title: miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction.
abstract: RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNA(miR)) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNA(miR)s resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNA(miR)s for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences.
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.|Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Swaroopa
authors.initials: S
authors.lastname: Guda
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.|Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Brendel
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.|Harvard Medical School, Boston, Massachusetts, USA.|Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Renella
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.|Stem Cell Program, Boston Children's Hospital, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Du
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.|Harvard Medical School, Boston, Massachusetts, USA.|Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Bauer
authors.affiliation: Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Canver
authors.affiliation: Genetic Perturbation Platform, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Grenier
authors.affiliation: Department of Molecular Biology & Genetics, College of Arts and Sciences, Cornell University, Ithaca, New York, USA.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Grimson
authors.affiliation: Harvard Medical School, Boston, Massachusetts, USA.|Howard Hughes Medical Institute, Boston, Massachusetts, USA.
authors.forename: Sophia C
authors.initials: SC
authors.lastname: Kamran
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.|Stem Cell Program, Boston Children's Hospital, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts, USA.
authors.forename: James
authors.initials: J
authors.lastname: Thornton
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Helen
authors.initials: H
authors.lastname: de Boer
authors.affiliation: Genetic Perturbation Platform, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Root
authors.affiliation: Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg Institute for Stem Cell Technology and Experimental Medicine, Heidelberg, Germany.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Milsom
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.|Harvard Medical School, Boston, Massachusetts, USA.|Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.|Howard Hughes Medical Institute, Boston, Massachusetts, USA.
authors.forename: Stuart H
authors.initials: SH
authors.lastname: Orkin
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.|Stem Cell Program, Boston Children's Hospital, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts, USA.
authors.forename: Richard I
authors.initials: RI
authors.lastname: Gregory
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.|Harvard Medical School, Boston, Massachusetts, USA.|Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Williams
mesh_terms: D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D002460:Cell Line; D019070:Cell Lineage / Q000235:genetics*; D041905:Erythroid Cells / Q000166:cytology / Q000378:metabolism; D005319:Fetal Hemoglobin / Q000096:biosynthesis* / Q000235:genetics*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D023061:Gene Order; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics*; D012097:Repressor Proteins; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D055545:gamma-Globins / Q000096:biosynthesis / Q000235:genetics
chemical_list: C486563:BCL11A protein, human; D002352:Carrier Proteins; D035683:MicroRNAs; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D055545:gamma-Globins; D005319:Fetal Hemoglobin