A60-bp sequence near the 5 end of the retroviral long terminal repeat (LTR) has been called a “negative control region”(ncr) because of its demonstrated inhibitory effect on expression [1]. The ncr has been shown to have a sequence element that binds the transcriptional factor YY-1, which may act in positive or negative ways. We had shown previously that deletion of the ncr fragment from a retroviral vector (MND) led to improved expression levels in murine embryonic stem (ES) cells and hematopoietic stem cells (HSC)[2, 3]. As reported in a recent issue of Molecular Therapy, Wahlers et al.[4] have identified another putative transcriptional factor binding site in the ncr of the LTR, for the nuclear factor of activated T cells—NFAT. They found that this NFAT-binding site has a positive effect on expression from retroviral vector LTR, in that its deletion by site-directed mutagenesis accounted for approximately 50% of the decreased expression caused by the ncr deletion (mean fluorescence intensity of an eGFP reporter). Thus, their findings would support retaining the ncr fragment in retroviral vectors because of the positive effects on expression from the NFAT site. We have made the converse analysis of the other major motif of the ncr region, the YY-1 site. We eliminated the YY-1 binding site from the MPSV LTR by site-directed mutagenesis to a sequence that abolished binding by YY-1 as measured by electrophoretic gel shift mobility assay (EMSA)(Figs. 1A and 1B). The vector with the eliminated YY-1 site (MYD-eGFP) consistently showed higher expression in F9 murine embryonal carcinoma cells than did the vector with an intact MPSV LTR (MD-eGFP), directly implicating the YY-1 site in suppression of expression (Fig. 1C). MYD-eGFP was expressed at a slightly greater level than the MND-eGFP vector, which has the entire ncr fragment deleted, removing both the YY-1 and the putative NFAT site (Fig. 1C). The majority of the studies of Wahlers et al. were done using the LTR from the spleen focus-forming virus (SFFV), which they have previously found to be the most active for gene expression in hematopoietic cells from among a group of LTRs from different murine retroviruses, such as MOMLV or MPSV [5]. As they point out, the SFFV LTR is unique among the LTRs studied in that it has an adjacent Sp1 site that interacts positively with the NFAT site. The other LTRs lack this Sp1 site and therefore their NFAT sites may be less active than the one from the SFFV LTR. In F9 EC cells, vectors with site-directed deletions exclusively in the putative NFAT motif (MND) did show a minimal decrease in expression compared to a vector with the intact MPSV LTR (MD-eGFP)(Fig. 1C). Together, our studies and those of Wahlers et al. suggest that removal of the ncr fragment has opposing positive and negative effects on expression, from elimination of the YY-1 and NFAT sites, respectively. The way to produce a vector to capitalize on these results for maximal expression would be by elimination of the YY-1 site and retention of the NFAT site, in a vector configuration such as MYD-eGFP.

However, it must be stated that the magnitude of the effects of the sequences from the ncr are relatively small. In murine gene transfer/BMT recipients, we compared directly expression by the MSCV and MND vectors; these vectors differ mainly by the presence of the ncr in the LTR of MSCV and its absence from MND (although MSCV also lacks one of the two LTR enhancer repeats present in MND). We could not detect significant differences in expression activity, with persistent stable expression seen from both vectors (Table 1). The in vivo studies by Wahlers et al. comparing expression by …