Rapid establishment of stable retroviral packaging cells and recombinant susceptible target cell lines employing novel transposon vectors derived from Sleeping Beauty

Abstract

Viral vector particles derived from murine leukemia virus (MLV) mediate highly efficient stable gene transfer used in gene therapeutic approaches and in the generation of transgenic cell lines. However, the establishment of stable viral packaging cells (VPCs) is a time-consuming challenge. To overcome this limitation, we successfully generated novel Sleeping Beauty-derived transposon vectors entailing envelope and packaging expression cassettes as well as a transfer vector. Upon multiplexed transposition in human cells, VPC bulk populations yielding titers of over 1 × 106 transduction-competent vectors were established within three weeks. In contrast, conventional plasmid-based establishment of VPCs, conducted in parallel, took much longer and yielded significantly lower vector productivity and vector fitness. The generated MLV vectors decorated with the envelope proteins of ecotropic MLV PVC-211mc mediated efficient transduction of Chinese hamster ovary (CHO) cells. Cell susceptibility was further elevated upon recombinant expression of the murine ecotropic receptor mCAT employing a transposon vector.