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Viral Vectors (viral + vector)
Selected AbstractsEffect of Protease Inhibitors on Yield of HSV-1-Based Viral VectorsBIOTECHNOLOGY PROGRESS, Issue 3 2000James B. Wechuck The ability to obtain high titer replication-defective herpes simplex virus (HSV) recombinant vectors will dramatically affect their use in gene therapy clinical trials. A variety of techniques and reagents have been employed to increase the overall yield of the vector. The effects of protease inhibitors on the yield of an HSV-1-based viral vector were examined. Experiments were conducted using a commercial protease inhibitor cocktail typically used in mammalian cell culture for protein production. Contrary to our expectation for enhanced vector yield, the results showed a dramatic reduction in vector yield. Moreover, it was found that AEBSF is the only component in the protease cocktail responsible for the low vector yield. On the basis of our hypothesis regarding the mode of action of AEBSF, we suggest that it should not be included in protease inhibitor cocktails designed for use in cultures aimed at production of viral vectors derived from HSV-1 or possibly several other vectors. [source] Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivoEXPERIMENTAL PHYSIOLOGY, Issue 1 2005Z. Shevtsova The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research. [source] Viral vectors carrying NR1 sequences injected into rat hippocampus interfered with learning and memoryJOURNAL OF NEUROCHEMISTRY, Issue 2002V. Cheli NMDA receptors are relevant to learning and memory as has been shown both by pharmacological and genetic manipulations. Gene knockouts are useful for investigating in vivo functions, but genetic deletions unrestricted in time or region, may lead to developmental defects or death. The challenge is to control expression with temporal and spatial restrictions in the brain. Viral vectors derived from herpes type-1 neurotropic virus are interesting candidates for it. To regulate gene expression of the NMDA receptor NR1 subunit, vectors carrying either sense NR1(+) or antisense NR1(,) sequences and that of the green fluorescent protein (GFP), were constructed. The protein or RNA expression were corroborated in cell culture by GFP autofluorescence, Western blots, immunofluorescence and RT-PCR, and in rat brain, by Western blots and GFP autofluorescence. The vectors were injected into the dorsal hippocampus of adult male Wistar rats. After 6 days each rat was trained and evaluated for both habituation to an open field and inhibitory avoidance to a foot-shock. The rats injected with GFP-NR1(+) vectors showed habituation and learned the inhibitory avoidance, like sham operated rats; while animals injected with GFP-NR1(,) vectors did not. The vectors were useful to modify endogenous gene expression at a defined period, in restricted regions, leading to investigate in vivo functions. NR1 subunit in the hippocampus is involved in mechanisms leading to habituation and to avoidance behaviour, since even a slight change in the availability of NR1 interfered with them. [source] A comparative analysis of constitutive and cell-specific promoters in the adult mouse hippocampus using lentivirus vector-mediated gene transferTHE JOURNAL OF GENE MEDICINE, Issue 11 2008Hitoshi Kuroda Abstract Background Viral vectors provide powerful tools for transgene delivery to the mammalian brain to assess the effects of therapeutic proteins, antisense RNAs or small interfering RNAs. A key advantage of such approaches is that specific brain regions implicated in a particular disease can be independently targeted. Methods To optimize transgene expression in sub-regions of the mouse hippocampus and with a view towards devising gene therapy strategies for Alzheimer's disease, we designed lentivirus-based reporter vectors bearing various promoters, including constitutive and cell-specific promoters. Furthermore, we devised methods allowing a side-by-side comparison of transgene expression levels in neural cells both in vitro and in vivo. Results Following stereotaxic injection into the adult mouse hippocampus, titer-adjusted lentiviral vectors bearing constitutive promoters resulted in robust and sub-region-specific transgene expression. Our results show that the human CMV-IE promoter resulted in efficient transgene expression in the entire hippocampus whereas transgene expression mediated by the hybrid hEF1,/HTLV promoter was limited mainly in the dentate gyrus and the CA2/3 region. Finally, the neuron-specific human synapsin I promoter was particularly effective in the dentate gyrus. Conclusions These findings indicate that subregion-specific transgene expression in the hippocampus can be achieved following lentivirus vector-mediated gene transfer. Copyright © 2008 John Wiley & Sons, Ltd. [source] Viral vectors as tools to model and treat neurodegenerative disordersTHE JOURNAL OF GENE MEDICINE, Issue 5 2005N. Déglon Abstract The identification of disease-causing genes in familial forms of neurodegenerative disorders and the development of genetic models closely replicating human central nervous system (CNS) pathologies have drastically changed our understanding of the molecular events leading to neuronal cell death. If these achievements open new opportunities of therapeutic interventions, including gene-based therapies, the presence of the blood-brain barrier and the post-mitotic and poor regenerative nature of the target cells constitute important challenges. Efficient delivery systems taking into account the specificity of the CNS are required to administer potential therapeutic candidates. In addition, genetic models in large animals that replicate the late stages of the diseases are in most cases not available for pre-clinical studies. The present review summarizes the potential of viral vectors as tools to create new genetic models of CNS disorders in various species including primates and the recent progress toward viral gene therapy clinical trials for the administration of therapeutic candidates into the brain. Copyright © 2005 John Wiley & Sons, Ltd. [source] Gene therapy clinical trials worldwide 1989,2004,an overviewTHE JOURNAL OF GENE MEDICINE, Issue 6 2004Michael L. Edelstein Abstract In 1989, Rosenberg et