Improved retroviral vectors for gene transfer and expression

Abstract

We describe a set of murine retrovirus-based vectors that include unique cloning sites for insertion of cDNAs such that the cDNA can be driven by either the retroviral long terminal repeat, the immediate early promoter of human cytomegalovirus, or the simian virus 40 early promoter. The vectors carry the neomycin phosphotransferase gene expressed from an alternate promoter as a selectable marker. These vectors have been constructed to prevent viral protein synthesis from the remaining viral sequences, to yield high-titer virus stocks after introduction into retrovirus packaging cells, and to eliminate homologous overlap with viral DNAs present in retrovirus packaging cells in order to prevent helper virus production. Methods for generating high-titer virus are described.

Figure 1. Retroviral vectors N2 and LNL6

Abbreviations are: LTR, retroviral long terminal repeat; SD, splice donor; SA, splice acceptor; ψ+, extended packaging signal; pA, polyadenylation site. Arrows indicate transcriptional start sites and direction of transcription.

Figure 2. Comparison of viral sequences in PA317 cells and the retroviral vectors

At top is a diagram of the replication-competent murine virus AM-MLV, which was made from MoMLV by replacing the env and part of the pol region of MoMLV with that of amphotropic virus 4070A (14). Sequences in the PA317 cells and in the vectors are aligned with the helper virus to show their origin. The vertical crosshatched box shows a 59-bp region of homology between the viral DNA in PA317 cells and the retroviral vectors where a homologous recombination event could add functional 5′ retroviral sequences to the defective helper virus DNA in PA317 cells. While additional homology exists at the 3′ end of ψ+, a recombination in this area would add 5′ viral sequences with a stop codon where the gag start codon should be, and thus would be non-functional. No homologous overlap exists at the 3′ end. Abbreviations are PBS− and PBS+, minus and plus strand primer binding sites respectively; pA, polyadenylation signal; SV40, the late polyadenylation signal from SV40; LTR, the retroviral long terminal repeat; ΔLTR, LTR with a deletion at the 5′ end; ψ+, retroviral packaging signal. Arrows indicate transcriptional start sites and direction of transcription.

Figure 3. Improved retroviral vectors

Arrows indicate transcriptional start sites and direction of transcription, pA indicates polyadenylation signals. Unique cloning sites for cDNA insertion are shown. The sequences in all vectors consist of the 5′ LTR and sequences through base 541 from MoMSV, base 566 to 1038 from MoMLV, non-retroviral sequences, and base 7774 through the 3′ LTR from MoMLV. Retroviral sequence numbers are as described (21). The neo sequences were from transposon Tn5 (3) and the protein-encoding region is crosshatched. The bacterial promoter was removed prior to insertion. SV indicates a Pvu II to Hind III fragment from SV40 containing the early promoter (8,20). CMV indicates a Bal I to Xma III fragment from human cytomegalovirus which contains the immediate early promoter (5). Unique BamH I sites separate the SV40 and CMV promoters from the neo gene sequences in LNSX and LNCX, respectively, allowing simple replacement of these promoters with other sequences.