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At least 667 records · Page 37Linked to original sources
PubMed2004

Rescue and sprouting of motoneurons following ventral root avulsion and reimplantation combined with intraspinal adeno-associated viral vector-mediated expression of glial cell line-derived neurotrophic factor or brain-derived neurotrophic factor.

Following avulsion of a spinal ventral root, motoneurons that project through the avulsed root are axotomized. Avulsion between, for example, L2 and L6 leads to denervation of hind limb muscles. Reimplantation of an avulsed root directed to the motoneuron pool resulted in re-ingrowth of some motor axons. However, most motoneurons display retrograde atrophy and subsequently die. Two neurotrophic factors, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), promote the survival of motoneurons after injury. The long-term delivery of these neurotrophic factors to the motoneurons in the ventral horn of the spinal cord is problematic. One strategy to improve the outcome of the neurosurgical reinsertion of the ventral root following avulsion would involve gene transfer with adeno-associated viral (AAV) vectors encoding these neurotrophic factors near the denervated motoneuron pool. Here, we show that AAV-mediated overexpression of GDNF and BDNF in the spinal cord persisted for at least 16 weeks. At both 1 and 4 months post-lesion AAV-BDNF- and -GDNF-treated animals showed an increased survival of motoneurons, the effect being more prominent at 1 month. AAV vector-mediated overexpression of neurotrophins also promoted the formation of a network of motoneuron fibers in the ventral horn at the avulsed side, but motoneurons failed to extent axons into the reinserted L4 root towards the sciatic nerve nor to improve functional recovery of the hind limbs. This suggests that high levels of neurotrophic factors in the ventral horn promote sprouting, but prevent directional growth of axons of a higher number of surviving motoneurons into the implanted root.

Animals↗
PubMed2005

Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems.

To develop nonviral gene vectors that are sufficient for clinical application, it is necessary to understand why and to what extent nonviral vectors are inferior to viral vectors, which in general show a more efficient transfection activity. This study describes a systematic and quantitative comparison of the cellular uptake and subsequent intracellular distribution (e.g., endosome/lysosome, cytosol, and nucleus) of exogenous DNA transfected by viral and nonviral vectors in living cells, using a combination of TaqMan PCR and a recently developed confocal image-assisted three-dimensionally integrated quantification method. As a model, adenovirus (Ad) and Lipofectamine Plus (LFN) were used for comparison since they are highly potent and widely used viral and nonviral vectors, respectively. The findings indicate that the efficiency of cellular uptake for LFN is significantly higher than that for Ad. Once taken up by a cell, Ad exhibited comparable endosomal escape and slightly higher nuclear transfer efficiency compared with LFN. In contrast, LFN requires 3 orders of magnitude more intranuclear gene copies to exhibit a transgene expression comparable to that of the Ad, suggesting that the difference in transfection efficiency principally arises from differences in nuclear transcription efficiency and not from a difference in intracellular trafficking between Ad and LFN.

Adenoviridae↗
PubMed2003

Components of gene therapy experimentation that contribute to relative risk.

Gene therapy is the purposeful delivery of genetic material to somatic cells for the purpose of treating disease or biomedical investigation. Either viral or non-viral vector methods can be used. The risk of collateral exposure of laboratory animal care personnel to gene therapy vectors is dependent on a number of factors. These factors are intrinsic to the gene therapy vector (the vehicle for genetic conveyance), product encoded by the genetic construct delivered, method of delivery, and immune status of the recipient. The component risks of gene therapy experiments can be analyzed to surmise the overall relative risk of the experiment. Knowledge of the components that contribute potential hazardous risk to a study can assist animal care staff in identifying area(s) where prudent practices should be focused. Gene therapy experiments involving viral vectors are generally performed at either biosafety level 2 or 3. The objective of this review is to report on various components of gene therapy experiments, focusing on characteristics of viral and non-viral vectors, to assist the laboratory animal science community in determining prudent biosafety practices.

Animal Technicians↗
PubMed2003

Non-viral and hybrid vectors in human gene therapy: an update.