al. performed the first human gene therapy trial when they used a retrovirus to introduce the gene coding for resistance to neomycin into human tumor-infiltrating lymphocytes before infusing them into five patients with advanced melanoma. This study demonstrated the feasibility of using retroviral gene transduction in humans and set the stage for further studies. Since then, over 900 clinical trials have been completed, are ongoing or have been approved worldwide. These trials have been designed to establish feasibility and safety, to demonstrate the reality of expression of therapeutic protein(s) in vivo by the genes transferred and, in some cases, to show therapeutic benefit. There is no single source of information that presents an overview of all the clinical trials undertaken worldwide. In 1997 we set up a database to bring all the information on clinical trials together as comprehensively and as globally as possible. The data were compiled and are regularly updated from official agency sources, the published literature, presentations at conferences and from information kindly provided by investigators or trial sponsors themselves. As of January 31, 2004, we have identified 918 trials in 24 countries. The USA accounts for two-thirds of these trials. Cancer is by far the most common disease indication, followed by inherited monogenic diseases, and cardiovascular diseases. Viral vectors have been the most frequently used vehicles for transferring genes into human cells, with retroviruses and adenoviruses representing the vast majority. Plasmid (naked) DNA and other non-viral vectors have been used in one-quarter of the trials. Over 100 distinct genes have been transferred. This article aims to provide a descriptive overview of the clinical trials that, to the best of our knowledge, have been or are being performed worldwide. Details of the data presented, including an interactive, searchable database that currently holds information on 918 trials, can be found on The Journal of Gene Medicine clinical trials website 1. Copyright © 2004 John Wiley & Sons, Ltd. [source] Insulin-like growth factor-1 and neurotrophin-3 gene therapy prevents motor decline in an X-linked adrenoleukodystrophy mouse model,ANNALS OF NEUROLOGY, Issue 1 2009Roberto Mastroeni MSc X-linked adrenoleukodystrophy (X-ALD) is the most common inherited peroxisomal disorder characterized by a progressive demyelination of the central nervous system. The marked loss of myelin and oligodendrocytes observed in the disease prompted us to evaluate the therapeutic potential of insulin-like growth factor-1 and neurotrophin-3, two potent inducers of myelin formation and oligodendrocyte survival. Viral vectors engineered to produce insulin-like growth factor-1 or neurotrophin-3 were administrated into the cerebrospinal fluid of an X-linked adrenoleukodystrophy mouse model. We show that viral-based, long-lasting delivery of insulin-like growth factor-1 and neurotrophin-3 significantly halts the progression of the disease and leads to potent protective effect against the demyelination process. Ann Neurol 2009;66:117,122 [source] NG2 proteoglycan-expressing cells of the adult rat brain: Possible involvement in the formation of glial scar astrocytes following stab woundGLIA, Issue 3 2005G. Alonso Abstract Stab wound lesion to the adult central nervous system induces strong proliferative response that is followed by the formation of a dense astroglial scar. In order to determine the origin of those astrocytes composing the glial scar, the cell proliferation marker bromodeoxyuridine (BrdU) was administered to lesioned rats that were fixed 3 h or 6 days later. At 3 h after the BrdU administration, labeled nuclei were frequently associated with either NG2+ cells or microglia/macrophages, but rarely with astrocytes expressing glial fibrillary acidic protein (GFAP). Six days later, by contrast, numerous BrdU-labeled nuclei were associated with astrocytes located along the lesion borders. After the injection of a viral vector of the green fluorescent protein (GFP) into the lesional cavity, GFP was preferentially detected within NG2- or GFAP-labeled cells when lesioned animals were fixed 1 or 6 days after the injections, respectively. The combined detection of glial markers within cells present in the lesioned area indicated that, although they rarely express GFAP, the marker of mature astrocytes, NG2+ cells located along the lesion borders frequently express nestin and vimentin, i.e., two markers of immature astrocytes. Lastly, chronic treatment of lesioned rats with dexamethasone was found to inhibit the proliferation of NG2+ cells present within the lesioned area and to subsequently alter the formation of a dense astroglial scar. Taken together, these data strongly suggest that following a surgical lesion, at least a portion of the astrocytes that constitute the glial scar are issued from resident NG2+ cells. © 2004 Wiley-Liss, Inc. [source] An In Vivo Model to Study Osteogenic Gene Regulation: Targeting an Avian Retroviral Receptor (TVA) to Bone With the Bone Sialoprotein (BSP) Promoter,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2005Ling Li Abstract To study bone development in vivo, a transgenic mouse model was established in which an avian retroviral receptor (TVA) gene driven by the BSP promoter was selectively expressed in skeletal tissues. The model was validated by showing suppressed BSP expression and delayed bone and tooth formation after infection with a virus expressing a mutated Cbfa1/Runx2 gene. Introduction: Tissue-specific expression of the avian retroviral (TVA) receptor can be used to efficiently target ectopic expression of genes in vivo. To determine the use of this approach for studies of osteogenic differentiation and bone formation at specific developmental stages, transgenic mice expressing the TVA receptor under the control of a 5-kb bone sialoprotein (BSP) promoter were generated. The mice were first analyzed for tissue-specific expression of the TVA gene and then, after infection with a viral construct, for the effects of a dominant-negative form of the Cbfa1/Runx2 transcription factor on bone formation. Materials and Methods: We first generated transgenic mice (BSP/TVA) in which the TVA gene was expressed under the control