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Gene Therapy Applications (gene + therapy_application)
Selected AbstractsIn vivo electroporation and ubiquitin promoter , a protocol for sustained gene expression in the lungTHE JOURNAL OF GENE MEDICINE, Issue 7 2006Amiq Gazdhar Abstract Background Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. Methods The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 µl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. Results Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932 ± 249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382 ± 318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989 ± 710 RLU/mg of total lung protein) with a peak at day 20 (2821 ± 2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. Conclusions These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved. Copyright © 2006 John Wiley & Sons, Ltd. [source] The skin as a biofactory for systemic secretion of erythropoietin: potential of genetically modified keratinocytes and fibroblastsEXPERIMENTAL DERMATOLOGY, Issue 6 2008Frank Scheidemann Abstract Background:, The skin is an interesting target tissue for gene therapy applications because of its ready accessibility. One possibility would be to utilize the genetically modified skin as a biofactory secreting a systemically needed product, such as erythropoietin (EPO). Methods:, Keratinocytes (KC) and fibroblasts (FB) were transduced with a retroviral vector encoding human EPO. Gene transfer efficiency was assessed by real-time PCR analysis and flow cytometry of transduced cells. In addition, EPO synthesis and secretion were analysed by quantifying the amount of RNA and secreted protein in both monolayer cultures and skin equivalents (SE). Results:, When cultured as a monolayer, EPO-KC synthesized significantly more EPO than EPO-FB, as shown by quantitatively measuring the amount of secreted protein and RNA. This correlated with an increased EPO-vector incorporation in KC compared with FB, demonstrated by determining both the percentage of transduced cells and the average transgene copy number per cell. In addition, in transduced cell cultures enriched to equally high percentages of EPO+ cells, KC showed a higher activity of EPO secretion than FB. Finally, when assembled in a SE, EPO-KC secreted significantly higher amounts of EPO than EPO-FB, although reduced secretory activity of EPO-KC monolayers grown in high calcium concentrations suggested that in stratified epidermis differentiated KC secrete less EPO than non-differentiated KC. Conclusion:, In summary, while both transduced KC and FB are able to synthesize and secrete human EPO, KC show higher potential in serving as possible target cells for therapeutic substitution with EPO, probably because of improved transduction rates and increased secretory activity. [source] Novel hepatic progenitor cell surface markers in the adult rat liver,HEPATOLOGY, Issue 1 2007Mladen I. Yovchev Hepatic progenitor/oval cells appear in injured livers when hepatocyte proliferation is impaired. These cells can differentiate into hepatocytes and cholangiocytes and could be useful for cell and gene therapy applications. In this work, we studied progenitor/oval cell surface markers in the liver of rats subjected to 2-acetylaminofluorene treatment followed by partial hepatectomy (2-AAF/PH) by using rat genome 230 2.0 Array chips and subsequent RT-PCR, immunofluorescent (IF), immunohistochemical (IHC) and in situ hybridization (ISH) analyses. We also studied expression of the identified novel cell surface markers in fetal rat liver progenitor cells and FAO-1 hepatoma cells. Novel cell surface markers in adult progenitor cells included tight junction proteins, integrins, cadherins, cell adhesion molecules, receptors, membrane channels and other transmembrane proteins. From the panel of 21 cell surface markers, 9 were overexpressed in fetal progenitor cells, 6 in FAO-1 cells and 6 are unique for the adult progenitors (CD133, claudin-7, cadherin 22, mucin-1, ros-1, Gabrp). The specificity of progenitor/oval cell surface markers was confirmed by ISH and double IF analyses. Moreover, study of progenitor cells purified with Ep-CAM antibodies from D-galactosamine injured rat liver, a noncarcinogenic model of progenitor cell activation, verified that progenitor cells expressed these markers. Conclusion: We identified novel cell surface markers specific for hepatic progenitor/oval cells, which offers powerful tool for their identification, isolation and studies of their physiology and pathophysiology. Our studies also reveal the mesenchymal/epithelial phenotype of these cells and the existence of species diversity in the hepatic progenitor cell identity. (HEPATOLOGY 2007;45:139,149.) [source] Activity of the matrix metalloproteinase-9 promoter in human normal and tumor cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004Cristina Morelli Matrix metalloproteinases (MMPs) belong to a family of proteins essential for those processes involving extracellular matrix degradation, such as embryonic development, morphogenesis, and tissue resorption and remodeling. Some members of this family play a crucial role also in tumor invasion. Most notably, MMP-9 is expressed in invasive tumors, and represents a key protein in brain tumor progression, whereas it is not expressed in adult normal tissues. The expression of the MMP-9, like other members of the family, is transcriptionally regulated. We, therefore, postulated that the MMP-9 promoter could be useful in driving selective expression of exogenous genes in tumor cells. This represents a key feature for gene therapy applications, since currently employed viral promoters induce severe organ toxicity, limiting the clinical benefits. In this study, we investigated the activity of the MMP-9 promoter in driving exogenous gene expression in human cell lines. High levels of reporter gene expression were detected in tumor derived cell lines, whereas the MMP-9 promoter activity in non-tumor cells was negligible. Furthermore, we show that tumor necrosis factor alpha (TNF,) is able to enhance considerably the MMP-9 promoter activity only in tumor cells. Since recent studies have indicated that MMP-9 enzymatic activity is detectable in the blood, it would be possible to screen potential responsive patients for a tumor gene therapy approach based on the MMP-9 promoter. Taken together these data suggest that MMP-9 promoter has the characteristics for transcritpionally targeted and inducible gene therapy applications. J. Cell. Physiol. 