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Home CCSU Theses & Dissertations Incorporation of Listeriolysin O into a ligand-based carrier system resulting in enhancement of...

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Incorporation of Listeriolysin O into a ligand-based carrier system resulting in enhancement of gene expression / Cherie Walton

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Identifier	Thesis 1509
Author	Walton, Cherie
Title	Incorporation of Listeriolysin O into a ligand-based carrier system resulting in enhancement of gene expression / Cherie Walton
Publisher	Central Connecticut State University
Date	1998
Resource Type	Master's Thesis
Notes	Receptor-mediated endocytosis as a route of gene delivery shows promise as it is a natural cellular process and allows targeting of ligands to specific cells. A carrier system consisting of two important components: a targetable ligand, asialoorosomucoid (ASOR), and polylysine (PL), a polycation has been found to bind DNA in a non-damaging manner. This conjugate, designated ASORPL, can target liver cells, be internalized via receptor-mediated endocytosis, and result in expression of the delivered gene. One of the limiting factors of this system is the trapping in endosomal compartments and subsequent degradation by lysosomal enzymes. Listeriolysin O is a toxin that allows for the escape of the bacteria Listeria monocytegenes from vesicles by undergoing a conformational change in response to an acid environment, as is seen in phagolysosomes/ endosomes. It was hypothesized that the ASORPL carrier system could be optimized if the endosomolytic properties of LLO could be incorporated. LLO was purified from a hypersecretor strain of Listeria monocytogenes using a spiral cartridge concentrator and column chromatography. ASORPL and DNA (CMV luciferase or nlacZ) were complexed by electrostatic interaction between the positive charge of polylysine and the negative charge of DNA. LLO was conjugated to ASORPL-DNA with the cleavable crosslinker N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP). After incubation with the ASORPL-DNA-LLO conjugate, HUH 7, asialogylcoprotein receptor positive liver cells, demonstrated a 26 times increase in gene expression over that of control (ASORPL-DNA without LLO). The incorporation of LLO did not interfere with cell specificity as the increase in expression could be competed out with the addition of excess ASOR. Also, SK Hep I, asialoglycoprotein negative cells, did not demostrate an increase in foreign gene expression. Similar experiments using the nlacZ gene also resulted in the elevation of B-galactosidase expression in receptor positive cells. This conjugate was then injected into mice to test the success of targeting and foreign expression in liver cells in vivo. Only a slight increase was detected. Studies reveal that serum may cause dissociation or degradation of the LLO from the ASORPL-DNA. Further research is needed to optimize the conjugate for in vivo therapy.
Subject	Proteins -- Physiological aspects DNA-binding proteins
Department	Department of Biomolecular Sciences
Advisor	King, Thomas R.
Type	Text
Digital Format	application/pdf
Language	eng
OCLC number	40103538
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Incorporation of Listeriolysin O into a ligand-based carrier system resulting in enhancement of gene expression / Cherie Walton

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