of a 4.9-kb mouse BSP promoter. The tissue-specific expression of the TVA gene was analyzed by RT-PCR, in situ hybridization, and immunohistochemistry and compared with the expression of the endogenous BSP gene. A 396-bp fragment of mutated Cbfa1/Runx2 (Cbfa1mu) encoding the DNA-binding domain was cloned into a RCASBP (A) viral vector, which was used to infect neonatal BSP/TVA mice. Results and Conclusion: Expression of the TVA receptor mRNA and protein in the transgenic mice was consistent with the expression of endogenous BSP. Four days after systemic infection with the Cbfa1mu-RCASBP (A) vector, RT-PCR analyses revealed that the expression of BSP mRNA in tibia and mandibles was virtually abolished, whereas a 30% reduction was seen in calvarial bone. After 9 days, BSP expression in the tibia and mandible was reduced by 45% in comparison with control animals infected with an empty RCASBP vector, whereas BSP expression in the membranous bone of calvariae was decreased ,15%. However, after 4 and 8 weeks, there was almost no change in BSP expression in any of the bone tissues. In comparison, a reduction in osteopontin expression was only observed 9 days after viral transfection in the three bones. Histomorphological examination revealed that bone formation and tooth development were delayed in some of the mice infected with mutated Cbfa1. These studies show that BSP/TVA transgenic mice can be used to target genes to sites of osteogenesis, providing a unique system for studying molecular events associated with bone formation in vivo. [source] A novel immunotherapy for superficial bladder cancer by intravesical immobilization of GM-CSFJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6b 2010Zhiming Hu Abstract In situ gene therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF) was demonstrated to successfully inhibit tumour cell growth in a mouse orthotopic bladder cancer model, but suffered from several disadvantages, such as limited efficiency for gene delivery, low expression efficiency of the transgene and the safety concern resulting from viral vector. In order to address the limits, a novel immunotherapy was developed attentively through immobilization of streptavidin-tagged bioactive GM-CSF on the biotinylated mucosal surface of bladder wall on the basis of both the unique property of streptavidin (SA) to bind rapidly and almost irreversibly to any biotin-linked molecule and the outstanding ability of biotin to be incorporated easily into the proteins on the cell surface. The mouse orthotopic model of MB49 bladder cancer was used to evaluate the feasibility and efficacy of the novel immunotherapy performed twice a week for 3 weeks. Briefly, 1 day after intravesical implantation of 1 × 106 MB49 tumour cells in C57BL/6 mouse, 100 ,l of 1 mg/ml NHS-PEO4-biotin was instilled and allowed to incubate in the bladder for 30 min., followed by intravesical instillation of 100 ,l of 0.15 mg/ml SA-GM-CSF bifunctional fusion protein and incubation for 1 hr. SA-GM-CSF fusion protein was shown to be immobilized efficiently and durably on the biotinylated mucosal surface of bladder wall. The bladder cancer incidence was dramatically decreased from 100% in the control group to 37.5% in the SA-GM-CSF group. Importantly, 70% of the SA-GM-CSF-cured mice were protected against a second intravesical wild-type MB49 tumour challenge, indicating that an effective anti-tumour immunity was generated against MB49 bladder cancer. Thus, the novel immunotherapy may be an attractive therapeutic alternative and should be evaluated in bladder cancer patients. [source] Effect of cell-based VEGF gene therapy on healing of a segmental bone defectJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2009Ru Li Fracture healing requires coordinated coupling between osteogenesis and angiogenesis in which vascular endothelial growth factor (VEGF) plays a key role. We hypothesized that targeted over-expression of angiogenic and osteogenic factors within the fracture would promote bone healing by inducing development of new blood vessels and stimulating/affecting proliferation, survival, and activity of skeletal cells. Using a cell-based method of gene transfer, without viral vector, 5.0,×,106 fibroblasts transfected with VEGF were delivered to a 10-mm bone defect in rabbit tibiae (Group 1) (n,=,9); control groups were treated with fibroblasts (Group 2) (n,=,7), or saline (Group 3) (n,=,7) only. After 12 weeks, eight tibial fractures healed in Group 1, compared to four each in Groups 2 and 3. In Group 1, ossification was seen across the entire defect; in Groups 2 and 3, the defects were fibrous and sparsely ossified. Group 1 had more positively stained (CD31) vessels than Groups 2 and 3. MicroCT 3-D showed complete bridging of the new bone for Group 1, but incomplete healing for Groups 2 and 3. MicroCT bone structural parameters showed significant differences between VEGF treatment and control groups (p,<,0.05). These results indicate that the cell-based VEGF gene therapy has significant angiogenic and osteogenic effects to enhance healing of a segmental defect in the long bone of rabbits. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:8,14, 2009 [source] Targeting murine small bowel and colon through selective superior mesenteric artery injectionMICROSURGERY, Issue 6 2010Stacy L. Porvasnik M.S. Administration of molecular, pharmacologic, or cellular constructs to the intestinal epithelium is limited by luminal surface mucosal barriers and ineffective intestinal delivery via systemic injection. Many murine models of intestinal disease are used in laboratory investigation today and would benefit specific modulation of the intestinal epithelium. Our aim was to determine the feasibility of a modified microsurgical approach to inject the superior mesenteric artery (SMA) and access the intestinal epithelium. We report the detailed techniques for selective injection of the SMA in a mouse. Mice were injected with methylene blue dye to grossly assess vascular distribution, fluorescent microspheres to assess biodistribution and viral vector to determine biological applicability. The procedure yielded good recovery with minimal morbidity. Tissue analysis revealed good uptake in the small intestine and colon. Biodistribution analysis demonstrated