Non-viral DNA vectors have several advantages over viral vectors. For example, virus production is expensive and there are safety concerns regarding viral manipulations. In addition, the size of the delivered plasmid is limited by the size of the viral capsid, whereas this is not a problem with non-viral vectors. The major disadvantage of using non-viral DNA delivery vectors, compared with their viral counterparts, is the low transfection efficiency. This has resulted in low levels of usage in clinical trials. Consequently, the majority of research into non-viral gene therapy has been focused on developing more efficient vectors.

Biolistics↗
PubMed2005

Intracellular routing of plasmid DNA during non-viral gene transfer.

Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic DNA in genetic and acquired human diseases. Whereas the lack of specific immune response favors the use of plasmid-cationic polymer complexes, the limited efficacy and short duration of transgene expression impose major hurdles in the application of non-viral gene delivery techniques. Here, we review the major cellular, metabolic and physico-chemical impediments that non-viral vectors encounter before plasmid DNA enters the nucleus. Following endocytosis of DNA-polycation complexes, a large fraction of the DNA is targeted to the lysosomes. Since the cytosolic release of heterologous DNA is a prerequisite for nuclear translocation, entrapment and degradation of plasmid DNA in endo-lysosomes constitute one of the major impediments to efficient gene transfer. Plasmid DNA that escapes the endo-lysosomal compartment encounters the diffusional and metabolic barriers of the cytoplasm, reducing greatly the number of intact plasmids that reach the nucleosol. Nuclear translocation of DNA requires either the disassembly of the nuclear envelope or active nuclear transport via the nuclear pore complex. A better understanding of the cellular and molecular basis of non-viral vector trafficking from the extracellular compartment into the nucleus may provide strategies to overcome those obstacles that limit the efficiency of gene delivery.

Animals↗
PubMed2000

Complete and long-term rescue of lesioned adult motoneurons by lentiviral-mediated expression of glial cell line-derived neurotrophic factor in the facial nucleus.

To date, delivery of neurotrophic factors has only allowed to transiently protect axotomized facial motoneurons against cell death. In the present report, long-term protection of these neurons was evaluated by continuously expressing the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) within the facial nucleus using a lentiviral vector system. The viral vector was injected unilaterally into the facial nucleus of 4-month-old Balb/C mice. In contrast to axotomy in other adult rodents, facial nerve lesion in these animals leads to a progressive and sustained loss and/or atrophy of >50% of the motoneurons. This model thus represents an attractive model to evaluate potential protective effects of neurotrophic factors for adult-onset motoneuron diseases, such as amyotrophic lateral sclerosis. One month after unilateral lentiviral vector injection, the facial nerve was sectioned, and the animals were killed 3 months later. Viral delivery of the GDNF gene led to long-term expression and extensive diffusion of GDNF within the brainstem. In addition, axotomized motoneurons were completely protected against cell death, because 95% of the motoneurons were present as demonstrated by both Nissl staining and choline acetyltransferase immunoreactivity. Furthermore, GDNF prevented lesion-induced neuronal atrophy and maintained proximal motoneuron axons, despite the absence of target cell reinnervation. This is the first evidence that viral-mediated delivery of GDNF close to the motoneuron cell bodies of the facial nucleus of adult mice can lead to complete and long-term protection against lesion-induced cell death.

Age Factors↗
PubMed1997

Development and characterization of cationic liposomes conjugated with HVJ (Sendai virus): reciprocal effect of cationic lipid for in vitro and in vivo gene transfer.

