replication-defective retroviruses engineered to serve as gene delivery vehicles. The use of retroviral vectors for human gene therapy requires careful screening of vector-producing cell lines and culture supernatants to ensure the absence of replication competent retrovirus (RCR) in clinical products. In this study we have examined several different culture assays routinely used to test for the presence of RCR. Results indicate that cocultivation of a vector-producing cell line with a permissive cell line can reproducibly detect a low level of contaminating RCR. RCR was detected less frequently in direct tests of cell-free culture supernatants from a contaminated vector-producing line. Further studies revealed that recombinant retroviral vector can interfere, to varying degrees, with the detection of low-level RCR in culture supernatants when a marker rescue assay, an extended mink S+L- assay or a PG-4 S+L- assay is used. Interference can be partially overcome by culturing the vector preparation with a permissive cell line for several days before testing on the indicator cell line. The interference phenomenon we have observed may also occur in other culture assays routinely used for the detection of RCR.

Animals↗
PubMed1991

Co-expression of hepatitis B virus antigens by a non-defective adenovirus vaccine vector.

Adenovirus type 7 vaccine strain was engineered to express foreign antigens from both the E3 early promoter in the E3 region and the major late promoter inserted between the E4 region and the right inverted terminal repeat. This multiple expression vector was used to express hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), and hepatitis B surface antigen (HBsAg). The gene inserted in the E3 region was derived from the core gene of the hepatitis B virus genome. When the precore region was present, an immunoreactive group of proteins with molecular weights ranging from 15,000 to 19,000 was secreted into the media. Velocity sedimentation centrifugation of media and lysates from cells infected with recombinants containing the core gene with the precore region resulted in peaks of HBeAg at the top of the gradient where authentic HBeAg should be found. In addition to the core gene in the E3 region, the surface antigen gene of hepatitis B virus was inserted behind the major late promoter in the E4 region resulting in an adeno-hepatitis recombinant virus capable of expressing both the core gene and the HBsAg cells. Cells infected with the adeno-hepatitis recombinants could also be stained with peroxidase-conjugates after reacting to antibody against HBcAg. Inoculation of dogs with the recombinant viruses which contained the core gene, with and without the precore sequence, resulted in a significant antibody response to HBcAg/HBeAg. The dogs also produced a significant antibody response to HBsAg as well as neutralizing antibody to adenovirus.

Adenoviruses, Human↗
PubMed2006

Virus-based gene therapy strategies for bone regeneration.

Gene therapy has emerged as a promising strategy for the repair and regeneration of damaged musculoskeletal tissues. Application of this paradigm to bone healing has shown enhanced efficacy in preclinical animal studies compared to conventional bone grafting approaches. This review discusses current and emerging virus-based genetic engineering strategies for the delivery of therapeutic molecules which promote skeletal regeneration. Viral gene delivery vectors are discussed in the context of bone repair in order to illustrate the challenges and applications of these methods with tissue-specific examples. Moreover the concepts discussed can be broadly applied to promote healing in a wide range of tissues. We also present important considerations involved in the application of these gene therapy techniques to a variety of osteogenic (e.g. bone marrow-derived cells) and non-osteogenic (e.g. fibroblasts and skeletal myoblasts) cell types. Criteria for the selection of regenerative molecules with soluble versus intracellular modes of action and emerging combinatorial approaches are also discussed. Overall, gene transfer technologies have the potential to overcome limitations associated with existing bone grafting approaches and may enable investigators to design therapies which more closely mimic the complex spatial and temporal cascade of proteins involved in endogenous bone development and repair.

Animals↗
PubMed2004

Targeted gene delivery to vascular tissue in vivo by tropism-modified adeno-associated virus vectors.

BACKGROUND: Gene therapy offers an unprecedented opportunity to treat diverse pathologies. Adeno-associated virus (AAV) is a promising gene delivery vector for cardiovascular disease. However, AAV transduces the liver after systemic administration, reducing its usefulness for therapies targeted at other sites. Because vascular endothelial cells (ECs) are in contact with the bloodstream and are heterogeneous between organs, they represent an ideal target for site-specific delivery of biological agents. METHODS AND RESULTS: We isolated human venous EC-targeting peptides by phage display and genetically incorporated them into AAV capsids after amino acid 587. Peptide-modified AAVs transduced venous (but not arterial) ECs in vitro, whereas hepatocyte transduction was significantly lower than with native AAV. Intravenous infusion of engineered AAVs into mice produced reduced vector accumulation in liver measured 1 hour and 28 days after injection and delayed blood clearance rates compared with native AAV. Peptide-modified AAVs produced enhanced uptake of virions in the vena cava with selective transgene expression. Retargeting was dose dependent, and coinfusion of either heparin or free competing peptides indicated that uptake was principally independent of native AAV tropism and mediated via the peptide. CONCLUSIONS: AAV tropism can be genetically engineered by use of phage display-derived peptides to generate vectors that are selective for the vasculature.

Animals↗
PubMed2003

Vaccinia vectors as candidate vaccines: the development of modified vaccinia virus Ankara for antigen delivery.

Vaccinia viruses engineered to express foreign genes are powerful vectors for production of recombinant proteins. Originating from highly efficacious vaccines securing world-wide eradication of smallpox, the most appealing use of vaccinia vectors is to serve as vaccine delivery system for heterologous antigens. Concerns about the safety of vaccinia virus have been addressed by the development of vectors based on attenuated viruses. One of them, modified vaccinia virus Ankara (MVA) can be considered as current vaccinia virus strain of choice for clinical investigation. Historical development and use of MVA as vaccine against smallpox allowed to establish an extraordinary safety profile. MVA can be used under conditions of biosafety level 1 because of its avirulence and its deficiency to productively grow in human cells. In recent years significant progress has been made with regard to the development of MVA vector technologies. Compared to replication competent vaccinia viruses, MVA provides similar levels of recombinant gene expression even in nonpermissive cells. In animal models, MVA vaccines have been found immunogenic and protective against various infectious agents including immunodeficiency viruses, influenza, parainfluenza, measles virus, flaviviruses, or plasmodium parasites. By now first data from clinical trials are becoming available. In this article we briefly review history of MVA and state-of-the art technologies with regard to generation of recombinant MVA vaccines, and describe the progress to develop MVA vector vaccines against important infectious diseases.

Animals↗
PubMed1985

[Vector plasmids and the strategy of molecular cloning for streptococci].

The experiments on elaboration of gene engineering methodology for streptococci are described. Two vectors were constructed for DNA cloning in streptococci on the basis of plasmids pSM19035 and pIP2501. Some of the plasmids occurred to be valuable vectors for molecular cloning in bacilli. Peculiarities of the transformation mechanism in streptococci were found to impede the molecular cloning. The recombination technique of cloning was successfully used in the streptococcal system.

Cloning, Molecular↗