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Sequences within the coding regions of clotting factor VIII and CFTR block transcriptional elongation
D D Koeberl1, C L Halbert, A Krumm
1Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA.
Insights
Low expression of clotting factor VIII (FVIII) and cystic fibrosis transmembrane conductance regulator (CFTR) cDNAs is due to transcriptional elongation blocks. These findings complicate gene therapy vector development.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- Biotechnology
Background:
- Clotting factor VIII (FVIII) and cystic fibrosis transmembrane conductance regulator (CFTR) cDNAs exhibit significantly lower expression levels compared to other cDNAs like clotting factor IX (FIX) when produced using expression vectors.
- This reduced expression poses a challenge for therapeutic applications and the development of effective gene therapy vectors.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the dramatically reduced expression of FVIII and CFTR cDNAs.
- To identify inhibitory sequences within FVIII cDNA and analyze their impact on gene expression.
- To determine the implications of these findings for gene therapy and protein production.
Main Methods:
- Localization of inhibitory sequences within FVIII cDNA (FVIII INS).
- Analysis of RNA degradation and stability.
- Nuclear run-on experiments to assess transcriptional initiation and elongation.
- Orientation and position dependency studies of the FVIII INS.
- Retroviral vector-based expression studies for both FVIII and CFTR.
Main Results:
- A 1.2-kb inhibitory sequence (FVIII INS) in FVIII cDNA reduced steady-state RNA levels by 30- to 100-fold, with stable RNA degradation.
- Nuclear run-on experiments revealed a block to transcriptional elongation, not initiation, for FVIII.
- The inhibitory effect of FVIII INS was dependent on its orientation and position relative to the promoter.
- Similar blocks to transcriptional elongation were observed within the CFTR cDNA using retroviral vectors, resulting in 1,000-fold decreased RNA levels.
Conclusions:
- Reduced expression of FVIII and CFTR is primarily caused by blocks to transcriptional elongation within their respective cDNAs.
- These blocks are influenced by the sequence's orientation and position within the expression vector.
- The identified transcriptional elongation blocks present significant hurdles for producing high levels of FVIII and CFTR proteins for therapeutic use and for developing efficient retroviral gene therapy vectors.
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.