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Transduction Efficiency (transduction + efficiency)
Selected AbstractsEfficient gene transfer in mouse neural precursors with a bicistronic retroviral vectorJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2001Isabelle A. Franceschini Abstract Gene transfer into neural precursors is a powerful approach to study the function of specific gene products during nervous system development. Here we describe a retrovirus-based methodology to transduce foreign genes into mouse neural precursors. We used a high-titer bicistronic retroviral vector that encodes a marker gene, placental alkaline phosphatase (plap), and a selection gene, neomycin phosphotransferase II (neoR), under the translational control of two retroviral internal ribosome entry segments. Transduction efficiency even without selection was up to 95% for multipotential neurospheres derived from embryonic striata and grown with basic fibroblast growth factor 2. Expression of plap and neoR was sustained with time in culture and upon differentiation into neurons, astrocytes, and oligodendrocytes, as shown by double immunofluorescence labeling with cell type-specific markers, Western blotting, and neomycin resistance. However, levels of plap were decreased in differentiated oligodendrocytes. Transduction with the same vector of neonatal oligodendrocyte precursors grown in oligospheres consistently resulted in a lower proportion of plap-immunoreactive cells and enhanced cell death in the absence of neomycin. However, plap expression was maintained in some differentiated oligodendrocytes expressing galactocerebroside or myelin basic protein. In that neurospheres can be easily expanded in vitro and factors enabling their differentiation into the three main central nervous system cell types are being elucidated, this methodology could be used in the future to produce large number of transduced, differentiated neural cells. J. Neurosci. Res. 65:208,219, 2001. © 2001 Wiley-Liss, Inc. [source] Tissue Engineering of Urethra Using Human Vascular Endothelial Growth Factor Gene-Modified Bladder Urothelial CellsARTIFICIAL ORGANS, Issue 2 2008Yong Guan Abstract:, Acquired or congenital abnormalities may lead to urethral damage or loss, often requiring surgical reconstruction. Urethrocutaneous fistula and strictures are common complications, due to inadequate blood supply. Thus, adequate blood supply is a key factor for successful urethral tissue reconstruction. In this study, urethral grafts were prepared by seeding rabbit bladder urothelial cells (UCs) modified with human vascular endothelial growth factor (VEGF165) gene in the decellularized artery matrix. A retroviral pMSCV-VEGF165 -GFP vector was cloned by insertion of VEGF open reading frame into the vector pMSCV-GFP (murine stem cell virus [MSCV]; green fluorescent protein [GFP]). Retrovirus was generated using package cell line 293T. Rabbit UCs were expanded ex vivo and modified with either MSCV-VEGF165 -GFP or control MSCV-GFP retrovirus. Transduction efficiency was analyzed by fluorescence-activated cell sorting. The expression of VEGF165 was examined by immunofluorescence, reverse transcript-polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay (ELISA). Decellularized rabbit artery matrix was seeded with genetically modified UCs and was subsequently cultured for 1 week prior to subcutaneous implantation into nude mice. Four weeks after implantation, the implants were harvested and analyzed by fluorescence microscopy, and by histologic and immunohistochemical staining. Ex vivo transduction efficiency of UCs was greater than 50% when concentrated retrovirus was used. The modified cells expressed both VEGF and GFP protein. Furthermore, the VEGF-modified UCs secreted VEGF in a time-dependent manner. Scanning electron microscopy and histochemical analysis of cross sections of the cultured urethral grafts showed that the seeded cells were attached and proliferated on the luminal surface of the decellularized artery matrix. In the subcutaneously implanted vessels, VEGF-modified cells significantly enhanced neovascularization and the formation of a urethral layer compared to GFP-modified cells. These results indicate that VEGF gene therapy may be a suitable approach to increase the blood supply in tissue engineering for treatment of urethral damage or