Impact of vector design on the potential activation of a cellular gene located downstream of the transcriptional direction of the vector.

The gray boxes in panel A show a randomly inserted retroviral transgene with a conventional LTR architecture. Here, the enhancer (E) and promoter (P) are terminally repeated, and the polyadenylation [p(A)] signal is weak; a splice donor is present in the retroviral untranslated region and another one within the transgene cDNA. The desired vector transcript is shown as line D; potential aberrant transcripts are numbered and shown as dotted lines. Either of the vector's SDs may interact with a splice acceptor (SA) of a downstream-located cellular gene to generate alternative splice products 1 and 2. Aberrant transcripts 1 to 3 result from lack of termination; transcript 4, from activation of the 3′ promoter of the vector; and transcript 5, from a distant action of the vector's enhancer on a cellular promoter (which may also be located upstream and/or in inverse orientation to the insertion). Transcripts similar to 1 and 4 have been detected in the case of leukemia following retroviral gene marking in mice.20 The hypothetical vector shown in panel B was designed to prevent aberrant transcripts by deleting the enhancer-promoter from the LTR, inserting a strong splice acceptor upstream of the vectors coding sequences, deleting SD of the cDNA, utilizing a strong p(A) signal, and flanking the transgene cassette with insulator (INS) sequences that prevent long-distance enhancer interactions. Secondary prevention strategies shown in panels C and D make use of coexpressed selectable marker genes (“Impact of vector design”).