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.

Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype

“…microRNA-adapted shRNAs (shRNA miR ) targeting BCL11A led to significantly increased target knockdown while avoiding nonsequence-specific cytotoxicity associated with pol III promoterdriven shRNAs (21). Here we show that knockdown of BCL11A unexpectedly and profoundly impairs long-term engraftment of both human and mouse HSCs in a sequence-specific fashion.…”

“…We previously identified highly effective shRNAs targeting the BCL11A mRNA (21). These shRNAs, targeting sequences of the mRNA that are conserved between mice and humans, were subsequently engineered into pol II-driven shRNA miR s in lentivirus vectors to reduce nonspecific toxicities related to shRNA overexpression (21)(22)(23).…”

“…These shRNAs, targeting sequences of the mRNA that are conserved between mice and humans, were subsequently engineered into pol II-driven shRNA miR s in lentivirus vectors to reduce nonspecific toxicities related to shRNA overexpression (21)(22)(23). In order to attain high-efficiency knockdown of BCL11A in hematopoietic cells, the shRNA miR was expressed from a strong SFFV promoter ( Figure 1A) mediating ubiquitous expression.…”

“…Figure 3A). The cells were transduced with the LCR or SFFV vector expressing an NT control and the 3 most potent shRNA miR s (shRNA miR 3, -5, and -8) as defined by previous in vitro studies with SFFV-shRNA miR (21). BCL11A protein expression was determined by immunoblot after 11 days of erythroid differentiation in Venus-positive cells; γ-globin mRNA and HbF levels were analyzed on day 18.…”

BCL11A frameshift mutation associated with dyspraxia and hypotonia affecting the fine, gross, oral, and speech motor systems

“…BCL11A has been shown to be a critical modulator of hemoglobin switching and HbF silencing (Basak et al, 2015; Funnell et al, 2015; Guda et al, 2015; Xu et al, 2013). In the murine brain, Bcl11a controls cell polarity, radial migration of upper cortical neurons, axon branching, and dendrite outgrowth (Kuo, Chen, & Hsueh 2010; Kuo, Hong, & Hsueh, 2009; Kuo, Hong, Chien, & Hsueh, 2010; Wiegreffe et al, 2015).…”