loss. [source] Detectable reporter gene expression following transduction of adenovirus and adeno-associated virus serotype 2 vectors within full-thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivoJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2010Kelly S. Santangelo Abstract This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adeno-associated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p,,,0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full-thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:149,155, 2010 [source] Scaleable purification process for gene therapy retroviral vectorsTHE JOURNAL OF GENE MEDICINE, Issue 4 2007Teresa Rodrigues Abstract Background Retroviral vectors (RVs) constitute one of the preferred gene therapy tools against inherited and acquired diseases. Development of scaleable downstream processes allowing purification under mild conditions and yielding viral preparations with high titer, potency and purity is critical for the success of clinical trials and subsequent clinical use of this technology. Methods A purification process for murine leukaemia virus (MLV)-derived vector supernatants was developed based on membrane separation and anion-exchange chromatography (AEXc). Initial clarification of the vector stocks was performed using 0.45 µm membranes followed by concentration with 500 kDa molecular weight cut-off (MWCO) membranes; further purification was performed by AEXc using a tentacle matrix bearing DEAE functional ligands. Finally, concentration/diafiltration was performed by 500 kDa MWCO membranes. To validate final product quality the process was scaled up 16-fold. Results Optimization of microfiltration membrane pore size and ultrafiltration transmembrane pressure allowed the recovery of nearly 100% infectious particles. Further purification of the RVs by AEXc resulted in high removal of protein contaminants while maintaining high recoveries of infectious vectors (77 ± 11%). Up-scaling of the process resulted in high titer vector preparations, 3.2 × 108 infectious particles (IP)/ml (85-fold concentration), with an overall recovery reaching 26%. The process yielded vectors with transduction efficiencies higher than the starting material and more than 99% pure, relative to protein contamination. Conclusions The combination of membrane separation and AEXc processes results in a feasible and scaleable purification strategy for MLV-derived vectors, allowing the removal of inhibitory contaminants thus yielding pure vectors with increased transduction efficiencies. Copyright © 2007 John Wiley & Sons, Ltd. [source] Ex vivo hypothermic recirculatory adenoviral gene transfer to the transplanted pig heartTHE JOURNAL OF GENE MEDICINE, Issue 7 2006Keiji Oi Abstract Background To facilitate the application of adenoviral gene therapy in clinical heart transplantation, we developed an ex vivo hypothermic recirculatory adenoviral gene transfer method to the transplanted pig heart. Methods Experimental animals were assigned into three groups; controls, 1 × 108 plaque-forming units (pfu)/ml group and 1 × 109 pfu/ml group. During the 30 min gene transfer perfusion, 200 ml of University of Wisconsin solution containing the adenoviral vector was recirculated through the coronary vessels. The myocardial temperature was maintained below 4 °C and the perfusion pressure was adjusted at 50 mmHg. Results Cardiac myocyte transduction efficiencies in the 1 × 108 pfu/ml group were 0.04% and 0.07%, whereas transduction efficiencies in the 1 × 109 pfu/ml group were widely distributed from 0.45% to 22.62%. The gene transduction efficiency increased with the virus titer. Additionally, no difference in the transduction efficiency was observed between different segments of the left ventricle. The current gene transfer method at 1 × 109 pfu/ml of adenovirus titer enabled homogeneous gene transduction into the transplanted pig heart up to a maximum of 22.62%. Conclusions This model can be applied to a large isolated heart and will greatly facilitate the investigation of gene therapy in large animal models of heart transplantation. Copyright © 2006 John Wiley & Sons, Ltd. [source] Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactorTHE JOURNAL OF GENE MEDICINE, Issue 12 2004Dao Pan Abstract Background One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency. Other