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Ex Vivo Gene Therapy (ex + vivo_gene_therapy)
Selected AbstractsEfficient hepatocyte engraftment and long-term transgene expression after reversible portal embolization in nonhuman primates,HEPATOLOGY, Issue 3 2009Ibrahim Dagher The feasibility of ex vivo gene therapy as an alternative to liver transplantation for the treatment of liver metabolic diseases needs to be analyzed in large animal models. This approach requires appropriate gene transfer vectors and effective hepatocyte engraftment. Lentiviral vectors have the ability to transduce nondividing differentiated cells, such as hepatocytes, and portal vein occlusion increases hepatocyte engraftment. We investigated whether reversible portal vein embolization combined with ex vivo lentivirus-mediated gene transfer is an effective approach for successful hepatocyte engraftment in nonhuman primates and whether the transgene remains expressed in the long term in transplanted hepatocytes in situ. Simian hepatocytes were isolated after left lobe resection, and the left and right anterior portal branches of animals were embolized with absorbable material. Isolated hepatocytes were labeled with Hoechst dye or transduced in suspension with lentiviruses expressing green fluorescent protein under the control of the human apolipoprotein A-II promoter and transplanted via the inferior mesenteric vein. The whole procedure was well tolerated. The embolized liver was revascularized within 2 weeks. The volume of nonembolized liver increased from 38.7% ± 0.8% before embolization to 55.9% ± 1% after embolization and hepatocytes significantly proliferated (10.5% ± 0.4% on day 3 after embolization). Liver repopulation after transplantation with Hoechst-labeled hepatocytes was 7.4% ± 1.2%. Liver repopulation was 2.1% ± 0.2% with transduced hepatocytes, a proportion similar to that obtained with Hoechst-labeled cells, given that the mean transduction efficacy of simian hepatocyte population was 34%. Transgene expression persisted at 16 weeks after transplantation. Conclusion: We have developed a new approach to improve hepatocyte engraftment and to express a transgene in the long term in nonhuman primates. This strategy could be suitable for clinical applications. (HEPATOLOGY 2009.) [source] Ex vivo bone morphogenetic protein-2 gene delivery using gingival fibroblasts promotes bone regeneration in ratsJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 3 2010Joong-Ho Shin Shin J-H, Kim K-H, Kim S-H, Koo K-T, Kim T-I, Seol Y-J, Ku Y, Rhyu I-C, Chung C-P, Lee Y-M. Ex vivo bone morphogenetic protein-2 gene delivery using gingival fibroblasts promotes bone regeneration in rats. J Clin Periodontol 2009; 37: 305,311. doi: 10.1111/j.1600-051X.2009.01522.x. Abstract Aim: The aim of the present study was to investigate bone regeneration following ex vivo bone morphogenetic protein-2 (BMP-2) gene delivery using human gingival fibroblasts (HGFs) in rat calvarial defects. Materials and Methods: An 8 mm craniotomy defect was created in Sprague,Dawley rats. The animals were divided into four groups: (1) non-grafted group, the defect was left empty; (2) collagen matrix group, the defect was filled with collagen matrix only; (3) HGF group, the defect was filled with non-transduced HGFs on collagen matrix; (4) BMP-2/HGF group, the defect was filled with BMP-2 gene-transduced HGFs on collagen matrix. Animals were sacrificed at 2 and 4 weeks after surgery, and micro-computed tomographic and histologic observations were performed. Results: The BMP-2/HGF group showed promoted osseous healing of calvarial defects, as compared with the other groups. At both 2 and 4 weeks, regenerated bone area was significantly greater in the BMP-2/HGF group than the other three groups. Quite a few number of transplanted HGFs were observed within the regenerated bone tissues. Conclusions: The results of this study suggest that ex vivo BMP-2 gene delivery induces prominent bone regeneration in vivo and HGFs may be useful as target cells for ex vivo gene therapy. [source] Phage ,C31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell linesTHE JOURNAL OF GENE MEDICINE, Issue 5 2006Yoshinori Ishikawa Abstract Background X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor , chain (,c) gene. Although ex vivo gene therapy, i.e., transduction of the ,c gene into autologous CD34+ cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the ,C31 integrase-based integration system using human T-cell lines, including the ,c-deficient ED40515(-). Methods A ,C31 integrase and a neor gene expression plasmid containing the ,C31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a ,c expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced ,c in an ED40515(-)-derived clone. Results Following co-introduction of the ,C31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated ,c gene exhibited stable expression of the ,c protein, with normal IL-2 signaling, as assessed by STAT5 activation. Conclusions This study supports the possible future use of this ,C31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1. Copyright © 2006 John Wiley & Sons, Ltd. [source] Local ex vivo gene therapy with bone marrow stromal cells expressing human BMP4 promotes endosteal bone formation in miceTHE JOURNAL OF GENE MEDICINE, Issue 1 2004Xiao S. Zhang Abstract Background Bone loss in osteoporosis is caused by an imbalance between resorption and formation on endosteal surfaces of trabecular and cortical bone. We investigated the feasibility of increasing endosteal bone formation in mice by ex vivo gene therapy with bone marrow stromal cells (MSCs) transduced with a MLV-based retroviral vector to express human bone morphogenetic protein 4 (BMP4). Methods We assessed two approaches for administering transduced MSCs. ,-Galactosidase (,-Gal) transduced C57BL/6J mouse MSCs were injected intravenously via tail vein or directly injected into the femoral bone marrow cavity of non-marrow-ablated syngenic recipient mice and bone marrow cavity engraftment was assessed. BMP4- or ,-Gal-transduced cells were injected into the femoral bone marrow cavity and effects on bone were evaluated by X-ray, peripheral quantitative computed tomography (pQCT), and histology. Results After tail-vein injection less than 20% of recipient mice contained ,-Gal-positive donor cells in femur, humerus or vertebra marrow cavities combined, and in these mice only 0.02,0.29% of injected cells were present in the bone marrow. In contrast, direct intramedullary injection was always successful and an average of 2% of injected cells were present in the injected femur marrow cavity 24 hours after injection. Numbers of donor cells decreased over the next 14 days. Intramedullary injection of BMP4-transduced MSCs induced bone formation. Trabecular bone mineral density (BMD) determined by pQCT increased 20.5% at 14 days and total BMD increased 6.5% at 14 days and 10.4% at 56 days. Conclusions The present findings support the feasibility of using ex vivo MSC-based retroviral gene therapy to induce relatively sustained new bone formation, with normal histological appearance, at endosteal bone sites. Copyright © 2004 John Wiley & Sons, Ltd. [source] Determination of the baculovirus transducing titer in mammalian cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Zun-Ren Chan Abstract Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4,7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments. © 2005 Wiley Periodicals, Inc. [source] | |||||||||||||