Analysis of charged polymer effects on recombinant retrovirus-mediated gene transfer success

Thesis (Ph.D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 2002.Includes bibliographical references (leaves 142-154).Poly-l-lysine and hexadimethrine bromide (polybrene) are cationic polymers which are frequently used to enhance the transduction efficiency of recombinant retroviruses in gene therapy experiments. Conversely, chondroitin sulfate proteoglycan is an anionic polymer endogenously present in retrovirus stocks which inhibits transduction efficiency. An experimental study was performed to determine the mechanisms of retrovirus transduction modulation by these charged polymers, and it was found that they were capable of increasing or decreasing the flux of virus particles onto the cell surface. These effects, and adsorption in general, were independent of the cellular receptor-virus envelope interaction which was believed to provide the driving force for initial virus attachment. In order to consider the feasibility of alternative driving forces for virus attachment, a mathematical model of adsorption was constructed taking into account the electrostatic properties of the system. The model predicted that either cationic polymer-mediated virus aggregation or membrane charge shielding could yield adsorption phenomenon consistent with the previous observations. An experimental study was undertaken to distinguish between these two potential mechanisms, and it was found that both were at work depending on the physicochemical characteristics of the cationic polymer. All cationic polymers were capable of charge shielding, however, only high molecular weight polymers (> 15 kD) could aggregate the virus. Anionic polymers, conversely, were found to inhibit transduction and adsorption via preventing cationic polymers from performing these functions.by Howard E. Davis, Jr.Ph.D