inadequacies of current transduction protocols are related to their multi-step procedures, e.g., using tissue-culture flasks, roller bottles or gas-permeable bags for clinical application. Methods In comparison with a conventional bag transduction protocol, a ,closed' hollow-fiber bioreactor system (HBS) was exploited to culture and transduce human peripheral blood CD34+ progenitor cells (PBPCMPS) from patients with mucopolysaccharidosis type I (MPS I) using an amphotropic retroviral vector based on a murine Moloney leukemia virus LN prototype. Both short-term colony-forming cell (CFC) and long-term culture initiating cell (LTCIC) assays were employed to determine transduction frequency and transgene expression in committed progenitor cells and primitive progenitors with multi-lineage potentials. Results A novel ultrafiltration-transduction method was established to culture and transduce enzyme-deficient PBPCMPS over a 5-day period without loss in viability and CD34 identity (n = 5). Significantly higher transduction efficiencies were achieved in primary CFC that derived from the HBS (5.8,14.2%) in comparison with those from gas-permeable bags (undetectable to 1.7%; p < 0.01). Up to 15-fold higher-than-normal enzyme activity was found in selected PBPCMPS -LP1CD transductants. Moreover, higher gene transfer (4.4-fold) and expression in very primitive progenitors were observed in products from the HBS compared with bag experiments as indicated by CFC derived from primitive LTCIC. Remarkably, with relatively modest gene transfer levels in LTCIC from HBS experiments, the expression of the IDUA transgene corrected the enzyme-deficiency in 5-week long-term cultures (LTC). Conclusions MPS I progenitor cells achieved normalized enzyme levels in LTC after transduction in a HBS system. These studies demonstrate the advantages of a bioreactor-transduction system for viral-mediated stem cell gene transfer. Copyright © 2004 John Wiley & Sons, Ltd. [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] 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] HDAC inhibitor valproic acid enhances tumor cell kill in adenovirus-HSVtk mediated suicide gene therapy in HNSCC xenograft mouse modelINTERNATIONAL JOURNAL OF CANCER, Issue 3 2010Vishal Kothari Abstract Safety, efficacy and enhanced transgene expression are the primary concerns while using any vector for gene therapy. One of the widely used vectors in clinical trials is adenovirus which provides a safe way to deliver the therapeutic gene. However, adenovirus has poor transduction efficiency in vivo since most tumor cells express low coxsackie and adenovirus receptors. Similarly transgene expression remains low, possibly because of the chromatization of adenoviral genome upon infection in eukaryotic cells, an effect mediated by histone deacetylases (HDACs). Using a recombinant adenovirus (Ad-HSVtk) carrying the herpes simplex thymidine kinase (HSVtk) and GFP genes we demonstrate that HDAC inhibitor valproic acid can bring about an increase in CAR expression on host cells and thereby enhanced Ad-HSVtk infectivity. It also resulted in an increase in transgene (HSVtk and GFP) expression. This, in turn, resulted in increased cell kill of HNSCC cells, following ganciclovir treatment in vitro as well as in vivo in a xenograft nude mouse model. [source] Chimeric HIV-1 and HIV-2 lentiviral vectors with added safety insurance,JOURNAL OF MEDICAL VIROLOGY, Issue 2 2007Geetanjali Sachdeva Abstract Lentiviruses are unique in their ability to infect both dividing and non-dividing cells. This makes the vectors derived from them particularly useful for gene transfer into non-dividing cells, including stem cells. Lentiviral vectors are becoming the vectors of choice for si/shRNA delivery. The utility of the lentiviral vectors will be enhanced if additional elements of safety are built into their design. One safety concern is the generation of replication competent virus by recombination. We reasoned that HIV-1 and HIV-2 hybrid or chimeric lentiviral vectors will have added safety insurance in this regard. This is based on the premise that HIV-1 and HIV-2 are dissimilar enough in sequence to curtail recombination, yet similar enough to complement functionally. For hybrid vectors, we found that both HIV-1 and HIV-2 transfer vector RNAs could be packaged to equivalent titer by the