Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia
Internal ribosome entry site (IRES) sequences have become a valuable tool in the construction of gene transfer and therapeutic vectors for multi-cistronic gene expression from a single mRNA transcript. The optimal conditions for effective use of this sequence to construct a functional expression vec...
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Format: | Article |
Language: | English |
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KeAi Communications Co., Ltd.
2019-03-01
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Series: | Non-coding RNA Research |
Online Access: | http://www.sciencedirect.com/science/article/pii/S246805401830088X |
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language |
English |
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Article |
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DOAJ |
author |
Faisal A. Al-Allaf Zainularifeen Abduljaleel Mohammad Athar Mohiuddin M. Taher Wajahatullah Khan Huseyin Mehmet Mukaddes Colakogullari Sophia Apostolidou Brian Bigger Simon Waddington Charles Coutelle Michael Themis Mohammed N. Al-Ahdal Futwan A. Al-Mohanna Zuhair N. Al-Hassnan Abdellatif Bouazzaoui |
spellingShingle |
Faisal A. Al-Allaf Zainularifeen Abduljaleel Mohammad Athar Mohiuddin M. Taher Wajahatullah Khan Huseyin Mehmet Mukaddes Colakogullari Sophia Apostolidou Brian Bigger Simon Waddington Charles Coutelle Michael Themis Mohammed N. Al-Ahdal Futwan A. Al-Mohanna Zuhair N. Al-Hassnan Abdellatif Bouazzaoui Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia Non-coding RNA Research |
author_facet |
Faisal A. Al-Allaf Zainularifeen Abduljaleel Mohammad Athar Mohiuddin M. Taher Wajahatullah Khan Huseyin Mehmet Mukaddes Colakogullari Sophia Apostolidou Brian Bigger Simon Waddington Charles Coutelle Michael Themis Mohammed N. Al-Ahdal Futwan A. Al-Mohanna Zuhair N. Al-Hassnan Abdellatif Bouazzaoui |
author_sort |
Faisal A. Al-Allaf |
title |
Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia |
title_short |
Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia |
title_full |
Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia |
title_fullStr |
Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia |
title_full_unstemmed |
Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia |
title_sort |
modifying inter-cistronic sequence significantly enhances ires dependent second gene expression in bicistronic vector: construction of optimised cassette for gene therapy of familial hypercholesterolemia |
publisher |
KeAi Communications Co., Ltd. |
series |
Non-coding RNA Research |
issn |
2468-0540 |
publishDate |
2019-03-01 |
description |
Internal ribosome entry site (IRES) sequences have become a valuable tool in the construction of gene transfer and therapeutic vectors for multi-cistronic gene expression from a single mRNA transcript. The optimal conditions for effective use of this sequence to construct a functional expression vector are not precisely defined but it is generally assumed that the internal ribosome entry site dependent expression of the second gene in such as cassette is less efficient than the cap-dependent expression of the first gene. Mainly tailoring inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector further in construction of optimised cassette for gene therapy of familial hypercholesterolemia. We tailored the size of the inter-cistronic spacer sequence at the 5′ region of the internal ribosome entry site sequence using sequential deletions and demonstrated that the expression of the 3′ gene can be significantly increased to similar levels as the cap-dependent expression of the 5’ gene. Maximum expression efficiency of the downstream gene was obtained when the spacer is composed of 18–141 base pairs. In this case a single mRNA transcriptional unit containing both the first and the second Cistron was detected. Whilst constructs with spacer sequences of 216 bp or longer generate a single transcriptional unit containing only the first Cistron. This suggests that long spacers may affect transcription termination. When the spacer is 188 bp, both transcripts were produced simultaneously in most transfected cells, while a fraction of them expressed only the first but not the second gene. Expression analyses of vectors containing optimised cassettes clearly confirm that efficiency of gene transfer and biological activity of the expressed transgenic proteins in the transduced cells can be achieved. Furthermore, Computational analysis was carried out by molecular dynamics (MD) simulation to determine the most emerges as viable containing specific binding site and bridging of 5′ and 3′ ends involving direct RNA-RNA contacts and RNA-protein interactions. These results provide a mechanistic basis for translation stimulation and RNA resembling for the synergistic stimulation of cap-dependent translation. Keywords: Internal ribosome entry site, IRES, Gene therapy, Inter-cistronic sequences, MD simulation, Protein structure modeling, Familial hypercholesterolemia |
url |
http://www.sciencedirect.com/science/article/pii/S246805401830088X |
work_keys_str_mv |
