Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes

Abstract

Cationic polymers, such as polybrene and protamine sulfate, are typically used to increase the efficiency of retrovirus-mediated gene transfer, however, the mechanism of their enhancement of transduction has remained unclear. As retrovirus transduction is fundamentally limited by the slow diffusion of virus to the target cell surface, we investigated the ability of polybrene to modulate this initial transport step. We compared the ability of both envelope (gp70) and capsid (p30) protein based assays to quantitate virus adsorption and found that p30 based assays were more reliable due to their ability to distinguish virus binding from free gp70 binding. Using the p30 based assay, we established that polybrene concentrations, which yielded 10-fold increases in transduction also, yielded a significant increase in virus adsorption rates on murine fibroblasts. Surprisingly, this enhancement, and adsorption in general, were receptor and envelope independent, as adsorption occurred equivalently on receptor positive and negative Chinese hamster ovary cells, as well as with envelope positive and negative virus particles. These findings suggest that the currently accepted physical model for early steps in retrovirus transduction may need to be reformulated to accommodate an initial adsorption step whose driving force does not include the retrovirus concentration, and the reclassification of currently designated ‘receptor’ molecules as fusion triggers. The implication of these findings with respect to the development of targeted retrovirus-mediated gene therapy protocols is discussed.

Introduction

Recombinant retroviruses are a widely used vector in human gene therapy clinical trials, yet despite advances in both the productivity of viral packaging cell lines, transduction efficiencies have remained unacceptably low for many applications [1], [2], [3], [4]. While the presence of endogenous inhibitors of transduction in virus stocks explains some of the difficulties encountered [5], [6], there exist basic biophysical constraints which limit the efficiency of retroviral gene transfer. Several groups have demonstrated that the slow diffusion and rapid inactivation of retroviruses are major contributors to the low observed efficiencies [7], [8], [9], [10]. With virus particles able to diffuse only a few microns before losing bioactivity, a large proportion of the initially active virus particles are inactive before they can interact with a target cell, fundamentally limiting achievable levels of transduction efficiency.

To circumvent these constraints, mechanical approaches, including centrifugation [8], [11], [12] and flow-through transduction [13], have been developed to increase the likelihood and frequency of active virus-cell interactions by adding a convective component to the mass transport of virus. While these strategies do yield enhanced transduction efficiencies, their expense and difficult scale-up limit their usefulness for large-scale experiments or in vivo clinical work. The classical approach to enhancing retrovirus transduction efficiency has been to add cationic polymers during the transduction process [14], [15], [16], however, the precise mechanism of their enhancement has remained unclear. As the cell and virus lipid membrane both possess a net-negative charge, it has been suggested that cationic polymers act by counteracting repulsive electrostatic effects, thereby enhancing adsorption [14], [17]. To date, however, there have been no reports demonstrating cationic polymer effects on virus binding with either the FACS-based immunofluorescence [18], [19] or any other virus binding assay [20], [21], [22].

In this report, we describe a new retrovirus-binding assay based upon quantitation of the amount of adsorbed capsid (p30) protein in cell lysates. Using this assay, we have been able to quantify retrovirus adsorption to target cells under conditions identical to those used in traditional transduction experiments. In initial experiments, we showed that p30 is the more reliable marker of virus adsorption as its detection is not confounded by the high levels of free gp70 we found to be present in retrovirus vector stocks. We used the p30 assay to study the mechanism of polybrene's enhancement and found that polybrene increased the relative rate of virus adsorption several-fold, and, surprisingly, that this enhanced adsorption could be demonstrated in the absence of both the virus envelope and cellular receptor for the virus. Based upon our findings, we propose a new model of retrovirus adsorption, which is diffusion-limited, receptor and tropism independent, and modulable by positively charged compounds. The implications of our findings for the development of targeted retrovirus-mediated gene therapy protocols is discussed.