HIV-1 packaging machinery. However, HIV-2 packaging machinery was unable to package HIV-1 transfer vector as well as it did HIV-2 transfer vector. This non-reciprocacity suggested that the requirement for HIV-2 vectors was more stringent and that for HIV-1 vectors more promiscuous. When the HIV-1 transfer vector was packaged with the chimeric packaging construct where the leader-gag region of HIV-2 was replaced with that of HIV-1 packaging construct, the titer of the vector went up. This suggests that at least some of the determinants of specificity for vector assembly reside in the leader-gag region. Incorporation of central polypurine tract (cPPT) and woodchuck post-transcriptional enhance element (WPRE) into the HIV-2 vectors had only modest effect on vector titer. Thus, chimeric lentiviral vectors with added safety features can be designed without compromising transduction efficiency. J. Med. Virol. 79:118,126, 2007. © 2006 Wiley-Liss, Inc. [source] Genetic marking with the ,LNGFR-gene for tracing goat cells in bone tissue engineering,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2004M. C. Kruyt Abstract The use of bone marrow derived stromal cells (BMSC's) for bone tissue engineering has gained much attention as an alternative for autologous bone grafting. Little is known however, about the survival and differentiation of the cells, especially in the clinical application. The aim of this study was to develop a method to trace goat BMSC's in vivo. We investigated retroviral genetic marking, which allows stable expression of the label with cell division. Goat BMSC's were subjected to an amphotropic envelope containing a MoMuLV-based vector expressing the human low affinity nerve growth factor receptor (,LNGFR). Labeling efficiency and effect on the cells were analyzed. Furthermore, transduced cells were seeded onto porous ceramic scaffolds, implanted subcutaneously in nude mice and examined after successive implantation periods. Flow cytometry indicated a transduction efficiency of 40,60%. Immunohistochemistry showed survival and subsequent bone formation of the gene-marked cells in vivo. Besides, marked cells were also found in cartilage and fibrous tissue. These findings indicate the maintenance of the precursor phenotype following gene transfer as well as the ability of the gene to be expressed following differentiation. We conclude that retroviral gene marking with ,LNGFR is applicable to trace goat BMSC's in bone tissue engineering research. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] Schwann cell delivery of neurotrophic factors for peripheral nerve regenerationJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 2 2010Srinivas Madduri Current treatments of injured peripheral nerves often fail to mediate satisfactory functional recovery. For axonal regeneration, neurotrophic factors (NTFs) play a crucial role. Multiple NTFs and other growth-promoting factors are secreted, amongst others, by Schwann cells (SCs), which also provide cellular guidance for regenerating axons. Therefore, delivery of NTFs and transplantation of autologous or genetically modified SCs with therapeutic protein expression have been proposed. This article reviews polymer-based and cellular approaches for NTF delivery, with a focus on SCs and strategies to modulate SC gene expression. Polymer-based NTF delivery has mostly resided on nerve conduits (NC). While NC have generally provided prolonged NTF release, their therapeutic effect has remained significantly below that achieved with autologous nerve grafts. Several studies demonstrated enhanced nerve regeneration using NC seeded with SCs. The SCs have sometimes been modified genetically using non-viral or viral vectors. Whereas non-viral vectors produced poor transgene delivery, adenoviral vectors mediated high transgene transduction efficiency of SCs. Further improvements of safety and transgene expression of adenoviral vector may lead to rapid translation of pre-clinical research to clinical trials. [source] Immobilized HIV-1 Tat protein promotes gene transfer via a transactivation-independent mechanism which requires binding of Tat to viral particlesTHE JOURNAL OF GENE MEDICINE, Issue 11 2009Filomena Nappi Abstract Background Retroviral transduction of cells is improved upon virus adsorption onto immobilized fibronectin (FN) fragments. Because HIV-1 