Sequences Within the Coding Regions of Clotting Factor VIII and CFTR Block Transcriptional Elongation

Abstract

The clotting factor VIII (FVIII) and cystic fibrosis transmembrane conductance regulator (CFTR) cDNAs have dramatically reduced levels of expression compared to clotting factor IX (FIX) and other cDNAs (100 and 1,000-fold lower, respectively), when produced in cells by using an expression vector. Part of the inhibitory signal in the FVIII cDNA has been localized to a 1.2-kb inhibitory sequence (FVIII INS), which decreased steady-state RNA levels from a retroviral vector by 30- to 100-fold. An analysis of RNA degradation indicated that the FVIII INS vector RNA is relatively stable. Nuclear run-on experiments with the FVIII INS vector demonstrated a low signal for FVIII, in contrast to the high signal for a FIX vector. The low signal for FVIII INS was not due to a decrease in transcriptional initiation. Thus, FVIII expression is reduced through a block to transcriptional elongation, as has been found in c-myc and other genes. We show that the inhibitory effect of FVIII INS is orientation dependent with regard to the promoter. In addition, the inhibitory effect is position dependent, because expression of FVIII INS sequence increased when it was moved 1 kb further from the promoter in a retroviral vector. Similar results were observed by using a retroviral vector for expression of the CFTR cDNA. The CFTR retroviral vector produced 1,000-fold decreased steady-state RNA levels, compared to the parent vector. Nuclear run-on analysis with the CFTR vector revealed a block to transcriptional elongation within the CFTR cDNA. The presence of blocks to transcriptional elongation within the FVIII and CFTR cDNAs complicates efforts to produce high levels of these proteins for therapeutic purposes and to develop high-titer retroviral expression vectors for human gene therapy. Koeberl et al. analyzed the control of expression for the FVIII and CFTR cDNAs in the context of retroviral expression vectors by utilizing RNA stability and nuclear run-on assays. Vector RNA containing FVIII INS was stable for at least 6 hr following inhibition of RNA synthesis by using actinomycin D. Nuclear run-on analyses revealed that FVIII INS vectors generate a higher prevalence of upstream transcripts compared to the FVIII INS sequence, indicating the presence of a block to transcriptional elongation. The block to transcriptional elongation in FVIII INS was found to be orientation-dependent. The FVIII INS inhibitory sequence was localized to several elements that have an additive inhibitory effect on expression. Like the FVIII cDNA, the expression of the CFTR cDNA is regulated at the level of transcriptional elongation. Decreased expression of the FVIII and CFTR cDNAs from retroviral vectors complicates efforts toward human gene therapy.