Quantitative analysis and characterization of intracellular gene delivery mechanisms

A goal for gene delivery research is to design vectors capable of (a) delivering transgenes to target cells, (b) yielding efficient gene expression, and (c) minimizing any immune, inflammatory, or cytotoxic response. Current research has focused on developing such vehicles using end gene expression as the benchmark. While transgene protein production is the overall objective of successful gene delivery, such qualitative treatment of gene delivery, especially for non-viral vectors, may result in unoptimized vectors and potential rate limiting steps unidentified. Quantitative analysis of the gene delivery pathway is essential for the characterization, comparison, and design of vectors. The complex nature of the mechanisms for gene delivery, particularly at the cellular level, contains multiple potentially rate limiting steps to successful gene expression. Through quantitative methodologies, vector efficacy can be related to molecular characteristics and specific processes within the gene delivery pathway. These potentially rate limiting steps include, but are not limited to, cell surface association, subcellular trafficking, endosomal escape, nuclear translocation, vector unpackaging, and gene expression. Design of synthetic gene delivery vectors seeks to develop molecular systems mimicking virus-like infection behavior, including cell membrane attachment and rapid internalization followed by endosomal escape, nuclear localization, and finally gene expression. To explore such opportunities for vector optimization and design, a model human hepatocellular carcinoma cell line by sets of transfection agents complexed with a plasmid. Time courses of plasmid numbers were determined both from whole cells and from isolated nuclei by real-time quantitative PCR.

Department dc:contributor.department

Massachusetts Institute of Technology. Biological Engineering Division.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2003

Author dc:creator

• Varga, Csanad M. (Csanad Mathias), 1976-

Advisor dc:contributor.advisor

• Douglas A. Lauffenburger.

• Biological Engineering Division.

Statement dc:rights

• MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/17587

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/17587