The aim of this study was to investigate the influence of cell cycle on transfection efficiency. Counterflow centrifugal elutriation was used which avoids possible side-effects from chemical treatment of cells. With this method, cell populations were fractionated by means of size and density, and fractions corresponding to discrete cell cycle phase-specific populations were transfected with various nonviral methods (Lipofectamine, TfpLys and TfPEI), adenovirus-enhanced transferrinfection (AVET system) and recombinant adenovirus. Transfection efficiency was found to be strongly dependent on the cell cycle stage at the time of transfection. Luciferase activity from cells transfected with polycation-or lipid-based transfection systems was 30-to more than 500-fold higher when transfection was performed during S or G2
Like viruses, intracellular bacteria stimulate their host cells to produce type I IFNs (IFN-α and IFN-β). In our study, we investigated the signals and molecules relevant for the synthesis of and response to IFN by mouse macrophages infected with Listeria monocytogenes. We report that IFN-β is the critical immediate-early IFN made during infection, because the synthesis of all other type I IFN, expression of a subset of infection-induced genes, and the biological response to type I IFN was lost upon IFN-β deficiency. The induction of IFN-β mRNA and the IFN-β-dependent sensitization of macrophages to bacteria-induced death, in turn, was absolutely dependent upon the presence of the transcription factor IFN regulatory factor 3 (IRF3). IFN-β synthesis and signal transduction occurred in macrophages deficient for TLR or their adaptors MyD88, TRIF, or TRAM. Expression of Nod2, a candidate receptor for intracellular bacteria, increased during infection, but the protein was not required for Listeria-induced signal transduction to the Ifn-β gene. Based on our data, we propose that IRF3 is a convergence point for signals derived from structurally unrelated intracellular pathogens, and that L. monocytogenes stimulates a novel TLR- and Nod2-independent pathway to target IRF3 and the type I IFN genes.
In many cases, nonviral particle-mediated gene delivery is highly dependent on the cell cycle status of transfected cells. Here we compare particle-mediated delivery with linear polyethylenimine (PEI) and physical transfer of DNA by electroporation with branched PEI and lipofection for their ability to transfect cells at different stages of the cell cycle. In contrast to other particle-mediated delivery methods (using Lipofectamine or branched PEI) linear PEI led to only small differences (within 1 log unit) in gene transfer between HeLa cells transfected in G1 and those in S/G2. Parallel transfections (lipofection or branched PEI) resulted in 2 to > 3 log-unit differences in luciferase expression between cells transfected in G1 and S/G2. Gene transfer by electroporation also revealed hardly any cell cycle dependence and displayed completely different expression kinetics. Reporter gene expression is already very high 3 hours after electroporation with roughly the same level of reporter gene expression in all cell cycle phases. We suggest that DNA electroporation and DNA transfection with linear PEI particles have improved nuclear import characteristics relative to the other tested DNA delivery systems.
Receptor-binding ligands have been incorporated into DNA/polyethylenimine (PEI) complexes to enhance cell binding and cellular internalization. This study characterizes receptor-mediated uptake of DNA/PEI complexes on a cellular basis. A novel assay based on flow cytometry was applied, discriminating between total cell-associated and extracellularly bound DNA complexes. Receptor-mediated uptake of ligand-containing DNA/PEI (molecular weight, 800 kd) complexes was found to occur quickly (within 1 hour), whereas unspecific uptake through adsorptive endocytosis is less efficient or requires extended periods to reach the same degree of internalization. Rapid, receptor-mediated internalization requires a small complex size; however, large, aggregated complexes show higher gene expression. Using PEI 25 kd conjugated to large proteins such as transferrin or antibodies, improper condensation with DNA leads to suboptimal uptake and gene expression, whereas partial replacement of ligand-PEI with unconjugated PEI increases both uptake and transfection. In contrast, the 8 kd protein epidermal growth factor conjugated to PEI 25 kd properly condenses DNA and mediates specific uptake into human adenocarcinoma (KB) cells. Modification of the complex surface with appropriate amounts of poly(ethylene glycol) (PEG) does not block ligand-mediated internalization. A higher degree of PEGylation reduces the internalization of transferrin or antibody-containing complexes to a level similar to that of ligand-free complexes. In contrast, epidermal growth factor "mediated uptake is less effected by excessive PEGylation.
BackgroundLow Density Lipoprotein (LDL) hypercholesterolemia, and its associated cardiovascular diseases, are some of the leading causes of death worldwide. The ability of proprotein convertase subtilisin/kexin 9 (PCSK9) to modulate circulating LDL cholesterol (LDLc) concentrations made it a very attractive target for LDLc management. To date, the most advanced approaches for PCSK9 inhibition are monoclonal antibody (mAb) therapies. Although shown to lower LDLc significantly, mAbs face functional limitations because of their relatively short in vivo half-lives necessitating frequent administration. Here, we evaluated the long-term efficacy and safety of PCSK9-specific active vaccines in different preclinical models.Methods and FindingPCSK9 peptide-based vaccines were successfully selected by our proprietary technology. To test their efficacy, wild-type (wt) mice, Ldlr
+/− mice, and rats were immunized with highly immunogenic vaccine candidates. Vaccines induced generation of high-affine PCSK9-specific antibodies in all species. Group mean total cholesterol (TC) concentration was reduced by up to 30%, and LDLc up to 50% in treated animals. Moreover, the PCSK9 vaccine-induced humoral immune response persisted for up to one year in mice, and reduced cholesterol levels significantly throughout the study. Finally, the vaccines were well tolerated in all species tested.ConclusionsPeptide-based anti-PCSK9 vaccines induce the generation of antibodies that are persistent, high-affine, and functional for up to one year. They are powerful and safe tools for long-term LDLc management, and thus may represent a novel therapeutic approach for the prevention and/or treatment of LDL hypercholesterolemia-related cardiovascular diseases in humans.
For systemic application, the physical and colloidal parameters of the transfection complexes, such as particle size, stability, and surface charge, determine DNA biodistribution, toxicity, and transfection efficacy. By controlling these parameters, DNA biodistribution and gene expression can be targeted to different organs.
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