RNA Trans-Splicing Targeting Endogenous β-Globin Pre-Messenger RNA in Human Erythroid Cells
| Autor: | John F. Tisdale, Brian Mozer, Kareem Washington, Luke P Skala, Lloyd G Mitchell, Lydia N. Raines, Naoya Uchida, Anna Shvygin, Matthew M. Hsieh, Charlotte Platner, Josiah Ballantine |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
RNA Splicing Trans-splicing Genetic Vectors Anemia Sickle Cell beta-Globins Biology Transfection Applied Microbiology and Biotechnology Trans-Splicing 03 medical and health sciences Exon Exon trapping Erythroid Cells hemic and lymphatic diseases Genetics RNA Precursors Humans Point Mutation Genetics (clinical) Research Articles Pharmacology Messenger RNA Splice site mutation Intron RNA Exons Genetic Therapy Molecular biology Introns 030104 developmental biology RNA splicing Molecular Medicine |
| Zdroj: | Human gene therapy methods. 28(2) |
| ISSN: | 1946-6544 |
| Popis: | Sickle cell disease results from a point mutation in exon 1 of the β-globin gene (total 3 exons). Replacing sickle β-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting β-globin pre-messenger RNA among human erythroid cells. Binding domains from random β-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34+ cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous β-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous β-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy. |
| Databáze: | OpenAIRE |
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