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At least 1,207 records · Page 67Linked to original sources
PubMed1987

Stability of a bacterial gene in a bovine papillomavirus-based shuttle vector maintained extrachromosomally in mammalian cells.

In order to analyse the stability of cloned genes in a viral vector we have constructed a shuttle vector based on bovine papillomavirus and the Escherichia coli gene lacZ. Propagation of this vector in mouse C127 cells and analysis of vector sequences in bacteria produced no detectable mutations in the lacZ gene in over 6137 clones analysed. This is 100-fold less than the mutation frequency observed when the same and similar target genes are replicated in monkey COS cells using a simian virus 40-based shuttle vector.

Animals↗
PubMed2004

Clinical trials in neurological disorders using AAV vectors: promises and challenges.

Currently, there are five phase I clinical trials of recombinant adeno-associated viral vectors for the treatment of neurological disorders that are approved or likely to be approved shortly. Two trials are testing different strategies to treat Parkinson's disease (PD), the third trial is aimed at treating Canavan's disease, a pediatric leukodystrophy, the fourth trial targets Alzheimer's disease (AD), and the fifth will attempt to target the lysosomal storage disorder, Batten's disease. All four clinical trials rely on the de novo expression of an enzyme or a trophic factor to correct neuropathology. Ironically, the theories used to choose enzymes for the two PD trials were widely divergent, whereas the enzymatic strategy used for one of the PD trials and the Canavan's trial have remarkable similarities. Other gene therapy treatment strategies for PD and other disorders, such as amyotrophic lateral sclerosis, are also on the horizon.

Clinical Trials as Topic↗
PubMed2000

[Protection against influenza by genetically engineered vaccines].

Viral vector recombinant DNA vaccines, so called genetically engineered vaccines, are introduced directly into cells to induce an antigen specific immune response. This is a new approach to vaccination that represents a new concept in immunotherapy, which attempts to induce a long-term effective, anti-specific, viral antigen immune response. These vaccines may also prove more potent than currently used vaccines when it comes to inducing cellular immune responses in addition to humoral immune response. In the case of influenza, an immune response with humoral characteristics was induced using an influenza hemagglutinin(HA) DNA vaccine. On the other hand, injection of influenza virus nucleoprotein(NP) induced a Th-1 type cellular immune response including CTL. Genetically engineered vaccines have the potential to effectively overcome the problem of unresponsiveness to currently used vaccines.

Animals↗
PubMed1997

Adenoviral/retroviral vector chimeras: a novel strategy to achieve high-efficiency stable transduction in vivo.

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. Realization of both goals with available vector systems has so far not been achieved. As a novel approach to solve this problem, we have developed a chimeric viral vector system that exploits favorable aspects of both adenoviral and retroviral vectors. In this schema, adenoviral vectors induce target cells to function as transient retroviral producer cells in vivo. The progeny retroviral vector particles can then effectively achieve stable transduction of neighboring cells. In this system, the nonintegrative adenoviral vector is rendered functionally integrative via the intermediate generation of an induced retroviral producer cell. Such chimeric vectors may now allow realization of the requisite goals for specific gene therapy applications.

Adenovirus Infections, Human↗
PubMed2003

Functional delivery of large genomic DNA to human cells with a peptide-lipid vector.

BACKGROUND: Nonviral gene transfer vectors have the potential to deliver much larger DNA constructs than current viral vectors but suffer from a low transfection efficiency. The LID vector, composed of Lipofectin (L), an integrin-targeting peptide (I) and DNA (D), is a highly efficient synthetic vector, both in vitro and in vivo, which may allow the transfer of genomic loci for gene therapy. METHODS: Transfection efficiencies were quantitated using the green fluorescent protein (GFP) reporter. Expression of a large genomic locus (NBS1 [Nijmegen breakage syndrome], encoding nibrin) was assessed by immunofluorescence. RESULTS: We report a systematic study of the parameters influencing delivery of BAC-based plasmids ranging in size from 12 to 242 kb using the LID vector. We showed 60% of cells were transfected with the smaller plasmids while plasmids up to 242 kb were consistently delivered to over 10% of cells. The number of transfected cells was related to number of plasmids in the transfection complex independent of plasmid size. Atomic force microscopy showed that LID particle size increased with plasmid size consistent with one plasmid molecule per particle. When LID vectors were used to deliver the NBS1 gene as a 143 kb construct to primary NBS cells, at least 57% of cells expressing GFP also expressed functional nibrin. CONCLUSIONS: We show that LID vectors represent a promising tool for the transfer of complete genomic loci.

