Current Issue : January - March Volume : 2013 Issue Number : 1 Articles : 7 Articles
Over the last 10-15 years, significant advances in vector design and delivery techniques have facilitated the development\r\nof nonviral approaches for the treatment of hemophilia. Despite these advancements, there remain several obstacles preventing\r\nthe successful application of these approaches in larger mammals such as dogs and humans. This review covers nonviral gene\r\ntherapy approaches using both in vivo gene delivery and ex vivo gene transfer. Plasmid-based approaches, as well as integrating\r\ntransposons are examined for efficacy, risks and limitations. Results are presented on the only human clinical trial in hemophilia that\r\nutilized nonviral approaches....
Ovarian cancer is the foremost cause of death from gynecological cancer in the developed world. In the USA\n27,000 new cases of ovarian cancer and 14,000 deaths are reported in 2010. About 80% of patients with ovarian\ncancer present with metastatic disease. The overall 5-year survival rate for women with cancer is 30%. The epithelial\ncells of the ovary constitute 1% of the total ovarian mass but constitute 90% of the ovarian neoplasms. Epithelial\novarian cancer (EOC) spreads initially by direct extensions into adjacent organs, especially the fallopian tubes, uterus\nand contralateral adnexa and occasionally the rectum, bladder and pelvic side wall. After direct extension, epithelial\novarian cancer frequently disseminates via transcoelmic route, with 70% of patients having peritoneal metastases\nat staging laparotomy. The correlation between molecular profiles and metastatic spread varies depending on\ntumor type and metastatic site and is combination of 2 models. First, tumors are genetically heterogeneous and\nthat metastases arise from clones with a genetically acquired metastatic phenotype and that the clonal genotype\ndetermines the final site of metastases. The second model is that metastatic cells are not a genetically primary tumor,\ninstead they arise as stochastic event, with a low but finite probability from tumor cell clones distinct from the primary\ntumor. Several cofactors, such as MMP-2/-9 inhibitor, TNF, lypmphotoxin a, Fas Ligand Fas L, APO3L, TRAIL,\ninterleukin -8 and P38 MAPK regulating ovarian cancer cells attachment to omentum and /or peritoneum have been\nidentified and would have noticeable clinical inhibition of the metastatic process, by enabling the identification of\ncellular or molecular targets that therapeutically viable. That would be able to block the steps necessary for ovarian\ncancer metastasis within the peritoneal cavity....
To evaluate the performance of the magnetic nanoparticles as gene transfer vector for breeding transgenic animals, we investigated\r\na new approach to deliver green fluorescent protein (GFP) gene to porcine kidney 15 (PK-15) and porcine embryonic\r\nfibroblast (PEF) cells using PEI-modified magnetic nanoparticles as gene vector. The morphology of the nanoparticles and\r\nnanoparticle/DNA complexes was characterized using scanning electron microscopy. It was found that the surface of the particles\r\nbecomes coarse and rough with increased average diameter, which implied the effective conjugating between nanoparticles\r\nwith DNA. The zeta potential of nanoparticle/DNA complexes drops down from +29.4mV to +23.1mV comparing with pure\r\nnanoparticles. Agarose gel electrophoresis experiments show that DNA plasmids can be protected effectively against degradation of\r\nexonuclease and endonuclease. The efficiency of gene delivery was affected by the mass ratio of nanoparticle/DNA and the amount\r\nof nanoparticle/DNA complexes. We confirm that the most optimal mass ratio of nanoparticle/DNA is 1 : 1 by conducting a\r\nseries of experiments. This work provides important experimental basis for the application of the magnetic nanoparticles on gene\r\ndelivery to porcine somatic cells, which is significant for the achieving of breeding new transgenic cloned pigs by using somatic\r\ncell nuclear transfer technique....
Background: Vascular endothelial growth factor (VEGF) is not only a potent angiogenic factor but it also promotes\r\naxonal outgrowth and proliferation of Schwann cells. The aim of the present study was to quantitatively assess\r\nreinnervation of musculocutaneous nerve (MCN) stumps using motor and primary sensory neurons after plasmid\r\nphVEGF transfection and end-to-end (ETE) or end-to-side (ETS) neurorrhaphy. The distal stump of rat transected\r\nMCN, was transfected with plasmid phVEGF, plasmid alone or treated with vehiculum and reinnervated following\r\nETE or ETS neurorrhaphy for 2 months. The number of motor and dorsal root ganglia neurons reinnervating the\r\nMCN stump was estimated following their retrograde labeling with Fluoro-Ruby and Fluoro-Emerald. Reinnervation\r\nof the MCN stumps was assessed based on density, diameter and myelin sheath thickness of regenerated axons,\r\ngrooming test and the wet weight index of the biceps brachii muscles.\r\nResults: Immunohistochemical detection under the same conditions revealed increased VEGF in the Schwann cells\r\nof the MCN stumps transfected with the plasmid phVEGF, as opposed to control stumps transfected with only the\r\nplasmid or treated with vehiculum. The MCN stumps transfected with the plasmid phVEGF were reinnervated by\r\nmoderately higher numbers of motor and sensory neurons after ETE neurorrhaphy compared with control stumps.\r\nHowever, morphometric quality of myelinated axons, grooming test and the wet weight index were significantly\r\nbetter in the MCN plasmid phVEGF transfected stumps. The ETS neurorrhaphy of the MCN plasmid phVEGF\r\ntransfected stumps in comparison with control stumps resulted in significant elevation of motor and sensory\r\nneurons that reinnervated the MCN. Especially noteworthy was the increased numbers of neurons that sent out\r\ncollateral sprouts into the MCN stumps. Similarly to ETE neurorrhaphy, phVEGF transfection resulted in significantly\r\nhigher morphometric quality of myelinated axons, behavioral test and the wet weight index of the biceps\r\nbrachii muscles.\r\nConclusion: Our results showed that plasmid phVEGF transfection of MCN stumps could induce an increase in\r\nVEGF protein in Schwann cells, which resulted in higher quality axon reinnervation after both ETE and ETS\r\nneurorrhaphy. This was also associated with a better wet weight biceps brachii muscle index and functional tests\r\nthan in control rats....
