MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'The Journal of Gene Medicine' source. Fri, 19 Mar 2010 17:01:42 +0100 http://www.medworm.com/index.php?rid=3369308&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1446 Targeted splice modulation of pre-mRNA transcripts by antisense oligonucleotides (AOs) can correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) arises as a result of mutations that interrupt the open-reading frame in the DMD gene encoding dystrophin such that dystrophin protein is absent, leading to fatal muscle degeneration. AOs have been shown to correct this dystrophin defect via exon skipping to yield functional dystrophin protein in animal models of DMD and also in DMD patients via intramuscular administration. To advance this therapeutic method requires increased exon skipping efficiency via an optimized AO sequence, backbone chemistry and additional modifications, and the improvement of methods for evaluating AO efficacy.In the present study, we ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3369308 Tue, 16 Mar 2010 00:00:00 +0100 3369308 http://www.medworm.com/index.php?rid=3365515&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1445 DNA vaccines ensure protective immunity against tumors in a variety of experimental models. The favorite target tumor-associated antigens have been those that are frequently expressed by human tumors, such as Her2. However, the efficacy of active vaccination is limited because Her2 is a self-tolerated antigen. Many strategies have been applied to increase the efficacy of DNA vaccination, such as fusion or co-administration of Her2 with cytokine and co-stimulatory molecules. GP96 is involved in innate and adaptive immune responses and evokes potent activation and maturation of dendritic cells along with increased secretion of pro-inflammatory cytokines. On the basis of previous studies, we expected the C-terminal of GP96 to act as a package and as a suitable substitute for both cytokine and... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3365515 Mon, 15 Mar 2010 00:00:00 +0100 3365515 http://www.medworm.com/index.php?rid=3334084&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1441 Nonviral gene therapy requires a high yield and a low cost production of eukaryotic expression vectors that meet defined criteria such as biosafety and quality of pharmaceutical grade. To fulfil these objectives, we designed a novel antibiotic-free selection system.The proposed strategy relies on the suppression of a chromosomal amber mutation by a plasmid-borne function. We first introduced a nonsense mutation into the essential Escherichia coli thyA gene, resulting in thymidine auxotrophy. The bacterial strain was optimized for the production of small and novel plasmids free of antibiotic resistance markers (pFARs) and encoding an amber suppressor t-RNA. Finally, the potentiality of pFARs as eukaryotic expression vectors was assessed by monitoring luciferase activities after electrotrans... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3334084 Fri, 05 Mar 2010 00:00:00 +0100 3334084 http://www.medworm.com/index.php?rid=3334083&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1442 Major complications with respect to the development of gene therapy treatments for hemophilia A include low factor VIII (fVIII) expression and humoral immune responses resulting in inhibitory anti-fVIII antibodies. We previously achieved sustained curative fVIII activity levels in hemophilia A mice after nonmyeloablative transplantation of genetically-modified hematopoietic stem cells (HSCs) encoding a B-domain deleted porcine fVIII (BDDpfVIII) transgene with no evidence of an immune response.Mouse HSCs were transduced using MSCV-based recombinant virus encoding BDDpfVIII and transplanted into hemophilia A mice. Transplanted mice were followed for donor cell engraftment, fVIII expression and activity, and generation of anti-fVIII immune response.We now show that: (i) the protein expressed ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3334083 Fri, 05 Mar 2010 00:00:00 +0100 3334083 http://www.medworm.com/index.php?rid=3300991&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1443 No Abstract. (Source: The Journal of Gene Medicine) The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3300991 Wed, 24 Feb 2010 16:29:17 +0100 3300991 http://www.medworm.com/index.php?rid=3300993&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1444 No Abstract. (Source: The Journal of Gene Medicine) The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3300993 Wed, 24 Feb 2010 00:00:00 +0100 3300993 http://www.medworm.com/index.php?rid=3300992&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1440 We recently expressed a potent and noncytotoxic short hairpin (sh)RNA directed against chemokine (c-c motif) receptor 5 (CCR5) using lentiviral mediated transduction of CD34+ hematopoietic progenitor cells (HPCs) and demonstrated the stable reduction of CCR5 expression in T-lymphocytes.In the present study, we further assessed the activity of the shRNA through HPC transduction and differentiation into macrophages derived from fetal liver CD34+ (FL-CD34+) HPCs. Transduced lentiviral vector encoding the human CCR5 shRNA was stably maintained in FL-CD34+ cells and in the terminally differentiated macrophages using macrophage colony-stimulating factor, granulocyte macrophage colony-stimulating factor, interleukin-3 and stem cell factor.Quantitative real-time polymerase chain reaction for CCR5 ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3300992 Wed, 24 Feb 2010 00:00:00 +0100 3300992 http://www.medworm.com/index.php?rid=3250280&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1439 [beta]2m-/Thy1+ bone marrow-derived hepatocyte stem cells (BDHSCs) isolated from the bone marrow of cholestatic rats by magnetic bead cell sorting consistently express characteristics of both stem and liver cells. These stem cells may be good vehicles for gene transfer. Administration of exogenous hepatocyte growth factor (HGF) may be potentially useful for the treatment of liver fibrosis. Because lentiviral vectors integrate stably into the host-cell genome of nondividing and dividing cells, it may efficiently transfect [beta]2m-/Thy1+ BDHSCs in vitro and secrete high-level HGF consistently. Transplantation of [beta]2m-/Thy1+ BDHSCs transduced with lentiviral vectors containing the HGF gene may reduce liver fibrosis in rats.Lentiviral vectors expressing HGF were constructed and used to tr... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3250280 Mon, 08 Feb 2010 00:00:00 +0100 3250280 http://www.medworm.com/index.php?rid=3246120&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1436 Dendritic cells (DCs) constitute an attractive target for immunotherapeutic approaches. Because DCs are largely refractory to transfection with plasmid DNA, several viral transduction protocols were established. The potential side-effects of lentiviral transduction on the phenotype and activation state of DCs left unstimulated after transduction have not been assessed. There is a need to analyse these parameters as a result of the requirement of using DCs with a low activation state for therapeutic strategies intended to induce tolerance.Lentivirally-transduced bone marrow (BM)-derived DCs (LV-DCs) in comparison with mock-transduced (Mock-DCs) and untreated DCs were analysed with regard to the induction of maturation processes on the RNA, protein and functional level. BM-DCs engineered to ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3246120 Sat, 06 Feb 2010 00:00:00 +0100 3246120 http://www.medworm.com/index.php?rid=3238568&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1435 Beta-defensin-2 (BD-2) plays an important role in host defense against pathogenic microbe challenge by its direct antimicrobial activity and immunomodulatory functions. The present study aimed to determine whether genetic up-regulation of rat BD-2 (rBD-2) could ameliorate chronic Pseudomonas aeruginosa lung infection in rats.Plasmid-encoding rBD-2 was delivered to lungs in vivo using linear polyethylenimine at 48 h before challenging with seaweed alginate beads containing P. aeruginosa. Macroscopic and histopathological changes of the lungs, bacterial loads, inflammatory infiltration, and the levels of cytokines/chemokines [interleukin (IL)-1[beta], tumor necrosis factor (TNF)-[alpha], kertinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2)] were measured at 3 and 7 d... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3238568 Thu, 04 Feb 2010 00:00:00 +0100 3238568 http://www.medworm.com/index.php?rid=3181597&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1432 The antigen-specific immune response is dependent not only on the properties of the antigens, but also on their encounter with antigen-presenting cells. A previous study showed that the spleen produced a large amount of transgenes after direct tissue injection of plasmid DNA. In addition, the spleen is the largest organ in the lymphatic system and contains a variety of types of immune cells, including lymphocytes, macrophages and dendritic cells. Thus, it can be a promising target for DNA vaccination.Tissue-dependent properties of transgene expression were examined using a plasmid vector expressing firefly luciferase. Mice received injections of pCMV-Luc into the dorsal skin or spleen followed by electroporation, and the luciferase activity was measured 6 h after injection. Then, plasmids ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3181597 Mon, 18 Jan 2010 00:00:00 +0100 3181597 http://www.medworm.com/index.php?rid=3178074&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1433 Efficient adenovirus (AdV)-mediated gene transfer is possible only in immature muscle or regenerating muscle, suggesting that a developmentally regulated event plays a major role in limiting AdV uptake in mature skeletal muscle. Previously, we showed that the expression of the primary coxsackie and adenovirus receptor (CAR) is severely down-regulated during muscle maturation and that, in muscle-specific CAR transgenic mice, there is significant enhancement of AdV-mediated gene transfer to mature skeletal muscle.To evaluate whether increasing CAR expression can also augment gene transfer to dystrophic muscle that has many regenerating fibers, we crossed CAR transgenics with dystrophin-deficient mice (mdx/CAR). We also tested a two-step protocol in which CAR levels were increased in the targ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3178074 Sat, 16 Jan 2010 00:00:00 +0100 3178074 http://www.medworm.com/index.php?rid=3178076&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1425 The objectives of the present study were to fully characterize and optimize the laminin-DNA-apatite composite layer in relation to the efficiency of gene transfer and to demonstrate the feasibility of the composite layer in the induction of cell differentiation.The laminin-DNA-apatite composite layer was prepared under various conditions. The efficiency of gene transfer on the resulting composite layer was evaluated using luciferase and ß-galactosidase gene expression assay systems. A laminin-DNA-apatite composite layer, prepared under the optimized condition using a plasmid including cDNA of nerve growth factor (NGF), was then applied to the neuron-like differentiation of PC12 cells.The laminin content of the laminin-DNA-apatite composite layer was found to be a dominant factor improving... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3178076 Fri, 15 Jan 2010 00:00:00 +0100 3178076 http://www.medworm.com/index.php?rid=3178075&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1426 Mesenchymal stem cells (MSC) are widely regarded as a promising tool for cellular therapy applications, and genetic modification by safe, liposome-based vectors may enhance their therapeutic potential.The present study describes the use of a cationic lipid vector (Lipofectamine 2000TM) to deliver genes to MSC isolated from a number of species in vitro and determined the characteristics of this vector system in terms of dose, toxicity and the time course of expression. In addition, the optimal use of a nuclear localization sequence (NLS) to enhance gene expression was explored.Lipofection of human MSC did not adversely affect their ability to differentiate into osteogenic- and adipogenic lineages. Although human and rat MSC were found to take up lipoplexes with relative efficiency, lower le... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3178075 Fri, 15 Jan 2010 00:00:00 +0100 3178075 http://www.medworm.com/index.php?rid=3170418&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1434 Vascular endothelial growth factor (VEGF) has mostly been tested to treat ischemic diseases, although the outcomes obtained are not satisfactory. Our hypothesis is that the local transient expression of VEGF and stem cell mobilizer granulocyte colony-stimulating factor (G-CSF) genes in ischemic limbs can complement their activities and be more efficient for limb recovery.Limb ischemia was surgically induced in mice and 50 µg of VEGF and/or G-CSF genes were locally transferred by electroporation. After 3-4 weeks, evidence of necrosis by visual inspection, capillary density, muscle mass, muscle force and hematopoietic cell mobilization were evaluated.After 4 weeks, 70% and 90% of the animals of the ischemic group (IG) and VEGF-treated group (VG), respectively, presented limb necrosis, in co... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3170418 Thu, 14 Jan 2010 00:00:00 +0100 3170418 http://www.medworm.com/index.php?rid=3144904&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1431 RNA interference is a powerful method for the knockdown of pathologically relevant genes. The in vivo delivery of siRNAs, preferably through systemic, nonviral administration, poses the major challenge in the therapeutic application of RNAi. Small interfering RNA (siRNA) complexation with polyethylenimines (PEI) may represent a promising strategy for siRNA-based therapies and, recently, the novel branched PEI F25-LMW has been introduced in vitro. Vascular endothelial growth factor (VEGF) is frequently overexpressed in tumors and promotes tumor growth, angiogenesis and metastasis and thus represents an attractive target gene in tumor therapy.In subcutaneous tumor xenograft mouse models, we established the therapeutic efficacy and safety of PEI F25-LMW/siRNA-mediated knockdown of VEGF. In bi... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3144904 Wed, 06 Jan 2010 00:00:00 +0100 3144904 http://www.medworm.com/index.php?rid=3144906&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1429 An improvement of retroviral infection has been postulated using a naturally occurring fragment of the abundant semen marker prostatic acidic phosphatase. This peptide, termed semen-derived enhancer of virus infection (SEVI), promotes HIV attachment to the target cells.In the present study, we examined whether SEVI would also enhance the infectivity of other viruses with different envelope proteins. We focused on retroviruses pseudotyped with envelopes that are commonly used for the genetic modification of cells, in particular, T cells and hematopoietic progenitor cells. Because the effect of SEVI is considered to be a result of its cationic properties, we compared SEVI with other cationic agents such as protamine sulfate and Polybrene.We found that SEVI increases the efficiency of gene tr... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3144906 Tue, 05 Jan 2010 00:00:00 +0100 3144906 http://www.medworm.com/index.php?rid=3144905&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1430 HD O is a low molecular weight pseudodendrimer containing oligoethylenimine and degradable hexanediol diacrylate diesters. DNA polyplexes display encouraging gene transfer efficiency in vitro and in vivo but also a limited stability under physiological conditions. This limitation must be overcome for further development into more sophisticated formulations.HD O polyplexes were laterally stabilized by crosslinking surface amines via bifunctional crosslinkers, bioreducible dithiobis(succimidyl propionate) (DSP) or the nonreducible analog disuccinimidyl suberate (DSS). Optionally, in a subsequent step, the targeting ligand transferrin (Tf) was attached to DSP-linked HD O polyplexes via Schiff base formation between HD O amino groups and Tf aldehyde groups, which were introduced into Tf by per... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3144905 Tue, 05 Jan 2010 00:00:00 +0100 3144905 http://www.medworm.com/index.php?rid=3113983&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1421 HIV-1 fails to successfully infect mouse T cells as a result of several blocks in the viral replication cycle. We investigated whether this also impacted on the use of HIV-1 derived lentiviral vectors for stable gene transfer into mouse T cells.Freshly isolated primary mouse T cells were immediately mixed with lentiviral vectors encoding an enhanced green fluorescent protein marker gene and transduction frequency was determined after 5 days of culture.Optimal transduction required both mouse T cell activation and cytokine support. Furthermore, transduction was also dependent upon the promoter chosen, with the rank order of potency being PGK > EF1 > SFFV > CMV. HIV-1 lentiviral vectors also efficiently transduced cytokine-stimulated T cells (in the absence of antibody driven T cell activati... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3113983 Wed, 23 Dec 2009 00:00:00 +0100 3113983 http://www.medworm.com/index.php?rid=3095608&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1424 Inhibiting MDM2 activity in tumors that express wild-type (wt) p53 but have high levels of MDM2 protein has been considered an attractive anticancer strategy for many years. Previous studies revealed that human ribosomal protein L23 (RPL23) inhibited MDM2-mediated p53 degradation and thus induced p53 levels as well as its activity, suggesting that it might be a candidate for use as a gene therapy for cancer. In the present study, we evaluated whether targeting this pathway could be of therapeutic value against human gastric carcinoma.Gastric cancer cell lines carrying wt or mutant p53 gene were infected with adenovirus expressing RPL23 (Ad-RPL23). Cell growth assay, flow cytometry assay and morphology were used to observe the effects of Ad-RPL23 infection on tumor cells, and further, the e... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3095608 Thu, 17 Dec 2009 00:00:00 +0100 3095608 http://www.medworm.com/index.php?rid=3038911&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1378 Drug resistance mediated by P-glycoprotein (P-gp) is one of the major reasons for the failure of rheumatoid arthritis (RA) therapy with disease modifying anti-rheumatic drugs and glucocorticoids. In the present study, we aimed to investigate the in vitro effectiveness of small interfering RNA (siRNA) to render rat fibroblast-like synoviocytes (FLS) susceptible to drugs. We also attempted the electroporation-mediated transfer of siRNA against multidrug resistance (MDR) genes into rat knee joints.FLS were transfected with siRNAs corresponding to MDR1a and MDR1b genes. FLS were treated with dexamethasone (DEX) and lipopolysaccharide. The mRNA and protein levels of tumor necrosis factor-[alpha], interleukin (IL)-6 and IL-1[beta] were measured. Both siRNAs were co-transduced into rat knee joint... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3038911 Mon, 30 Nov 2009 00:00:00 +0100 3038911 http://www.medworm.com/index.php?rid=3038910&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1412 Various approaches have been used to improve the efficacy of DNA vaccination, including the incorporation of molecular adjuvants. Because the CD40 ligand-CD40 interaction plays a major role in initiating immune responses, we sought to develop a molecular adjuvant targeting this interaction.We immunized mice with a foot-and-mouth disease virus DNA vaccine, pcD-VP1, together with a CD40-expressing plasmid, pcD-CD40. We found that pcD-CD40 induced anti-CD40 antibodies, which temporally correlated with the augmented production of anti-VP1 antibody. pcD-CD40 similarly augmented the humoral response of another DNA vaccine that targets hepatitis B virus, and passive transfer of anti-CD40 antisera also showed a similar effect. Furthermore, the pcD-CD40-elicited anti-CD40 antibodies were able to ac... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3038910 Mon, 30 Nov 2009 00:00:00 +0100 3038910 http://www.medworm.com/index.php?rid=3032936&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1418 Gene therapy has been used to treat a variety of health problems, but transfection inefficiency and the lack of safe vectors have limited clinical progress. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapy. The present study aimed to synthesize chitosan-alginate nanoparticles that can be used as carriers of the pAcGFP1-C1 plasmid and to use these nanoparticles with an ultrasound protocol to achieve high efficiency gene transfection.Chitosan was complexed with alginate and the pAcGFP1-C1 plasmid at different charge ratios to create chitosan-alginate-DNA nanoparticles (CADNs). The average particle size and loading efficiency were measured. Plasmid DNA retardation and integrity were analysed on 1% agarose gels.... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3032936 Fri, 27 Nov 2009 00:00:00 +0100 3032936 http://www.medworm.com/index.php?rid=3025532&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1414 Electroporation is a physical method used to transfer molecules into cells and tissues. Clinical applications have been developed for antitumor drug delivery. Clinical trials of gene electrotransfer are under investigation. However, knowledge about how DNA enters cells is not complete. By contrast to small molecules that have direct access to the cytoplasm, DNA forms a long lived complex with the plasma membrane and is transferred into the cytoplasm with a considerable delay.To increase our understanding of the key step of DNA/membrane complex formation, we investigated the dependence of DNA/membrane interaction and gene expression on electric pulse polarity and repetition frequency.We observed that both are affected by reversing the polarity and by increasing the repetition frequency of p... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3025532 Wed, 25 Nov 2009 00:00:00 +0100 3025532 http://www.medworm.com/index.php?rid=3025531&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1416 Radiation-induced salivary hypofunction is a common side-effect of treatment for head and neck cancers. Patients suffer significant morbidity and there is no suitable conventional therapy. We are conducting a Phase I clinical trial, using a first-generation serotype 5 adenoviral (Ad5) vector encoding human aquaporin-1 (AdhAQP1) to treat such patients. One week after the administration of AdhAQP1 to an enrolled, generally healthy patient, E1-containing adenovirus was detected in parotid saliva.The real-time quantitative polymerase chain reactuion (PCR) was used to measure the Ad5 E1 gene and AdhAQP1 in saliva and serum. PCR and sequencing were used to characterize viral/vector DNA extracted from saliva. The presence of infectious adenovirus was assessed by the inoculation of A549 cells with... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3025531 Wed, 25 Nov 2009 00:00:00 +0100 3025531 http://www.medworm.com/index.php?rid=3025535&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1413 When assessing the efficacy of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy, we routinely evaluate gene transfer in the mouse nose and measure transfection efficiency by assessing transgene-specific mRNA using the real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction. TaqMan is traditionally used to quantify expression in whole tissue homogenates, which in the nose would contain many cells types, including respiratory and olfactory epithelium. Only the respiratory epithelium is a satisfactory model for human airway epithelium and therefore CFTR gene transfer should be specifically assessed in respiratory epithelial cells (RECs).We have compared laser microdissection, pronase digestion and nasal brushing for: (i) the ability to enrich RECs from... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3025535 Tue, 24 Nov 2009 00:00:00 +0100 3025535 http://www.medworm.com/index.php?rid=3025534&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1415 We have previously shown that a novel synthetic peptide for ocular delivery (POD) can efficiently compact DNA and deliver it to cells in vitro. This observation prompted us to develop use of POD as a nonviral vector in vivo.POD peptide was modified using poly(ethylene) glycol (PEG-POD) and used to compact DNA into nanoparticles that were then analysed using electron microscopy, dynamic light scattering, and fluorescent labeling. Transfection efficiency and localization were determined 48 h post-injection into the subretinal space of the mouse eye using luciferase and LacZ, respectively. Efficiency of ocular transfection was compared to two other PEGylated peptides: PEG-TAT and PEG-CK30.PEG-POD can compact DNA and form discrete nanoparticles of approximately 136 nm that can penetrate and tr... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3025534 Tue, 24 Nov 2009 00:00:00 +0100 3025534 http://www.medworm.com/index.php?rid=3025533&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1417 We have previously developed lipopolythiourea lipids as neutral DNA condensing agents for systemic gene delivery. Optimization of the lipopolythiourea structure led to efficient transfecting agents. To further evaluate these lipids, we investigated the internalization process of the thiourea lipoplexes and their intracellular mechanism of transfection versus that of cationic lipoplexes.The MTT test was used for cytotoxicity assessment. Transfection efficiency was determined by luciferase read-out. Permeation to propidium iodide and enhanced green fluorescent protein was evaluated by flow cytometry. Kinetics of internalization and DNA release were monitored by confocal microscopy with labelled DNA. Endocytosis inhibitors were used to study the mechanisms of lipoplex internalization.Although... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=3025533 Tue, 24 Nov 2009 00:00:00 +0100 3025533 http://www.medworm.com/index.php?rid=2957867&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1406 The nuclear envelope that encloses the nucleus is a significant barrier to non-viral vectors and shrouds the relationship between the trafficking of plasmid DNA to the nucleus and expression of an encoded transgene. Here, we use a novel single cell approach to quantify nuclear import of plasmid DNA following non-viral transfection and correlate this with reporter gene expression.Through the fractionation of intact nuclei from HeLa cells, the intranuclear copy number of plasmid DNA was quantified after transfection with either polyethylenimine (PEI) or LipofectAMINE2000 (LFA). Importantly, the use of a reporter protein that is incorporated into chromatin and retained in isolated nuclei permits analysis of gene expression by flow cytometry to be compared with nuclear plasmid delivery.PEI was... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2957867 Wed, 04 Nov 2009 00:00:00 +0100 2957867 http://www.medworm.com/index.php?rid=2933532&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1407 In the present study, we evaluated the safety of CIGB-230, a novel vaccine candidate based on the mixture of a plasmid for DNA immunization, expressing hepatitis C virus (HCV) structural antigens, with a recombinant HCV Core protein.Fifteen HCV chronically-infected volunteers with detectable levels of HCV RNA genotype 1b, who were nonresponders to previous treatment with interferon plus ribavirin, were intramuscularly injected with CIGB-230 on weeks 0, 4, 8, 12, 16 and 20. Individuals were also immunized at weeks 28, 32 and 36 with a recombinant vaccine against hepatitis B. Adverse events were recorded and analyzed. Blood samples were taken every 4 weeks up to month 12 for hematological, biochemical, virological and immunological analysis.All patients completed the treatment with CIGB-230.... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2933532 Wed, 28 Oct 2009 00:00:00 +0100 2933532 http://www.medworm.com/index.php?rid=2933531&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1408 Asthma is a chronic disease characterized by airway inflammation caused by the dysregulated production of cytokines secreted by allergen-specific type 2 T helper (Th2) cells. Although the Th1-promoting cytokine, interleukin (IL)-12, is capable of inhibiting Th2-driven allergen-induced airway changes in mice, IL-12 also aggravates the Th1-driven inflammatory pulmonary pathology. Further, IL-10 was found to exert both anti-inflammatory and immunoregulatory activities. To avoid the side-effects of IL-12, we hypothesized that the low-dose expression of IL-10 with concomitant IL-12 administration in the airway may represent a more effective therapy for allergic airway diseases. Thus, the present study explored the immunomodulatory and therapeutic effects of IL-10 combined with IL-12 in airway i... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2933531 Wed, 28 Oct 2009 00:00:00 +0100 2933531 http://www.medworm.com/index.php?rid=2933530&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1409 Rheumatoid arthritis (RA) is a complex disease characterized by autoimmune inflammation and joint destruction. Despite recent advances in RA treatment, current therapies require further improvement to overcome adverse events and ineffectiveness in some cases. By targeting different pathways/molecules using drug combinations, a better treatment can be obtained, whereas adverse events are reduced. In order to develop a new treatment option, the present study employs a gene therapy-based combination therapy using doxycycline and human alpha-1 antitrypsin (hAAT).DBA/1 mice were immunized with type II collagen to induce arthritis. Four weeks before immunization, they received a doxycycline containing diet and a single injection of adeno-associated virus vector expressing hAAT under the control ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2933530 Wed, 28 Oct 2009 00:00:00 +0100 2933530 http://www.medworm.com/index.php?rid=2907683&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1405 The application of polyethylenimine (PEI) in gene delivery has been severely limited by significant cytotoxicity that results from a nondegradable methylene backbone and high cationic charge density. It is therefore necessary to develop novel biodegradable PEI derivates for low-toxic, highly efficient gene delivery.A series of novel cationic copolymers with various charge density were designed and synthesized by grafting different kinds of oligoethylenimine (OEI) onto a determinate multi-armed poly(L-glutamic acid) backbone. The molecular structures of multi-armed poly(L-glutamic acid)-graft-OEI (MP-g-OEI) copolymers were characterized using nuclear magnetic resonance, viscosimetry and gel permeation chromatography. Moreover, the MP-g-OEI/DNA complexes were measured by a gel retardation as... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2907683 Mon, 19 Oct 2009 23:00:00 +0100 2907683 http://www.medworm.com/index.php?rid=2893464&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1404 Cardiac gene transfer may serve as a novel therapeutic approach for heart disease. Numerous serotypes of recombinant adeno-associated virus (rAAV) have been identified with variable tropisms to cardiac tissue.Both in vitro and in vivo experiments were undertaken to compare cardiac tropisms of rAAV-2, 5, 7, 8 and 9. For the in vitro studies, 107 vector genome (vg) of rAAV-2, 5, 7, 8 or 9 were used to transduce both rat neonatal cardiac myocytes (RNCM) and fibroblasts (RNCF). For the in vivo studies, 4 × 1010 vg of rAAV-2, 5, 7, 8 or 9, and 4 × 1011 vg of rAAV8 or 9 were administered in 5-day-old rats via a relatively non-invasive intracardiac injection. One and two months post-administration, green fluorescent protein (GFP) expression in tissues was visualized and GFP mRNA was quantified ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2893464 Wed, 14 Oct 2009 23:00:00 +0100 2893464 http://www.medworm.com/index.php?rid=2822997&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1403 The present study aimed to evaluate the antitumor effectiveness of systemic interleukin (IL)-12 gene therapy in murine sarcoma models, and to evaluate its interaction with the irradiation of tumors and metastases. To avoid toxic side-effects of IL-12 gene therapy, the objective was to achieve the controlled release of IL-12 after intramuscular gene electrotransfer.Gene electrotransfer of the plasmid pORF-mIL12 was performed into the tibialis cranialis in A/J and C57BL/6 mice. Systemic release of the IL-12 was monitored in the serum of mice after carrying out two sets of intramuscular IL-12 gene electrotransfer of two different doses of plasmid DNA. The antitumor effectiveness of IL-12 gene electrotransfer alone or in combination with local tumor or lung irradiation with X-rays, was evaluat... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2822997 Tue, 22 Sep 2009 23:00:00 +0100 2822997 http://www.medworm.com/index.php?rid=2822996&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1392 Genetic modification of capsid proteins by peptide insertion has created the possibility of using adeno-associated viral (AAV) vectors for receptor specific gene transfer (AAV targeting). The most common site used for insertion in AAV serotype 2 capsids are amino acid positions 587 and 588 located at the second highest capsid protrusion. Reasoning that peptide insertions at the most exposed position augments target receptor interaction, we explored position 453 as a new insertion site.Position 453 was identified in silico. Capsid mutants carrying the model ligand RGD-4C in position 453 with and without R585A/R588A substitutions were compared with respective mutants carrying the ligand in position 587. The accessibility of the inserted ligand was determined by an enzyme-linked immunosorbent... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2822996 Tue, 22 Sep 2009 23:00:00 +0100 2822996 http://www.medworm.com/index.php?rid=2822995&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1401 Gene therapy represents a new treatment paradigm for HIV that is potentially delivered by a safe, once-only therapeutic intervention.Using mathematical modelling, we assessed the possible impact of autologous haematopoietic stem cell (HSC) delivered, anti-HIV gene therapy. The therapy comprises a ribozyme construct (OZ1) directed to a conserved region of HIV-1 delivered by transduced HSC (OZ1+HSC). OZ1+HSC contributes to the CD4+ T lymphocyte and monocyte/macrophage cell pools that preferentially expand under the selective pressure of HIV infection. The model was used to predict the efficacy of OZ1 in a highly active antiretroviral therapy (HAART) naïve individual and a HAART-experienced individual undergoing two structured treatment operations. In the standard scenario, OZ1+HSC was taken... