Gene Transfer Efficiency (gene + transfer_efficiency)

Distribution by Scientific Domains

Selected Abstracts

The skin as a biofactory for systemic secretion of erythropoietin: potential of genetically modified keratinocytes and fibroblasts

Frank 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]

In vivo gene marking of rhesus macaque long-term repopulating hematopoietic cells using a VSV-G pseudotyped versus amphotropic oncoretroviral vector

Patricia 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]

Timing of Human Insulin-Like Growth Factor-1 Gene Transfer in Reinnervating Laryngeal Muscle,

Hideki Nakagawa MD
Abstract Objectives/Hypothesis The authors have designed a rat laryngeal paralysis model to study gene transfer strategies using a muscle-specific expression system to enhance local delivery of human insulin-like growth factor-1 (hIGF-1). In preliminary studies, a nonviral vector containing the ,-actin promoter and human hIGF-1 sequence produced both neurotrophic and myotrophic effects 1 month after single injection of plasmid formulation into paralyzed rat thyroarytenoid muscle in vivo. Based on these findings, it is hypothesized that the effects of hIGF-1 will enhance the results of laryngeal muscle innervation procedures. The timing of gene delivery relative to nerve repair is likely to be important, to optimize the results. Study Design Prospective analysis. Methods The effects of nonviral gene transfer for the delivery of hIGF-1 were evaluated in rats treated immediately following recurrent laryngeal nerve transection and repair and in rats receiving a delayed treatment schedule, 30 days after nerve transection and repair. Gene transfer efficiency was determined using polymerase chain reaction and reverse transcriptase,polymerase chain reaction techniques. Muscle fiber diameter, motor endplate length, and percentage of motor endplates with nerve contact were examined to assess hIGF-1 trophic effects. Results Compared with reinnervated untreated control samples, both early and delayed hIGF-1 transfer resulted in significant increase in muscle fiber diameter. Motor endplate length was significantly decreased and nerve/motor endplate contact was significantly increased following delayed gene transfer, but not after early treatment. Conclusion We infer from results of the study that delayed hIGF-1 gene transfer delivered by a single intramuscular injection will enhance the process of muscle reinnervation. The clinical relevance of these findings supports the future application of gene therapy using nonviral vectors for management of laryngeal paralysis and other peripheral nerve injuries. [source]

Characterisation of a P140K mutant O6 -methylguanine-DNA-methyltransferase (MGMT)-expressing transgenic mouse line with drug-selectable bone marrow

Belinda A. Kramer
Abstract Background Gene transfer of the P140K mutant of O6 -methylguanine-DNA-methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) provides a mechanism for drug resistance and the selective expansion of gene-modified cells in vivo. Possible clinical applications for this strategy include chemoprotection to allow dose escalation of alkylating chemotherapy, or combining MGMT(P140K) expression with a therapeutic gene in the treatment of genetic diseases. Our aim is to use MGMT(P140K)-driven in vivo selection to develop allogeneic micro-transplantation protocols that rely on post-engraftment selection to overcome the requirement for highly toxic pre-transplant conditioning, and to establish and maintain predictable levels of donor/recipient chimerism. Methods Using stably transfected murine embryonic stem (ES) cells, we have generated a C57BL/6 transgenic mouse line with expression of MGMT(P140K) within the hematopoietic compartment for use as a standard source of donor HSC in such models. Functional characterisation of transgene expression was carried out in chemotherapy-treated transgenic mice and in allogeneic recipients of transgenic HSC. Results Expression of the transgene provided chemoprotection and allowed in vivo selection of MGMT(P140K)-expressing cells in transgenic mice after exposure to O6 -benzylguanine (BG) and N,N,-bis(2-chloroethyl)- N -nitrosourea (BCNU). In an allogeneic transplant experiment in which transgenic HSC were engrafted into 129 strain recipients following low intensity conditioning (Busulfan, anti-CD8, anti-CD40Ligand), MGMT(P140K)-expressing cells could be selected using chemotherapy. Conclusions This MGMT(P140K) transgenic mouse line provides a useful source of drug-selectable donor cells for the development of non-myeloablative allogeneic transplant models in which variation in transplant conditioning elements can be investigated independently of gene transfer efficiency. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Novel two-stage screening procedure leads to the identification of a new class of transfection enhancers

Birgit Neukamm
Abstract Background Non-viral gene transfer efficiency is low as compared to viral vector systems. Here we describe the discovery of new drugs that are capable of enhancing non-viral gene transfer into mammalian cells using a novel two-stage screening procedure. Methods First, potential candidates are preselected from a molecular library at various concentrations by a semi-automated yeast transfection screen (YTS). The maximal transfection efficiency of every positive drug is subsequently determined in independent experiments at the optimal concentration and compared to the inhibitory effect of the drug on cell growth (IC50). In a subsequent mammalian cell transfection screen (MTS), the maximal transfection efficiency and the IC50 are determined for all preselected drugs using a human cell line and a luciferase reporter gene construct. Results Employing our novel system we have been able to identify a new class of transfection enhancers, the tricyclic antidepressants (i.e. doxepin, maprotiline, desipramine and amoxapine). All positive drugs enhanced gene transfer in both yeast and human cell lines, but lower concentrations were sufficient for mammalian cells. With a triple combination of doxepin, amoxapine and chloroquine we obtained a transfection efficiency that exceeded that of chloroquine, one of the best-known transfection enhancers of mammalian cells, by nearly one order of magnitude. Conclusions Non-viral gene transfer efficiency can be increased significantly using new transfection enhancers that are identified by a novel, semi-automated two-stage screening system employing yeast cells in the first and specific human target cells in the second round. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Deficiency of oncoretrovirally transduced hematopoietic stem cells and correction through ex vivo expansion