Tat possesses the same functional domains that lead to increased transduction efficiency in FN by colocalization of bound virus and cells, we hypothesized that Tat could enhance gene transfer by a similar mechanism. Methods Single-cycle replication retro- or lentivirus carrying green fluorescent protein or cloramphenicol acetyltransferase as reporter genes were added to wells coated with Tat or Tat peptides. Wells were extensively washed to remove unbound virus and levels of transduction were detected by measuring reporter gene expression. Virus adsorption to immobilized Tat was measured using a p24 antigen capture assay. Results Immobilized Tat efficiently binds retro- and lentiviral particles and mediates virus transmission at virus input doses that were otherwise unable to transduce susceptible cells. Virus adsorption to Tat is not mediated by envelope glycoprotein (Env) because immobilized Tat binds and retains vesicular stomatitis virus G (VSV-G) pseudotypes as well as envelope-free particles. HIV-1 Env or VSV-G are required for Tat-assisted transduction, which is abrogated by an antibody blocking the HIV-1 Env,CD4 interaction. Tat-assisted transduction is mediated by the cysteine-rich region of Tat, which is known to be essential for Tat transactivation activity. However, Tat transactivation is not required for Tat-assisted transduction, as indicated by the enhancement of transduction by transactivation-silent Tat mutants. Conclusions Immobilized Tat promotes virus transduction by a transactiva- tion-independent mechanism, which requires binding of virus to Tat. Recombinant Tat or Tat fragments provide a new method to increase efficiency of retro- and lentiviral based gene transfer and gene therapy. Copyright © 2009 John Wiley & Sons, Ltd. [source] Ex vivo hypothermic recirculatory adenoviral gene transfer to the transplanted pig heartTHE JOURNAL OF GENE MEDICINE, Issue 7 2006Keiji Oi Abstract Background To facilitate the application of adenoviral gene therapy in clinical heart transplantation, we developed an ex vivo hypothermic recirculatory adenoviral gene transfer method to the transplanted pig heart. Methods Experimental animals were assigned into three groups; controls, 1 × 108 plaque-forming units (pfu)/ml group and 1 × 109 pfu/ml group. During the 30 min gene transfer perfusion, 200 ml of University of Wisconsin solution containing the adenoviral vector was recirculated through the coronary vessels. The myocardial temperature was maintained below 4 °C and the perfusion pressure was adjusted at 50 mmHg. Results Cardiac myocyte transduction efficiencies in the 1 × 108 pfu/ml group were 0.04% and 0.07%, whereas transduction efficiencies in the 1 × 109 pfu/ml group were widely distributed from 0.45% to 22.62%. The gene transduction efficiency increased with the virus titer. Additionally, no difference in the transduction efficiency was observed between different segments of the left ventricle. The current gene transfer method at 1 × 109 pfu/ml of adenovirus titer enabled homogeneous gene transduction into the transplanted pig heart up to a maximum of 22.62%. Conclusions This model can be applied to a large isolated heart and will greatly facilitate the investigation of gene therapy in large animal models of heart transplantation. Copyright © 2006 John Wiley & Sons, Ltd. [source] In vivo gene marking of rhesus macaque long-term repopulating hematopoietic cells using a VSV-G pseudotyped versus amphotropic oncoretroviral vectorTHE JOURNAL OF GENE MEDICINE, Issue 4 2004Patricia A. Shi Abstract Background Gene transfer efficiency into primitive hematopoietic cells may be limited by their expression of surface receptors allowing vector entry. Vectors pseudotyped with the vesicular stomatitis virus (VSV-G) envelope do not need receptors to enter cells, and therefore may provide superior transduction efficiency. Methods Using a competitive repopulation model in the rhesus macaque, we examined in vivo gene marking levels of blood cells transduced with two vectors: (i) a VSV-G pseudotyped retrovirus and (ii) a conventional amphotropic retrovirus. The VSV-G vector, containing the human glucose-6-phosphate dehydrogenase (G6PD) gene, was constructed for treatment of severe hemolytic anemia caused by G6PD deficiency. Three myeloablated animals were transplanted with peripheral blood CD34+ cells, half of