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2 results for “transfection efficiency”
Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells
<p>Raw data of the publication "Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells".</p> <p>Abstract: Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal<br> neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor<br> degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell<br> growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated,<br> often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are<br> costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene<br> therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor<br> (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression<br> of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system<br> that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a<br> low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated<br> the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as<br> primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed<br> by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected<br> in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination<br> with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while<br> ensuring high transfection efficiency and long-term transgene expression in RPE cells.</p>
Core-shell structured chitosan-polyethylenimine nanoparticles for gene delivery: Improved stability, cellular uptake, and transfection efficiency
<p>Gene therapy has emerged as a promising treatment option for various acquired and inherited diseases. The delivery of nucleic acids relies on so-called vectors that condense and encapsulate their cargo, generating stable nano-sized particles. Especially non-viral gene delivery systems are of increasing interest. However, accomplishing therapeutic levels of transgene expression and limited tolerability of these systems remain a challenge. Therefore, we investigate in the present study the improvement of nucleic acid delivery using depolymerized chitosan – polyethylenimine DNA core complexes (dCS-PEI/DNA). These core complexes are further entrapped into a variety of dCS-based shells, functionalized with poly(ethylene glycol) (PEG) spacers conjugated to ionic moieties (amino or carboxylate groups) and cell penetrating peptides. This modular approach allowed to evaluate the effect of the shell functional components on the physico-chemical particle characteristics and biological effects <em>in vitro</em>. The optimized ternary complex combines a core-dCS-LPEI/DNA complex with a shell consisting of dCS-PEG-COOH, which resulted in improved encapsulation of nucleic acid, accelerated cellular uptake, enhanced transfection efficiency, and superior transfection potency in human hepatoma HuH-7 cells and mouse primary hepatocytes. Effects on transgene expression are confirmed <em>in vivo</em> in wild-type mice following retrograde intrabiliary infusion. After administration to mice of only 100 ng complexed nanovector DNA, ternary complexes induce a high reporter gene signal for three days. We conclude that ternary core-shell structured particles comprising functionalized chitosan are a promising gene delivery technology for both <em>in vitro</em> as well as <em>in vivo </em>applications. The modular design will facilitate the development of chemically modified derivatives.</p>
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