some escape from the intestine with accumulation mainly in the liver. This microsurgical procedure provides an effective and efficient method for delivery of agents to the small intestine and colon, including biological agents. © 2010 Wiley-Liss, Inc. Microsurgery 30:487,493, 2010. [source] Conferring cadmium resistance to mature tobacco plants through metal-adsorbing particles of tomato mosaic virus vectorPLANT BIOTECHNOLOGY JOURNAL, Issue 3 2006Yoshinori Shingu Summary Tomato mosaic virus vectors were designed that produced, by a translational readthrough, a fusion protein consisting of coat protein and metal-binding peptide, as a result of which particles were expected to present the metal-binding peptides on their surface. When inoculated in plants, they were expected to replicate and form a metal-adsorbing artificial sink in the cytoplasm, so as to reduce metal toxicity. Vectors were constructed harbouring sequences encoding various lengths of polyhistidine as a metal-binding peptide. One of the vectors, TLRT6His, which contains a 6 × histidine sequence, moved systemically in tobacco plants, and its particles were shown to retain cadmium ions by an in vitro assay. When a toxic amount of cadmium was applied, the toxic effect was much reduced in TLRT6His-inoculated tobacco plants, probably as a result of cadmium adsorption by TLRT6His particles in the cytosol. This shows the possible use of an artificial sink for metal tolerance and the advantage of employing a plant viral vector for phytoremediation. [source] Retrograde adenoviral vector targeting of nociresponsive pontospinal noradrenergic neurons in the rat in vivoTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2009Patrick W. Howorth Abstract The spinal dorsal horn receives a dense innervation of noradrenaline-containing fibers that originate from pontine neurons in the A5, locus coeruleus (LC), and A7 cell groups. These pontospinal neurons are believed to constitute a component of the endogenous analgesic system. We used an adenoviral vector with a catecholaminergic-selective promoter (AVV-PRS) to retrogradely label the noradrenergic neurons projecting to the lumbar (L4,L5) dorsal horn with enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein (mRFP). Retrogradely labeled neurons (145 ± 12, n = 14) were found in A5-12%, LC-80% and A7-8% after injection of AVV-PRS-EGFP to the dorsal horn of L4,L5. These neurons were immunopositive for dopamine ,-hydroxylase, indicating that they were catecholaminergic. Retrograde labeling was optimal 7 days after injection, persisted for over 4 weeks, and was dependent on viral vector titer. The spinal topography of the noradrenergic projection was examined using EGFP- and mRFP-expressing adenoviral vectors. Pontospinal neurons provide bilateral innervation of the cord and there was little overlap in the distribution of neurons projecting to the cervical and lumbar regions. The axonal arbor of the pontospinal neurons was visualized with GFP immunocytochemistry to show projections to the inferior olive, cerebellum, thalamus, and cortex but not to the hippocampus or caudate putamen. Formalin testing evoked c-fos expression in these pontospinal neurons, suggesting that they were nociresponsive (A5-21%, LC-16%, and A7-26%, n = 8). Thus, we have developed a viral vector-based strategy to selectively, retrogradely target the pontospinal noradrenergic neurons that are likely to be involved in the descending control of nociception. J. Comp. Neurol. 512:141,157, 2009. © 2008 Wiley-Liss, Inc. [source] Combining angiogenic gene and stem cell therapies for myocardial infarctionTHE JOURNAL OF GENE MEDICINE, Issue 9 2009Jennifer Pons Abstract Background Transplantation of stem cells from various sources into infarcted hearts has the potential to promote myocardial regeneration. However, the regenerative capacity is limited partly as a result of the low survival rate of the transplanted cells in the ischemic myocardium. In the present study, we tested the hypothesis that combining cell and angiogenic gene therapies would provide additive therapeutic effects via co-injection of bone marrow-derived mesenchymal stem cells (MSCs) with an adeno-associated viral vector (AAV), MLCVEGF, which expresses vascular endothelial growth factor (VEGF) in a cardiac-specific and hypoxia-inducible manner. Methods MSCs isolated from transgenic mice expressing green fluorescent protein and MLCVEGF packaged in AAV serotype 1 capsid were injected into mouse hearts at the border of ischemic area, immediately after occlusion of the left anterior descending coronary, individually or together. Engrafted cells were detected and quantified by real-time polymerase chain reaction and immunostaining. Angiogenesis and infarct size were analyzed on histological and immunohistochemical stained sections. Cardiac function was analyzed by echocardiography. Results We found that co-injection of AAV1-MLCVEGF with MSCs reduced cell loss. Although injection of MSCs and AAV1-MLCVEGF individually improved cardiac function and reduced infarct size, co-injection of MSC and AAV1-MLCVEGF resulted in the best improvement in cardiac function as well as the smallest infarct among all groups. Moreover, injection of AAV1-MLCVEGF induced neovasculatures. Nonetheless, injection of MSCs attracted endogenous stem cell homing and increased scar thickness. Conclusions Co-injection of MLCVEGF and MSCs in ischemic hearts can result in better cardiac function and MSC survival, compared to their individual injections, as a result of the additive effects of each therapy. Copyright © 2009 John Wiley & Sons, Ltd. [source] Restricted transgene persistence after lentiviral vector-mediated fetal gene transfer in the pregnant rabbit modelTHE JOURNAL OF GENE MEDICINE, Issue 9 2008Rafael Moreno Abstract Background Prenatal gene transfer may enable early causal intervention for the treatment or prevention of many devastating diseases. Nevertheless, permanent correction of most inherited disorders requires a sustained level of expression from the therapeutic transgene, which could theoretically be achieved with integrating vectors. Methods Rabbit fetuses received 8.5 × 106 HIV-based recombinant lentivirus particles containing the enhanced green fluorescent protein (EGFP) transgene by