Cell Cycle Proteins↗
PubMed2001

Differential neuroprotection from human heat shock protein 70 overexpression in in vitro and in vivo models of ischemia and ischemia-like conditions.

We previously showed that overexpressing the 70-kDa inducible heat shock protein in primary astrocyte cultures and in a rodent stroke model using viral vectors resulted in protection from ischemia and ischemia-like injury. However, viral transfection could potentially provoke a stress response itself; therefore, we examined whether transgenic mice constitutively expressing human heat shock protein 70 were protected from ischemic insults. Astrocyte cultures from brains of heat shock protein 70 transgenic mice were resistant to hydrogen peroxide injury in a dose-dependent fashion, but were less resistant to hypoglycemia and oxygen-glucose deprivation. Because hydrogen peroxide exposure and glucose deprivation are partially dependent on glutathione levels, we determined whether heat shock protein 70 transgenic cultures had altered glutathione levels under normal growth conditions. However, there was no significant difference in glutathione levels between heat shock protein 70 transgenic and wildtype astrocytes. Hippocampal, but not cortical neuron cultures from these same transgenic mice were also protected against oxygen-glucose deprivation and glutamate toxicity. In an in vivo model of permanent focal cerebral ischemia, there was no significant difference in infarct size assessed 24 h postinsult. These results suggest that heat shock protein 70 protects against some but not all kinds of central nervous system injury. The protective effects may be related to the nature and severity of the insults, as well as subpopulations of brain cells and dose-dependent effects of HSP70 overexpression.

Animals↗
PubMed1980

Sensitive immunological detection of translation products in SV40 plaques.

A simple and sensitive in situ radioimmunoassay, using simian virus 40 (SV40) proteins as a model, has been developed for the detection of specific translation products of foreign genes cloned in viral vectors. This assay is based on the coupling of all proteins in viral plaques to diazophenylthioether (DPT)-paper. Specific proteins bound to the filter are detected by autoradiography after sequential incubation with (i) unfractionated and unlabeled specific antiserum and (ii) 125I-labeled protein A from Staphylococcus aureus. This assay detects SV40-specific proteins in individual plaques as early as 42 h after infection and its sensitivity limit is below 5 x 10(8) molecules of the major SV40 capsid protein, VP1.

Antigens, Neoplasm↗
PubMed2003

A new helper phage and phagemid vector system improves viral display of antibody Fab fragments and avoids propagation of insert-less virions.

Phage display technology (PDT) is a powerful method for isolating functional gene products such as antigen-specific monoclonal antibodies (mAbs). To improve the effectiveness of PDT, we sought to optimize display of Fab-g3p (antibody fragment fused with viral gene 3 protein) on phagemid virions and to optimize the yield of such phage. To do so, we constructed a novel helper phage, Phaberge, having a conditional deficiency in g3p production. Unlike most other published g3p-deficient helper phage, Phaberge is produced at high levels, 10(11) PFU/ml. As compared to g3p-sufficient helper phage, Phaberge caused a 5-20-fold increase in display level. Another novel feature is that Phaberge only packages insert-containing, not insert-less, phagemid into infectious virions. This should prove useful in preserving quality of phagemid libraries during propagation. In addition, other parameters were also found to affect production of phagemid virions. In particular, the choice of bacterial host cell, phagemid construct and growth temperature had a substantial impact on display levels, but generally no effect on number of phagemid virions produced. In short, we have established a set of parameters that improve production and quality of phagemid virions which we expect to facilitate the isolation of mAbs or other gene products by PDT.

