No abstract

A semi-replication-competent retroviral (s-RCR) vector system in which the gag-pol and env (GALV FMG, gibbon ape leukemia virus fusogenic membrane glycoprotein) genes were split into two separate packageable vectors was developed. These vectors are more efficient than replication-defective retroviral (RDR) vectors in gene delivery and have a higher transgene capacity than replication-competent retroviral (RCR) vectors. For the gag-pol vector construction, internal ribosomal entry site-enhanced green fluorescent protein (IRES-EGFP) was introduced downstream of the gag-pol sequence of the previously constructed MoMLV-10A1-EGFP vector to generate MoMLV-gag-pol-IRES-EGFP. For env vector construction, GALV FMG was inserted into the pCLXSN vector to generate pCLXSN-GALV FMG-IRES-EGFP. MoMLV-gag-pol-IRES-EGFP and pCLXSN-GALV FMG-IRES-EGFP were co-transfected into 293T cells to generate s-RCR viruses. These viruses propagated EGFP and induced syncytium formation due to the cytotoxicity of GALV FMG. To improve the cytotoxicity of s-RCR vector system, GALV FMG or the fusogenic envelope G glycoprotein of the vesicular stomatitis virus (VSV-G) was inserted into gag-pol vector. Co-transfection of MoMLV-gag-pol-IRES-GALV FMG + MoMLV-EGFP or MoMLV-VSV-G + pCLXSN-GALV FMG-IRES-EGFP in 293T cells induced stronger syncytium formation than s-RCR vectors (MoMLV-gag-pol-IRES-EGFP + pCLXSN-GALV FMG-IRES-EGFP). In addition, s-RCR stocks collected from transfected 293T cells induced syncytium formation in the human cancer cell lines HT1080 and TE671. Hence, the s-RCR vector systems developed in this study are useful tools for cancer gene therapy.