Today, nonviral gene transfer vectors attract more attention as a therapeutic strategy for human diseases, because viral vectors such as adenoviral and herpes viral vectors have been proven to have problems, especially in immunogenicity and cytotoxicity. However, the main limitation of nonviral vectors has been low efficiency of gene expression. To overcome this defect, we have developed a new class of transfection vehicles, HVJ-cationic liposomes. The use of the cationic lipid DC-cholesterol facilitates efficient entrapment of negatively charged macromolecules (plasmid DNA, oligodeoxynucleotides, and proteins) and efficient interaction with negatively charged plasma membranes of cultured cells in vitro. Moreover, the fusogenic envelope proteins of hemagglutinating virus of Japan (HVJ) enhance delivery of the enclosed materials into cells. Using firefly luciferase as a marker, we optimized the liposome formula. As a result, we have succeeded in obtaining 100-800 times higher gene expression in vitro than with the conventional HVJ-anionic liposomes. However, in vivo gene transfer experiments have revealed that the use of cationic lipid instead of anionic lipid reduced the transgene expression dramatically in organs such as muscle and liver. We further discovered that the use of anionic liposomes with a viral-mimic king lipid composition increased transfection efficiency by several times in vivo. We conclude that the alternative usage of transfer vectors, for example, HVJ-anionic liposomes for in vivo delivery to extended areas of organs and HVJ-cationic liposomes for in vitro delivery (and possibly for in vivo delivery to a restricted area of organs), is of significance.

Animals↗
PubMed2004

In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium.

We have developed an efficient, versatile, and user-friendly viral engineering and expression system that is based on in planta assembly of functional viral vectors from separate pro-vector modules. With this new system, instead of supplying a plant cell with a complete viral vector as a mature viral particle, an RNA or a linear DNA molecule, we use agrobacteria to deliver various modules that are assembled inside the cell with the help of a site-specific recombinase. The resulting DNA is transcribed, and undesired elements such as recombination sites are spliced out, generating a fully functional RNA replicon. The proposed protocol allows us, by simply treating a plant with a mixture of two or more agrobacteria carrying specific prefabricated modules, to rapidly and inexpensively assemble and test multiple vector/gene combinations, without the need to perform the various engineering steps normally required with alternative protocols. The process described here is very fast (expression requires 3-4 days); it provides very high protein yield (up to 80% of total soluble protein); more than before, it is carried out using in vivo manipulations; it is based on prefabricated genetic modules that can be developed/upgraded independently; and it is inherently scalable.

DNA, Bacterial↗
PubMed2001

Sindbis viral-mediated expression of Ca2+-permeable AMPA receptors at hippocampal CA1 synapses and induction of NMDA receptor-independent long-term potentiation.

Gene manipulation in order to artificially express a particular gene in neurons in the central nervous system is a powerful tool for the analysis of brain function. Sindbis viral vectors have been developed to express high levels of foreign genes in postmitotic brain neurons with little transfection of glial cells. In this study, we expressed the gene encoding the unedited GluR2 (GluR-B) subunit of the AMPA-type glutamate receptor that forms inwardly rectifying and Ca2+-permeable channels, in rat CA1 hippocampal neurons in slice cultures using Sindbis viral vectors. The pyramidal cell layer of the CA1 region was injected with recombinant Sindbis viruses encoding both enhanced green fluorescent protein (GFP) and unedited GluR2. The GFP fluorescence from CA1 neurons could be detected as early as 6 h and reached a maximal level about 48 h postinfection. The inwardly rectifying and Ca2+-permeable AMPA receptors were expressed in most CA1 pyramidal cells expressing GFP. These AMPA receptors expressed by gene transfer were involved in fast excitatory neurotransmission elicited by electrical stimulation of the Schaffer collaterals in the stratum radiatum. Tetanic stimulation of Schaffer collaterals induced NMDA receptor-independent, long-term potentiation due to Ca2+ influx through the newly expressed AMPA receptors in the area densely stained with GFP. Thus, the combined use of Sindbis viral vectors with the GFP reporter allowed physiological examination of the roles of a specific gene product in synaptic function in well-characterized brain neurons.

Animals↗
PubMedDate not supplied

Screening of novel excipients for improving the stability of retroviral and adenoviral vectors.