which were transduced with the VSV-G vector and the other half with the amphotropic vector. Results In all animals post-transplantation, levels of in vivo marking in circulating granulocytes and mononuclear cells were similar: 1% or less with both vectors. In one animal, the human G6PD enzyme transferred by the VSV-G vector was expressed in erythrocytes, early after transplantation, at a level of 45% of the endogenous rhesus G6PD protein. Conclusions In a clinically relevant animal model, we found similar in vivo marking with a VSV-G pseudotyped and a standard amphotropic oncoretroviral vector. Amphotropic receptor expression may not be a limiting factor in transduction efficiency, but VSV-G pseudotypes possess other practical advantages that may make them advantageous for clinical use. Copyright © 2004 John Wiley & Sons, Ltd. [source] Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L -lysine) block copolymer (PEG-PLL)THE JOURNAL OF GENE MEDICINE, Issue 4 2004Hiromichi Katakura Abstract Background Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L -lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. Methods A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. Results We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. Conclusions PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity. Copyright © 2004 John Wiley & Sons, Ltd. [source] Exploration of target molecules for prostate cancer gene therapyTHE PROSTATE, Issue 11 2007Kazuhiro Suzuki Abstract BACKGROUND Focusing on Adv-FZ33, a modified adenovirus in which a synthetic 33-amino-acid immunoglobulin G-binding domain was inserted into the adenoviral fiber protein, we tried to identify suitable target molecules for prostate cancer-specific gene therapy. METHODS Hybridomas were established from mice immunized with prostate cancer cell lines. The hybridomas were screened using Adv-FZ33 to create monoclonal antibodies (mAbs) that induced high gene transfer efficiency for PC-3 cells. Furthermore, we identified target antigens of the mAbs by immunoprecipitation and mass spectrometry, and investigated the expression of target molecules by flow cytometry and immunocytochemistry. RESULTS Using Adv-FZ33, we established four different mouse mAbs that increased transduction efficiency for PC-3. The target antigens identified were Ep-CAM, CD155, HAI-1, and Na,K-ATPase ,1. These antigens were expressed in several cancer cell lines, including prostate cancer. Human prostatic myofibroblast cells lacked expression of Ep-CAM and HAI-1. CONCLUSIONS We established anti-Ep-CAM mAb and anti- HAI-1 mAbs. Gene transduction via Ep-CAM and HAI-1 may be a novel strategy for treatment of prostate cancer. Prostate 67: 1163,1173, 2007. © 2007 Wiley-Liss, Inc. [source] Tissue Engineering of Urethra Using Human Vascular Endothelial Growth Factor Gene-Modified Bladder Urothelial CellsARTIFICIAL ORGANS, Issue 2 2008Yong Guan Abstract:, Acquired or congenital abnormalities may lead to urethral damage or loss, often requiring surgical reconstruction. Urethrocutaneous fistula and strictures are common complications, due to inadequate blood supply. Thus, adequate blood supply is a key factor for successful urethral tissue reconstruction. In this study, urethral grafts were prepared by seeding rabbit bladder urothelial cells (UCs) modified with human vascular endothelial growth factor (VEGF165) gene in the decellularized artery matrix. A retroviral pMSCV-VEGF165 -GFP vector was cloned by insertion of VEGF open reading frame into the vector pMSCV-GFP (murine stem cell virus [MSCV]; green fluorescent protein [GFP]). Retrovirus was generated using package cell line 293T. Rabbit UCs were expanded ex vivo and modified with either MSCV-VEGF165 -GFP or control MSCV-GFP retrovirus. Transduction efficiency was analyzed by fluorescence-activated cell sorting. The expression of VEGF165 was examined by immunofluorescence, reverse transcript-polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay (ELISA). Decellularized rabbit artery matrix was seeded with genetically modified UCs and was subsequently cultured for 1 week prior to subcutaneous implantation into nude mice. Four weeks after implantation, the implants were harvested and analyzed by fluorescence microscopy, and by histologic and immunohistochemical staining. Ex vivo transduction efficiency of UCs was greater than 