Disruption of Endosomal Membrane by Cationic Vectors Drives Endosomal Release and Enables Successful Gene Delivery
dc.contributor.author | Vaidyanathan, Sriram | |
dc.date.accessioned | 2016-06-10T19:31:20Z | |
dc.date.available | NO_RESTRICTION | |
dc.date.available | 2016-06-10T19:31:20Z | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016 | |
dc.identifier.uri | https://hdl.handle.net/2027.42/120751 | |
dc.description.abstract | The potential of gene therapy to treat congenital disorders is hindered by the lack of safe and effective delivery agents (vectors). More effective viral vectors possess several safety concerns but the safer non-viral vectors are less effective in inducing gene expression. Understanding how vector-DNA complexes (polyplexes) are internalized and transported to the nucleus would help make effective non-viral vectors. Several processes including 1) endosomal release, 2) transport within specific intracellular pathways, 3) protection of DNA from nucleases and 4) transport into the nucleus have been identified to affect gene delivery. However, the relative importance of these processes and their relationship to vector properties are unknown. Our experiments indicate that the endosomal release of polyplexes, due to the disruption of endosomal membranes mediated by the intercalation of free cationic vectors, is critical for successful gene delivery. This model was developed using two studies that 1) tracked the transport of intact DNA in cells and 2) quantified vector-cell membrane interactions. Specifically, present imaging techniques cannot distinguish functional intact DNA from the vast majority of degraded DNA. To distinguish intact DNA from degraded DNA, we used a novel DNA oligo nucleotide molecular beacon (OMB) labeled with a dye pair that exhibits Forester Resonance Energy Transfer (FRET). We observed that the fraction of cells displaying release of intact DNA from endosomes quantitatively predicted the fraction of cells displaying gene expression for effective (jetPEI) and ineffective cationic vectors (G5-PAMAM). Moreover, intact OMB delivered with G5-PAMAM and confined to endosomes could be released by the subsequent addition of L-PEI, with a corresponding 10-fold increase in transgene expression. Thus, microscopy studies indicated that free cationic vectors drive endosomal release. Moreover, experiments quantifying vector/cell membrane interactions further showed that the ability of free vectors , not polyplexes, to permeabilize cell membranes predicted successful gene expression. In summary, our experiments provide a novel new strategy that emphasizes the role of cell membrane-cationic vector interactions in driving successful gene expression. | |
dc.language.iso | en_US | |
dc.subject | Gene delivery | |
dc.subject | Gene therapy | |
dc.subject | Endosomal escape | |
dc.subject | Drug delivery | |
dc.subject | Proton sponge | |
dc.subject | siRNA delivery | |
dc.title | Disruption of Endosomal Membrane by Cationic Vectors Drives Endosomal Release and Enables Successful Gene Delivery | |
dc.type | Thesis | en_US |
dc.description.thesisdegreename | PhD | |
dc.description.thesisdegreediscipline | Biomedical Engineering | |
dc.description.thesisdegreegrantor | University of Michigan, Horace H. Rackham School of Graduate Studies | |
dc.contributor.committeemember | Banaszak Holl, Mark M | |
dc.contributor.committeemember | Orr, Bradford G | |
dc.contributor.committeemember | Kennedy, Robert T | |
dc.contributor.committeemember | Sept, David Samuel | |
dc.contributor.committeemember | Shea, Lonnie David | |
dc.subject.hlbsecondlevel | Biomedical Engineering | |
dc.subject.hlbsecondlevel | Biological Chemistry | |
dc.subject.hlbsecondlevel | Chemistry | |
dc.subject.hlbsecondlevel | Science (General) | |
dc.subject.hlbtoplevel | Engineering | |
dc.subject.hlbtoplevel | Health Sciences | |
dc.subject.hlbtoplevel | Science | |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/120751/1/svaidy_1.pdf | |
dc.owningcollname | Dissertations and Theses (Ph.D. and Master's) |
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