In the past decade there has been an increase in the application of viral vectors in the laboratory and clinical trials of human gene therapy, retroviral and adenoviral vectors among the most used. However, the limited stability of these vectors creates problems in the design of experiments, transport, and storage. Vectors stored at -80 degrees C must be quickly shipped on dry ice, which is somewhat cumbersome. Alternatively, viral vectors can be preserved in a lyophilized form. However, loss of viral activity during lyophilization can also be a serious problem. In this report we identify novel candidate formulations containing new compatible solutes, ectoin, hydroxyectoin, and firoin, that allow better stability of retroviral and adenoviral vectors during storage. For retroviral vectors, the maximum stabilization for long-term storage was achieved through lyophilization followed by storage at -20 degrees C using a formulation of Tris buffer pH 7.2 containing firoin (0.5 M), a half-life of 340 days being obtained. Adenoviral vectors storage at -80 degrees C in solution using Tris buffer pH 8.0 with firoin was the best method for long-term storage, with a half-life exceeding 1 year.

Adenoviridae↗
PubMed2000

Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer: a phase I/II clinical trial.

INTRODUCTION AND OBJECTIVES: Animal studies have indicated that the use of syngeneic dendritic cells that have been transfected ex vivo with DNA for tumor-specific antigen results in tumor regression and decreased number of metastases. Additional studies have also suggested the possibility to modulate the dendritic cells in vivo either by 'naked' DNA immunization or by injecting replication-deficient viral vectors that carry the tumor-specific DNA. Using the prostate- specific membrane antigen (PSMA) as a target molecule, we have initiated a clinical trial for immunotherapy of prostate cancer. The primary objective of the study was to determine the safety of the PSMA vaccine after repeated intradermal injections. METHODS: We have included the extracellular human PSMA DNA as well as the human CD86 DNA into separate expression vectors (PSMA and CD86 plasmids), and into a combined PSMA/CD86 plasmid. In addition, the expression cassette from the PSMA plasmid was inserted into a replication deficient adenoviral expression vector. Twenty-six patients with prostate cancer were entered into a phase I/II toxicity-dose escalation study, which was initiated in spring 1998. Immunizations were performed intradermally at weekly intervals. Doses of DNA between 100 and 800 microg and of recombinant virus at 5x10(8) PFUs per application were used. RESULTS AND CONCLUSION: No immediate or long-term side effects following immunizations have been recorded. All patients who received initial inoculation with the viral vector followed by PSMA plasmid boosts showed signs of immunization as evidenced by the development of a delayed-type hypersensitivity reaction after the PSMA plasmid injection. In contrast, of the patients who received a PSMA plasmid and CD86 plasmid, only 50% showed signs of successful immunization. Of the patients who received PSMA plasmid and soluble GM-CSF, 67% were immunized. However, all patients who received the PSMA/CD86 plasmid and sGM-CSF became immunized. The patients who did not immunize during the first round were later successfully immunized after a boost with the viral vector. The heterogeneity of the medical status and the presence in many patients of concomitant hormone therapy does not permit unequivocal interpretation of the data with respect to the effectiveness of the therapy. However, several responders, as evidenced by a change in the local disease, distant metastases, and PSA levels, can be identified. A phase II clinical study to evaluate the effectiveness of the therapy is currently underway.

Adenoviridae↗
PubMed1998

[Cationic liposomes in gene delivery].

Cationic liposomes have been extensively explored as gene delivery vector for several reasons. It is because disadvantages of viral vectors include risk of replication, possible immunogenicity, and the difficulty of obtaining a large quantity of viral vectors. Currently, a variety of cationic components for liposome formulations have been developed. The components are broadly divided into two classes based on the chemical structure of hydrophobic moieties: long aliphatic (saturated or unsaturated) hydrocarbons and cholesterol ring. A variety of hydrophilic moieties also include tertiary amines, ammonium salt and spermine. The role of liposomes is to condense DNA to form complexes with high affinity to cell surfaces where possible fusion or destabilization of the membrane and/or endocytosis are involved. However, at present, little structure-activity relationships are known. Some vectors are on clinical trials approved by NIH.

Cations↗
PubMed2004

Oral administration of a mite allergen expressed by zucchini yellow mosaic virus in cucurbit species downregulates allergen-induced airway inflammation and IgE synthesis.