50% when concentrated retrovirus was used. The modified cells expressed both VEGF and GFP protein. Furthermore, the VEGF-modified UCs secreted VEGF in a time-dependent manner. Scanning electron microscopy and histochemical analysis of cross sections of the cultured urethral grafts showed that the seeded cells were attached and proliferated on the luminal surface of the decellularized artery matrix. In the subcutaneously implanted vessels, VEGF-modified cells significantly enhanced neovascularization and the formation of a urethral layer compared to GFP-modified cells. These results indicate that VEGF gene therapy may be a suitable approach to increase the blood supply in tissue engineering for treatment of urethral damage or loss. [source] Engineering of Vascular Grafts With Genetically Modified Bone Marrow Mesenchymal Stem Cells on Poly (Propylene Carbonate) GraftARTIFICIAL ORGANS, Issue 12 2006Jun Zhang Abstract:, Bone marrow mesenchymal stem cells (MSCs) have demonstrated their pluripotency to differentiate into different cell lineages and may be an alternative cell source for vascular tissue engineering. The objective of this study is to create small diameter vessels by seeding and culture of genetically modified MSCs onto a synthetic polymer scaffold produced by an electrospinning technique. A tubular scaffold (2 mm in diameter) with a microstructure of nonwoven fibers was produced by electrospinning of poly (propylene carbonate) (PPC). Rat MSCs obtained from bone marrow were expanded in culture and modified with vasculoprotective gene endothelial nitric oxide synthase (eNOS) or marker gene green fluorescent protein (GFP). These MSCs were seeded onto the electrospun fibrous grafts (internal diameter = 2 mm), and cultured in 5% CO2 at 37°C. The growth of MSCs in the scaffold was analyzed with scanning electron microscopy (SEM) and hematoxylin and eosin (H&E) staining. The gene transfer and transgenic gene expression were examined with fluorescence-activated cell sorting (FACS), immunochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot. The production of nitric oxide (NO) by the engineered vessels was measured with an NO detection kit. Our data showed that the seeded cells integrated with the microfibers of the scaffold to form a three-dimensional cellular network, indicating a favorable interaction between this synthetic PPC scaffold with MSCs. High transduction efficiency was obtained with the use of concentrated retrovirus in the gene transfection of MSCs. The eNOS gene transcripts and protein were detected in the grafts seeded with eNOS-modified MSCs by RT-PCR and immunochemical staining. The amount of NO produced by grafts seeded with eNOS-modified MSCs was comparable to that produced by native blood vessels, and it was significantly higher than that in the grafts seeded with nonmodified MSCs. In summary, the vascular graft produced by culture of eNOS gene-modified MSCs onto the electrospun tubular scaffolds shows promising results in terms of function. The use of MSCs and therapeutic genes in tissue engineering of blood vessels could be helpful in improving vessel regeneration and patency. [source] Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 9ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Matthew Mitchell Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8,Å resolution using synchrotron radiation and belonged to the trigonal space group P32, with unit-cell parameters a = b = 251.0, c = 640.0,Å. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetric unit capsid have been determined by molecular-replacement methods and structure determination is in progress. [source] Mathematical model of the rate-limiting steps for retrovirus-mediated gene transfer into mammalian cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Venkata S. Tayi Abstract A quantitative understanding of the process of retrovirus-mediated gene transfer into mammalian cells should assist the design and optimization of transduction protocols. We present a mathematical model of the process that incorporates the essential rate-limiting transduction steps including diffusion, convection and decay of viral vectors, their binding at the cell surface and entry into the cell cytoplasm, reverse transcription of uncoated RNA to form DNA intermediates, transport of the latter through the cytosol to the cell nucleus