Hematopoietic SIN Lentiviral Micro RNA-Mediated Silencing of BCL11A: Pre-Clinical Evidence for a Sickle Cell Disease Gene-Therapy Trial

Abstract 753 Sickle cell disease (SCD) is caused by a mutation in the β-globin protein, leading to the polymerization of hemoglobin in deoxygenated conditions. The transcription factor BCL11A is a key regulator of developmental silencing of human fetal (γ-) globin, and also critical to repressing γ-globin in adult erythroid cells. A Bcl11a null mouse model carrying a transgenic YAC with a humanized β-globin locus (β-YAC) displays increased levels of fetal hemoglobin (HbF) in adult erythrocytes, and crossing these animals with a SCD murine model abolishes the SCD phenotype. BCL11A therefore constitutes a genetically validated target to induce HbF and reduce erythrocyte “sickling”, which would be predicted to ameliorate the phenotype of SCD patients. However, defective lymphoid development has been observed in Bcl11a genetic null mice, suggesting potential toxicities of BCL11A knockdown. We generated self-inactivating lentiviral vectors (LV) integrating miR-223 microRNA-based inhibitory shRNAs against BCL11A/Bcl11a. Since future clinical applications will need to balance efficacy and potential side effects, LVs were engineered to allow the comparison of effects of high level and ubiquitous versus erythroid lineage-restricted versus inducible expression of miRNA targeting BCL11A. LV backbones therefore included either a strong, viral LTR promoter/enhancer (SFFV-LV), a β-globin locus control region with the endogenous β-globin promoter (LCR-LV), or a tetracycline-inducible promoter (TET-LV). We performed assays to quantify transgenic miRNA expression and demonstrated that the BCL11A knockdown and induction of fetal globin gene output correlated with the expression of targeting miR223-based shRNA. Transduction at low MOI (=2) of murine hematopoietic stem cells (HSC) with LVs carrying the abovementioned regulatory elements leads to long-term engraftment and transgene expression in-vivo. Mice transplanted with SFFV-LV show fluorescent marking up to 70% across myeloid, lymphoid and erythroid lineages. The maximal BCL11A/Bcl11a mRNA and protein knock-down observed in primary hematopoietic cells in-vitro and in-vivo was 70%. This was confirmed in FACS-sorted bone marrow B-lymphoid (B220+) and erythroid progenitors (Terr119+/CD71+) and peripheral blood leukocytes at 4 months post-transplant. BCL11A/Bcl11a knockdown induced fetal globin gene expression depending on the vector backbone and targeting shRNA sequence employed. With SFFV-LV, we observed a 5–20 fold upregulation of fetal globin gene (γ/(ϵ+γ+β)) output in mice transplanted with HSCs containing the humanized β-YAC transgene. With TET-LV, the induction was dose-dependent and maximally caused a 150-fold increase in murine ϵγ-globin gene expression in-vitro. Human HSC transduced (MOI=2) with the LCR-LV and differentiated in-vitro resulted in a 3-fold increase of γ-globin mRNA in erythrocytes. SCD patient-derived HSC, which were transduced with LCR-LV (MOI=5) and transplanted into immunodeficient NSG mice, resulted in peripheral human erythrocytes that showed a reversal of the hemoglobin switch with a maximal induction 10% HbF as measured by flow cytometry. In a human ex-vivo B-lymphoid differentiation assay, SFFV-LV transduced (MOI=2) HSC populations with a 70% BCL11A knock-down showed no difference versus control in total cell numbers or in the sequential acquisition of CD43, CD19 and IgM (corresponding to physiological differentiation from common lymphoid progenitor to immature B-lymphocyte), thus showing no evidence for a differentiation block. Since IFN-response gene activation has been described with shRNA silencing and could potentially lead to HSC exhaustion, we quantified ISG20, ISG56 and OAS1 mRNA levels in human HSCs after miR-223-based SFFV-LV transduction (MOI=2). We observed less IFN-response gene activation in miR223-based SFFV-LV transduced HSC than in non-miRNA-based shRNA SFFV-LV transduced controls. In summary, our pre-clinical data demonstrates the potential efficacy of hematopoietic miRNA-mediated BCL11A/Bcl11a silencing to induce the expression of fetal hemoglobin in murine and human model systems, including primary cells. At the levels of BCL11A knock-down obtained, we did not observe any B-lymphoid toxicity. These results support the translation of LV-based miRNA-mediated BCL11A silencing into the clinical setting. Disclosures: No relevant conflicts of interest to declare.