Antibodies, Monoclonal↗
PubMed1999

Polymer conjugates for tumour targeting and intracytoplasmic delivery. The EPR effect as a common gateway?

Tumour capillaries are frequently hyperpermeable compared with normal vasculature, and thus they offer a much sought-after gateway for targeted delivery of cancer chemotherapy. Phase I clinical trials reported recently describe the first synthetic polymer-drug conjugate to be tested in man. N-(2-Hydroxypropyl)methacrylamide copolymer-doxorubicin (PK1, FCE 28068) displayed antitumour activity in chemotherapy-refractory patients, considerably reduced toxicity compared with doxorubicin, and evidence of tumour-selective targeting. With increasing understanding of the vector- and tumour-related factors that govern vascular permeability, non-viral vectors are being designed for tumour-selective targeting and subsequent intracytoplasmic delivery of macromolecular medicines such as genes, antisense oligonucleotides, proteins and peptides.

Journal Article↗
PubMed2006

Challenges and opportunities in dystrophin-deficient cardiomyopathy gene therapy.

The last decade has evidenced unprecedented progress in gene therapy of Duchenne and Becker muscular dystrophy (DMD and BMD) skeletal muscle disease. Cardiomyopathy is a leading cause of morbidity and mortality in both patients and carriers of DMD, BMD and X-linked dilated cardiomyopathy. However, there is little advance in heart gene therapy. The gene, the vector, vector delivery, the target tissue and animal models are five fundamental components in developing an effective gene therapy. Intensive effort has been made in optimizing gene transfer vectors and methods. Systemic and/or local delivery of recombinant adeno-associated viral vector have resulted in widespread transduction in the rodent heart. The current challenge is to define other parameters that are essential for a successful gene therapy such as the best candidate gene(s), the optimal expression level and the target tissue. This review focuses on these long-ignored aspects and points out future research directions. In particular, we need to address whether all or only some of the recently developed mini- and microgenes are protective in the heart, whether partial correction can lead to whole heart function improvement, whether over-expression is hazardous and whether correcting skeletal muscle disease can slow down or stop the progression of cardiomyopathy. Discussion is also made on whether the current mouse models can meet these research needs.

Animals↗
PubMed2003

Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodeficiency virus vaccine and localization in the central nervous system.

Replication-defective adenovirus (ADV) vectors represent a promising potential platform for the development of a vaccine for AIDS. Although this vector is typically administered intramuscularly, it would be desirable to induce mucosal immunity by delivery through alternative routes. In this study, the immune response and biodistribution of ADV vectors delivered by different routes were evaluated. ADV vectors expressing human immunodeficiency virus type 1 (HIV-1) Gag, Pol, and Env were delivered intramuscularly or intranasally into mice. Intranasal immunization induced greater HIV-specific immunoglobulin A (IgA) responses in mucosal secretions and sera than in animals with intramuscular injection, which showed stronger systemic cellular and IgG responses. Administration of the vaccine through an intranasal route failed to overcome prior ADV immunity. Animals exposed to ADV prior to vaccination displayed substantially reduced cellular and humoral immune responses to HIV antigens in both groups, though the reduction was greater in animals immunized intranasally. This inhibition was partially overcome by priming with a DNA expression vector expressing HIV-1 Gag, Pol, and Env before boosting with the viral vector. Biodistribution of recombinant adenovirus (rADV) vectors administered intranasally revealed infection of the central nervous system, specifically in the olfactory bulb, possibly via retrograde transport by olfactory neurons in the nasal epithelium, which may limit the utility of this route of delivery of ADV vector-based vaccines.

AIDS Vaccines↗
PubMed2006

ApoE gene therapy to treat hyperlipidemia and atherosclerosis.

