Papers by Keyword: Gene Therapy

Abstract: Pentose phosphate (PP) pathway, which is ubiquitously present in all living organisms, is one of the major metabolic pathways associated with glucose metabolism. The most important functions of this pathway includes the generation of reducing equivalents in the form of NADPH for reductive biosynthesis, and production of ribose sugars for the biosynthesis of nucleotides, amino acids, and other macromolecules required by all living cells. Under normal conditions of growth, PP pathway is important for cell cycle progression, myelin formation, and the maintenance of the structure and function of brain, liver, cortex and other organs. Under diseased conditions, such as in cases of many metabolic, neurological or malignant diseases, pathological mechanisms augment due to defects in the PP pathway genes. Adoption of alternative metabolic pathways by cells that are metabolically abnormal, or malignant cells that are resistant to chemotherapeutic drugs often plays important roles in disease progression and severity. Accordingly, the PP pathway has been suggested to play critical roles in protecting cancer or abnormal cells by providing reduced environment, to protect cells from oxidative damage and generating structural components for nucleic acids biosynthesis. Novel drugs that targets one or more components of the PP pathway could potentially serve to overcome challenges associated with currently available therapeutic options for many metabolic and non-metabolic diseases. However, careful designing of drugs is critical that takes into the accounts of cell’s broader genomic, proteomic and metabolic contexts under consideration, in order to avoid undesirable side-effects. In this review, we discuss the role of PP pathway under normal and abnormal physiological conditions and the potential of the PP pathway as a target for new drug development to treat metabolic and non-metabolic diseases.

Abstract: Fermentation, a process traditionally known for the anaerobic conversion of sugar to carbon dioxide and alcohol by yeast, now refers to an industrial process of manufacturing a wide variety of metabolites and biomaterials by using microorganisms or mammalian cells in a controlled culture environment. Fermentation can be performed in batch mode, continuous mode or in a combinatory, fed-batch mode, depending on the product of interest. Fermentation technology has long been known for the production of various medically important products such as antibiotics, solvents such as ethanol, intermediary compounds such as citric acid, probiotics such as yoghurt etc. New generation fermentation products include anti-viral drugs, therapeutic recombinant proteins and DNA, and monoclonal antibodies. Apart from the drugs, fermentation is also used for the commercial production of materials required for the development of diagnostic kits, drug delivery vehicles and medical devices. Fermentation technology remains at the heart of rapidly growing biopharmaceutical industry today, which is expected to expand even more in the days ahead, in parallel with the progress in novel, targeted drug discovery.

Abstract: Protein tyrosine phosphatase receptor-type O (PTPRO) has been described in several forms of cancer as a new member of the PTP family. The tumor suppressor function of PTPRO was evaluated by design and synthesis the 10-23 deoxyribozyme (DRz), thio-modified DRz (DRz-s) and antisense oligonucleotide (asON) of the PTPRO genomic mRNA to detect the catalytic cleavage activity. Firstly, the cDNA fragment of PTPRO gene was amplified from total cellular RNA of the HepG2.2.15 cells by reverse transcription PCR (RT-PCR). Subsequently, the fragments were cloned to pcDNA3.1(+) plasmids and generated a recombinant plasmids, then sifted the positive recombinant plasmids out to amplify. The expression vector of PTPRO mRNA was obtained in vitro transcription by using T7 RNA polymerase. The results of transfection indicated that when PTPRO mRNA gamyed with deoxyribozyme which activity enhanced, so DRz-s were detected with more intensive specific catalytic cleavage activity than DRz by cells transfecting. And the asON wasn't detected with the property.

Abstract: Gene therapy has the ability to treat diseases by delivering exogenous DNA into the nuclei of target cells to express a therapeutic protein. In intravenous gene delivery, free oligonucleotides and DNA are rapidly degraded by serum nucleases in the blood before they can reach the target site. Therefore, the main goal for gene therapy is not just to obtain cellular expression of an exogenous gene per se, but also to develop non-toxic and efficient carriers. Because of its abundant positive surface charges, PEI is able to condense the negatively charged DNA into PEI/DNA complexes with a net positive charge. PEG facilitates the formation of polyplexes with improved solubility, reduced aggregation, lower cytotoxicity, and possibly decreases opsonization with serum proteins in the bloodstream. Also, dexamethasone is the potent ligand of the glucocorticoid receptor which facilitates the transfer into nucleus, and it is known to enlarge the nuclear pore complexes. In this study, PEG-Glu-PEI-Dexa was synthesized as a kind of biodegradable polycation for gene delivery.

Abstract: An efficient and safe delivery system of RNA interfering is required for clinical application of gene therapy. The study aimed to develop Fe2O3-based nanoparticles for gene delivery to overcome the disadvantages of polyethyleneimine (PEI) or cationic liposome as gene carrier including the cytotoxicity caused by positive charge and aggregation in the cells surface. PEI-capped Fe2O3 nanoparticles are successfully manufactured utilizing Fe2O3 as core, PEI as carapace, which bind miRNA at an appropriate weight ratio by electrostatic interaction and result in well-dispersed nanoparticles. The synthesized GFP tag with miR-26a expression plasmid was used for monitoring transfection efficiency in HepG2 cells. The nanocomplex exhibited higher transfection efficiency and lower cytotoxicity in HepG2 cells than the PEI/DNA complex and commercially available liposome. The delivery resulted in a significantly upregulation of miR-26a in HepG2 cells. Our results offer an alternate delivery system for RNA interfering that can be used on any gene of interest.

