Effects of a second intron on recombinant MFG retroviral vector

Abstract

The retroviral vectors based on an MFG-type backbone have superior expression characteristics, in part, due to the presence of the retroviral chimeric intron (MFG intron). We tested the hypothesis that inclusion of a second intron in MFG vectors may influence packaging and/or LTR-driven transgene expression. We constructed two MFG retroviral vectors, MFG/hFIXc and MFG/hFIXm2, containing human factor IX (hFIX) cDNA without and with a 0.3-kb hFIX intron, respectively. When tested with primary mouse myoblasts or HepG2 cells in culture for transient expression activity, pMFG/hFIXm2 plasmid produced two-to-three fold higher hFIX than pMFG/hFIXc. These vectors produced equivalent retroviral titers from packaging cells. In transduced cells, the splicing of the MFG intron in the retroviral transcripts occured at a similar efficiency; however, MFG/hFIXc virus gave two-fold higher hFIX expression than that of the MFG/hFIXm2 viral infection. Analyses of MFG/hFIXm2 virion RNA and transduced cell genomic DNA suggested that, although the hFIX intron containing viral RNA are packaged, these viruses fail to integrate their transgenes into the genome of transduced cells, suggesting a block at the reverse transcription and/or integration steps. Similar results were also obtained with the prototype vectors, LIXcSN and LIXm2SN, lacking the MFG intron. Together, these results suggest that a hFIX cDNA sequence in the retroviral vectors performs better over hFIX intron-containing minigene.