VIRAL INTERFERENCE DURING SIMULTANEOUS TRANSDUCTION WITH 2 INDEPENDENT HELPER-FREE RETROVIRAL VECTORS

The ability to stably transduce a single cell with two independent ret roviral vectors would have distinct advantages for gene therapy, We de termined that cells can be transduced with two distinct retroviral vec tors and have quantitated transduction efficiencies in cells infected sequentially and simultaneously, Two amphotropic, helper virus-free, r etroviral vectors, a murine Moloney sarcoma virus-based vector contain ing the nuclear P-galactosidase and neomycin resistance genes (MMSVn b eta-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 seq uentially, Analytical flow cytometry was utilized to determine retrovi ral transduction efficiency by assessing the percentage of cells trans duced by either one or both retroviruses, in the absence of selection, Simultaneous retroviral transductions were infrequent events, In addi tion, transduction of previously infected cells (sequential transducti ons) occurred at lower than expected frequencies, Our data suggest tha t there is quantifiable viral interference in sequential retroviral tr ansductions, This interference occurs by a mechanism that appears to b e independent of the amphotropic retroviral receptor, Thus, such dual transductions will likely require in vitro selection or the use of a s ingle retrovirus which contains both desired genes on the same genome.