Year: 2006
Type: article
Abstract: Uptake of plasmid vectors through the plasma membrane of a cell is a one of the barriers to efficient non-viral gene delivery. To facilitate uptake, we explored the use of ‘oligosaccharide engineering’to install non-native “receptors” in the glycocalyx of mammalian cells for use as “tags” for the targeted delivery of cationic polymer- DNA nanoparticles. In this technique, which exploits the permissivity of the sialic acid biosynthetic pathway for synthetic N-acetylmannosamine (ManNAc) analogs, mammalian cells metabolically process these non-natural monosaccharides to the corresponding sialic acids and display them on the termini of cell surface oligosaccharides. Of relevance to gene delivery, N- thioglycolyl-D-mannosamine peracetate (Ac5ManNTGc) is a novel analog containing a pendant thiol group. Upon surface display as the N-thioglycolyl-neuraminic acid (Neu5TGc) form of sialic acid, the carbohydrate-expressed thiol groups are highly accessible compared to their protein counterparts, thereby facilitating selective chemical reactions with complementary functional groups.1 In this work, we tested the ability of Neu5TGc-displayed thiols to enhance the non-viral gene delivery process by derivatizing the surface of cationic polymer-DNA nanoparticles with maleimide functional groups. Maleimide groups selectively form covalent bonds with thiol groups; consequently the additional surface-exposed thiol groups presented on Ac5ManNTGc-treated cells are expected to efficiently capture the maleimide-decorated DNA-nanoparticles and, in turn, improve the probability of uptake and eventual gene expression. To test this model, human embryoid-body derived cells (hEBD) and human mesenchymal stem cells (hMSC) were cultured in medium containing (10μM) of Ac5ManNTGc for 3 days. The treated hEBD cells and hMSC showed a 15-fold and 2–3 fold increase in cell-surface thiol expression, respectively, compared to controls that were either untreated, or treated with a ManNAc analog that did not contain thiol groups. Ac5ManNTGc- treated cells transfected with maleimide-decorated chitosan-DNA nanoparticles showed an increase in Luciferase expression, as compared to untreated cells. By manipulating the glycosylation pathway to create synthetic sugar moieties on the cell surface, this cell engineering approach provides a model to study the ligand-“receptor” interactions that would have implications on targeted drug and gene delivery. 1. Sampathkumar, S.-G., Li, A.V., Jones, M.B., Sun, Z. & Yarema, K.J. Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology. Nature Chemical Biology Advance Online Publication, 12 March 2006, doi:10.1038/nchembio1770 (2006).
Source: Molecular Therapy
Institutions: Johns Hopkins University, Duke University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
OpenAlex ID: W1968103256