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2822995 Tue, 22 Sep 2009 23:00:00 +0100 2822995 http://www.medworm.com/index.php?rid=2822994&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1402 Adeno-associated virus (AAV) is an ideal gene therapy vector and is non-immunogenic in many small animal models. The stable gene expression commonly seen in murine models does not necessarily translate to nonhuman primates and higher-order species, highlighting the need for a better understanding of immune activation to these vectors. One capsid variant, AAVrh32.33, demonstrates a unique phenotype in murine muscle, reminiscent of what is often seen in higher-order species. AAVrh32.33 generates a strong CD8+ T-cell response to both capsid and encoded transgene antigens in a manner independent of transgene product or major histocompatability complex haplotype, making it an ideal candidate for studying immune activation to AAV in the mouse.To map the H-2b and H-2d dominant epitopes of the AAV... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2822994 Tue, 22 Sep 2009 23:00:00 +0100 2822994 http://www.medworm.com/index.php?rid=2795635&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1390 Baculovirus, which is widely utilized as an excellent tool for the production of recombinant protein in insect cells, has recently emerged as a novel and attractive gene delivery vehicle for mammalian cells. Alphavirus, such as Semliki Forest virus (SFV), has also received considerable attention for use as expression vectors because of its self-replicating property. In the present study, we investigated the characterization of recombinant baculovirus incorporating a hybrid cytomegalovirus (CMV) promoter/SFV replicon.Recombinant baculovirus containing the hybrid CMV promoter/SFV replicon was constructed. Using enhanced green fluorescence protein (EGFP) as the reporter gene, gene delivery efficiencies and the ability to express heterogenous protein in mammalian cells were evaluated. Optimal ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2795635 Mon, 14 Sep 2009 23:00:00 +0100 2795635 http://www.medworm.com/index.php?rid=2795634&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1391 Gene therapy for hemophilia A with adeno-associated virus (AAV) vectors involves difficulties in the efficient expression of factor VIII (FVIII) and in antibody formation against transgene-derived FVIII.AAV8 vectors carrying the canine B domain deleted FVIII (cFVIII) gene under the control of the ubiquitous [beta]-actin promoter, the liver-specific human [alpha]1 anti-trypsin promoter (HAAT) and the liver-specific hepatic control region (HCR) enhancer/human [alpha]1 anti-trypsin promoter complex (HCRHAAT) were used for the expression of cFVIII in FVIII deficient (fviii-/-) mice.Addition of the hepatic control region enhancer element to the HAAT promoter successfully augmented HAAT promoter activity without loss of liver-specificity in vivo. Using this enhancer/promoter complex, a high cFVI... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2795634 Mon, 14 Sep 2009 23:00:00 +0100 2795634 http://www.medworm.com/index.php?rid=2795633&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1394 The ability to deliver plasmid DNA (pDNA) to specific cells in vivo is crucial for achieving efficient targeted transfection with nonviral vectors. We previously used stealth liposomes containing the chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA3-DTDA) to target delivery of antigen and cytokines to immune cells in vivo. In the present study, we utilized liposomes containing NTA3-DTDA and the ionizable aminolipid 1,2-dioleoyl-3-dimethyl-ammonium-propane (DODAP) to incorporate pDNA into complexes for targeting to cells.Liposomes containing DODAP, NTA3-DTDA and helper lipids were acidified (pH 5.5) and mixed with pDNA to form complexes. These lipoplexes were neutralized and engrafted with His-tagged molecules for targeting to extracellular receptors. Targeted transfection eff... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2795633 Mon, 14 Sep 2009 23:00:00 +0100 2795633 http://www.medworm.com/index.php?rid=2795632&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1396 Amphiphilic triblock copolymers such as the polyethylene oxide-polypropylene oxide-polyethylene oxide L64 (PEO13-PPO30-PEO13) significantly increase transgene expression after injection of DNA/polymer mixtures into skeletal muscles. To better understand the way such copolymers act, we studied the behaviour of different poloxamers, including L64, both in vitro and in vivo.The in vitro and in vivo transfection activity of five copolymers that differ either by their molecular weight or by their hydrophilic/hydrophobic balance was evaluated. Furthermore, we also studied the membrane permeabilizing properties of the poloxamers.The results obtained indicate that, after intramuscular administration of DNA/poloxamer formulations, all five compounds were able to significantly increase the expressio... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2795632 Mon, 14 Sep 2009 23:00:00 +0100 2795632 http://www.medworm.com/index.php?rid=2795631&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1397 Recombinant adenoviruses (rAd) are well-characterized viral vectors and have been studied in many human diseases. However, there are no detailed methods for transferring genes to the stomach using rAd.Gastric epithelial cells were infected with rAd encoding green fluorescence protein (AdGFP) for different times, or with AdGFP that had been incubated in artificial gastric juice at different pH values for 1 h. Gene expression was detected by fluorescence microscope and flow cytometry. Mice were infected via oral administration with rAd encoding red fluorescence protein and [beta]-galactosidase (AdRFP-lacZ) or rAd encoding mouse interleukin-17 (AdmIL-17), and tissues were collected at the indicated times after infection. LacZ expression in different tissues was detected by X-gal staining and ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2795631 Mon, 14 Sep 2009 23:00:00 +0100 2795631 http://www.medworm.com/index.php?rid=2795630&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1395 Many studies aimed at retargeting adenovirus (Ad) rationally focus on genetic modification of fiber, which is the primary receptor-binding protein of Ad. Retargeted fibers ultimately require functional validation in the viral context.Lentiviral vectors (LV) were used to express fiber variants in cells. Infections with a fiber gene-deleted Ad vector yielded fiber-pseudotyped viruses. An enzyme-linked immunosorbent assay and slot blot-based assays probed target binding-ability of retargeted fibers. Differential treatments with an alkylating agent prior to western blot analysis allowed for examination of intra- and extracellular redox states of fibers.In the present study, LV-based fiber-pseudotyping of Ad is presented as an accelerated means to test new fibers. LV-mediated gene transfer yiel... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2795630 Mon, 14 Sep 2009 23:00:00 +0100 2795630 http://www.medworm.com/index.php?rid=2743254&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1386 The present study aimed to enhance the specific anti-caries immunity induced by DNA prime-protein boost strategy for an A-P fragment of a cell-surface protein antigen of Streptococcus mutans (PAc).BALB/c mice were immunized with DNA prime-protein boost, DNA-DNA or protein-protein regimens by the intranasal route, using combinations of plasmid vector (pCIA-P) that express PAc protein and a pure secretec recombinant PAc protein (rPAc). Then, a gnotobiotic mouse model was constructed 2 weeks after the last immunization, and specific immune responses in vivo and their protection against dental caries were observed.The present study revealed stronger antibody responses in the DNA prime-protein boost group compared to those elicited by either DNA-DNA vaccination or protein-protein vaccination. I... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2743254 Fri, 28 Aug 2009 23:00:00 +0100 2743254 http://www.medworm.com/index.php?rid=2707987&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1387 DNA vaccination is a strategy that has been developed primarily to elicit protective immunity against infection and cancer.DNA vaccine was used, in conjunction with an immunosuppressant, to tolerize harmful autoimmunity.Immunization of C57BL/6 mice with MOG35-55, a myelin oligodendrocyte glycoprotein-derived peptide, and FK506 (Tacrolimus) as a tolerogenic adjuvant stimulated regulatory dendritic cells, induced antigen-specific regulatory T cells (Treg), and protected the animals from subsequent induction of experimental autoimmune encephalomyelitis (EAE). After EAE induction, there were fewer lymphocytes, including fewer T helper 17 cells, and more Treg infiltrating the spinal cord in the immunized mice compared to in control mice. Furthermore, at the peak of the EAE manifestation, CD4 T ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2707987 Mon, 17 Aug 2009 23:00:00 +0100 2707987 http://www.medworm.com/index.php?rid=2707988&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1385 Malignant gliomas remain refractory to treatment despite advances in chemotherapy and surgical techniques. Viral vectors developed to treat gliomas have had low transduction capabilities, limiting their use. Gliomas over-express CD46, CD80, and CD86, all of which bind adenovirus serotype 3.To increase the infectivity and replication of oncolytic vectors in malignant brain tumors, we created a conditionally replicating adenovirus, CRAd-Survivin-5/3, which contains a survivin promoter-driving E1A and a chimeric fiber consisting of adenovirus serotype 3 knob.In vitro, this modified CRAd showed ten- to 100-fold increased cytotoxicity against glioma cells. Ex vivo analysis of primary glioblastoma multiforme samples infected with CRAd-Survivin-5/3 showed an increase in cytotoxicity of 20-30% com... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2707988 Sun, 16 Aug 2009 23:00:00 +0100 2707988 http://www.medworm.com/index.php?rid=2687449&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1373 Rapid clearance of adenoviruses from blood by macrophage lineage cells of the liver and spleen, and binding to platelets, hinder their successful systemic use for cancer gene therapy. Vitamin K dependent coagulation factors are important mediators for the adenovirus liver tropism. Here we aim to determine the effects of coagulation factor, thrombocyte and liver macrophage (Kupffer cell) ablation on biodistribution of serotype 5 adenoviruses in mice with orthotopic breast tumors.Prior to intravenous injection of adenoviruses, mice bearing orthotopic breast tumors were pretreated with warfarin to inhibit vitamin K dependent coagulation factor synthesis, an anti-platelet antibody causing thrombocytopenia or an inhibitor of the Kupffer cell scavenger receptor or their combination. Virus availa... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2687449 Mon, 10 Aug 2009 23:00:00 +0100 2687449 http://www.medworm.com/index.php?rid=2680991&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1383 We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation. Copyright © 2009 John Wiley & Sons, Ltd. (Source: The Journal of Gene Medicine) The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2680991 Fri, 07 Aug 2009 23:00:00 +0100 2680991 http://www.medworm.com/index.php?rid=2666398&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1382 We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR.We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient.Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells w... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2666398 Mon, 03 Aug 2009 23:00:00 +0100 2666398 http://www.medworm.com/index.php?rid=2666399&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1381 Retroviral transduction of cells is improved upon virus adsorption onto immobilized fibronectin (FN) fragments. Because HIV-1 Tat possesses the same functional domains that lead to increased transduction efficiency in FN by colocalization of bound virus and cells, we hypothesized that Tat could enhance gene transfer by a similar mechanism.Single-cycle replication retro- or lentivirus carrying green fluorescent protein or cloramphenicol acetyltransferase as reporter genes were added to wells coated with Tat or Tat peptides. Wells were extensively washed to remove unbound virus and levels of transduction were detected by measuring reporter gene expression. Virus adsorption to immobilized Tat was measured using a p24 antigen capture assay.Immobilized Tat efficiently binds retro- and lentivira... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2666399 Sun, 02 Aug 2009 23:00:00 +0100 2666399 http://www.medworm.com/index.php?rid=2647976&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1377 Efficient protection of dopaminergic neurons against a subsequent 6-hydroxydopamine lesion by glial cell line-derived neurotrophic factor (GDNF) gene delivery has been demonstrated. By contrast, the neurorestorative effects of GDNF administered several weeks after the toxin have been less characterized. In particular, whether these were permanent or dependent on the continuous presence of GDNF remains elusive.A tetracycline-inducible adeno-associated virus (AAV)-1 vector expressing human GDNF cDNA was administered unilaterally in the rat striatum 5 weeks after 6-hydroxydopamine. Rats were treated with doxycycline (dox) or untreated from the day of vector injection until sacrifice (4 or 14 weeks). A sub-group was dox-treated for 7 weeks then untreated until 14 weeks. The motor behavior was ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2647976 Tue, 28 Jul 2009 23:00:00 +0100 2647976 http://www.medworm.com/index.php?rid=2647977&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1376 Ultrasound (US) is a novel and effective tool for the local delivery of genes into target tissues. US can temporarily change the permeability of a cell membrane and thus enhance the delivery of naked DNA into cells. In the present study, the efficiencies of gene expression mediated by US delivery in orthotopic liver tumor, subcutaneous tumor and muscle tissue were evaluated by changing the contrast agent concentrations and US exposure durations.Plasmid DNA coding for luciferase, interleukin-12 or enhanced green fluorescence protein was mixed with SonoVue® and injected intratumorally or intramuscularly. The injection sites were then exposed to US (20% duty cycle and 0.4 W/cm2 intensity).The results obtained showed that the optimal condition was 50% SonoVue® for tumors and 30% for muscle, ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2647977 Mon, 27 Jul 2009 23:00:00 +0100 2647977 http://www.medworm.com/index.php?rid=2641056&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1368 Cystic fibrosis (CF) is caused by a defect in cystic fibrosis transmembrane conductance regulator (CFTR) activity, often resulting in an incurable airway disease. Gene therapy into the conducting airway epithelium is a potential cure for CF; however, most gene vectors do not result in long-lived expression, and require re-dosing. Perversely, intrinsic host immune responses can then block renewed gene transfer.To investigate whether persistent gene expression could be achieved after a single dosing event, thus avoiding the issue of blocking host responses, we used a gene transfer protocol that combined an airway pretreatment using lysophosphatidylcholine with a human immunodeficiency virus type-1 (vesicular stomatitis virus G pseudotype) derived lentiviral vector to test whether an integrat... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2641056 Sun, 26 Jul 2009 23:00:00 +0100 2641056 http://www.medworm.com/index.php?rid=2637087&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1374 Various strategies have been examined to improve both transfection efficiency and cytotoxicity of polyethylenimine (PEI), a widely used polycationic nonviral gene vector. In the present study, we sought to improve PEI transfection efficiency by combining the osmotic burst mechanism for lysing endocytotic vesicles with the lipid depletion mechanism, which was accomplished by maintaining buffering capacity at the same time as adding a lipid-absorbing hydrophobic shell.PEI was altered via the substitution of various percentages of its primary amines with carboxylate-terminated short, moderate and long alkyl chains, by reaction with bromoacetic, 6-bromohexanoic, 10-bromodecanoic and 16-bromohexadecanoic acids. Modified polymers were complexed with plasmid and the particle size and zeta potenti... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2637087 Fri, 24 Jul 2009 23:00:00 +0100 2637087 http://www.medworm.com/index.php?rid=2633262&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1369 This study assessed whether a Sca-1+ cell-based ex vivo gene transfer strategy, which has been shown to promote robust endosteal bone formation with a modified fibroblast growth factor-2 (FGF2) gene, can be extended to use with bone morphogenetic protein (BMP)2/4 hybrid gene.Sublethally irradiated recipient mice were transplanted with lentiviral (LV)-BMP2/4-transduced Sca-1+ cells. Bone parameters were monitored by pQCT and µCT. Gene expression was assessed by the real-time reverse transcriptase-polymerase chain reaction.Recipient mice of LV-BMP2/4-transduced Sca-1+ cells yielded high engraftment and increased BMP4 mRNA levels in marrow cells; but exhibited only insignificant increases in serum and bone alkaline phosphatase activity compared to control mice. pQCT and µCT analyses of femu... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2633262 Wed, 22 Jul 2009 23:00:00 +0100 2633262 http://www.medworm.com/index.php?rid=2619635&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1372 Infantile-onset glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) causes death early in childhood from cardiorespiratory failure in the absence of effective treatment, whereas late-onset Pompe disease causes a progressive skeletal myopathy. The limitations of enzyme replacement therapy could potentially be addressed with adeno-associated virus (AAV) vector-mediated gene therapy.AAV vectors containing tissue-specific regulatory cassettes, either liver-specific or muscle-specific, were administered to 12- and 17-month-old Pompe disease mice to evaluate the efficacy of gene therapy in advanced Pompe disease. Biochemical correction was evaluated through acid [alpha]-glucosidase (GAA) activity and glycogen content analyses of the heart and skeletal muscle. Western blotting, u... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2619635 Mon, 20 Jul 2009 23:00:00 +0100 2619635 http://www.medworm.com/index.php?rid=2616414&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1370 Lentiviral vectors (LV) offer several advantages over other gene delivery vectors. Their potential for the integration and long-term expression of therapeutic genes renders them an interesting tool for gene and cell therapy interventions. However, large-scale LV production remains an important challenge for the translation of LV-based therapeutic strategies to the clinic. The development of robust processes for mass production of LV is needed.A suspension-grown HEK293 cell line was exploited for the production of green fluorescent protein-expressing LV by transient polyethylenimine (PEI)-based transfection with LV-encoding plasmid constructs. Using third-generation packaging plasmids (Gag/Pol, Rev), a vesicular stomatitis virus G envelope and a self-inactivating transfer vector, we employe... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2616414 Sun, 19 Jul 2009 23:00:00 +0100 2616414 http://www.medworm.com/index.php?rid=2616413&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1371 Dendritic cell (DC)-based vaccines have become a promising modality in cancer immunotherapy. However, their ability to initiate tumor antigen-specific T cell immunity is limited in various negative-feedback mechanisms. The rapid down-regulation of chemokines, such as the interferon inducible protein of 10 kDa (IP-10), which chemoattracts activated antigen-specific CD8+ T cells, would represent negative-feedback regulation. Therefore, we attempted to improve DC vaccine potency by introducing the IP-10 gene retrovirally aiming to replenish the chemoattractive activity of DCs.We introduced IP-10 gene into DC2.4 cells, referred to as DC-IP10, using a retroviral system. Nonsecretable mIP-10-expressing DCs (DC-mIP10) were also prepared to evaluate the effects of secretion in IP-10-mediated modul... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2616413 Sun, 19 Jul 2009 23:00:00 +0100 2616413 http://www.medworm.com/index.php?rid=2555174&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1365 A variety of synthetic carriers, such as cationic polymers and lipids, have been used as nonviral carriers for small interfering RNA (siRNA) delivery. Although siRNA polyplexes and lipoplexes exhibited good gene silencing efficiencies, they often showed serious cytotoxicities, which are not useful for clinical applications. A double-stranded RNA binding cellular protein with highly specific siRNA binding property and noncytotoxicity was used for siRNA delivery.A double-stranded RNA binding domain (dsRBD) of human double-stranded RNA activated protein kinase R was genetically produced and utilized to complex siRNA for intracellular delivery. For characterization of the siRNA/dsRBD complexes, decomplexation assay and RNase protection assay were performed. Cytotoxicity and target gene inhibit... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2555174 Mon, 29 Jun 2009 23:00:00 +0100 2555174 http://www.medworm.com/index.php?rid=2555172&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1352 Small interfering RNA (siRNA) has been recognized as a new therapeutic drug to treat various diseases by inhibition of oncogene or viral gene expression. Because hyaluronic acid (HA) has been described as a biocompatible biomaterial, we tested the nanoparticles formed by electrostatic complexation of negatively-charged HA and cationic poly L-arginine (PLR) for siRNA delivery systems.Different electrostatic complexes of HA and PLR (HPs) were formulated: HP101 with 50% (w/w) HA and HP110 with 9% (w/w) HA.Gel retardation assays showed that HP101 and HP110 could form complexes with siRNAs. The diameters of these complexes were less than 200 nm. Cellular delivery efficiency of siRNAs by HPs depended on cell surface CD44 density. The HP-mediated delivery of siRNAs was highest in WM266.4 cells fo... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2555172 Mon, 29 Jun 2009 23:00:00 +0100 2555172 http://www.medworm.com/index.php?rid=2555173&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1367 Intronic sequences have the potential to improve gene expression in eukaryotes by a variety of mechanisms. In this context, human [beta]-globin (hBG) introns were inserted into the human factor IX (hFIX) cDNA in cytomegalovirus (CMV)-regulated plasmids. The resulting construct was then used for further expression analysis in vitro.Seven hFIX-expressing plasmids with different combinations of the two hBG introns and the Kozak element were constructed and used for a systematic expression analysis in cultured Chinese hamster ovary (CHO) cells. In parallel, the hBG intronic sequences were analysed for the presence of possible regulatory elements.All the constructed plasmids resulted in transient expression of the hFIX. However, the coagulation activities varied according to the particular cons... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2555173 Sun, 28 Jun 2009 23:00:00 +0100 2555173 http://www.medworm.com/index.php?rid=2520121&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1366 Cell and tissue-specific DNA delivery can be achieved by derivatizing vehicles with a targeting ligand for certain receptor. CXCR4 is a receptor of chemokine stromal cell-derived factor (SDF)-1 and viral protein viral macrophage inflammatory protein (vMIP)-II. It is expressed on some types of stem and cancer cells. The present study aimed to design and characterize the group of CXCR4 targeted peptides for receptor-mediated gene delivery. We focused on bifunctional peptide carriers: two derived from N-terminal sequences of SDF-1 and one from vMIP-II.Three synthetic chemokine-derived peptides, designated long CDP (KPVSLSYRSPSRFFESH-K9-biotin), short CDP (KPVSLSYR-K9-biotin) and viral CDP (D-LGASWHRPDK-K9-biotin), were evaluated for gene delivery to CXCR4 positive and negative cells. Oligolys... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2520121 Thu, 25 Jun 2009 23:00:00 +0100 2520121 http://www.medworm.com/index.php?rid=2500627&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1355 Liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM). Recent advances in the knowledge about the cellular, molecular and genetic aspects of fibrosis have opened a new era of research on liver cirrhosis. A transcription factor, Sp1, originally described as a ubiquitous transcription factor, is involved in the basal expression of ECM genes and may be important in the fibrotic processes.The chronic hepatic damage received intraperitoneal injection of carbon tetrachloride (2 mg/kg) dissolved in corn oil (1 : 3 ratio) three times a weekly for 8 weeks. The delivery of decoy oligodeoxynucleotide (ODN) was performed by injection of 10 µg of scrambled decoy ODN or 10 µg of ring type (R)-Sp1 decoy ODN through the mouse tail vein. All animals of each group wer... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2500627 Wed, 24 Jun 2009 23:00:00 +0100 2500627 http://www.medworm.com/index.php?rid=2500633&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1357 Although human islet transplantation is a promising approach for treating type I diabetes, its success is limited as a result of the poor survival rate of transplanted islets. Expression of a growth factor gene to promote revascularization and silencing of pro-apoptotic genes before transplantation may improve the outcome of islet transplantation.In the present study, we constructed bipartite plasmid vectors to co-express a vascular endothelial growth factor (VEGF) cDNA and short hairpin (sh)RNA targeting inducible NO synthase (iNOS) gene. First, we screened shRNA sequences against human iNOS by transfecting plasmids encoding shRNA targeting different start sites of human iNOS. Then, the effect of different promoters [such as H1, U6 and cytomegalovirus (CMV)] and micro RNA backbones on gen... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2500633 Tue, 23 Jun 2009 23:00:00 +0100 2500633 http://www.medworm.com/index.php?rid=2500632&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1361 Nonviral gene therapy of chronic degenerative diseases such as Parkinson's disease (PD) is a great challenge as a result of the low tranfection efficiency of nonviral gene vectors. We previously constructed a lactoferrin (Lf)-modified vector, which was demonstrated to be potential for brain gene delivery both in vitro and in vivo. In the present study, this type of vector was applied to load human glial cell line-derived neurotrophic factor gene (hGDNF).A rat PD model was constructed by the unilateral lesion of striatum using 6-hydroxydopamine (6-OHDA). Lf-modified nanoparticles (NPs) were prepared and characterized. Neuroprotective effects of Lf-modified NPs were examined in the 6-OHDA-lesioned PD model via a regimen of multiple dosing intravenous administrations.The size of Lf-modified N... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2500632 Tue, 23 Jun 2009 23:00:00 +0100 2500632 http://www.medworm.com/index.php?rid=2500631&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1362 Transplantation of stem cells from various sources into infarcted hearts has the potential to promote myocardial regeneration. However, the regenerative capacity is limited partly as a result of the low survival rate of the transplanted cells in the ischemic myocardium. In the present study, we tested the hypothesis that combining cell and angiogenic gene therapies would provide additive therapeutic effects via co-injection of bone marrow-derived mesenchymal stem cells (MSCs) with an adeno-associated viral vector (AAV), MLCVEGF, which expresses vascular endothelial growth factor (VEGF) in a cardiac-specific and hypoxia-inducible manner.MSCs isolated from transgenic mice expressing green fluorescent protein and MLCVEGF packaged in AAV serotype 1 capsid were injected into mouse hearts at the... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2500631 Tue, 23 Jun 2009 23:00:00 +0100 2500631 http://www.medworm.com/index.php?rid=2500629&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1363 Cystic fibrosis (CF) respiratory epithelia are characterized by a defect Cl- secretion and an increased Na+ absorption through epithelial Na+ channels (ENaC). The present study aimed to find an effective inhibitor of human ENaC with respect to replacing amiloride therapy for CF patients. Therefore, we developed specific antisense oligonucleotides (AON) that efficiently suppress Na+ hyperabsorption by inhibiting the expression of the [alpha]-ENaC subunit.We heterologously expressed ENaC in oocytes of Xenopus laevis for mass screening of AON. Additionally, primary cultures of human nasal epithelia were transfected with AON and were used for Ussing chamber experiments, as well as biochemical and fluorescence optical analyses.Screening of several AON by co-injection or sequential microinjectio... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2500629 Mon, 15 Jun 2009 23:00:00 +0100 2500629 http://www.medworm.com/index.php?rid=2500628&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1364 One method for the delivery of therapeutic proteins to the spinal cord is to inject nonviral gene vectors including plasmid DNA into the cerebrospinal fluid (CSF) that surrounds the spinal cord (intrathecal space). This approach has produced therapeutic benefits in animal models of disease and several months of protein expression; however, there is little information available on the immune response to these treatments in the intrathecal space, the relevance of plasmid CpG sequences to any plasmid-induced immune response, or the effect of this immune response on transgene expression.In the present study, coding or noncoding plasmids were delivered to the intrathecal space of the lumbar spinal region in rats. Lumbosacral CSF was then collected at various time points afterwards for monitorin... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2500628 Mon, 15 Jun 2009 23:00:00 +0100 2500628 http://www.medworm.com/index.php?rid=2467214&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1354 In previous studies, we showed that gene activated matrices (GAMs) containing nonviral vectors successfully deliver genes to neurons after optic nerve and spinal cord injury. In the present study, we evaluated whether adenoviral vectors delivered within a GAM increase the efficiency of local gene delivery to injured CNS neurons. Lyophilized GAMs containing collagen and adenoviral vectors were assessed in vitro and in vivo.We evaluated viral vector stability, release kinetics and efficiency of transduction for this GAM formulation in vitro using the quantitative polymerase chain reaction (qPCR), flow cytometry and fluorescence microscopy. Using PCR, reverse transcriptase-PCR and confocal microscopy, we assessed viral DNA retrograde axonal transport, green fluorescent protein (GFP) expressio... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2467214 Tue, 09 Jun 2009 04:00:00 +0100 2467214 http://www.medworm.com/index.php?rid=2500630&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1358 This Response refers to JGM 1286, which was published in The Journal of Gene Medicine 11:2. Please follow the link below to view the Letter to the Editor. (Source: The Journal of Gene Medicine) The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2500630 Mon, 08 Jun 2009 23:00:00 +0100 2500630 http://www.medworm.com/index.php?rid=2467217&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1351 We describe and characterize an alternative retroviral packaging system derived from the RD114 retrovirus.RD114-derived recombinant retroviruses were produced transiently by transfection of 293T cells, and viral titers were assessed on TE671 cells by measuring the percentage of infected green fluorescent protein (GFP) positive cells by fluorescence-activated cell sorter (FACS) analysis. Purified human hematopoietic cells (lymphocytes and CD34+ cells) were activated and transduced on retronectin-coated plates. Two days later, the percentage of GFP positive cells was evaluated by FACS analysis.We demonstrate that RD114 viral particles could package MLV transfer vectors, and that, in addition to its natural envelope, RD114 cores could be efficiently pseudotyped by the Gibbon ape leukemia, the... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2467217 Mon, 08 Jun 2009 04:00:00 +0100 2467217 http://www.medworm.com/index.php?rid=2467216&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1353 The low stability of gammaretroviral and lentiviral vectors affects their production, making high quality clinical preparations a difficult goal to achieve. Recently, our laboratory has shown that the main inactivation mechanism for both these vectors is the loss of their capacity to perform reverse transcription. The present study aimed to increase the stability of gammaretroviral and lentiviral at 37 °C and at 4 °C.Inactivation studies were performed with gammaretroviral and lentiviral vectors at 37 and 4 °C, with and without several stabilizing compounds. The residual viral infectivity and reverse transcription capacity of these samples were tested.The results obtained demonstrate that it is possible to increase the stability of reverse transcription and the infectivity stability of ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2467216 Mon, 08 Jun 2009 04:00:00 +0100 2467216 http://www.medworm.com/index.php?rid=2467215&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1356 The poor prognosis associated with ovarian cancer is primarily the result of delayed diagnosis and the lack of an effective treatment for advanced disease. Use of novel immunotherapy strategies are being evaluated that work to enhance local and systemic immune responses against cancer cells and can possibly work together with traditional cytotoxic chemotherapy regimens to produce more effective treatment options.In the present study, we describe a gene-based therapy whereby the anticancer cytokine interleukin-12 gene (pmIL-12) is formulated with a synthetic polymeric delivery vehicle (PPC) and administered intraperitoneally into a mouse model of disseminated ovarian cancer.The administration of pmIL-12/PPC in tumor-bearing mice was associated with a shift towards a Th1 immune state, includ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2467215 Mon, 08 Jun 2009 04:00:00 +0100 2467215 http://www.medworm.com/index.php?rid=2426455&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1348 The type I interferon (IFN) response to DNA/cationic liposome complex, or lipoplex, has been reported in cultured cells, but little is known about the response in vivo. Studies of the pro-inflammatory cytokine response to lipoplex have shown the importance of the unmethylated CpG dinucleotide (CpG motif) and its receptor, Toll-like receptor (TLR)-9.CpG- and non-CpG lipoplex consisting of CpG- or non-CpG plasmid DNA, respectively, and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol liposomes were intravenously injected into mice. IFN-[beta] and interleukin (IL)-6 in the serum and organs were measured by the enzyme-linked immunosorbent assay. The involvement of TLR9, phagocytic cells and the spleen in the responses was evaluated using TLR9-/-, clodronate liposome-tr... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2426455 Thu, 21 May 2009 04:00:00 +0100 2426455 http://www.medworm.com/index.php?rid=2421799&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1343 In vivo adeno-associated virus (AAV) delivery to adult liver results in sustained expression of the transgene. However, it has been suggested that AAV delivery to the newborn liver may result in transient expression. In the present study, we analysed transgene expression after AAV8 delivery of a therapeutic or a marker gene to newborn rat liver.Recombinant AAV 8 vectors carrying either the human UGT1A1 cDNA or the lacZ gene were injected intravenously in 2-day-old Gunn or Wistar rats. Serum bilirubin level was recorded in Gunn rats and [beta]-galactosidase expression was monitored by immunohistochemistry or enzyme activity. The molecular forms of AAV genome were analysed by the polymerase chain reaction and Southern blotting in whole liver and by the quantitative polymerase chain reaction ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2421799 Tue, 19 May 2009 04:00:00 +0100 2421799 http://www.medworm.com/index.php?rid=2421798&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1345 Targeted gene transduction in vivo is the ultimate preferred method for gene delivery. We previously developed targeting lentiviral vectors that specifically recognize cell surface molecules with conjugated antibodies and mediate targeted gene transduction both in vitro and in vivo. Although effective in some experimental settings, the conjugation of virus with antibodies is mediated by the interaction between protein A and the Fc region of antibodies, which is not as stable as covalent conjugation. We have now developed a more stable conjugation strategy utilizing the interaction between avidin and biotin.We inserted the biotin-adaptor-peptide, which was biotinylated by secretory biotin ligase at specific sites, into our targeting envelope proteins, enabling conjugation of the pseudotyped... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2421798 Tue, 19 May 2009 04:00:00 +0100 2421798 http://www.medworm.com/index.php?rid=2421797&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1347 The use of ultrasound (US)-facilitated gene therapy is increasing rapidly as a result of its high specificity and non-invasiveness. However, the acoustic parameters that produce the most efficient transfection have not been established. The present study investigated the effects of time parameters [including pulsing strategy (on- and off-times), exposure duration, pore opening time and expression duration] of US-facilitated gene transfection.Cervical cancer cells (HeLa cells) cultured with pCMViLUC plasmids were exposed to 1-MHz pulsed US, and gene transfection efficiency and cell viability were assessed. The ability of in vivo transfection by ultrasound using different pulsing strategy was also evaluated.For a constant total exposure time, the transfection was always better for a longer o... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2421797 Tue, 19 May 2009 04:00:00 +0100 2421797 http://www.medworm.com/index.php?rid=2421796&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1346 The genomic integration of a lentiviral vector developed for the treatment of Wiskott-Aldrich syndrome (WAS) was assessed by localizing the vector insertion sites (IS) in a murine model of gene therapy for the disease.Transduced hematopoietic progenitor cells were transplanted into mice or cultured in vitro. The IS were determined in the genomic DNA from blood, the bone marrow of the animals and from cultured cells.Sequencing vector-genomic DNA junctions yielded more than 150 IS of which 50-70% were located in transcription units. To obtain additional sequences from the population of cultured cells, we used a vector-tag concatenation technique providing 190 additional IS. Altogether, the profiles confirmed the bias for integration in transcription units. The vector did not congregate as ho... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2421796 Tue, 19 May 2009 04:00:00 +0100 2421796 http://www.medworm.com/index.php?rid=2411713&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1341 Hepatic insulin gene therapy (HIGT) produces near-normal glycemia in diabetic rats. Hepatic insulin production is expected to stimulate glycogen storage. However, the effect of HIGT on hepatic glycogen metabolism in vivo is unknown.After administration of an adenoviral vector capable of inducing glucose responsive insulin production from hepatocytes, we evaluated circulating hormones, cytokines, hepatic gene expression and hepatic glycogen content in diabetic CD-1 mice receiving intravenous streptozotocin. Nondiabetic mice and diabetic mice treated with empty adenovirus served as controls.Peripheral concentrations of human insulin in HIGT mice were less than concentrations of mouse insulin among controls. However, expression of insulin responsive genes in HIGT livers indicated a significan... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2411713 Sat, 16 May 2009 01:06:41 +0100 2411713 http://www.medworm.com/index.php?rid=2411716&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1333 Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding the possible promoter choices and eventually allowing cell type specific down-regulation of target genes.In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2411716 Mon, 11 May 2009 04:00:00 +0100 2411716 http://www.medworm.com/index.php?rid=2411715&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1339 Targeting gene therapy vectors that can home in on desired cell and tissue types in vivo comprise the ultimate gene delivery system. We have previously developed targeting lentiviral vectors by pseudotyping vectors with modified Sindbis virus envelope proteins. The envelope protein contains the Fc-binding region of protein A (ZZ domain), so the virus can be conjugated with antibodies. The conjugated antibody mediates specific transduction of the cells and tissues expressing the target antigens, both in vitro and in vivo. However, more stable conjugation of targeting molecules would be optimal for use in immunocompetent animals, as well as in humans.We inserted integrin-targeting peptides into two sites of the targeting envelope proteins and determined whether the peptides serve as receptor... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2411715 Mon, 11 May 2009 04:00:00 +0100 2411715 http://www.medworm.com/index.php?rid=2411714&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1344 Inherited apolipoprotein (Apo) A-I deficiency is an orphan disorder characterized by high-density lipoprotein (HDL)-cholesterol deficiency and premature atherosclerosis. Constitutive over-expression of ApoA-I might provide a means to treat this disease. The present study provides a comprehensive evaluation of adeno-associated virus (AAV)-mediated ApoA-I gene delivery to express human (h)ApoA-I and correct the low HDL-cholesterol phenotype associated with ApoA-I deficiency.In an effort to maximize AAV-mediated gene expression, we performed head-to-head comparisons of recombinant AAVs with pseudotype capsids 1, 2, 6 and 8 administered by different routes with the use of five different liver-specific promoters in addition to cytomegalovirus as single-stranded or as self-complementary (sc) AAV... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2411714 Mon, 11 May 2009 04:00:00 +0100 2411714 http://www.medworm.com/index.php?rid=2401934&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1342 Control of inflammation is essential for the clinical management of many common human diseases. However, there are few generally applicable strategies to convert an abnormal intracellular signal into a gene expression that leads to normalization of the intracellular environment. Recently, we proposed a novel strategy termed D-RECS (i.e. drug or gene delivery system responding to cellular signals) to convert an intracellular signal to transgene expression. In the present study, we applied this concept to inflammatory cells using I[kappa]-B kinase as a signal molecule that triggers the gene expression.Candidate cationic substrates of I[kappa]-B kinase (IKK)[beta] were synthesized and their reactivity was investigated. Then, polymers grafted with these peptides were prepared by radical polyme... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2401934 Sun, 10 May 2009 04:00:00 +0100 2401934 http://www.medworm.com/index.php?rid=2395701&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1340 Salivary glands have been proposed as target organs for gene therapy. They secrete endogenous, as well as transgenic proteins, in a polarized manner. Transgene-encoded regulated pathway proteins primarily follow the regulated pathway in rat salivary glands and are secreted into saliva in an exocrine manner. Conversely, constitutive pathway proteins generally are secreted more basolaterally and thus follow the endocrine route. In the present study, we studied in vivo the sorting of the mouse immunoglobulin G2b Fc fragment, which is physiologically secreted via the constitutive pathway.Adenoviral vectors encoding the Fc fragment and human growth hormone were delivered into rat and mouse submandibular glands in vivo to compare their serum-to-saliva distribution. We also compared the intracell... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2395701 Thu, 07 May 2009 04:00:00 +0100 2395701 http://www.medworm.com/index.php?rid=2373483&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1338 There has been considerable interest in researching the regulatory mechanisms that control transgene expression. In particular, there is impetus to investigate the intranuclear mechanisms for gene expression in order to improve the transfection efficiency of nonviral gene carriers.To clarify the direct relationship between DNA decondensation and gene expression, plasmid DNA encoding Keima-Red fluorescent protein was doubly-labeled by a pair of donor-acceptor fluorescent dyes (fluorescein and Cy3), and transfected to HuH-7 cells using nonviral gene carriers: polyethylenimine (polyplex) and LipofectAMINE 2000 (lipoplex). Fluorescence resonance energy transfer analysis between the two dyes represents the condensation state of the pDNA. The intranuclear trafficking and condensation state of th... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2373483 Wed, 29 Apr 2009 04:00:00 +0100 2373483 http://www.medworm.com/index.php?rid=2373485&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1331 Bone morphogenetic protein-2 (BMP-2) is believed to participate in bone healing and regeneration. Previous studies using BMP-2 in clinical applications have encountered difficulties that include the lack of an efficient, safe and simple delivery system, and expensive proteins and matrices. The gene transfer approach is a promising option for utilizing BMP-2. Viral vector-mediated gene transfer is efficient, but safety concerns prevent its clinical application for common diseases. Sonoporation is a simple and inexpensive method that only requires a plasmid and a sonoporation device.We used a plasmid-based human BMP-2 construct (pCAGGS-BMP-2) and examined the induction of bone in the skeletal muscle of mice after plasmid transfer by transcutaneous sonoporation. First, an in vitro study was p... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2373485 Mon, 27 Apr 2009 04:00:00 +0100 2373485 http://www.medworm.com/index.php?rid=2373484&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1337 Efficient and continuous expression of a therapeutic transgene is a key factor for improving the efficacy of gene therapy. Some insulators are known to contribute to continuous high-level expression of a therapeutic transgene.Using the human AAVS1 insulator (DHS) found in the AAVS1 DNase I hypersensitive site, chicken [beta]-globin insulator (cHS4) and sea urchin arylsufatase insulator (Ars), we newly constructed three recombinant adeno-associated virus vectors (rAAV) and examined their capability of transducing the mouse quadriceps muscle.DHS increased transgene expression from the human elongation factor 1[alpha] promoter (EF) by 1000-fold, up to the high level achieved by the human cytomegalovirus immediate early promoter/enhancer (CMV), which comprises an extremely strong promoter for ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2373484 Mon, 27 Apr 2009 04:00:00 +0100 2373484 http://www.medworm.com/index.php?rid=2363016&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1332 Adenovirus type 5 (Ad5) is widely used as a vehicle for vaccine delivery in the treatment of infectious disease and cancer. However, the efficacy of Ad5 vectors has been limited in humans because exposure to Ad5 infections results in most adults having neutralizing antibodies against Ad5. To overcome this limitation, the hexon epitope present in the fifth hypervariable region of Ad5 was modified.To evaluate the ability of Ad5 vectors encoding the HIV env protein to induce Ag-specific immune responses in the face of pre-existing anti-Ad5 immunity, mice were administrated intramuscularly with the Ad-Luc vector, and then vaccinated with parental or hexon-modified Ad5 vectors (Ad-HisHIV, Ad-END/AAAHIV or Ad-HIV) at week 8. HIV-specific cell-mediated immune responses were detected through a com... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2363016 Fri, 24 Apr 2009 04:00:00 +0100 2363016 http://www.medworm.com/index.php?rid=2352549&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1334 The adeno-associated virus (AAV) has many safety features that favor its use in the treatment of arthritic conditions; however, the conventional, single-stranded vector is inefficient for gene delivery to fibroblastic cells that primarily populate articular tissues. This has been attributed to the inability of these cells to convert the vector to a double-stranded form. To overcome this, we evaluated double-stranded self-complementary (sc) AAV as a vehicle for intra-articular gene delivery.Conventional and scAAV vectors were used to infect lapine articular fibroblasts in culture to determine transduction efficiency, transgene expression levels, and nuclear trafficking. scAAV containing the cDNA for interleukin (IL)-1 receptor antagonist (Ra) was delivered to the joints of naïve rabbits an... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2352549 Tue, 21 Apr 2009 04:00:00 +0100 2352549 http://www.medworm.com/index.php?rid=2352548&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1329 RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences.In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV-1 molecular clone an... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2352548 Tue, 21 Apr 2009 04:00:00 +0100 2352548 http://www.medworm.com/index.php?rid=2338400&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1328 Cerepro® (sitimagene ceradenovec) is an adenoviral vector containing herpes simplex virus thymidine kinase gene (HSV-tk), which is being developed for the treatment of high-grade glioma with oral ganciclovir (GCV). The nonclinical safety and biodistribution of Cerepro® were assessed following intravenous (i.v.) or intracerebral (i.c.) injection.Crl : WI(GLX/BRL/Han) rats (n = 198) were injected i.c. or i.v. with Cerepro® or vehicle control, with GCV by intraperitoneal (i.p.) injection to selected groups. Safety was assessed by observation of animal behaviour and post mortem histology. Antibody response was assessed, and biodistribution measured using the quantitative polymerase chain reaction (PCR) and reverse transcriptase-PCR in blood and tissues.Following i.v. or i.c. injection, ther... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2338400 Sat, 18 Apr 2009 04:00:00 +0100 2338400 http://www.medworm.com/index.php?rid=2321215&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1327 Adeno-associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long-term therapy is the development of immune responses against the vector or transgene product.We evaluated cellular and humoural responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression.Following subretinal administration of vector, splenocytes and T-cells from draining lymph nodes showed minimal activation following stimulation by co-culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2321215 Fri, 10 Apr 2009 04:00:00 +0100 2321215 http://www.medworm.com/index.php?rid=2321216&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1326 Conditionally replicative adenovirus (CRAd) provides a promising strategy for solid tumor therapy. However, relatively few studies have been addressed on hematopoietic malignancies. We previously found that ZD55, a serotype 5 (Ad5)-based, E1B 55-kDa deleted CRAd, inhibited leukemic cell growth and induced apoptosis. In the present study, we employed SG235, a new CRAd with both an E1B 55-kDa deletion and an Ad5/F35 chimeric fiber, for the treatment of B-cell tumors.CRAd SG235 was engineered not to express adenovirus E1B 55-kDa gene, and the wild-type Ad5 fiber was replaced by a chimeric Ad5/35 fiber containing an Ad5 tail, an Ad35 shaft and an Ad35 knob. Using in vitro and in vivo experiments, the infectivity and selective cytotoxicity of SG235 on B-cell tumor lines were evaluated. Apoptosi... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2321216 Wed, 01 Apr 2009 04:00:00 +0100 2321216 http://www.medworm.com/index.php?rid=2321217&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1325 The success of gene therapy for inherited neurodegenerative diseases such as metachromatic leukodystrophy (MLD) depends on the development of efficient gene delivery throughout the brain guarded by the blood-brain barrier and achieves distribution of the deficient enzyme throughout the brain. Direct injection of viral vector into the brain parenchyma is too invasive and may not be sufficient to treat the entire brain. As an alternative approach, we examined the feasibility of intrathecal (IT) injection of adeno-associated viral vector serotype 1 (AAV1).AAV1 vector expressing arylsulfatase A (ASA) and green fluorescence protein (GFP) was intrathecally injected into ASA knockout MLD model mice. Expression of GFP was assessed by fluorescence microscopy and immunohistochemical methods, whereas... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2321217 Tue, 31 Mar 2009 04:00:00 +0100 2321217 http://www.medworm.com/index.php?rid=2294199&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1322 p27kip1 (p27), a cyclin-dependent kinase inhibitor (CDKI), is an important regulator of cell cycle progression and a putative tumor suppressor gene, and plays an important role in the inhibition of genesis and progression of several kinds of cancers. The present study aimed to evaluate the anti-tumor effects of p27 gene therapy by a nonviral gene delivery strategy on pulmonary metastatic tumors.A recombinant plasmid composed of a p27 sequence was constructed and identified; it was then formulated with condensing agent protamine sulfate and entrapped into cationic liposomes. The resulting lipid-polycation-DNA complexes (LPD) were prepared into lyophilized forms. 5 × 105 of CT26 colorectal adenocarcinoma cells were inoculated into female Balb/c mice via the tail vein to establish lung tumor... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2294199 Fri, 27 Mar 2009 04:00:00 +0100 2294199 http://www.medworm.com/index.php?rid=2294202&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1312 We report the design of an optimized and extended NF[kappa]B DNA binding sequence that promotes an efficient plasmid nuclear import.On the basis of structural studies, the 5[prime]-CTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGGACTTTCCAGG-3[prime] segment (termed 3NF) comprising three 10-bp [kappa]B sites (GGGACTTTCC) separated by a 5-bp optimized spacer (AGCTG) was selected for its capacity to ensure the best structural fit with NF[kappa]B and to fix simultaneously three proteins. Plasmids encoding luciferase and bearing this sequence (3NF-plasmids) were constructed and their nuclear import and gene expression efficiencies compared with that of plasmids containing classical [kappa]B motifs.A high luciferase expression was associated with plasmids containing one (p3NF-luc) or two (p3NF-luc-3NF) 3NF s... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2294202 Thu, 26 Mar 2009 04:00:00 +0100 2294202 http://www.medworm.com/index.php?rid=2294201&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1319 Previously, using an adenoviral vector, we showed that miniature pigs could provide a valuable and affordable large animal model for pre-clinical gene therapy studies to correct parotid gland radiation damage. However, adenoviral vectors lead to short-term transgene expression and, ideally, a more stable correction is required. In the present study, we examined the suitability of using a serotype 2 adeno-associated viral (AAV2) vector to mediate more stable gene transfer in the parotid glands of these animals.Heparan sulfate proteoglycan was detected by immunohistochemistry. [beta]-galactosidase expression was determined histochemically. An AAV2 vector encoding human erythropoietin (hEpo) was administered via Stensen's duct. Salivary and serum hEpo levels were measured using an enzyme-link... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2294201 Thu, 26 Mar 2009 04:00:00 +0100 2294201 http://www.medworm.com/index.php?rid=2294200&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1321 A promising strategy to enhance axon regeneration is to employ short interfering (si)RNA targeting either RhoA or p75NTR, which are components of a signalling cascade triggered by growth inhibitory ligands. However, it is important to profile the biological impact of siRNA on cell homeostasis in order to develop safe and effective therapies.We used microarray and quantitative reverse transcriptase-polymerase chain reaction techniques to analyse the transcriptional effects of siRNA against p75NTR and RhoA in neuronal cell line and primary cultures.Expression analysis showed that primary rat dorsal root ganglion cells were up to 279-fold more sensitive than nerve growth factor-differentiated PC12 cells in detecting innate immune responses to siRNA. The sequence and method of synthesis of siR... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2294200 Thu, 26 Mar 2009 04:00:00 +0100 2294200 http://www.medworm.com/index.php?rid=2281685&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1320 Dexamethasone is a potent glucocorticoid with anti-inflammatory effects. Dexamethasone can protect ischemic cardiomyocytes from apoptosis. To apply the anti-apoptotic effect of dexamethasone to ischemic disease gene therapy, dexamethasone-conjugated polyethylenimine (PEI-Dexa) was synthesized and evaluated as an anti-apoptotic gene carrier.PEI-Dexa was synthesized with low molecular weight polyethylenimine (PEI2K, 2 kDa). The transfection efficiency and cytotoxicity of PEI-Dexa were evaluated by luciferase assay and the MTT assay. To evaluate the anti-apoptotic effect, PEI-Dexa/DNA complex was transfected into cells and the cells were treated with H2O2. Cell viability and apoptosis level were measured by the MTT assay and caspase-3 assay, respectively.A transfection assay into H9C2 rat car... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2281685 Sun, 22 Mar 2009 04:00:00 +0100 2281685 http://www.medworm.com/index.php?rid=2267935&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1317 Nonviral gene transfer generally suffers from short-term expression of transgenes. We have previously demonstrated that plasmids with reduced CpG content exhibited a more prolonged expression of murine interferon (IFN)-[beta] or IFN-[gamma], which was effective in inhibiting metastatic tumor growth. A further extension of the duration of transgene expression could be achieved by controlling the number and location of CpG motifs in plasmid DNA.Luciferase-expressing plasmids with differing CpG content were injected into the tail vein of mice by the hydrodynamic injection method. The effects of CpG content on the duration of transgene expression were examined, focusing on cytosine methylation and pro-inflammatory cytokines. Based on the findings, IFN-[gamma]-expressing plasmids were construct... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2267935 Mon, 16 Mar 2009 04:00:00 +0100 2267935 http://www.medworm.com/index.php?rid=2267933&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1318 Diabetes mellitus is a common comorbidity of atherosclerosis. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of the angiogenic response to hypoxia.We studied the effects of adenoviral vectors expressing a constitutively active HIF-1[alpha] hybrid (Ad2/HIF-1[alpha]/VP16) or vascular endothelial growth factor (Ad2/VEGF) on collateral development and vascular leakiness in a diabetic rat model of hindlimb ischemia.After the removal of the right femoral artery, the mRNA levels of VEGF, angiopoietin-1 and angiopietin-4 in the calf muscles, as measured by Taqman reverse transcriptase-polymerase chain reaction, were transiently elevated in Zucker lean (ZL) but not Zucker diabetic fatty (ZDF) rats. The angiographic score, as determined by post-mortem angiography, was significantly lower... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2267933 Mon, 16 Mar 2009 04:00:00 +0100 2267933 http://www.medworm.com/index.php?rid=2267937&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1314 Schistosomiasis causes liver and intestinal damage and can be very debilitating. The pairing of a male worm with a female worm residing in the gynaecophoral canal of male plays a critical role in the development of female parasite. Because the male specific gynaecophoral canal protein of Schistosoma japonicum (SjGCP) is found in significant quantities in the adult female worm after pairing, it could play an important role in parasite pairing.In the present study, three small interfering (si)RNA duplexes targeting the SjGCP gene were designed, synthesized and the silencing effects were evaluated in vitro as well as in mice infected with S. japonicum in vivo.In vitro studies using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR revealed the red... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2267937 Fri, 13 Mar 2009 04:00:00 +0100 2267937 http://www.medworm.com/index.php?rid=2255682&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1313 Mesenchymal stem cells (MSCs) are a promising vehicle for targeted cancer gene therapy because of their potential of tumor tropism. For efficient therapeutic application, we developed retroviral vector-producing MSCs that enhance tumor transduction via progeny vector production.Rat bone marrow-derived MSCs were nucleofected with the proviral plasmids (vesicular stomatitis virus-G protein-pseudotyped retroviral vector components) (VP-MSCs) or pLTR plasmid alone (non-VP-MSCs). The luciferase-based in vivo imaging system was used to assess gene expression periodically. To evaluate the anticancer effects, we administered MSCs expressing herpes simplex virus-thymidine kinase (HSV-tk) into the left ventricular cavity of nude mice engrafted with 9L glioma cells subcutaneously.In vivo imaging reve... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2255682 Thu, 12 Mar 2009 04:00:00 +0100 2255682 http://www.medworm.com/index.php?rid=2239346&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1309 Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis. A soluble form of Flt-1, a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidence suggests the applicability of sFlt-1 in tumor suppression. In the present study, we have developed and tested strategies targeted specifically to VEGF for the treatment of ascites formation.As an initial strategy, we produced recombinant sFLT-1 in the baculovirus expression system and used it as a trap to sequester VEGF in the murine ascites carcinoma model. The effect of the treatment on the weight of the animal, cell number, ascites volume and proliferating endothelial cells was studied. The second strategy involved, producing Ehrlich ascites tumor (EAT) cells stably transfected with ve... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2239346 Fri, 06 Mar 2009 05:00:00 +0100 2239346 http://www.medworm.com/index.php?rid=2231605&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1305 Glycogen storage disease type II (GSDII) or Pompe disease is an inherited disease of glycogen metabolism caused by a lack of functional lysosomal acid [alpha]-glucosidase (GAA). Affected individuals store glycogen in lysosomes resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe form. Even if enzyme replacement therapy (ERT) has already proven some efficacy, its results remain heterogeneous in skeletal muscle, especially in cross reactive immunological material (CRIM)-negative patients. We investigated for the first time the use of hematopoietic stem cell (HSC) gene therapy in a murine model of GSDII.Deficient HSC were transduced with a lentiviral vector expressing human GAA or enhanced green fluorescent protein (GFP) under the control of the retroviral... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2231605 Wed, 04 Mar 2009 05:00:00 +0100 2231605 http://www.medworm.com/index.php?rid=2231604&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1310 Supercoiled topology of transfected plasmid DNA (pDNA) is critical for transgene expression in mammalian cells. In the present study, we analysed transgene expression of transfected supercoiled pDNA concatemers.Jurkat T cells were transfected with a supercoiled 4.7-kb monomeric and, in parallel, a 9.4-kb dimeric pEGFP plasmid concatemer using electroporation. The absolute amounts of pDNA delivered into the cytoplasm and the nucleus were quantified by quantitative real-time polymerase chain reaction. Further, the number and mean fluorescent intensity (MFI) of enhanced green fluorescent protein (EGFP) expressing cells and the relative amounts of TOTO-1 fluorescently-labeled pDNA associated with the cell, located in the cytoplasm, and in the nucleus, were analysed by flow cytometry.For both c... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2231604 Wed, 04 Mar 2009 05:00:00 +0100 2231604 http://www.medworm.com/index.php?rid=2231603&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1311 The present study aimed to determine the efficiency and safety of baculovirus-mediated intravitreal gene transfer in rabbit eye and to compare its efficiency with adenovirus. We also studied how an intravitreal injection of vectors producing vascular endothelial growth factor D (VEGF-D) impacts the vasculature of rabbit eye.Baculoviral (BacVEGF-D) or adenoviral VEGF-D (AdVEGF-D) were administered intravitreally into the right eye at different doses (108, 109 and 1010 IU/ml) to 24 animals. Left eyes were injected with control viruses. To determine how long transgene expression lasted, we injected BacVEGF-D or BacLacZ to the vitreous humour of 11 animals and followed them for 4 weeks. Vitreous samples were taken after sacrifice for enzyme-linked immunosorbent assays and eyes were removed and... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2231603 Wed, 04 Mar 2009 05:00:00 +0100 2231603 http://www.medworm.com/index.php?rid=2221886&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1308 The cytotoxic T lymphocyte antigen 4 (CTLA4) represents an attractive ligand for use in the targeting of antigens to dendritic cells (DCs). Studies have shown that CTLA4 targeted DNA vaccines induced accelerated and increased antibody responses compared to nontargeted vaccines. However, little is known about the molecular events on DCs after transfection with targeted DNA vaccines.Here we constructed a green fluorescent protein (GFP)-labeled targeted anti-caries plasmid pMGJGLU/GFP and a GFP-labeled nontargeted anti-caries plasmid pCDGLU/GFP, compared the antibody responses they induced in mice, and investigated the gene expression profiles of DCs transfected with pMGJGLU/GFP and pCDGLU/GFP by gene array analysis.The data obtained showed that pMGJGLU/GFP induced accelerated and increased s... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2221886 Sat, 28 Feb 2009 05:00:00 +0100 2221886 http://www.medworm.com/index.php?rid=2221888&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1306 Polymer coating of adenovirus type 5 (Ad5) particles produces a 'stealth' Ad5 (sAd5) that confers protection from immune recognition, blocks receptor-mediated uptake, and favours uptake into pinocytic cells.In mixed cultures of primary adult rat dorsal root ganglion neurones (DRGN), rat C6 glioma cells, A9 non-Coxsackie and Ad Receptor (CAR)- and CAR-expressing fibroblasts, reporter gene expression after sAd5 pinocytotic uptake was monitored using the green fluorescent protein (gfp) gene, and viral particle trafficking and polymer coat dismantling was followed using Yoyo-1 tagged Ad5 DNA and Texas Red (TR) to label the coat.sAd5.gfp was pinocytosed by significantly higher proportions of neurones, than other cells, but GFP was not expressed. The TR-labelled coat remained co-localised with t... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2221888 Thu, 26 Feb 2009 05:00:00 +0100 2221888 http://www.medworm.com/index.php?rid=2221887&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1307 The expression of Moloney murine leukemia virus (Mo-MLV) gag proteins is sufficient to generate retrovirus-like particles (retroVLPs) that can be used as antigen-display platforms by pseudotyping with heterologous envelope proteins or by insertion of epitopes in structural constituents. To circumvent the in vitro production of such retroVLPs, we used DNA plasmids generating recombinant retroVLPs (plasmo-retroVLPs) as immunogens. We previously demonstrated that plasmo-retroVLPs induce significantly better antigen-specific T cell responses and antiviral immune protection than plasmids bearing a single mutation preventing retroVLPs assembly. In the present study, we investigated the possibility of using such plasmo-retroVLPs in prime-boost immunization strategies for hepatitis C virus (HCV) v... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2221887 Thu, 26 Feb 2009 05:00:00 +0100 2221887 http://www.medworm.com/index.php?rid=2187023&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1294 Developing vectors that target specifically to disease sites after systemic injection is an important goal in gene therapy research.We prepared fluorescent DNA polyplexes ([les]150 nm in diameter) comprising plasmid DNA condensed with poly(L-lysine) and coated with a multivalent reactive copolymer based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). These polyplexes were then surface modified with a recombinant P-selectin glycoprotein ligand-1 immunoglobulin chimera (rPSGL-Ig) previously investigated as a selectin antagonist in clinical studies.Five minutes after jugular vein injection of these polyplexes, fluorescence accumulation in inflamed cremasteric venules of C57BL6 mice was more than eight-fold higher than that observed after injection of Fc-blocked control polyplexes. Fluores... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2187023 Sun, 15 Feb 2009 05:00:00 +0100 2187023 http://www.medworm.com/index.php?rid=2166923&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1297 We describe the construction, characterization and use of a cellular genomic reporter assay for MCM expression that will potentially identify therapeutic pharmacological agents for methylmalonic aciduria treatment.Homologous recombination was used to insert an enhanced green fluorescent protein (EGFP) cassette inframe before the last codon of exon 13 of the MCM gene (MUT) in a BAC clone. The construct was used to generate stable HeLa cell lines. EGFP expression was measured by flow cytometry and the real-time reverse transcriptase-polymerase chain reaction was used to quantify changes in MUT gene mRNA levels.The genomic reporter assay used to screen a selection of compounds. Cisplatin, zidovudine and adefovir were found to increase the levels of MCM mRNA and EGFP expression, providing supp... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2166923 Sun, 08 Feb 2009 05:00:00 +0100 2166923 http://www.medworm.com/index.php?rid=2159964&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1300 Porcine circovirus type 2 (PCV2) is the primary cause of an emerging swine disease, postweaning multisystemic wasting syndrome, that is responsible for economic losses. To develop an effective vaccine for PCV2, we evaluated a heterologous prime-boost vaccine approach, using a gene gun-mediated naked DNA vector as a priming and modified vaccinia virus ankara (MVA) as a booster, in Balb/c mice.Three open reading frames (ORF) of PCV2 viral samples from infected pigs were amplified, and gene gun-mediated DNA priming vaccination was performed followed by boosts with MVA vectors expressing the same ORFs of PCV2. After vaccination, mice were challenged with PCV2 virus, and virus titers in the lungs and lymph nodes were measured.The combination of ORF-2 and -3 in this gene-based vaccine strategy r... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2159964 Thu, 05 Feb 2009 05:00:00 +0100 2159964 http://www.medworm.com/index.php?rid=2159966&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1298 Granulocyte-colony-stimulating factor (GM-CSF) is a pleiotropic factor for hematopoiesis that stimulates myeloblasts, monoblasts and mobilization of bone marrow stem cells. Therefore, the GM-CSF gene is a potential candidate for vessel formation and tissue remodeling in the treatment of ischemic diseases.A new mouse limb ischemia was established by surgery and gene transfer was performed by injection of 100 µg of a plasmid carrying GM-CSF. Muscle force and weight, histology, capillary density, circulating stem cells and monocytes were determined after 3-4 weeks.More than 60% of nontreated ischemic animals showed gangrene below the heel after 4 weeks, whereas the GM-CSF gene-treated animals showed only darkening of nails or toes. These animals demonstrated a full recovery of the affected m... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2159966 Wed, 04 Feb 2009 05:00:00 +0100 2159966 http://www.medworm.com/index.php?rid=2159965&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1299 Baculovirus is an effective vector for gene delivery into primary chondrocytes and repeated baculovirus transduction (i.e. supertransduction) appears to be promising for prolonging transgene expression, but how supertransduction may influence baculovirus-mediated gene delivery is unknown.We first investigated whether prior baculovirus transduction suppressed the ensuing transgene expression mediated by the supertransduced baculovirus, and then examined whether baculovirus triggered the expression of various cytokines. Whether interferon-[alpha] and -[beta] (IFN-[alpha]/[beta]) suppressed the transgene expression as well as the pivotal step responsible for the attenuated transgene expression were examined.Baculovirus transduction of chondrocytes elicited an immediate yet transient expressio... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2159965 Wed, 04 Feb 2009 05:00:00 +0100 2159965 http://www.medworm.com/index.php?rid=2150696&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1293 Fulminant liver failure can cause extreme mortality due to the lack of effective and targeting therapeutics for the disease. Novel therapeutics using antisense technology require an efficient and safe delivery system with Kupffer cell targeting ability.We explored the capacity of galactosylated low molecular weight chitosan (GLC) to efficiently mediate the antisense oligonucleotide (ASO) TJU-2755 into Kupffer cells, enhance the effect of the oligonucleotides on the suppression of tumor necrosis factor (TNF)-[alpha] and prolong the active time of the antisense drug in vivo. The protective and therapeutic effect of ASO/GLC in the animal model of D-galactosamine/lipopolysaccharide-induced fulminant hepatitis was tested.ASOs delivered by GLC were concentrated in Kupffer cells and more potent i... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2150696 Mon, 02 Feb 2009 05:00:00 +0100 2150696 http://www.medworm.com/index.php?rid=2150695&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1304 Gene delivery using synthetic vectors is frequently based on cationic compounds such as polyethyleneimine (PEI). However, few data have been published on the ability of PEI to mediate transgene expression in muscle tissue. Besides cationic vectors, there is increasing interest focusing on amphiphilic copolymers as gene carriers into striated muscles, although their mechanism of action is unknown.Plasmid DNA was associated with three different polymers: the cationic polyethyleneimine and two amphiphilic copolymers displaying few (tetronic 304) or no charges (pluronic L64). The resulting formulations were investigated by dynamic light scattering, laser doppler velocimetry, gel retardation assay and transmission electron microscopy. The toxicity and efficiency of the carriers were assessed in... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2150695 Mon, 02 Feb 2009 05:00:00 +0100 2150695 http://www.medworm.com/index.php?rid=2150694&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1303 Dominant-negative transforming growth factor [beta] type II receptor (T[beta]RII[Delta]cyt) is a protein that blocks transforming growth factor (TGF-[beta]) signaling. Because the consequences of blocking TGF-[beta] have not been completely elucidated in liver fibrosis, we analysed the effects of adenoviral delivery of T[beta]RII[Delta]cyt on profibrogenic genes and matrix metalloproteinase (MMP) proteins, as well as on TGF-[beta] signal repressor SKI-like oncogene (SnoN), in cultured hepatic stellate cells (HSCs) and in a rat model of liver fibrosis.To induce liver fibrosis, rats were treated with thioacetamide for 7 weeks and administrated once with Ad-T[beta]RII[Delta]cyt or Ad-[beta]gal through the iliac vein. Fibrosis was measured by morphometric analysis. We evaluated SnoN by western... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2150694 Mon, 02 Feb 2009 05:00:00 +0100 2150694 http://www.medworm.com/index.php?rid=2115208&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1296 Gene editing is potentially a powerful technology for introducing genetic changes by using short single-stranded DNA oligonucleotides (ssODNs). However, their efficiency is reduced by the mismatch repair system, especially MSH2, which may suppress gene editing, although findings vary depending on readout and type of oligonucleotide used. Additionally, successfully edited cells are reported to arrest at the S- or G2-phase. In the present study, we evaluate whether a novel ssODN design and down-regulation of MSH2 expression allows the isolation of replicating gene-edited cells.Cultured Chinese hamster ovary cells expressing mutated enhanced green fluorescent protein were targeted with ssODNs of varying design, all capable of restoring fluorescence, which allows the monitoring of correction e... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2115208 Tue, 20 Jan 2009 05:00:00 +0100 2115208 http://www.medworm.com/index.php?rid=2110217&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1290 Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood, ex vivo transduction of the gene of interest into them, and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor, time and money, while enhancing HSCs viability, transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes, in which reverse transcription of viral DNA is not completed.In the present study, we tested the possibility of targeting CD34+ progenitor cells wi... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2110217 Fri, 16 Jan 2009 05:00:00 +0100 2110217 http://www.medworm.com/index.php?rid=2110216&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1295 Intramuscular injection is a popular and effective approach to administer naked plasmid for transgene expression. The use of an adjuvant can provide a straightforward approach for enhancing transgene expression.Expression plasmid was formulated with various concentrations of trehalose for injection into the skeletal muscles of C57BL/6 mice. The effects of trehalose on gene dosage and the duration of transgene expression were assessed. The levels of transgene expression were indicated by levels of luciferase expression of the homogenized whole skeletal muscle or by histological X-gal staining of [beta]-galactosidase expression. Trehalose was also added to serum to examine the ability of protecting the DNA from degradation.It was found that an optimal trehalose concentration of 10 mM will ac... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2110216 Fri, 16 Jan 2009 05:00:00 +0100 2110216 http://www.medworm.com/index.php?rid=2100502&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1289 Expression cassettes can be inserted at several positions into recombinant adenoviral genomes but the implications of this choice for transgene expression level have not been determined. Knowledge of the relative expression levels of transgenes inserted at different sites in the adenoviral genome is of particular significance for transgene expression monitoring approaches that rely on the concordant expression of a marker transgene inserted elsewhere in the viral genome.Three expression cassettes, each comprising a cytomegalovirus promoter driving one of three marker peptides [serum carcinoembryonic antigen (sCEA), beta subunit of human chorionic gonadotropin ([beta]hCG) or human sodium iodide symporter (hNIS)], were inserted into E1, E3 or E4 cloning sites in a recombinant adenoviral vect... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2100502 Tue, 13 Jan 2009 05:00:00 +0100 2100502 http://www.medworm.com/index.php?rid=2100503&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1288 Antisense-mediated exon skipping is a putative treatment for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs), the disrupted DMD reading frame is restored, allowing generation of partially functional dystrophin and conversion of a severe Duchenne into a milder Becker muscular dystrophy phenotype. In vivo studies are mainly performed using 2[prime]-O-methyl phosphorothioate (2OMePS) or morpholino (PMO) AONs. These compounds were never directly compared.mdx and humanized (h)DMD mice were injected intramuscularly and intravenously with short versus long 2OMePS and PMO for mouse exon 23 and human exons 44, 45, 46 and 51.Intramuscular injection showed that increasing the length of 2OMePS AONs enhanced skipping efficiencies of human exon 45, but decreased efficiency for... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2100503 Mon, 12 Jan 2009 05:00:00 +0100 2100503 http://www.medworm.com/index.php?rid=2073153&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1287 Infected wounds present a major complication in patients with diabetes. Staphylococcus aureus is the most common single isolate in diabetic wounds. Human beta-defensin (hBD)-3 is antimicrobial active and appears to play a key role in the immune response. The present study aimed to analyse the effect of hBD-3 expression in a model of infected diabetic wounds.Excisional wounds were created on the backs of Yorkshire pigs and Ad5-CMV-hBD-3 vectors were microseeded. Wounds were inoculated with S. aureus, covered with a polyurethane chamber and analysed for transgene expression, bacterial infection, re-epithelialization, wound contraction, wound fluid production and blood vessel formation.hBD-3-treated wounds showed a total bacterial load of 2.1 × 108 colony-forming units (CFU)/g tissue, versus... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2073153 Sat, 03 Jan 2009 05:00:00 +0100 2073153 http://www.medworm.com/index.php?rid=2051952&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1285 Eosinophilia plays the major role in the pathogenesis of asthma and correlates with the up-regulation of eotaxin, which, together with interleukin (IL)-5, is important for differentiation, chemo-attraction, degranulation, and survival of eosinophils in local tissue. In a previous study, we found that administration of lentivirus-delivered short hairpin RNA (shRNA) to suppress the expression of IL-5 inhibited airway inflammation. The present study aimed to investigate the role of eotaxin shRNA and the synergistic effect of eotaxin and IL-5 shRNAs on airway inflammation in an ovalbumin (OVA)-induced murine model of asthma.Lentivirus-delivered shRNAs were used to suppress the expression of eotaxin and/or IL-5 in local tissue in an OVA-induced murine asthma model.Intra-tracheal administration ... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2051952 Sat, 20 Dec 2008 05:00:00 +0100 2051952 http://www.medworm.com/index.php?rid=2051953&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1284 Human adenovirus serotype 41 (Ad41) is a natural pathogen of the digestive tract and can cause gastroenteritis. There has been interest in reconstructing Ad41 as a gene delivery vector targeting the gastrointestinal tract, which is hampered by its fastidiousness.An Ad41 E1B55K-transduced 293 cell line (293E12) was established as the packaging cell line. A backbone plasmid (pAdbone41) and a shuttle plasmid (pSh41-CMV) were constructed based on the Ad41 genome. Replication-defective adenovirus (Ad41-GFP) was rescued in 293E12 after being transfected with the linearized adenoviral plasmid, which was generated by homologous recombination of pAdbone41 and the shuttle plasmid carrying the GFP gene in Escherichia coli strain BJ5183. The packaging ability of 293E12, the stability of the Ad41-GFP g... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2051953 Thu, 18 Dec 2008 05:00:00 +0100 2051953 http://www.medworm.com/index.php?rid=2021177&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1283 For adenovirus vectors derived from human serotype 5 (Ad5), the efficiency and safety after intravascular delivery is hindered by their sequestration in nontarget tissues, predominantly the liver. The latter is largely dictated by adenovirus binding to blood coagulation zymogens. In addition, several target cells, such as endothelial and smooth muscle cells, are difficult to transduce by Ad5 due to the low expression of the primary coxsackie-adenovirus receptor (CAR). Therefore, alternative adenovirus serotypes are being explored.In the present study, we assessed the tropism of mouse adenovirus type 1 (MAV-1), a nonhuman adenovirus for which cellular attachment is CAR-independent.The typical replication of MAV-1 in endothelial cells as observed in vivo was not reflected in elevated attachm... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2021177 Tue, 09 Dec 2008 05:00:00 +0100 2021177 http://www.medworm.com/index.php?rid=2021180&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1276 The transthyretin (TTR) amyloidosis is an incurable fatal inherited disease that is characterized by progressive peripheral and autonomic neuropathy. It is caused by missense amyloidogenic mutations in the TTR gene that destabilize the native tetrameric state and lead to the cytotoxic misfolded monomeric state. One interesting variant (T119M) stabilizes heterotetramers with amyloidogenic TTR and, in the reported heterozygous individuals, protects the carriers from disease. In the present study, we characterize in vitro and in vivo the ectopic expression of the human T119M mutant, termed a transsuppressor for TTR amyloid disease.Lentiviral vectors encoding wild or mutant forms of human TTR were constructed and transduced to the human hepatocellular carcinoma cell line, HepG2, or mice. Heter... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2021180 Mon, 08 Dec 2008 05:00:00 +0100 2021180 http://www.medworm.com/index.php?rid=2021179&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1280 Nitric oxide (NO·) derived from donor drugs has been shown to be an effective chemosensitizer in vitro. We investigated the combination of inducible nitric oxide synthase (iNOS) gene transfer, driven by a strong constitutive promoter (cytomegalovirus; CMV) with the DNA cross-linking agent cisplatin in mouse and human tumour cell lines.Proof of principal experiments were performed in the radiation-induced fibrosarcoma-1 (RIF-1) murine cell line. Cells were transfected with constitutively expressed CMV/iNOS plasmid DNA using a cationic lipid vector, before exposure to cisplatin. In vivo efficacy was determined in an intradermal RIF-1 tumour model, with intraperitoneal administration of cisplatin. Additionally, treatment potential was investigated in various human tumour cell lines including... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2021179 Mon, 08 Dec 2008 05:00:00 +0100 2021179 http://www.medworm.com/index.php?rid=2021178&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1277 A monoclonal antibody (mAb) 2F5 binds to the membrane-proximal external region (MPER) of the transmembrane subunit gp41 of human immunodeficiency virus type 1 (HIV-1) is known to broadly neutralize HIV-1 strains. The Adenovirus type 5 vector (Ad5) has been widely applied for HIV-1 vaccine, and hexon hypervariable region 5 (HVR5) is exposed on viral surface and easily target host immune responses against Ad5.We constructed a recombinant adenovirus type 5 vector (rAd5) with a 2F5-binding epitope (ELDKWA) of MPER on Ad5-HVR5. In addition, we developed rAd5 encoding the HIV-1IIIB envelope (Env) gene for the induction of Env-specific cellular immunity.The virus titers of the constructed rAd5 were similar to that of the parental Ad5 vector. Furthermore, high-dose immunization of rAd5 induced Env... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2021178 Mon, 08 Dec 2008 05:00:00 +0100 2021178 http://www.medworm.com/index.php?rid=2004627&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1281 We proposed to exploit hypoxia-inducible factor (HIF)-1[alpha] overexpression in prostate tumours and use this transcriptional machinery to control the expression of the suicide gene cytosine deaminase (CD) through binding of HIF-1[alpha] to arrangements of hypoxia response elements. CD is a prodrug activation enzyme, which converts inactive 5-fluorocytosine to active 5-fluorouracil (5-FU), allowing selective killing of vector containing cells.We developed a pair of vectors, containing either five or eight copies of the hypoxia response element (HRE) isolated from the vascular endothelial growth factor (pH5VCD) or glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (pH8GCD) gene, respectively. The kinetics of the hypoxic induction of the vectors and sensitization effects were evalua... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=2004627 Wed, 03 Dec 2008 05:00:00 +0100 2004627 http://www.medworm.com/index.php?rid=1998604&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1282 Japanese encephalitis (JE) is a serious infection disease throughout southern and eastern Asia. The design and development of safer and more efficacious vaccines against Japanese encephalitis virus (JEV) is a matter of high priority. Recently, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein was described as an attractive gene-delivery vehicle in mammalian cells and a potential vector for vaccine development. In the present study, we constructed recombinant pseudotype baculovirus encoding the Japanese encephalitis virus (JEV) envelope (E) protein and demonstrated that it could elicit high protective immunity in mice.Recombinant pseudotype baculovirus (BV-G-E) was generated by inserting JEV E gene fragment into pFastBac-VSV/G vector. BALB/c mice were immunized with BV-G-... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1998604 Mon, 01 Dec 2008 05:00:00 +0100 1998604 http://www.medworm.com/index.php?rid=1998605&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1275 Small interfering RNA (siRNA) delivery is a promising approach for the treatment of cardiovascular diseases. Matrix metalloproteinase (MMP) 2 over-expression in the arterial wall has been implicated in restenosis after percutaneous coronary intervention, as well as in spontaneous atherosclerotic plaque rupture. We hypothesized that in vivo local delivery of siRNA targeted at MMP2 (MMP2-siRNA) in the balloon-injured carotid artery of hypercholesterolemic rabbits may lead to inhibition of MMP2 expression.Two weeks after balloon injury, 5 µmol/l of Tamra-tagged MMP2-siRNA, scramble siRNA or saline was locally injected in the carotid artery and incubated for 1 h.Fluorescent microscopy studies showed the circumferential uptake of siRNA in the superficial layers of neointimal cells. MMP2 mRNA l... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1998605 Fri, 28 Nov 2008 05:00:00 +0100 1998605 http://www.medworm.com/index.php?rid=1989653&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1269 The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood.We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs.The differential modulation of a high number o... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1989653 Thu, 27 Nov 2008 05:00:00 +0100 1989653 http://www.medworm.com/index.php?rid=1974447&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1273 We present a novel targeted lipopolymeric vector, which improves significantly the levels of luciferase gene expression in the liver upon i.v. administration. Targeted-NP2 particles showed a five- and 12-fold higher transfection activity in the liver compared to non-targeted (plain) complexes or naked pCMV DNA, respectively. On the other hand, BNL tumor-bearing animals treated with AF-NP1 containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice, without signs of recurrence. High levels of IL-12 and interferon-[gamma] were detected in the sera of treated animals. Mice survival also improved considerably. Tumor treatment with AF-NP2 formulations lead only to a retardation in the tumor growth.In the present study,... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1974447 Fri, 21 Nov 2008 05:00:00 +0100 1974447 http://www.medworm.com/index.php?rid=1974448&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1272 It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufacturing process. Several challenges encountered in the gene therapy field have emphasized the need for the development of novel platforms that allow the recovery of gene vectors and enable efficient transfection of cells. The use of plasmid DNA-based therapeutics relies on procedures that efficiently purify the supercoiled (sc) plasmid isoform. Plasmid DNA (pDNA) purification strategies that use amino acids as immobilized ligands have recently yielded interesting results.The present study describes a strategy that uses arginine-chromatography to specifically purify sc pDNA from other isoforms and Escherichia coli impurities present in a clarified lysate.Control ana... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1974448 Wed, 19 Nov 2008 05:00:00 +0100 1974448 http://www.medworm.com/index.php?rid=1951244&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1265 We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg-Dmdmdx-4Cv/J (4CV) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame.A series of 2[prime]-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels.Compared to the original mdx mouse studies, induction of exon skipping from the 4CV dystrophin mRNA was... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1951244 Wed, 12 Nov 2008 05:00:00 +0100 1951244 http://www.medworm.com/index.php?rid=1951243&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1268 Interleukin (IL)-13, overproduced in the skin of atopic dermatitis (AD), has been shown to play an essential role in the pathogenesis of the disease. Thus, inhibition of IL-13 production should provide a key step to alleviate disease conditions of the atopic skin. In the present study, IL-13 antisense oligonucleotide (ASO) was designed and formulated with cationic elastic liposome (cEL) to improve transdermal delivery.ASOs were generated against murine IL-13 mRNA (+4 to + 23) and complexed with cEL. Physicochemical properties of IL-13 ASO/cEL complex were examined by DNA retardation and DNase I protection assay. An in vitro inhibition study was performed in T-helper 2 (Th2) cells and cytotoxicity was tested by the XTT assay. The in vivo effect of IL-13 ASO/cEL complex was tested in a murin... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1951243 Wed, 12 Nov 2008 05:00:00 +0100 1951243 http://www.medworm.com/index.php?rid=1951242&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1271 Japanese encephalitis (JE) is a serious infection and disease in southern and eastern Asia. The design and development of safer and more efficacious vaccines against Japanese encephalitis virus (JEV) is a high-priority target in the world. Recently, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) was described as an attractive gene delivery vehicle in mammalian cells and a potential vector for vaccine development. In the present study, we constructed a recombinant pseudotype baculovirus encoding the JEV envelope (E) protein and demonstrated that it could elicit high protective immunity in mice.Recombinant pseudotype baculovirus (BV-G-E) was generated by inserting JEV E gene fragment into pFastBac-VSV/G vector. BALB/c mice were immunized with BV-G-E and challenge... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1951242 Wed, 12 Nov 2008 05:00:00 +0100 1951242 http://www.medworm.com/index.php?rid=1946672&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1270 The extremely poor prognosis of patients with pancreatic ductal adenocarcinoma indicates the need for novel therapeutic approaches. The growth arrest and DNA damage-inducible (Gadd) gene Gadd45a is a member of a group of genes that are induced by DNA damaging agents and growth arrest signals.We evaluated the biological activity of Gadd45a in pancreatic ductal adenocarcinoma cancer-derived cell lines and assessed the efficacy of a combined treatment with adenoviral-mediated expression of Gadd45a (Ad-G45a) and anticancer drug (Etoposide, cisplatin, 5-fluorouracil, respectively) for the PANC1 cell line.Gadd45a is variously expressed in cell lines derived from pancreatic ductal adenocarcinoma cancer and adenoviral-mediated expression of Gadd45a (Ad-G45a) in these cells results in apoptosis via... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1946672 Tue, 11 Nov 2008 05:00:00 +0100 1946672 http://www.medworm.com/index.php?rid=1946674&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1266 Nuclear factor-kappa B (NF-[kappa]B) is well known for playing a pivotal role in restenosis after percutaneous coronary intervention (PCI).This is the first report to demonstrate an effect of NF-[kappa]B decoy oligodeoxynucleotides (ODN) to prevent restenosis after PCI after a 4-year observation using a coronary computed tomography (CT) scan. We showed that the decoy treatment suppressed neointimal formation after stent implantation compared to that in the same artery.Thus, for the first time, we demonstrate the clinical usefulness of the CT scan to reveal the effects of NF-[kappa]B decoy ODN transfer after PCI. Copyright © 2008 John Wiley & Sons, Ltd. (Source: The Journal of Gene Medicine) The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1946674 Mon, 10 Nov 2008 05:00:00 +0100 1946674 http://www.medworm.com/index.php?rid=1946673&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1267 DNA vaccines offer unique potential for generating protective and therapeutic immunity against infectious and malignant diseases. Unfortunately, rapid degradation and poor cellular uptake has significantly limited the efficacy of 'naked' plasmid DNA vaccines. We have previously described stabilized plasmid lipid particles (SPLP) as effective nonviral gene delivery vehicles for the transfection of tumours at distal sites following intravenous administration. Based on their low toxicity and favourable transfection profile following systemic administration, we investigate SPLP as gene delivery vehicles for the generation of a systemically administered genetic vaccine.The uptake of SPLP and their ability to transfect splenic antigen presenting cells (APC) following systemic administration is a... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1946673 Mon, 10 Nov 2008 05:00:00 +0100 1946673 http://www.medworm.com/index.php?rid=1879502&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1261 Purine nucleoside phosphorylase (PNP) deficiency causes the accumulation of toxic purine metabolites and lethal T cell immune defects, which might be corrected by expressing PNP by transplanting bone marrow (BM) cells transduced with lentiviral vectors containing the human PNP gene (lentiPNP).Lymphocytes from a single PNP-deficient patient as well as lymphocytes, fibroblasts and BM from PNP-deficient (PNP - /-) mice were transduced with lentiPNP. Female PNP - /- mice were transplanted with lentiPNP transduced BM cells from male PNP - /- mice or normal BM.LentiPNP transduction significantly increased PNP expression in PNP-deficient human lymphocytes, murine lymphocytes, fibroblasts and BM cells. LentiPNP transduction also significantly improved the proliferation of PNP - /- murine lymphocyt... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1879502 Thu, 16 Oct 2008 04:00:00 +0100 1879502 http://www.medworm.com/index.php?rid=1853408&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1257 We previously demonstrated that a new lentiviral vector derived from nonpathogenic simian immunodeficiency virus (SIVagm) was efficient and safe for long-lasting retinal gene transfer, and that it provided the significant therapeutic effect of expressing human pigment epithelium-derived factor (hPEDF) in Royal College of Surgeons (RCS) rats. In the present study, to obtain a more pronounced outcome, we assessed the potential synergistic effect of the simultaneous gene transfer of hPEDF and human fibroblast growth factor-2 (hFGF-2) by improved third-generation SIV on RCS rats and retinal degeneration slow (rds) mice, because the former targets the primary neurons, including photoreceptor cells (PCs), whereas the latter is effective for targeting secondary neural cells, including Muller cell... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1853408 Mon, 06 Oct 2008 04:00:00 +0100 1853408 http://www.medworm.com/index.php?rid=1853407&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1262 We describe the successful modification of adenovirus vectors with large polysaccharides by reductive amination. The particles were efficiently modified, physically intact and, importantly, detargeted from the natural Coxsackie and adenovirus receptor/integrin pathway in vitro. In addition, they exhibited significantly decreased transduction of muscle after local delivery and of liver after systemic delivery in mice. However, despite the modification of 60% of capsid surface amino groups, mannanylated particles were unable to evade neutralizing anti-Ad5 antibodies.Mannanylated vectors are a paradigm for a novel class of glycoviruses modified with large polysaccharides. Vector promiscuity as one of the important hurdles for Ad-mediated gene transfer could be significantly decreased in vivo,... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1853407 Mon, 06 Oct 2008 04:00:00 +0100 1853407 http://www.medworm.com/index.php?rid=1824233&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1258 To overcome the extracellular barriers in gene delivery and direct gene delivery to target tissues, substrate-mediated transfection, which sustains the release of naked DNA or vector/DNA complexes, and also supports cell growth, has been developed.In the present study, polyamidoamine (PAMAM) dendrimer/DNA complexes encapsulated functional biodegradable polymer films for substrate-mediated gene delivery were prepared. To maintain the activity of DNA during dehydration, the dendrimer/DNA complexes were encapsulated in a water soluble polymer, poly [alpha],[beta]-[N-(2-hydroxyethyl)-L-aspartamide], and then deposited on or sandwiched in functional polymer films with a fast degradation rate to mediate gene transfection. The in vitro gene transfections of pGL3-Luc and pEGFP-C1 plasmids in HEK29... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1824233 Thu, 25 Sep 2008 04:00:00 +0100 1824233 http://www.medworm.com/index.php?rid=1824237&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1212 Numerous synthetic cationic vectors have been synthesized and are successfully used for in vitro gene transfer but an excess of positive charges can lead to cytotoxicity and does not enable specific transfection.We decided to develop alternative molecular systems consisting of neutral, colloidally stable bioassemblies equipped with ligands for specific cell targeting. Consequently, we directed our efforts toward the development of a multimodular non-viral gene delivery system consisting of a condensed core of DNA with cationic liposomes of bis(guanidinium)-tren-cholesterol and an external corona of poly(ethylene oxide) stretches harbored by the steric stabilizers used to stabilize lipoplexes colloidally. A ligand capable of cell targeting by receptor-mediated endocytosis was covalently lin... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1824237 Wed, 24 Sep 2008 04:00:00 +0100 1824237 http://www.medworm.com/index.php?rid=1824236&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1248 The involvement of surface molecules in HIV-1-derived lentivirus (LV)-mediated transduction of airway epithelial cells has not been studied so far. The present study aimed to evaluate the role of glycosaminoglycans (GAGs) in gene transfer mediated by a third generation vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped LV vector in an in vitro model of polarized airway epithelial cells.Human bronchial (16HBE-S1) and tracheal (CFT1-C2) epithelial cells were grown either on plastic or on filters and transduced with the LV vector polypurine tract (PPT)-green fluoresecent protein (GFP). Zonula Occludens (ZO)-1, a marker of tight junction, and GAG localization were assessed by cytofluorimetry and confocal microscopy. Soluble GAGs and removal of cell surface GAGs were used to affect L... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1824236 Wed, 24 Sep 2008 04:00:00 +0100 1824236 http://www.medworm.com/index.php?rid=1824235&cid=s_33648_50_f&fid=33648&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fjgm.1250 Genetic manipulation of the allograft is an attractive approach to prevent the graft against chronic deterioration through stable expression of immunomodulatory or protective genes. However, the best strategy for prevention of chronic allograft deterioration remains unclear.The efficacies of adeno-associated viral vector-mediated stable expression of indoleamine 2,3-dioxygenase (IDO), cytotoxic T-lymphocyte associated antigen 4 with human immunoglobulin G1 (CTLA4Ig) or interleukin-10 (IL-10) in the prevention of chronic allograft deterioration were compared in a rat heart transplantation model.Transduction of grafts with IL-10 significantly prolonged allograft survival, whereas transduction of grafts with IDO did not improve graft survival compared to controls. Analysis of long-term surviv... The Journal of Gene Medicine journals http://www.medworm.com/rss/comments.php?id=1824235 Wed, 24 Sep 2008 04:00:00 +0100 1824235