intrahepatic, intra-amniotic or intraperitoneal injection at 22 days of gestation. Provirus presence and transgene expression in rabbit tissues were evaluated at both 1.5 and 16 weeks post- in utero intervention by polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively. Moreover, we assessed persistence of EGFP by immunohistochemistry. Enzyme-linked immunosorbent assays confirmed the development of antibodies specific against both the viral vector and the reporter protein. Results Regardless of the route of administration employed, lentiviral vector-based in utero gene transfer was safe and reached 85% of the intervened fetuses at birth. However, the integrated provirus frequency was significantly reduced to 50% of that in young rabbits at 16 weeks post-treatment. In these animals, EGFP expression was evident in many tissues, including cytokeratin 5-rich basal cells from stratified and pseudostratified epithelia, suggesting that the lentiviral vector might have reached progenitor cells. Conversely, we identified the presence of immune-inflammatory infiltrates in several EGFP-expressing tissues. Moreover, almost 70% of the lentiviral vector-treated rabbits elicited a humoral immune response against the viral envelope and/or the EGFP. Conclusions At two-thirds gestational age, the adaptive immune system of the rabbit appears a relevant factor limiting transgene persistence and expression following lentiviral vector-mediated in utero gene transfer. Copyright © 2008 John Wiley & Sons, Ltd. [source] An inventory of shedding data from clinical gene therapy trialsTHE JOURNAL OF GENE MEDICINE, Issue 10 2007Ellen A. M. Schenk-Braat Abstract Viruses are the most commonly used vectors for clinical gene therapy. The risk of dissemination of a viral vector into the environment via excreta from the treated patient, a phenomenon called shedding, is a major safety concern for the environment. Despite the significant number of clinical gene therapy trials that have been conducted worldwide, there is currently no overview of actual shedding data available. In this article, an inventory of shedding data obtained from a total of 100 publications on clinical gene therapy trials using retroviral, adenoviral, adeno-associated viral and pox viral vectors is presented. In addition, the experimental set-up for shedding analysis including the assays used and biological materials tested is summarized. The collected data based on the analysis of 1619 patients in total demonstrate that shedding of these vectors occurs in practice, mainly determined by the type of vector and the route of vector administration. Due to the use of non-quantitative assays, the lack of information on assay sensitivity in most publications, and the fact that assay sensitivity is expressed in various ways, general conclusions cannot be made as to the level of vector shedding. The evaluation of the potential impact and consequences of the observations is complicated by the high degree of variety in the experimental design of shedding analysis between trials. This inventory can be supportive to clinical gene therapy investigators for the establishment of an evidence-based risk assessment to be included in a clinical protocol application, as well as to national regulatory authorities for the ongoing development of regulatory guidelines regarding gene therapy. Copyright © 2007 John Wiley & Sons, Ltd. [source] Delivery of an Adenoviral Vector to the Crushed Recurrent Laryngeal Nerve,THE LARYNGOSCOPE, Issue 6 2003Adam Rubin MD Abstract Objectives Objectives were to create a model of recurrent laryngeal nerve injury for testing the efficacy of potential therapeutic viral gene therapy vectors and to demonstrate that remote injection of a viral vector does not cause significant additional neuronal injury. Study Design Animal model. Methods Rats were randomly assigned to three groups of 10 animals each. In group I, the recurrent laryngeal nerve was crushed. In group II, the nerve was crushed and then injected with an adenoviral vector containing no transgene. In group III, the nerve was identified but was not crushed. Rats were killed at 1 week, and their larynges and brainstems were cryosectioned in 15-,m sections. Laryngeal cryosections were processed for acetylcholine histochemical analysis (motor endplates) followed by neurofilament immunoperoxidase (nerve fibers). Percentage of nerve,endplate contact was determined and compared between groups. Fluorescent in situ hybridization was performed on brainstem sections from rats in group II to confirm the presence of virus. Results No significant difference in percentage of nerve,endplate contact exists between the two crushed-nerve groups (groups I and II) (P = .88). The difference between both crushed-nerve groups and the group with noncrushed nerves (group III) was highly significant (P <.0001). The presence of virus was confirmed in group II rats. Conclusions Crush provides a significant measurable injury to the recurrent laryngeal nerve and may be used as a model to explore therapeutic interventions for nerve injury. The remote injection of viral vector did not cause significant additional neuronal injury. Remote delivery of viral vectors to the central nervous system holds promise in the treatment of recurrent laryngeal nerve injury and central nervous system diseases. [source] Capturing of cell culture-derived modified Vaccinia Ankara virus by ion exchange and pseudo-affinity membrane adsorbersBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Michael W. Wolff Abstract Smallpox is an acute, highly infectious viral disease unique to humans, and responsible for an estimated 300,500 million deaths in the 20th century. Following successful vaccination campaigns through the 19th and 20th centuries, smallpox was declared eradicated by the World Health Organization in 1980. However, the threat of using smallpox as a biological weapon prompted efforts of some governments to produce smallpox vaccines for emergency preparedness. An additional aspect for the interest in smallpox virus is its potential use as a platform technology for vector vaccines. In particular, the latter requires a high safety level for routine applications. IMVAMUNE®, a third generation smallpox vaccine based on the attenuated Modified Vaccinia Ankara (MVA) virus, demonstrates superior safety compared to earlier generations and represents therefore an interesting choice as viral vector. Current downstream production processes of Vaccinia virus and MVA are mainly based on labor-intensive centrifugation and filtration methods, requiring expensive nuclease treatment in order to achieve sufficient low host-cell DNA levels for human vaccines. This study compares different ion exchange and pseudo-affinity membrane adsorbers (MA) to capture chicken embryo fibroblast cell-derived MVA-BN® after cell homogenization and clarification. In parallel, the overall performance of classical bead-based resin chromatography (Cellufine® sulfate and Toyopearl® AF-Heparin) was investigated. The two tested pseudo-affinity MA (i.e., sulfated cellulose and heparin) were superior over the applied ion exchange MA in terms of virus yield and contaminant depletion. Furthermore, studies confirmed an expected increase in productivity resulting from the increased volume throughput of MA compared to classical bead-based column chromatography methods. Overall virus recovery was ,60% for both pseudo-affinity MA and the Cellufine® sulfate resin. Depletion of total protein ranged between 86% and 102% for all tested matrices. Remaining dsDNA in the product fraction varied between 24% and 7% for the pseudo-affinity chromatography materials. Cellufine® sulfate and the reinforced sulfated cellulose MA achieved the lowest dsDNA product contamination. Finally, by a combination of pseudo-affinity with anion exchange MA a further reduction of host-cell DNA was achieved. Biotechnol. Bioeng. 2010. 105: 761,769. © 2009 Wiley Periodicals, Inc. [source] Effect of Protease Inhibitors on Yield of HSV-1-Based Viral VectorsBIOTECHNOLOGY PROGRESS, Issue 3 2000James B. Wechuck The ability to obtain high titer replication-defective herpes simplex virus (HSV) recombinant vectors will dramatically affect their use in gene therapy clinical trials. A variety of techniques and reagents have been employed to increase the overall yield of the vector. The effects of protease inhibitors on the yield of an HSV-1-based viral vector were examined. Experiments were conducted using a commercial protease inhibitor cocktail typically used in mammalian cell culture for protein production. Contrary to our expectation for enhanced vector yield, the results showed a dramatic reduction in vector yield. Moreover, it was found that AEBSF is the only component in the protease cocktail responsible for the low vector yield. On the basis of our hypothesis regarding the mode of action of AEBSF, we suggest that it should not be included in protease inhibitor cocktails designed for use in cultures aimed at production of viral vectors derived from HSV-1 or possibly several other vectors. [source] Immune responses to gene therapy vectors in the context of corneal transplantationACTA OPHTHALMOLOGICA, Issue 2009T RITTER Purpose The genetic engineering of grafts or cells prior to transplantation is an attractive approach to protect the graft from allogeneic rejection. Virus vector-based gene therapy is a promising method for successful ex-vivo gene transfer however, the induction of an immune response against gene-modified tissues raises concern. Methods Different virus families (Adenovirus, Retrovirus, Adeno-associated virus, Herpesvirus) have been studied as gene therapy vehicles for the delivery of therapeutic molecules. Moreover, different serotypes or envelope proteins have been used to modulate transduction efficiencies of target cells or to evade pre-existing immunity. Results Here we review gene therapeutic applications using viral vectors in the context of cornea transplantation. Both local and systemic expression of immunomodulatory molecules have led to the prevention of corneal graft rejection. However, different results have been obtained with regard to the induction of immune responses after local or systemic expression of the gene therapy vector. Not surprisingly over-expression of anti-inflammatory molecules not only modulated allograft rejection but also influenced the immune response against the viral vector and virally transduced cells. Conclusion Recent clinical trials indicate that the application of viral vectors in ophthalmology is promising however, the generation of immune responses against the viral vector or virally transduced cells are still a serious obstacle for a broader application of gene therapy. Supported by Deutsche Forschungsgemeinschaft (DFG Pl 150/14-1 and Ri 764/10-1) and Science Foundation of Ireland (SFI 06/RFP/BIC056 and SFI 07/IN.1/B925) [source] Gene therapy in epilepsyEPILEPSIA, Issue 1 2009Véronique Riban Summary Results from animal models suggest gene therapy is a promising new approach for the treatment of epilepsy. Several candidate genes such as neuropeptide Y and galanin have been demonstrated in preclinical studies to have a positive effect on seizure activity. For a successful gene therapy-based treatment, efficient delivery of a transgene to target neurons is also essential. To this end, advances have been made in the areas of cell transplantation and in the development of recombinant viral vectors for gene delivery. Recombinant adeno-associated viral (rAAV) vectors in particular show promise for gene therapy of neurological disorders due to their neuronal tropism, lack of toxicity, and stable persistence in neurons, which results in robust, long-term expression of the transgene. rAAV vectors have been recently used in phase I clinical trials of Parkinson's disease with an excellent safety profile. Prior to commencement of phase I trials for gene therapy of epilepsy, further preclinical studies are ongoing including evaluation of the therapeutic benefit in chronic models of epileptogenesis, as well as assessment of safety in toxicological studies. [source] Haematopoietic progenitor cells from the common marmoset as targets of gene transduction by retroviral and adenoviral vectorsEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2001Hitoshi Hibino Abstract: To establish a new non-human primate model for human cytokine and gene therapy, we characterized lymphocytes and haematopoietic progenitor cells of the small New World monkey, the common marmoset. We first assessed the reactions of marmoset