BACKGROUND: Sublingual-swallow immunotherapy in house dust mite-related asthma has a good safety profile and improves respiratory function and bronchial hyperreactivity. Zucchini yellow mosaic virus (ZYMV) is envisaged as a promising viral vector for expressing large quantity of foreign proteins in cucurbit species. OBJECTIVE: We sought to investigate whether oral feeding of dust mite allergen expressed by ZYMV in a cucurbit species can suppress allergen-induced inflammation and IgE synthesis. METHODS: An infectious plant virus clone, p35SZYMV2-26, that contains the full-length cDNA to the genomic RNA of a Taiwan isolate of ZYMV, driven by the cauliflower mosaic virus 35S promoter, was engineered as an in vivo viral vector to express Dermatophagoides pteronyssinus group 5 allergen (Der p 5) in cucurbit species. Female BALB/c mice were intraperitoneally sensitized with Escherichia coli bacteria-expressed Der p 5 and orally treated with the virus-expressed Der p 5 (vDer p 5) extracted from the recombinant virus-infected squash plants. Der p 5-specific immunoglobulins were measured by ELISA, and bronchoalveolar lavage assays were used to measure airway inflammation. RESULTS: Infectivity assays and immunoblotting revealed that large quantities of free-form vDer p 5 are produced in the recombinant virus-infected squash plants. The recombinant virus carried and expressed the Der p 5 allergen in squash plants for at least 1 year after numerous passages. In animal tests, squash extract containing vDer p 5 inhibited Der p 5-specific IgE synthesis and airway inflammation. CONCLUSION: Our results suggest that oral feeding with allergen produced by the plant viral vector provides a novel approach for the therapy of allergic asthma.

Administration, Oral↗
PubMed2003

Single HSV-amplicon vector mediates drug-induced gene expression via dimerizer system.

A variety of viral vectors have been used to deliver genes into various tissues. Most have typically relied on either viral or cell-specific mammalian promoters to express transgenes. More recently, regulated promoter systems have been developed to fine-tune gene expression. Due to limited transgene capacity in most viral vectors, regulatory elements are typically subcloned into two separate vectors, which must be delivered simultaneously to a target cell. Here, we have cloned all the components of the rapamycin-based "dimerizer" system into the pantropic HSV-amplicon vector and used it to deliver and regulate red fluorescent protein (RFP) expression in cultured cells in a drug-dose-dependent manner. 293T/17 cells infected at an m.o.i. of 1 transducing unit/cell and induced with 20 nM rapamycin resulted in a 25-fold increase in RFP mRNA levels after 24 h as assessed by quantitative RT-PCR. However, due to a reduced ability to detect RFP optically, only a 5-fold induction in the number of RFP-expressing cells was noted by FACS analysis 48 h after infection. Further, there was at least 100-fold variation in the levels of RFP in individual, infected cells in the induced state. Gene induction in several neuronal models, including primary cell culture and organotypic cultures, as well as in rodent brain, was observed.

Animals↗
PubMed2006

A heterologous system for assembly of retroviral gene vectors: intracellular budding in yeast?

