Viral Interference During Simultaneous Transduction with Two Independent Helper-Free Retroviral Vectors

Abstract

The ability to stably transduce a single cell with two independent retroviral vectors would have distinct advantages for gene therapy. We determined that cells can be transduced with two distinct retroviral vectors and have quantitated transduction efficiencies in cells infected sequentially and simultaneously. Two amphotropic, helper virus-free, retroviral vectors, a murine Moloney sarcoma virus-based vector containing the nuclear β-galactosidase and neomycin resistance genes (MMSVnβ-gal/neo^R) and a Harvey virus-derived vector containing the human multidrug resistance gene (HaMDR) were introduced into NIH-3T3 cells, pig keratinocytes, and primary pig fibroblasts simultaneously and sequentially. Analytical flow cytometry was utilized to determine retroviral transduction efficiency by assessing the percentage of cells transduced by either one or both retroviruses, in the absence of selection. Simultaneous retroviral transductions were infrequent events. In addition, transduction of previously infected cells (sequential transductions) occurred at lower than expected frequencies. Our data suggest that there is quantifiable viral interference in sequential retroviral transductions. This interference occurs by a mechanism that appears to be independent of the amphotropic retroviral receptor. Thus, such dual transductions will likely require in vitro selection or the use of a single retrovirus which contains both desired genes on the same genome.

Overview summary

The ability to transduce cells with multiple retroviral vectors has several advantages in gene therapy. In this study two independent, amphotropic, retroviral vectors, produced in the same packaging cell line, were utilized to co-transduce single cells simultaneously and sequentially. To evaluate if transduction utilizing two independent retroviral vectors is a feasible approach for gene therapy, the dual transduction efficiencies were determined and compared to the transduction efficiency of a single retroviral vector. Using analytical flow cytometry, Walker et al. demonstrate that simultaneous transduction frequency is low and that transduction of a single cell with a replication-defective retroviral vector appears to impede transduction of the same cell with a second (sequential transduction) retroviral vector.