bone marrow (BM) and peripheral blood (PB) cells to mouse anti-human monoclonal antibodies (mAbs) for the purpose of isolating marmoset lymphocytes and haematopoietic progenitor cells. Both cell fractions stained with CD4 and CD8 mAbs were identified as lymphocytes by cell proliferation assay and morphological examination. Myeloid-specific mAbs such as CD14 and CD33 did not react with marmoset BM and PB cells. No available CD34 and c-kit mAbs could be used to purify the marmoset haematopoietic progenitor cells. Furthermore, we studied the in vitro transduction of the bacterial ,-galactosidase (LacZ) gene into CFU-GM derived from marmoset BM using retroviral and adenoviral vectors. The transduction efficiency was increased by using a mixed culture system consisting of marmoset BM stromal cells and retroviral producer cells. It was also possible to transduce LacZ gene into marmoset haematopoietic progenitor cells with adenoviral vectors as well as retroviral vectors. The percentage of adenovirally transduced LacZ-positive clusters was 15% at day 4 (multiplicity of infection=200), but only 1,2% at day 14. The differential use of viral vector systems is to be recommended in targeting different diseases. Our results suggested that marmoset BM progenitor cells were available to examine the transduction efficiency of various viral vectors in vitro. [source] Immunisation with BCG and recombinant MVA85A induces long-lasting, polyfunctional Mycobacterium tuberculosis -specific CD4+ memory T lymphocyte populationsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2007Natalie Abstract In the search for effective vaccines against intracellular pathogens such as HIV, tuberculosis and malaria, recombinant viral vectors are increasingly being used to boost previously primed T cell responses. Published data have shown prime-boost vaccination with BCG-MVA85A (modified vaccinia virus Ankara expressing antigen 85A) to be highly immunogenic in humans as measured by ex vivo IFN-, ELISPOT. Here, we used polychromatic flow cytometry to investigate the phenotypic and functional profile of these vaccine-induced Mycobacterium tuberculosis (M.tb) antigen 85A-specific responses in greater detail. Promisingly, antigen 85A-specific CD4+ T cells were found to be highly polyfunctional, producing IFN-,, TNF-,, IL-2 and MIP-1,. Surface staining showed the responding CD4+ T cells to be relatively immature (CD45RO+ CD27intCD57,); this observation was supported by the robust proliferative responses observed following antigenic stimulation. Furthermore, these phenotypic and functional properties were independent of clonotypic composition and epitope specificity, which was maintained through the different phases of the vaccine-induced immune response. Overall, these data strongly support the use of MVA85A in humans as a boosting agent to expand polyfunctional M.tb -specific CD4+ T cells capable of significant secondary responses. [source] Nucleofection: a new, highly efficient transfection method for primary human keratinocytes,EXPERIMENTAL DERMATOLOGY, Issue 4 2005Jörg H. W. Distler Abstract:, Transfection is an essential tool for numerous in vitro applications including studies of gene expression, promoter analysis, and intracellular signaling pathways and also for therapeutic strategies such as tissue engineering and gene therapy. However, transfection of primary cells including keratinocytes with common methods such as calcium phosphate, DEAE-dextran, liposome-mediated transfer, electroporation or viral vectors is problematic because of low transfection efficiency and the induction of terminal differentiation. Here we analyzed the use of nucleofection, a new, electroporation-based transfection method that enables the DNA to enter directly the nucleus, for the transfection of keratinocytes. Several different conditions were tested and optimized, resulting in a final transfection efficiency of 56% in primary human epidermal keratinocytes. This efficiency is superior to all non-viral transfection methods reported so far. The number of non-viable keratinocytes after nucleofection was low, varying between 14 and 16%. In contrast to other transfection protocols, nucleofection did not induce terminal differentiation in the transfected keratinocytes. In addition, nucleofection is a fast method, because the results can be analyzed within 7 h. In summary, nucleofection is a fast, easy and highly effective alternative for the transfection of primary human keratinocytes, which offers new opportunities for various research applications. [source] Therapeutic angiogenesis and vasculogenesis for tissue regenerationEXPERIMENTAL PHYSIOLOGY, Issue 3 2005Paolo Madeddu Therapeutic angiogenesis/vasculogenesis holds promise for the cure of ischaemic disease. The approach postulates the manipulation of spontaneous healing response by supplementation of growth factors or transplantation of vascular progenitor cells. These supplements are intended to foster the formation of arterial collaterals and promote the regeneration of damaged tissues. Angiogenic factors are generally delivered in the form of recombinant proteins or by gene transfer using viral vectors. In addition, new non-viral methods are gaining importance for their safer profile. The association of growth factors with different biological activity might offer distinct advantages in terms of efficacy, yet combined approaches require further optimization. Alternatively, substances with pleiotropic activity might be considered, by virtue of their ability to target multiple mechanisms. For instance, some angiogenic factors not only stimulate the growth of arterioles and capillaries, but also inhibit vascular destabilization triggered by metabolic and oxidative stress. Transplantation of endothelial progenitor cells was recently proposed for the treatment of peripheral and myocardial ischaemia. Progenitor cells can be transplanted either without any preliminary conditioning or after ex vivo genetic manipulation. Delivery of genetically modified progenitor cells eliminates the drawback of immune response against viral vectors and makes feasible repeating the therapeutic procedure in case of injury recurrence. It is envisioned that these new approaches of regenerative medicine will open unprecedented opportunities