Background Transduction with recombinant Human Immunodeficiency Virus (HIV) -1 derived lentivirus vectors is a multi-step process initiated by surface attachment and subsequent receptor-directed uptake into the target cell. We previously reported the retention of vesicular stomatitis virus G protein (VSV-G) pseudotyped particles on murine progenitor cells and their delayed cell-cell transfer. Methods To examine the underlying mechanism in more detail we used a combination of approaches focused on investigating the role of receptor-independent factors in modulating attachment. Results Studies of synchronized transduction herein reveal cell-type specific rates of vector particle clearance with substantial delays during particle entry into murine hematopoietic progenitor cells. The observed uptake kinetics from the surface of the 1° cell correlate inversely with the magnitude of transfer to 2° targets, corresponding with our initial observation of preferential cell-cell transfer in the context of brief vector exposures. We further demonstrate that vector particle entry into cells is associated with the cell–type specific abundance of extracellular matrix fibronectin. Residual particle – ECM binding and 2° transfer can be competitively disrupted by heparin exposure without affecting murine progenitor homing and repopulation. Conclusions While cellular attachment factors, including fibronectin, aid gene transfer by colocalizing particles to cells and disfavoring early dissociation from targets, they also appear to stabilize particles on the cell surface. Our study highlights the inadvertent consequences for cell entry and cell-cell transfer.
Preclinical evidence and clinical trials speak to the therapeutic potential of retrovirus vectors for the heritable genetic modification of cells. Careful evaluation of the antecedent risks is critical to move these applications forward. Others previously demonstrated the persistence of intact vector particles on the surface of target cells. Inadvertent particle transfer after in vivo applications could lead to the transduction of bystander tissues, or provoke immunological responses. We recently demonstrated prolonged adherence of VSV-G pseudotyped, HIV-1 derived lentivirus particles after ex vivo transduction culture of murine hematopoietic target cells (1°) with subsequent transduction of secondary (2°) targets in vitro and in vivo. Extended particle adherence is independent of Env pseudotype and routine wash procedures (Pan et al., J Virol. Jan 2007). We hypothesized that unwanted carryover could be minimized by disrupting the vector particle attachment to 2° cells while maintaining uptake to 1° targets. Initial studies indicated that the transduction of 1° targets at 4°C (to prevent uptake) for up to 6 hours followed by serial PBS washes and subsequent direct co-culture with fibroblasts resulted in undiminished 2° gene transfer compared to transduction at 37°C. Conversely, post-transduction exposure to escalating concentrations of citric acid resulted in a systematic decrease in both 1° and 2° gene transfer rates. This is consistent with separable mechanisms for pH sensitive VSV-G mediated uptake of particles in 1° targets and the receptor independent attachment responsible for carryover and 2° transduction, respectively. Glycosaminoglycans, including heparin, quantitatively bind to pseudotyped vector particles. We found that exposure of particles to heparin effectively abrogated subsequent transduction of cells by disrupting attachment. Remarkably, serial heparin washes at the conclusion of transduction had only minimal effects on gene transfer to 1° targets, but resulted in a two-log reduction in 2° gene transfer. Increases in the concentration of protamine sulfate (a polycation) during transduction partly reversed the effect of heparin (a polyanion), demonstrating the residual impact of electrostatic interactions on attachment of retrovirus particles from the 1° cell. In further studies we showed that trypsin washes following vector exposure incompletely cleaved 1° cell surface bound particles while pronase effectively degraded cell surface bound particles in a dose dependent manner, abrogating carryover. Because pronase at high concentrations also compromised cell surface epitope integrity we studied the expression of chemokine receptor (CXCR) 4, both a critical mediator of progenitor cell homing to the bone marrow and a representative protease-sensitive surface molecule. These experiments revealed a dose dependent degradation of CXCR4 on the cell surface of 1° target cells and rapid regeneration within three hours, critical for applications involving the injection of ex vivo modified hematopoietic cells. In conclusion, our results demonstrate that select wash procedures can disrupt the ability of virus particles to bind secondary targets, degrade residual surface bound particles and reduce gene transfer to inadvertent 2° targets in vitro by up to 99%. These studies are important first steps in understanding and limiting inadvertent carryover in the context of gene therapy while maximizing target cell transduction.
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