David Bryder
Abstract Background Extensive efforts to develop hematopoietic stem cell (HSC) based gene therapy have been hampered by low gene marking. Major emphasis has so far been directed at improving gene transfer efficiency, but low gene marking in transplanted recipients might equally well reflect compromised repopulating activity of transduced cells, competing for reconstitution with endogenous and unmanipulated stem cells. Methods The autologous settings of clinical gene therapy protocols preclude evaluation of changes in repopulating ability following transduction; however, using a congenic mouse model, allowing for direct evaluation of gene marking of lympho-myeloid progeny, we show here that these issues can be accurately addressed. Results We demonstrate that conditions supporting in vitro stem cell self-renewal efficiently promote oncoretroviral-mediated gene transfer to multipotent adult bone marrow stem cells, without prior in vivo conditioning. Despite using optimized culture conditions, transduction resulted in striking losses of repopulating activity, translating into low numbers of gene marked cells in competitively repopulated mice. Subjecting transduced HSCs to an ex vivo expansion protocol following the transduction procedure could partially reverse this loss. Conclusions These studies suggest that loss of repopulating ability of transduced HSCs rather than low gene transfer efficiency might be the main problem in clinical gene therapy protocols, and that a clinically feasible ex vivo expansion approach post-transduction can markedly improve reconstitution with gene marked stem cells. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactor

Dao 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]

An efficient targeted radiotherapy/gene therapy strategy utilising human telomerase promoters and radioastatine and harnessing radiation-mediated bystander effects

Marie Boyd
Abstract Background Targeted radiotherapy achieves malignant cell-specific concentration of radiation dosage by tumour-affinic molecules conjugated to radioactive atoms. Combining gene therapy with targeted radiotherapy is attractive because the associated cross-fire irradiation of the latter induces biological bystander effects upon neighbouring cells overcoming low gene transfer efficiency. Methods We sought to maximise the tumour specificity and efficacy of noradrenaline transporter (NAT) gene transfer combined with treatment using the radiopharmaceutical meta-[131I]iodobenzylguanidine ([131I]MIBG). Cell-kill was achieved by treatment with the ,-decay particle emitter [131I]MIBG or the ,-particle emitter [211At]MABG. We utilised our novel transfected mosaic spheroid model (TMS) to determine whether this treatment strategy could result in sterilisation of spheroids containing only a small proportion of NAT-expressing cells. Results The concentrations of [131I]MIBG and [211At]MABG required to reduce to 0.1% the survival of clonogens derived from the TMS composed of 100% of NAT gene-transfected cells were 1.5 and 0.004 MBq/ml (RSV promoter), 8.5 and 0.0075 MBq/ml (hTR promoter), and 9.0 and 0.008 MBq/ml (hTERT promoter), respectively. The concentrations of radiopharmaceutical required to reduce to 0.1% the survival of clonogens derived from 5% RSV/NAT and 5% hTERT/NAT TMS were 14 and 23 MBq/ml, respectively, for treatment with [131I]MIBG and 0.018 and 0.028 MBq/ml, respectively, for treatment with [211At]MABG. Conclusions These results indicate that the telomerase promoters have the capacity to drive the expression of the NAT. The potency of [211At]MABG is approximately three orders of magnitude greater than that of [131I]MIBG. Spheroids composed of only 5% of cells expressing NAT under the control of the RSV or hTERT promoter were sterilised by radiopharmaceutical treatment. This observation is indicative of bystander cell-kill. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Lactosylated polyethylenimine for gene transfer into airway epithelial cells: role of the sugar moiety in cell delivery and intracellular trafficking of the complexes

Stéphanie Grosse
Abstract Background As we have previously shown that lactosylated polyethylenimine (PEI) is the most efficient glycosylated PEI for gene transfer into human airway epithelial cells in primary culture, we have studied here the role of the lactose residue in the enhancement of gene transfer efficiency observed with lactosylated PEI as compared with unsubstituted PEI in immortalized (,CFTE29o- cells) and primary human airway epithelial cells. Methods and results After three transfections of 1 h performed daily, 60% of ,CFTE29o- cells were transfected with lactosylated PEI, whereas 25% of cells were transfected with unsubstituted PEI (p < 0.05). Cell viability was 1.8-fold greater with lactosylated PEI as compared with unsubstituted PEI (p < 0.05). As assessed by flow cytometry, the cellular uptake of lactosylated complexes was greater than that of complexes made with unsubstituted PEI (p < 0.05) and involved mostly a receptor-mediated endocytosis. The study of the intracellular trafficking in airway epithelial cells of complexes showed an endosomal and lysosomal accumulation of lactosylated complexes. In the presence of a proton pump inhibitor, the level of lactosylated and unsubstituted PEI-mediated gene expression was reduced more than 20-fold, whereas the cell viability increased to almost 100%. For both complexes, a nuclear localization was observed for less than 5% of intracellular complexes. Conclusions Our results show that the greater gene transfer efficiency observed for lactosylated complexes may be attributed to a higher amount of lactosylated complexes incorporated by airway epithelial cells and a lower cytotoxicity that might be related to reduced endosomolytic properties. However, the lactose residues substituting the PEI did not promote the entry of the plasmid into the nucleus. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Exploration of target molecules for prostate cancer gene therapy

THE PROSTATE, Issue 11 2007
Kazuhiro 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]