title: RNA Trans-Splicing Targeting Endogenous β-Globin Pre-Messenger RNA in Human Erythroid Cells.
abstract: 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.
authors.affiliation: 1 Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) , Bethesda, Maryland.
authors.forename: Naoya
authors.initials: N
authors.lastname: Uchida
authors.affiliation: 2 Genetics and Human Genetics, Howard University , Washington, DC.
authors.forename: Kareem N
authors.initials: KN
authors.lastname: Washington
authors.affiliation: 3 Office of Research Integrity , Office of the Assistant Secretary for Health, Rockville, Maryland.
authors.forename: Brian
authors.initials: B
authors.lastname: Mozer
authors.affiliation: 1 Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) , Bethesda, Maryland.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Platner
authors.affiliation: 1 Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) , Bethesda, Maryland.
authors.forename: Josiah
authors.initials: J
authors.lastname: Ballantine
authors.affiliation: 1 Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) , Bethesda, Maryland.
authors.forename: Luke P
authors.initials: LP
authors.lastname: Skala
authors.affiliation: 1 Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) , Bethesda, Maryland.
authors.forename: Lydia
authors.initials: L
authors.lastname: Raines
authors.affiliation: 1 Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) , Bethesda, Maryland.
authors.forename: Anna
authors.initials: A
authors.lastname: Shvygin
authors.affiliation: 1 Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) , Bethesda, Maryland.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Hsieh
authors.affiliation: 4 RetroTherapy , Bethesda, Maryland.
authors.forename: Lloyd G
authors.initials: LG
authors.lastname: Mitchell
authors.affiliation: 1 Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) , Bethesda, Maryland.
authors.forename: John F
authors.initials: JF
authors.lastname: Tisdale
mesh_terms: D000755:Anemia, Sickle Cell / Q000235:genetics / Q000628:therapy*; D041905:Erythroid Cells / Q000378:metabolism / Q000473:pathology; D005091:Exons / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics / Q000627:therapeutic use; D006801:Humans; D007438:Introns / Q000235:genetics; D017354:Point Mutation; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics; D020040:Trans-Splicing / Q000235:genetics*; D014162:Transfection; D055544:beta-Globins / Q000235:genetics*
chemical_list: D012322:RNA Precursors; D055544:beta-Globins
keywords: RNA trans-splicing; lentiviral vector; β-globin gene