199: 126,133, 2004© 2003 Wiley-Liss, Inc. [source] Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surfaceJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 4 2010Irina V. Balyasnikova Abstract Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright © 2009 John Wiley & Sons, Ltd. [source] Construction of Polycation-Based Non-Viral DNA Nanoparticles and Polyanion Multilayers via Layer-by-Layer Self-Assembly,MACROMOLECULAR RAPID COMMUNICATIONS, Issue 20 2005Kefeng Ren Abstract Summary: The multilayers of polycation-based non-viral DNA nanoparticles and biodegradable poly(L -glutamic acid) (PGA) were constructed by a layer-by-layer (LbL) technique. Poly(ethyleneimine) (PEI) was used to condense DNA to develop non-viral DNA nanoparticles. AFM, UV-visible spectrometry, and TEM measurements revealed that the PEI-DNA nanoparticles were successfully incorporated into the multilayers. The well-structured, easily processed multilayers with the non-viral DNA nanoparticles may provide a novel approach to precisely control the delivery of DNA, which may have great potential for gene therapy applications in tissue engineering, medical implants, etc. A TEM image of the cross section of a (PGA/PEI-DNA nanoparticle)20 multilayer. [source] Lack of RNA,DNA oligonucleotide (chimeraplast) mutagenic activity in mouse embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2005Aristides D. Tagalakis Abstract There are numerous reports of the use of RNA,DNA oligonucleotides (chimeraplasts) to correct point mutations in vitro and in vivo, including the human apolipoprotein E gene (ApoE). Despite the absence of selection for targeting, high efficiency conversion has been reported. Although mainly used to revert deleterious mutations for gene therapy applications, successful use of this approach would have the potential to greatly facilitate the production of defined mutations in mice and other species. We have attempted to create a point mutation in the mouse ApoE gene by microinjection of chimeraplast into the pronuclei of 1-cell mouse eggs. Following transfer of microinjected eggs we analysed 139 E12.5 embryos, but obtained no evidence for successful conversion. Mol. Reprod. Dev. 71: 140,144, 2005. © 2005 Wiley-Liss, Inc. [source] Adenoviral vectors for gene transfer and therapyTHE JOURNAL OF GENE MEDICINE, Issue S1 2004Christoph Volpers Abstract Due to the very efficient nuclear entry mechanism of adenovirus and its low pathogenicity for humans, adenovirus-based vectors have become gene delivery vehicles that are widely used for transduction of different cell types, especially for quiescent, differentiated cells, in basic research, in gene therapy applications, and in vaccine development. As an important basis for their use as gene medicine, adenoviral vectors can be produced in high titers, they can transduce cells in vivo with transgenes of more than 30 kb, and they do not integrate into the host cell genome. Recent advances in the development of adenoviral vectors have brought considerable progress on issues like target cell specificity and tropism modification, long-term expression of the transgene, as well as immunogenicity and toxicity in vivo, and have suggested that the different generations of non-replicative and replicative vectors available today will each suit best for certain applications. Copyright © 2004 John Wiley & Sons, Ltd. [source] Tricalcium phosphate nanoparticles enable rapid purification, increase transduction kinetics, and modify the tropism of mammalian virusesBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Imke A.J. Dreesen Abstract Adenoviral, adeno-associated viral, and retroviral particles are chosen as gene delivery shuttles in more than 50% of all gene therapy clinical trials. Bulk availability of clinical-grade viral particles and their efficiency to transduce the therapeutic cargo into specific target cells remain the most critical bottlenecks in gene therapy applications to date. Capitalizing on the flame-spray technology for the reproducible economic large-scale production of amorphous tricalcium phosphate nanoparticulate powders (ATCP), we designed a scalable ready-to-use gravity-flow column set-up for the straightforward concentration and purification of transgenic adenoviral, adeno-associated viral, and lentiviral particles. Specific elution buffers enabled rapid release of viral particles from the ATCP matrix of the column and provided high-titer virus preparations in an unsurpassed period of time. The interaction of ATCP with adenoviral, adeno-associated viral, and lentiviral particles in solution increased the transduction kinetics of several mammalian cell lines in culture. The nanoparticles were also able to modify the tropism of murine leukemia virus (MLV) towards transduction of human cells. Based on these findings, we believe that the use of flame-spray tricalcium phosphate nanoparticles will lead to important progress in the development of future gene therapy initiatives. Biotechnol. Bioeng. 2009;102: 1197,1208. © 2008 Wiley Periodicals, Inc. [source] | |||||||||||||