Abstract: Calcium phosphate is a natural biomineral and therefore possesses an excellent biocompatibility due to its chemical similarity to human hard tissue (bone and teeth). Calcium phosphate nanoparticles can be precipitated under controlled conditions and used as carrier in biological systems, e.g. to transfer nucleic acids or drugs. Such nanoparticles can also be suitably functionalized with fluorescing dyes, polymeric agents, pro-drugs or activators. The small monodisperse nanoparticles only mildly influence the intracellular calcium level and therefore are not toxic for cells.

Abstract: The development of skin tissue engineering provides a noninvasive method for skin restoration. Unfortunately, the lack of a vascular plexus leads to greater time for vascularization compared with native skin autografts and contributes to graft failure. Our purpose was to construct tissue-engineered skin with VEGF- modified human bone marrow mesenchymal stem cells (hMSCs) as well as acellular dermal matrix(ADM) in vitro , Thus by increased vascular endothelial growth factor expression, which could prospectively improve vascularization of tissue-engineered skin for wound healing applications. To reach this aim, hMSCs were isolated and cultured with density gradient centrifugation combined with attachment culture method in vitro. Liposome- mediated gene transfer was used to generate a population of hMSCs overexpressing the gene encoding VEGF165. Then VEGF- modified hMSCs were seeded onto the surface of ADM. The experimental results showed that ADM we prepared has good compatibility with MSCs, the cells in ADM grew and proliferated well in vitro and the tissue - engineered skin with VEGF- modified hMSCs and ADM has been successfully constructed.

Abstract: Novel hybrid biomaterial of gelatin-siloxane nanoparticles (GS NPs), with positive surface potential and lower cytotoxicity, was synthesized through a 2-step sol-gel process. The pDNA-GS NPs complex was formulated with high encapsulation efficiency, and exhibited and efficient transfection in vitro. We thus envision that the GS NPs material could serve as non-viral gene vectors for gene therapy.

Abstract: In this study, we constructed a novel drug delivery system for realizing the combination of gene therapy and chemotherapy by co-loading 5-FU and antisense EGFR (epidermal growth factor receptor) plasmids in biodegradable PLGA/O-CMC (O-carboxmethyl-chitosan) nanoparticles. This novel kind of nanoparticle was characterized by dynamic light scattering for size, size distribution and zeta potential, and scanning electron microscopy (SEM) and transmission electronic microscopy (TEM) for morphological properties and structures. Drug encapsulation efficiency and drug release kinetics under in vitro conditions were also measured. At the same time, the MTT assay, the TUNEL technique and immunohistochemical staining were used to investigate the antitumor activity of these multi-functional nanoparticles on human glioma cells. It was concluded that the 5-FU and plasmid encapsulation efficiencies were as high as 94.5% and 95.7%, and the 5-FU release activity from nanoparticles could be sustained for as long as three weeks. Both the MTT assay and the TUNEL method illustrated that these multi-functional nanoparticles had cytotoxicities as high as 93.5% and could induce apoptosis in most glioma cells. Immunohistochemical staining proved that plasmids on the surface of nanoparticles could transfect gliomas cells, verified by a decline in the expression level of EGFR protein by the glioma cells. Therefore, this novel delivery system for drugs and genes provides another therapeutic pathway for cancer and needs further research.

Abstract: Recent study shows that endothelial progenitor cells (EPCs) and gene therapy technologies are effective strategies in the inhibition of stenosis and thrombus formation and improving the patency rate of the vascular graft in vivo. In this study, rat EPCs were cultured from bone marrow, and plated in fibronectin-coated plates with EBM-2 medium. Bone marrow mesenchymal stem cells (MSCs) were cultured with alpha minimum essential medium ( -MEM). After two weeks, EPCs were immunohistochemically characterized using antibodies specific for endothelial cells. Retroviral vectors pMSCV-eNOS, pMSCV-tPA, pMSCV-LacZ and pMCSV-GFP were constructed. Retroviral particles were produced using packaging cell line 293T cells. Gene transfer was carried out by exposing cells to virus solution for 6 hours in the presence of 8µg/ml polybrene. For constructing vessels, MSCs and EPCs were seeded on fibronectin coated ePTFE graft in tissue culture condition for 2-4 weeks. The attachment and growth of cells were analyzed with scanning electron microscopy (SEM). Our data showed that the EPCs expressed VEGF, Lectin BS-1, RECA-1, indicating they are endothelial lineage. The concentrated retroviral particles showed many folds higher transduction efficiency to NIH 3T3 cells than the commercial reagent Fugene. SEM data showed dense attachment of MSCs on the graft surface. MSCs/EPCs co-culture gave much better cell coverage on the graft than culture of EPCs alone.