Introduction: Sj�¶grenâ��s syndrome (SjS) is a systemic autoimmune disease characterized by decreased salivary and\r\nlacrimal gland secretions, resulting in severe dry mouth and dry eyes. Recent studies have suggested that TH17\r\ncells and its signature cytokine IL-17 are involved in the underlying pathogenic mechanisms leading to destructive\r\ninflammation and autoimmunity. In the present study, we examined whether IL-27, a natural inhibitor of TH17\r\nactivity, could down-regulate or reverse SjS in C57BL/6.NOD-Aec1Aec2 mice, a model of primary-SjS.\r\nMethods: Recombinant serotype 2 adeno-associated viral (AAV2) vectors expressing either IL-27 (rAAV2-IL27) or\r\nLacZ (rAAV2-LacZ) were injected into 6 or 14 week-old C57BL/6.NOD-Aec1Aec2 mice. Changes in IL-27, IL-17, and\r\nIL-10 cytokine levels in peripheral blood were determined by ELISAs, while flow cytometry analyses were used to\r\nquantify cytokine-positive splenocytes. Histological assessment of salivary glands, anti-nuclear autoantibody (ANA)\r\nstaining, and stimulated saliva flow rates were used to profile SjS disease severity.\r\nResults: Mice systemically treated with intravenous rAAV2-IL27 injections at either 6 or 14 weeks of age exhibited\r\nlong-term elevated levels of serum IL-27 with concomitantly reduced levels of IL-17 compared with sera from mice\r\ninjected with rAAV2-LacZ or saline out to 20 weeks post-inoculation. Most importantly, disease profiles revealed\r\nthat rAAV2-IL27 treatment had little effect on lymphocytic focus (LF) scores, but resulted in structural changes in LF,\r\nlower titers of ANAs with changes in staining patterns, and a less severe clinical disease as determined by saliva\r\nflow rates.\r\nConclusions: These data support the concept that IL-27, when provided exogenously, can induce a suppressive\r\neffect on SjS development and thus may be an effective therapeutic agent for regulating TH17 pro-inflammatory\r\nactivity in autoimmune diseases where the TH17 system has been shown to play an important role in their\r\npathogenesis....
Hemophilia A and B are rare incurable hereditary diseases due to deficiencies in clotting factor VIII (FVIII)\r\nand factor IX (FIX), respectively. These genetic defects result in potentially life-threatening, uncontrolled bleeding\r\nepisodes. Current treatment by protein substitution therapy does not constitute a cure making gene therapy an\r\nattractive alternative. Lentiviral vectors (LVs) have many distinctive features that make them especially well suited for\r\nFVIII or FIX gene delivery. This includes the lack of vector-specific pre-existing immunity, their ability to permanently\r\ntransduce both dividing and non-dividing cells and their capacity to readily accommodate FIX and FVIII expression\r\ncassettes, consistent with their packaging capacity of 10 kb. LVs have been used to achieve sustained therapeutic\r\nclotting factor expression levels and hemostatic correction in preclinical hemophilic mouse models. The liver has been\r\nthe target organ of choice for direct in vivo LV transduction of FVIII or FIX genes, resulting in sustained therapeutic\r\neffects. Nevertheless, the potential development of neutralizing antibodies to the clotting factors following ex vivo\r\nor in vivo gene therapy with LVs can preclude long-term phenotypic correction. These risks can be minimized by\r\npreventing ectopic expression in antigen-presenting cells. LVs are well suited to deliver the clotting factor genes into\r\nhematopoietic stem/progenitor cells, allowing for stable FVIII or FIX expression upon hematopoietic reconstitution.\r\nIn addition, therapeutic FVIII and FIX expression levels have been achieved in vivo after transplantation of lentivirally\r\ntransduced endothelial and mesenchymal stem/progenitor cells. Current challenges relate primarily to the translation\r\nof these findings to larger preclinical animal models and ultimately to patients suffering from hemophilia....
X-linked severe combined immunodeficiency (SCID-X1), caused by a defect of the cytokine receptor common\r\ngamma chain (?c), has been successfully treated by gene therapy in the clinic. However, the occurrence of leukemia\r\nin several patients preceded by loss of oligoclonality revealed that treatment is associated with a risk inherent to\r\nthe genetic modification of hematopoietic stem cells. In this study, we developed a safety approach that allows\r\nthe specific elimination of gene-modified cells. For this, a small peptide sequence (myc-tag) was introduced into\r\nthe murine ?c protein. Cells expressing the modified chain can be detected with a myc-specific antibody by flow\r\ncytometry and are effectively depleted in vitro in the presence of complement factors. Further, thymic-derived T\r\ncells from mice reconstituted with myc-tagged ?c-transduced bone marrow stem cells can be depleted by antibody\r\nadministration in vivo. Similarly, specific complement-mediated lysis was observed for human T cells expressing\r\nthe human myc-tagged ?c. In a cell proliferation assay, the modified cytokine receptor chain showed no functional\r\nimpairment compared to the wild-type chain. In sum, we show proof-of-principle of a safety mechanism for SCID-X1\r\ngene therapy that would allow elimination of gene-corrected cells in a patient upon observation of monoclonal\r\noutgrowth....
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