Optimization of Bcl11a Knockdown By miRNA Scaffold Embedded Shrnas Leading to Enhanced Induction of Fetal Hemoglobin in Erythroid Cells for the Treatment of Beta-Hemoglobinopathies

Repression of the BCL11A protein could represent a therapeutic target for beta-hemoglobinopathies, as its knock-down has been shown to induce the expression of the fetal HBG (y-globin) gene ultimately leading to increased levels of the fetal hemoglobin tetramer (HbF, a2y2). In mice, Bcl11a is a key repressor of the murine HBG homolog Hbb-y. RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via pol III promoters has been used to modulate gene expression in a variety of mammalian cell types. However, we found a negative impact of Bcl11a knockdown on hematopoietic stem cells (HSCs), limiting the repopulation efficiency and long-term engraftment after genetic modification, which is a major impediment for its translation into human therapeutic applications. To achieve lineage-specific targeting of mRNAs in an attempt to reduce HSC toxicity, expression of shRNAs via pol II promoters is required, necessitating embedding the shRNA in mammalian microRNA (shRNAmir) sequences for expression and processing. To achieve optimal knockdown of the Bcl11a transcription factor in erythroid progenitor and precursor cells, we first compared the efficiency of mRNA modulation via pol III (U6-promoter) vs pol II (SFFV-promoter) based lentiviral vectors. We demonstrate a 100-1000 fold lower Hbb-y induction using shRNAmir vs pol III mediated shRNA vector backbones due to reduced Bcl11a knockdown efficiency. In order to understand the molecular basis for these differences, small RNA sequence analysis was performed on murine erythroleukemia cells (MEL) cells transduced by multiple shRNA–shRNAmir pairs. We show that shRNAs expressed via a U6 promoter yield guide strand sequences which differ by a 2-4 nt shift compared to pol II driven (shRNAmir) mature guide strand sequences. RNA sequencing demonstrated that the stretch of uridines making up part of the pol III termination signal is transcribed and included at the 3’ end of the shRNA. This results in the generation of mature guide strand sequences with an alternative seed sequence compared to the predicted sequence and compared to miRNA embedded shRNAs. The difference in the seed sequences between the two expression systems strongly influences the efficacy of target gene knockdown, leading to reduced knockdown in pol II based vectors. We engineered a 4bp shift into guide strands of shRNAmirs that resulted in a faithfully processed shRNA sequence (a mature guide strand sequence identical to U6-driven sh-RNAs) and improved knock-down efficiency of Bcl11a at the protein level in most cases. The improved knockdown of Bcl11a was associated with a 100-300-fold enhancement of Hbb-y induction in MEL cells. Based on these results, we propose a modified strategy for the prospective design of shRNAmirs derived from shRNA screens in pol III vector backbones to achieve lineage-specific regulation of target genes. Targeted expression of shRNAmiRs to the erythroid compartment driven by a b-globin promoter/LCR element circumvented the detrimental effect on HSC engraftment, while still mediating efficient BCL11A knockdown, leading to high y-globin induction and formation of substantial amounts of fetal hemoglobin in human CD34-derived erythroid cells in vitro. Disclosures No relevant conflicts of interest to declare.