for the care of life-threatening diseases. [source] Somatic Gene Transfer: Implications for Cardiovascular ControlEXPERIMENTAL PHYSIOLOGY, Issue 6 2000S. Kasparov There is a great need for closer integration between physiologists and molecular biologists. These two research fields might soon form a single research discipline under the umbrella of molecular physiology. One of the areas where this interaction may be particularly fruitful is with somatic gene transfer using replication-deficient viral vectors. We applied this approach to study the central control of the cardiovascular system at the level of the nucleus of the solitary tract (NTS). We assess critically this new methodology as applied to experiments in an integrative environment (such as the whole animal). The usefulness of in vivo gene transfer is illustrated by an experiment where viral gene delivery helped to circumvent the problem imposed by an absence of a specific pharmacological blocker of the enzyme, endothelial nitric oxide synthase (eNOS). The pros and cons of using adenoviral vectors as opposed to conventional pharmacological approaches are discussed. We conclude that the use of adenoviruses to manipulate genes offers a new avenue for physiologists studying neuronal mechanisms in integrative models. [source] Gene therapy for haemophilia,yes, but,with non-viral vectors?HAEMOPHILIA, Issue 3 2009A. LIRAS Summary., High-purity plasma-derived and recombinant factors are currently safe and efficient treatment for haemophilia. The mid-term future of haemophilia treatment will involve the use of modified recombinant factors to achieve advantages such as decreased immunogenicity in inhibitor formation and enhanced efficacy as a result of their longer half-life. In the long-term, gene therapy and cell therapy strategies will have to be considered. Achievements in cell therapy to date have been using embryonic stem cells and hepatic sinusoidal endothelial cells. Current gene therapy strategies for haemophilia are based on gene transfer using adeno-associated viruses and non-viral vectors. Gene therapy for haemophilia is justified because it is a chronic disease and because a very regular factor infusion is required that may involve fatal risks and because it is very expensive. Haemophilia is a very good candidate for use of gene therapy protocols because it is a monogenic disease, and even low expression is able to achieve reversion from a severe to a moderate phenotype. The current trends in haemophilia using adeno-associated viral vectors are safe but also involve immunogenicity problems. The other alternatives are non-viral vectors. There have been in recent years relevant advances in non-viral transfection that raise hope for considering this possibility. Several research groups are opting for this experimental alternative. An expression over 5%, representing a moderate phenotype, for a few months with a high safety, regarding vector, transfected cells, and implantation procedure, would already be a great success. This may represent an intermediate protocol in which the expression levels and times obtained are lower and shorter respectively as compared to viral vectors, but which provide a potential greater patient safety. This may more readily win acceptance among both patients and haematologists because fatal events in the past due to HIV/HCV infection may constrain the implementation of viruses as vectors. [source] Gene delivery of Cu/Zn-superoxide dismutase improves graft function after transplantation of fatty livers in the ratHEPATOLOGY, Issue 6 2000Thorsten G. Lehmann Oxygen-derived free radicals play a central role in reperfusion injury after organ transplantation, and fatty livers are particularly susceptible. Endogenous radical scavengers such as superoxide dismutase (SOD) degrade these radicals; however, SOD is destroyed rapidly when given exogenously. Therefore, an adenoviral vector encoding the Cu/Zn-SOD gene (Ad.SOD1) was used here to test the hypothesis that organ injury would be reduced and survival increased in a rat model of transplantation of fatty livers. Donors received chow diet (untreated), high-fat diet, or ethanol-containing high-fat diet. Some of the ethanol-fed donors were infected either with the gene lacZ encoding bacterial ,-galactosidase (Ad.lacZ), or Ad.SOD1. After liver transplantation, SOD activity and protein expression in liver, survival, histopathology, release of transaminases, free radical adducts in bile, and activation of NF-,B, I,B kinase (IKK), Jun-N-terminal kinase (JNK), and TNF, were evaluated. Ad.SOD1 treatment increased survival dramatically, blunted transaminase release, and reduced necrosis and apoptosis significantly. Free radical adducts were increased two-fold in the ethanol group compared with untreated controls. Ad.SOD1 blunted this increase and reduced the activation of NF-,B. However, release of TNF, was not affected. Ad.SOD1 also blunted JNK activity after transplantation. This study shows that gene therapy with Ad.SOD1 protects marginal livers from failure after transplantation because of decreased oxygen radical production. Genetic modification of fatty livers using viral vectors represents a new approach to protect marginal grafts against primary nonfunction. [source] Viral-mediated expression of a constitutively active form of CREB in hippocampal neurons increases memoryHIPPOCAMPUS, Issue 3 2009Leonardo Restivo Abstract Synaptic activity-dependent phosphorylation of the transcription factor cAMP response element binding protein (CREB) leads to CREB-dependent gene transcription, a process thought to underlie long-term hippocampal synaptic plasticity and memory formation. We previously reported that increasing CREB activity in glutamatergic neurons enhances synaptic plasticity and neuronal excitability. Whether these modifications are sufficient to promote hippocampal-dependent memory formation was not determined. Here, we provide direct evidence that a brief increase in CREB-dependent transcription in either CA1 or DG neurons, using in vivo viral vectors, is sufficient to boost memory for contextual representations, as tested in the contextual fear conditioning task, without affecting motor, pain, or anxiety behaviors. © 2008 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||