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doaj-e7460ad473a841058174a80c260fe2412021-02-02T02:06:58ZengKeAi Communications Co., Ltd.Non-coding RNA Research2468-05402019-03-0141114Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemiaFaisal A. Al-Allaf0Zainularifeen Abduljaleel1Mohammad Athar2Mohiuddin M. Taher3Wajahatullah Khan4Huseyin Mehmet5Mukaddes Colakogullari6Sophia Apostolidou7Brian Bigger8Simon Waddington9Charles Coutelle10Michael Themis11Mohammed N. Al-Ahdal12Futwan A. Al-Mohanna13Zuhair N. Al-Hassnan14Abdellatif Bouazzaoui15Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi Arabia; Science and Technology Unit, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi Arabia; Molecular Diagnostics Unit, Department of Laboratory and Blood Bank, King Abdullah Medical City, Makkah, 21955, Saudi Arabia; Gene Therapy Research Group, Department of Molecular and Cell Medicine, Faculty of Medicine, Imperial College London, South Kensington, London, SW7 2AZ, UK; Institute of Reproductive and Developmental Biology, Division of Clinical Sciences, Faculty of Medicine, Imperial College London, London, W12 0NN, UK; Corresponding author. Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P. O. Box 18802, Makkah 21955, Saudi Arabia.Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi Arabia; Science and Technology Unit, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi Arabia; Corresponding author. Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P. O. Box 18802, Makkah 21955, Saudi Arabia.Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi Arabia; Science and Technology Unit, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi ArabiaDepartment of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi Arabia; Science and Technology Unit, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi ArabiaDepartment of Basic Sciences, College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Sciences, PO Box 3124, Riyadh, 11426, Saudi ArabiaInstitute of Reproductive and Developmental Biology, Division of Clinical Sciences, Faculty of Medicine, Imperial College London, London, W12 0NN, UKInstitute of Reproductive and Developmental Biology, Division of Clinical Sciences, Faculty of Medicine, Imperial College London, London, W12 0NN, UKInstitute of Reproductive and Developmental Biology, Division of Clinical Sciences, Faculty of Medicine, Imperial College London, London, W12 0NN, UKGene Therapy Research Group, Department of Molecular and Cell Medicine, Faculty of Medicine, Imperial College London, South Kensington, London, SW7 2AZ, UKGene Therapy Research Group, Department of Molecular and Cell Medicine, Faculty of Medicine, Imperial College London, South Kensington, London, SW7 2AZ, UKGene Therapy Research Group, Department of Molecular and Cell Medicine, Faculty of Medicine, Imperial College London, South Kensington, London, SW7 2AZ, UKGene Therapy Research Group, Department of Molecular and Cell Medicine, Faculty of Medicine, Imperial College London, South Kensington, London, SW7 2AZ, UKDepartment of Infection and Immunity, King Faisal Specialist Hospital & Research Center, Riyadh, 11211, Saudi ArabiaDepartment of Cell Biology, King Faisal Specialist Hospital and Research Center, Riyadh, 11211, Saudi ArabiaDepartment of Medical Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, 11211, Saudi ArabiaDepartment of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi Arabia; Science and Technology Unit, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi ArabiaInternal ribosome entry site (IRES) sequences have become a valuable tool in the construction of gene transfer and therapeutic vectors for multi-cistronic gene expression from a single mRNA transcript. The optimal conditions for effective use of this sequence to construct a functional expression vector are not precisely defined but it is generally assumed that the internal ribosome entry site dependent expression of the second gene in such as cassette is less efficient than the cap-dependent expression of the first gene. Mainly tailoring inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector further in construction of optimised cassette for gene therapy of familial hypercholesterolemia. We tailored the size of the inter-cistronic spacer sequence at the 5′ region of the internal ribosome entry site sequence using sequential deletions and demonstrated that the expression of the 3′ gene can be significantly increased to similar levels as the cap-dependent expression of the 5’ gene. Maximum expression efficiency of the downstream gene was obtained when the spacer is composed of 18–141 base pairs. In this case a single mRNA transcriptional unit containing both the first and the second Cistron was detected. Whilst constructs with spacer sequences of 216 bp or longer generate a single transcriptional unit containing only the first Cistron. This suggests that long spacers may affect transcription termination. When the spacer is 188 bp, both transcripts were produced simultaneously in most transfected cells, while a fraction of them expressed only the first but not the second gene. Expression analyses of vectors containing optimised cassettes clearly confirm that efficiency of gene transfer and biological activity of the expressed transgenic proteins in the transduced cells can be achieved. Furthermore, Computational analysis was carried out by molecular dynamics (MD) simulation to determine the most emerges as viable containing specific binding site and bridging of 5′ and 3′ ends involving direct RNA-RNA contacts and RNA-protein interactions. These results provide a mechanistic basis for translation stimulation and RNA resembling for the synergistic stimulation of cap-dependent translation. Keywords: Internal ribosome entry site, IRES, Gene therapy, Inter-cistronic sequences, MD simulation, Protein structure modeling, Familial hypercholesterolemiahttp://www.sciencedirect.com/science/article/pii/S246805401830088X |