and, finally, nuclear import and integration of the delivered DNA into the target cell genome. Cell and virus population balances are used to account for the kinetics of multiple vector infections which influence the transduction efficiency and govern the integrated copy number. The mathematical model is validated using gibbon ape leukemia virus envelope pseudotyped retroviral vectors and K562 target cells. Viral intermediate complexes derived from the internalized retroviral vectors are found to remain stable inside the K562 cells and the cytoplasmic trafficking time is consistent with the time scale for retrovirus uncoating, reverse transcription and transport to the cell nucleus. The model predictions of transduction efficiency and integrated copy number agree well with experimental data for both static (i.e., standard gravity) and centrifugation-based gene transfer protocols. The formulation of the model can also be applied to transduction protocols involving lenti- or foamy-viruses and so should prove to be useful for the optimization of several types of gene transfer processes. Biotechnol. Bioeng. 2010;105: 195,209. © 2009 Wiley Periodicals, Inc. [source] Up-regulation of integrin ,3 in radioresistant pancreatic cancer impairs adenovirus-mediated gene therapyCANCER SCIENCE, Issue 10 2009Takuya Egami Adenovirus-mediated gene therapy is a promising approach for the treatment of pancreatic cancer. We previously reported that radiation enhanced adenovirus-mediated gene expression in pancreatic cancer, suggesting that adenoviral gene therapy might be more effective in radioresistant pancreatic cancer cells. In the present study, we compared the transduction efficiency of adenovirus-delivered genes in radiosensitive and radioresistant cells, and investigated the underlying mechanisms. We used an adenovirus expressing the hepatocyte growth factor antagonist, NK4 (Ad-NK4), as a representative gene therapy. We established two radioresistant human pancreatic cancer cell lines using fractionated irradiation. Radiosensitive and radioresistant pancreatic cancer cells were infected with Ad-NK4, and NK4 levels in the cells were measured. In order to investigate the mechanisms responsible for the differences in the transduction efficiency between these cells, we measured expression of the genes mediating adenovirus infection and endocytosis. The results revealed that NK4 levels in radioresistant cells were significantly lower (P < 0.01) than those in radiosensitive cells, although there were no significant differences in adenovirus uptake between radiosensitive cells and radioresistant cells. Integrin ,3 was up-regulated and the Coxsackie virus and adenovirus receptor was down-regulated in radioresistant cells, and inhibition of integrin ,3 promoted adenovirus gene transfer. These results suggest that inhibition of integrin ,3 in radioresistant pancreatic cancer cells could enhance adenovirus-mediated gene therapy. (Cancer Sci 2009; 100: 1902,1907) [source] Efficient Gene Transduction by RGB-fiber Modified Recombinant Adenovirus into Dendritic CellsCANCER SCIENCE, Issue 3 2001Rumiko Asada-Mikami Dendritic cells (DC) are important antigen-presenting cells in the development of an anti-tumor T cell response. To extend the range of current immuno/gene therapies, we tested luciferase-expressing RGD-adenovirus (Ad) (Ad5lucRGD)-mediated transduction into DC. Phenotypically characterized DC were generated from peripheral blood CD14+ cells by incubation with granulocyte-macrophage colony-stimulating factor, interleukin-4 and tumor necrosis factor a. On the 7th day of culture, the cells became mature DC with a CD1a+, CD11c+, CD80+, CD83+, CD86+, human leukocyte antigen (HLA)-DR+, CD14, phenotype. The expression of (,,,3 integrin was enhanced on day 3 and returned to the basal level on day 7. We then compared the transduction efficiency of an AdSlucRGD system to that using conventional Ad, in cells harvested on days 1, 3 and 7 of culture. Luciferase activity was negligible in AdCMVLuc, but remarkable in cells processed with Ad5lucRGD. Activity was maximal in cells that had been cultured for 3 days. Recombinant Ad5 fiber knob protein blocked AdCMVLuc- and Ad5lucRGD-mediated gene transduction by 90% and 20%, respectively. Surface markers and cytokine production were not affected by Ad5lucRGD-mediated transduction. [source] |