Mammalian retroviral gene vectors are of particular importance in gene therapy because of their efficient chromosomal integration and resulting stable maintenance of transgenes. These vectors are currently produced in mammalian cells, with low yield and at substantial expense. Therefore, a more efficient heterologous production system for retroviral vectors would be desirable. With their impressive track record in biotechnology, various yeast species appear like ideal organisms to generate retroviral vectors. Typically, retroviral vector particles emerge from mammalian cells after budding at the plasma membrane. However, in yeast, viral budding at the plasma membrane is blocked by the cell wall. At the same time, mass production of enveloped viral vectors in yeast protoplasts is technically challenging. Recent reports indicated the generation of infectious virions via intracellular budding for some combinations of retroviruses and mammalian cells. Relying on these data I hypothesise that a successful assembly of the retroviral transducing particles can be accomplished intracellularly inside yeast cells with a normal cell wall. Firstly, it is possible that some of the intracellular yeast compartments have all the necessary host factors that are required for successful RNA packaging, budding and maturation of infectious retroviral vector particles. Secondly, it might be possible to improve intracellular viral vector production by artificially targeting viral cores to bud at specific intracellular vesicular structures using appropriate targeting or retention signals. A suitable envelope protein, conferring infectivity and specific cellular tropism to the vector particles, can be expressed in yeast or, alternatively, 'bald' viral particles without envelope protein can be produced in yeast and later complexed with a desired envelope protein in vitro. Retroviral budding on yeast intracellular membranes can rely on the same host factors that are used by yeast retrotransposons driving intracellular formation of non-infectious virus-like particles. It is likely that optimal vector packaging system can be found as a result of the dedicated screening of the various retroviral vectors against an array of yeast species. The central implication of the hypothesis is that budding of retroviral vector particles inside yeast cells, with no need for obtaining and maintaining yeast spheroplasts or protoplasts, can be an efficient and economical method of mass production of these valuable gene therapy vectors.

Biotechnology↗
PubMed2004

Integration and long-term expression in xenografted human glioblastoma cells using a plasmid-based transposon system.

Gene therapy has the potential to become an effective component of cancer treatment by transferring genes that cause immunomodulation or tumor cell death or that inhibit angiogenesis into tumor cells or tumor-associated stroma. Viral vectors have been the primary gene transfer vehicles used for intratumoral gene transfer to date. Plasmid-based vectors may be safer and more scalable than viral vectors. However, attempts at plasmid-based intratumoral gene transfer have been met with transient expression and poor gene transfer efficiency. Here we report integration and long-term expression of reporter genes in human glial tumors, growing in nude mice, using the Sleeping Beauty (SB) transposon system. A two-plasmid system was used, in which linear polyethylenimine was complexed with a GFP, NEO, or luciferase transposon plasmid and a SB transposase-expressing plasmid. SB-mediated transposition led to chromosomal integration of the NEO transgene in roughly 8% of tumor cells. SB-mediated insertions were cloned from the genomes of glial tumor cells to provide molecular proof of transposase-mediated integration. Luciferase studies showed that SB facilitated long-term expression of the transgene in glial tumors. SB-mediated intratumoral gene transfer is a novel, nonviral technique that could be used to augment conventional therapy for glioblastoma or other cancers.

Animals↗
PubMed2001

Cell-specific delivery of genes with glycosylated carriers.

Cationic liposomes and polymers have been accepted as effective non-viral vectors for gene delivery with low immunogenicity unlike viral vectors. However, the lack of organ or cell specificity sometimes hampers their application and the development of a cell-specific targeting technology for them attracts great interest in gene therapy. In this review, the potential of cell-specific delivery of genes with glycosylated liposomes or polymers is discussed. Galactosylated liposomes and poly(amino acids) are selectively taken up by the asialoglycoprotein receptor-positive liver parenchymal cells in vitro and in vivo after intravenous injection. DNA-galactosylated cationic liposome complexes show higher DNA uptake and gene expression in the liver parenchymal cells in vitro than DNA complexes with bare cationic liposomes. In the in vitro gene transfer experiment, galactosylated liposome complexes are more efficient than DNA-galactosylated poly(amino acids) complexes but they have some difficulties in their biodistribution control. On the other hand, introduction of mannose residues to carriers resulted in specific delivery of genes to non-parenchymal liver cells. These results suggest advantages of these glycosylated carriers in cell-specific targeted delivery of genes.

Amino Acid Sequence↗
PubMed1998

Muscle maturation: implications for gene therapy.

Skeletal muscle is a promising target tissue for gene therapy, for both muscle and non-muscle disorders. A variety of methods have been studied to transfer genes into skeletal muscle, including retroviral, adenoviral and herpes simplex viral vectors. However, various factors impede muscle-based viral gene therapy. Here, we discuss why some viral vectors cannot efficiently transduce mature muscle fibers, and describe some new approaches to overcome this barrier.

Animals↗