Atherosclerosis is the leading cause of death in industrialized countries and is becoming an increasingly worldwide risk to health. Apolipoprotein E (ApoE) is a blood circulating protein with pleiotropic atheroprotective properties that has emerged as a strong candidate for treating hypercholesterolemia and cardiovascular disease. In this review, we discuss the major developments in both viral and non-viral vectors aimed at achieving efficient delivery and sustained expression of an ApoE transgene. The technological advances in engineering viruses include cross-packaging to generate different serotypes of recombinant adeno-associated virus, and the use of multiple-deleted and helper-dependent recombinant adenovirus vectors to minimize immune responses and to package genomic loci. Non-viral ApoE delivery systems, including plasmids and cell-based therapy are also described in this review. Finally, a radical alternative to gene addition that has the potential for permanent cure in many genetic diseases--'targeted gene editing'--is reviewed. This technology uses synthetic oligonucleotides to correct underlying point mutations in situ and has been evaluated for repairing dysfunctional APOE genes.

Animals↗
PubMed1998

Characterization of intrastriatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and human GTP-cyclohydrolase I in a rat model of Parkinson's disease.

To achieve local, continuous L-DOPA delivery in the striatum by gene replacement as a model for a gene therapy for Parkinson's disease, the present studies used high titer purified recombinant adeno-associated virus (rAAV) containing cDNAs encoding human tyrosine hydroxylase (hTH) or human GTP-cyclohydrolase I [GTPCHI, the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis] or both to infect the 6-OHDA denervated rat striatum. Striatal TH and GTPCHI staining was observed 3 weeks after rAAV transduction, with little detectable perturbation of the tissue. Six months after intrastriatal rAAV transduction, TH staining was present but apparently reduced compared with the 3 week survival time. In a separate group of animals, striatal TH staining was demonstrated 1 year after rAAV transduction. Double staining studies using the neuronal marker NeuN indicated that >90% of rAAV-transduced cells expressing hTH were neurons. Microdialysis experiments indicated that only those lesioned animals that received the mixture of MD-TH and MD-GTPCHI vector displayed BH4 independent in vivo L-DOPA production (mean approximately 4-7 ng/ml). Rats that received the hTH rAAV vector alone produced measurable L-DOPA (mean approximately 1-4 ng/ml) only after receiving exogenous BH4. L-Aromatic amino acid decarboxylase blockade, but not 100 mM KCl-induced depolarization, enhanced L-DOPA overflow, and animals in the non-hTH groups (GTPCHI and alkaline phosphatase) yielded minimal L-DOPA. Although elevated L-DOPA was observed in animals that received mixed hTH and hGTPCHI rAAV vectors, there was no reduction of apomorphine-induced rotational behavior 3 weeks after intrastriatal vector injection. These data demonstrate that purified rAAV, a safe and nonpathogenic viral vector, mediates long-term striatal hTH transgene expression in neurons and can be used to successfully deliver L-DOPA to the striatum.

Animals↗
PubMedDate not supplied

Recent advances in gene therapy of GI and liver diseases.

Gene therapy toward the digestive organs has made substantial progress. The strategies applied include ex vivo and in vivo delivery. The ex vivo pathway does not require a tissue-specific vector while the in vivo pathway is advantageous for tissues not obtainable or hard to culture. The specific vectors of gene delivery for the liver and gastrointestinal tract include (1) viral vectors: retrovirus, adenovirus, and adeno-associated virus are the three most common currently used. (2) liposomes: when cationic substances are incorporated, DNA can be coated on the surface and carried into cells. (3) receptor-mediated gene therapy: natural and physiological and tissue-specific ligands were incorporated and modified to carry foreign DNA into the cells. They are tissue-specific and relatively nontoxic. The innovations focus on the prolongation of gene expression and a more efficient gene delivery.

Gastrointestinal Diseases↗
PubMedDate not supplied

Designing gene delivery vectors for cardiovascular gene therapy.

Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy.

Adenoviridae↗
PubMed2005

Gene therapy for the prevention of ischemia/reperfusion injury in organ transplantation.

