Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia

• DOI: 10.1016/j.ncrna.2018.11.005
• Publication Date: 2018-11-22T11:23:07Z
• Authors: Faisal A. Al-Allaf; Zainularifeen Abd...; Mohammad Athar; Mohiuddin M. Taher; Wajahatullah Khan; Huseyin Mehmet; Mukaddes Colakogu...; Sophia Apostolidou; Brian Bigger; Simon Waddington; Charles Coutelle; Michael Themis; Mohammed N. Al-Ahdal; Futwan A. Al-Mohanna; Zuhair N. Al-Hassnan; Abdellatif Bouazz...
• Keywords: Sequence (biology); Expression cassette

Internal ribosome entry site (IRES) sequences have become a valuable tool in the construction of gene transfer and therapeutic vectors for multi-cistronic expression from single mRNA transcript. The optimal conditions effective use this sequence to construct functional vector are not precisely defined but it is generally assumed that internal dependent second such as cassette less efficient than cap-dependent first gene. Mainly tailoring inter-cistronic significantly enhances IRES bicistronic further optimised therapy familial hypercholesterolemia. We tailored size spacer at 5' region using sequential deletions demonstrated 3' can be increased similar levels Maximum efficiency downstream was obtained when composed 18-141 base pairs. In case transcriptional unit containing both Cistron detected. Whilst constructs with 216 bp or longer generate only Cistron. This suggests long spacers may affect transcription termination. When 188 bp, transcripts were produced simultaneously most transfected cells, while fraction them expressed Expression analyses cassettes clearly confirm biological activity transgenic proteins transduced cells achieved. Furthermore, Computational analysis carried out by molecular dynamics (MD) simulation determine emerges viable specific binding bridging ends involving direct RNA-RNA contacts RNA-protein interactions. These results provide mechanistic basis translation stimulation RNA resembling synergistic translation.