miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction.

Mol Ther
Authors
Keywords
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.

Year of Publication
2015
Journal
Mol Ther
Volume
23
Issue
9
Pages
1465-74
Date Published
2015 Sep
ISSN
1525-0024
URL
DOI
10.1038/mt.2015.113
PubMed ID
26080908
PubMed Central ID
PMC4817887
Links
Grant list
K08 DK093705 / DK / NIDDK NIH HHS / United States
U01HL117720-01 / HL / NHLBI NIH HHS / United States
U01 HL117720 / HL / NHLBI NIH HHS / United States
Howard Hughes Medical Institute / United States
F30DK103359-01A1 / DK / NIDDK NIH HHS / United States
F30 DK103359 / DK / NIDDK NIH HHS / United States