Current Issue : October - December Volume : 2019 Issue Number : 4 Articles : 5 Articles
There is an increased interest in plasmid DNA as therapeutics. This is evident in the number\nof ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic,\nhigh yield and high level of supercoiling are required. From the bioprocessing point of view, the\nsupercoiling level potentially has an impact on the ease of downstream processing. We approached\nmeeting these requirements through plasmid engineering...............................
With the rapid progress of genetic engineering and gene therapy, theWorld Anti-Doping\nAgency has been alerted to gene doping and prohibited its use in sports. However, there is no\nstandard method available yet for the detection of transgenes delivered by recombinant adenoviral\n(rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV\nvectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene\nwas delivered in mice through intravenous injection or local muscular injection. After five days, stool\nand whole blood samples were collected, and total DNA was extracted. As additional experiments,\nwhole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv\nvector. Transgene fragments from different DNA samples were analyzed using semi-quantitative\nPCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene\nfragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool\nDNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a\ncombination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used\nin this model. These results could accelerate the development of detection methods for gene doping....
Human mesenchymal stem cells (hMSCs) show enormous potential in regenerative\nmedicine and tissue engineering. However, current use of hMSCs in clinics is still limited because\nthere is no appropriate way to control their behavior in vivo, such as differentiation to a desired\ncell type. Genetic modification may provide an opportunity to control the cells in an active manner.\nOne of the major hurdles for genetic manipulation of hMSCs is the lack of an effcient and safe gene\ndelivery system. Herein, biocompatible calcium phosphate (CaP)-based nanoparticles stabilized\nwith a catechol-derivatized hyaluronic acid (dopa-HA) conjugate were used as a carrier for gene\ntransfection to hMSCs for improved differentiation. Owing to the specific interactions between HA\nand CD44 of bone marrow-derived hMSCs, dopa-HA/CaP showed significantly higher transfection\nin hMSCs than branched polyethylenimine (bPEI, MW 25 kDa) with no cytotoxicity. The co-delivery\nof a plasmid DNA encoding bone morphogenetic protein 2 (BMP-2 pDNA) and micro RNA 148b\n(miRNA-148b) by dopa-HA/CaP achieved significantly improved osteogenic differentiation of hMSCs....
The pancreas is a glandular organ that functions in the digestive system and endocrine\nsystem of vertebrates. The most common disorders involving the pancreas are diabetes, pancreatitis,\nand pancreatic cancer. In vivo gene delivery targeting the pancreas is important for preventing or\ncuring such diseases and for exploring the biological function of genes involved in the pathogenesis\nof these diseases. Our previous experiments demonstrated that adult murine pancreatic cells can\nbe efficiently transfected by exogenous plasmid DNA following intraparenchymal injection and\nsubsequent in vivo electroporation using tweezer-type electrodes. Unfortunately, the induced gene\nexpression was transient. Transposon-based gene delivery, such as that facilitated by piggyBac (PB),\nis known to confer stable integration of a gene of interest (GOI) into host chromosomes, resulting\nin sustained expression of the GOI. In this study, we investigated the use of the PB transposon\nsystem to achieve stable gene expression when transferred into murine pancreatic cells using the\nabove-mentioned technique. Expression of the GOI (coding for fluorescent protein) continued for at\nleast 1.5 months post-gene delivery. Splinkerette-PCR-based analysis revealed the presence of the\nconsensus sequence TTAA at the junctional portion between host chromosomes and the transgenes;\nhowever, this was not observed in all samples. This plasmid-based PB transposon system enables\nconstitutive expression of the GOI in pancreas for potential therapeutic and biological applications....
During the last decade, important advances have occurred regarding understanding of the\npathogenesis and treatment of rheumatoid arthritis (RA). Nevertheless, response to treatment is not\nuniversal, and choosing among different therapies is currently based on a trial and error approach.\nThe specific patientâ??s genetic background influences the response to therapy for many drugs: In this\nsense, genomic studies on RA have produced promising insights that could help us find an effective\ntherapy for each patient. On the other hand, despite the great knowledge generated regarding the\ngenetics of RA, most of the investigations performed to date have focused on identifying common\nvariants associated with RA, which cannot explain the complete heritability of the disease. In this\nregard, rare variants could also contribute to this missing heritability as well as act as biomarkers\nthat help in choosing the right therapy. In the present article, different aspects of genetics in the\npathogenesis and treatment of RA are reviewed, from large-scale genomic studies to specific rare\nvariant analyses. We also discuss the shared genetic architecture existing among autoimmune diseases\nand its implications for RA therapy, such as drug repositioning....
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