Current Issue : October - December Volume : 2016 Issue Number : 4 Articles : 5 Articles
Background: Application of genetically modified bone marrow concentrates in articular cartilage lesions is a\npromising approach to enhance cartilage repair by stimulating the chondrogenic differentiation processes in sites\nof injury.\nMethod: In the present study, we examined the potential benefits of transferring the proliferative and\npro-chondrogenic basic fibroblast growth factor (FGF-2) to human bone marrow aspirates in vitro using the\nclinically adapted recombinant adeno-associated virus (rAAV) vectors to monitor the biological and chondrogenic\nresponses over time to the treatment compared with control (lacZ) gene application.\nResults: Effective, significant FGF-2 gene transfer and expression via rAAV was established in the aspirates relative\nto the lacZ condition (from ~ 97 to 36 pg rhFGF-2/mg total proteins over an extended period of 21 days).\nAdministration of the candidate FGF-2 vector led to prolonged increases in cell proliferation, matrix synthesis, and\nchondrogenesis but also to hypertrophic and terminal differentiation in the aspirates.\nConclusion: The present evaluation shows the advantages of rAAV-mediated FGF-2 gene transfer to conveniently\nmodify bone marrow concentrates as a future approach to directly treat articular cartilage lesions, provided that\nexpression of the growth factor is tightly regulated to prevent premature hypertrophy in vivo....
The herpes simplex virus thymidine kinase/ganciclovir (HSV TK/GCV) system is one of\nthe best studied cancer suicide gene therapy systems. Our previous study showed that caspase 3\nexpression was upregulated and bladder tumor growth was significantly reduced in rats treated\nwith a combination of Bifidobacterium (BF) and HSV TK/GCV (BF-rTK/GCV). However, it was raised\nwhether the BF-mediated recombinant thymidine kinase combined with ganciclovir (BF-rTK/GCV)\nwas safe to administer via venous for cancer gene therapy. To answer this question, the antitumor\neffects of BF-rTK/GCV were mainly evaluated in a xenograft nude mouse model bearing MKN-45\ngastric tumor cells. The immune response, including analysis of cytokine profiles, was analyzed to\nevaluate the safety of intramuscular and intravenous injection of BF-rTK in BALB/c mice. The results\nsuggested that gastric tumor growth was significantly inhibited in vivo by BF-rTK/GCV. However,\nthe BF-rTK/GCV had no effect on mouse body weight, indicating that the treatment was safe for\nthe host. The results of cytokine profile analysis indicated that intravenous injection of a low dose\nof BF-rTK resulted in a weaker cytokine response than that obtained with intramuscular injection.\nFurthermore, immunohistochemical analysis showed that intravenous administration did not affect\nthe expression of immune-associated TLR2 and TLR4. Finally, the BF-rTK/GCV inhibited vascular\nendothelial growth factor (VEGF) expression in mouse model, which is helpful for inhibiting of tumor\nangiogenesis. That meant intravenous administration of BF-rTK/GCV was an effective and safe way\nfor cancer gene therapy....
Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are\namong the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and\nnon-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce\nstable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a\nnon-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and\nthis SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression\nof the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without\nthe S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore,\nSouthern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion,\nthe minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that\nSNIL vectors have the potential for use as a gene transfer tool....
Transplantation of a donor cornea to restore vision is the most frequently performed transplantation\nin the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft\nas they maintain corneal transparency and avoid graft failure due to corneal opaqueness.\nGiven the characteristic of being a monolayer and in direct contact with culture medium during\ncultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches\nprior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent\na promising tool for gene therapy of CEC. However, high vector titers are needed to achieve\nsufficient gene expression. One of the rate-limiting steps for transgene expression is the\nconversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA.\nThis step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this\nstudy was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors\nin CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein\n(GFP) as transgene were used. Both in CEC and in donor corneas, transduction with\nscAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference\nin transduction efficiency decreased with increasing vector titer. In most cases, only\nhalf the vector titer of scAAV2 was required for equal or higher gene expression rates than\nthose of ssAAV2. In human donor corneas, GFP expression was 64.7�±11.3%(scAAV) and\n38.0�±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained\ntheir viability and showed regular morphology. Working together with regulatory authorities,\na translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of\ncorneal allografts during cultivation and transplantation could therefore become more\nrealistic....
Background: Gene therapy has the potential to provide long-term production of therapeutic proteins in the joints of\nosteoarthritis (OA) patients. The objective of this study was to analyse the therapeutic potential of disease-inducible\nexpression of anti-inflammatory interleukin-10 (IL-10) in the three-dimensional micromass model of the human\nsynovial membrane.\nMethods: Synovial tissue samples from OA patients were digested and the cells were mixed with Matrigel to obtain\n3D micromasses. The CXCL10 promoter combined with the firefly luciferase reporter in a lentiviral vector was used to\ndetermine the response of the CXCL10 promoter to tumour necrosis factor alpha (TNF-�±), interleukin-1�² (IL-1�²) and\nlipopolysaccharide (LPS). The effects of recombinant IL-10 on gene expression were determined by quantitative PCR.\nThe production of IL-10 from the CXCL10p-IL10 vector and the effects on pro-inflammatory cytokine production were\nassessed by multiplex ELISA.\nResults: Micromasses made from whole synovial membrane cell suspensions form a distinct surface composition\ncontaining macrophage and fibroblast-like synoviocytes thus mimicking the synovial lining. This lining can be\ntransduced by lentiviruses and allow CXCL-10 promoter-regulated transgene expression. Adequate amounts of IL-10\ntransgene were produced after stimulation with pro-inflammatory factors able to reduce the production of synovial\nIL-1�² and IL-6.\nConclusions: Synovial micromasses are a suitable model to test disease-regulated gene therapy approaches and\nthe CXCL10p-IL10 vector might be a good candidate to decrease the inflammatory response implicated in the\npathogenesis of OA....
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