Introduction of gene therapy into molecular medicine has been gaining increasing interest. Although treatment of various diseases e.g. monogenetic defects or cancer by using gene transfer technologies has been extensively probed, the clinical success has been limited. However, recent experimental data suggest that gene therapy may represent an attractive and powerful approach in preventing ischemia/reperfusion injury as well as organ rejection in transplant recipients. Easy and selective access to the donor organ facilitates the reduction of potentially harmful systemic side effects of gene therapy vectors. By introducing anti-apoptotic or cytoprotective genes, these studies focused on the protection of the transplant from the apoptotic cell death. In addition, down-regulation of adhesion molecules and/or blockade of gene expression in the graft itself also ameliorated ischemia/reperfusion injury. This review summarizes the current progress on gene therapy application in combating ischemia-reperfusion injury in organ transplantation. Although the use of viral vectors is emphasized, non-viral gene transfer techniques are also discussed. Future development of novel, low-immunogenic vectors should further contribute to the minimization of ischemia/reperfusion injury, and thus to the overall success of organ transplantation.

Adenoviridae↗
PubMed1990

BSMV genome mediated expression of a foreign gene in dicot and monocot plant cells.

Barley stripe mosaic virus (BSMV) possesses a tripartite genome composed of RNAs alpha, beta and gamma that have been cloned into in vitro transcription vectors from which infectious transcripts can be obtained. The BSMV genome has been engineered here to serve as an expression vector in plant protoplasts. Open reading frame (ORF) b of RNA beta, encoding a non-structural protein, was replaced by the firefly luciferase (luc) reporter gene to yield RNA beta 2-luc. In the presence of both RNAs alpha and gamma, RNA beta 2-luc mediated efficient expression of the luc gene upon transfection into tobacco and maize protoplasts. This expression ranged from 20- to 123-fold higher than the luciferase activity obtained from transfection with a luc gene mRNA. Replication of RNA beta and its derivatives i.e. 'minus' strand synthesis, was confirmed by Northern analysis, indicating that the high level of luc gene expression using RNA beta 2-luc resulted from RNA amplification. ORFa of RNA beta, encoding the coat protein, was also replaced by the luc gene to yield RNA beta 1-luc. Although transfection of RNA beta 1-luc alone produces luciferase efficiently, neither 'minus' strand synthesis nor further increase of luciferase activity was observed in the presence of RNAs alpha and gamma, or RNAs alpha, beta and gamma, suggesting that the deleted sequences within ORFa are cis-acting for replication of RNA beta. Our results demonstrate that a foreign gene can be expressed by replacement of a viral non-structural protein gene that is essential for virus multiplication in plants, leading to a potential strategy for virus 'containment' with use of 'disarmed' plant viral vectors.

Animals↗
PubMed1997

Hepatic drug delivery and gene therapy.

On September 21-22, 1995, an international meeting entitled "Targeting of Novel Therapeutics to the Liver and GI Tract" was held at the Natcher Conference Center on the campus of the National Institutes of Health. The conference was sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases through the Division of Digestive Diseases and Nutrition and Digestive Diseases Interagency Coordinating Committee (DDICC). Section 440A of Public Law 94-562 in 1976 created the DDICC for the purpose of coordinating the digestive disease-related research activities of relevant federal health agencies into a coordinated program aimed at combating digestive diseases. As part of this federal effort, an assessment of the "state of the art" for targeted drug therapeutics to the liver and gene therapy was undertaken through the conference, cochaired by Dr. Mark Zern (Thomas Jefferson Medical College) and Dr. Flossie Wong-Staal (University of California, San Diego, CA). The conference was divided into four sessions: Session I was Vectors and Techniques; Session II was Liver and Metabolic Diseases; Session III was Hepatitis and GI Disease; and Session IV was Approaches for HIV Infection. This summary focuses on the new technologies and the studies directly pertaining to liver disease. Table 1 lists the techniques and their applications. Table 2 describes viral vectors that have been employed for the purpose of hepatic gene therapy. Table 3 summarizes the studies presented as posters at the conference.

DNA Repair↗