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      A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids

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          Abstract

          Synthetic vectors based on reducible polycations consisting of histidine and polylysine residues (HIS RPCs) were evaluated for their ability to deliver nucleic acids. Initial experiments showed that RPC-based vectors with at least 70% histidine content mediated efficient levels of gene transfer without requirement for the endosomolytic agent chloroquine. Significant gene transfer was observed in a range of cell types achieving up to a 5-fold increase in the percentage of transfected cells compared to 25 kDa PEI, a gold standard synthetic vector. In contrast to 25 kDa PEI, HIS RPCs also mediated efficient transfer of other nucleic acids, including mRNA encoding green fluorescent protein in PC-3 cells and siRNA directed against the neurotrophin receptor p75 NTR in post-mitotic cultures of rat dorsal root ganglion cell neurons. Experiments to elevate intracellular glutathione and linear profiling of cell images captured by multiphoton fluorescent microscopy highlighted that parameters such as the molecular weight and rate of cleavage of HIS RPCs were important factors in determining transfection activity. Altogether, these results demonstrate that HIS RPCs represent a novel and versatile type of vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids. This should enable different or a combination of therapeutic strategies to be evaluated using a single type of polycation-based vector.

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          Most cited references54

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          Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis.

          The relatively high transfection efficiency of polyethylenimine (PEI) vectors has been hypothesized to be due to their ability to avoid trafficking to degradative lysosomes. According to the proton sponge hypothesis, the buffering capacity of PEI leads to osmotic swelling and rupture of endosomes, resulting in the release of the vector into the cytoplasm. The mechanism of PEI-mediated DNA transfer was investigated using quantitative methods to study individual steps in the overall transfection process. In addition to transfection efficiency, the cellular uptake, local pH environment, and stability of vectors were analyzed. N-Quaternized (and therefore non-proton sponge) versions of PEI and specific cell function inhibitors were used to further probe the proton sponge hypothesis. Both N-quaternization and the use of bafilomycin A1 (a vacuolar proton pump inhibitor) reduced the transfection efficiency of PEI by approximately two orders of magnitude. Chloroquine, which buffers lysosomes, enhanced the transfection efficiency of N-quaternized PEIs and polylysine by 2-3-fold. In contrast, chloroquine did not improve the transfection efficiency of PEI. The measured average pH environment of PEI vectors was 6.1, indicating that they successfully avoid trafficking to acidic lysosomes. Significantly lower average pH environments were observed for permethyl-PEI (pH 5.4), perethyl-PEI (pH 5.1), and polylysine (pH 4.6) vectors. Cellular uptake levels of permethyl-PEI and perethyl-PEI vectors were found to be 20 and 90% higher, respectively, than that of parent PEI vectors, indicating that the reduction in transfection activity of the N-quaternized PEIs is due to a barrier downstream of cellular uptake. A polycation/DNA-binding affinity assessment showed that the more charge dense N-quaternized PEIs bind DNA less tightly than PEI, demonstrating that poor vector unpackaging was not responsible for the reduced transfection activity of the N-quaternized PEIs. The results obtained are consistent with the proton sponge hypothesis and strongly suggest that the transfection activity of PEI vectors is due to their unique ability to avoid acidic lysosomes. Copyright (c) 2004 John Wiley & Sons, Ltd.
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            siRNAs: applications in functional genomics and potential as therapeutics.

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              P75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp.

              In inhibiting neurite outgrowth, several myelin components, including the extracellular domain of Nogo-A (Nogo-66), oligodendrocyte myelin glycoprotein (OMgp) and myelin-associated glycoprotein (MAG), exert their effects through the same Nogo receptor (NgR). The glycosyl phosphatidylinositol (GPI)-anchored nature of NgR indicates the requirement for additional transmembrane protein(s) to transduce the inhibitory signals into the interior of responding neurons. Here, we demonstrate that p75, a transmembrane protein known to be a receptor for the neurotrophin family of growth factors, specifically interacts with NgR. p75 is required for NgR-mediated signalling, as neurons from p75 knockout mice are no longer responsive to myelin and to each of the known NgR ligands. Blocking the p75-NgR interaction also reduces the activities of these inhibitors. Moreover, a truncated p75 protein lacking the intracellular domain, when overexpressed in primary neurons, attenuates the same set of inhibitory activities, suggesting that p75 is a signal transducer of the NgR-p75 receptor complex. Thus, interfering with p75 and its downstream signalling pathways may allow lesioned axons to overcome most of the inhibitory activities associated with central nervous system myelin.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Research
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                2005
                2005
                24 May 2005
                : 33
                : 9
                : e86
                Affiliations
                Molecular Neuroscience Group, Department of Medicine, University of Birmingham Birmingham, B15 2TT, UK
                1School of Chemistry, University of Birmingham B15 2TT, UK
                2Department of Clinical Pharmacology, University of Oxford Oxford, OX2 6HE, UK
                3Department of Pharmaceutical Sciences, Wayne State University Detroit, MI 48202, USA
                4Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School Boston, MA 02129, USA
                5Department of Engineering Science, University of Oxford Oxford, OX1 3PJ, UK
                Author notes
                *To whom correspondence should be addressed. Tel: +44 121 627 2331; Fax: +44 121 472 0499; Email: mread@ 123456globalnet.co.uk
                Article
                10.1093/nar/gni085
                1140087
                15914665
                fea06a8a-a346-46a1-8269-4d2cfc1f96f5
                © The Author 2005. Published by Oxford University Press. All rights reserved

                The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@ 123456oupjournals.org

                History
                : 17 December 2004
                : 10 February 2005
                : 09 May 2005
                Categories
                Methods Online

                Genetics
                Genetics

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