Targeted reduction of let-7a miRNA increases fetal hemoglobin in human adult erythroblasts

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Authors

• JF de Vasconcellos
• C Byrnes
• YT Lee
• M Kaushal
• JM Allwardt
• A Rabel
• JL Miller

Abstract

Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with hemoglobinopathies. Previous studies showed that let-7 miRNAs are regulated during the fetal-to-adult transition in the erythroid lineage and reduced expression of let-7 in adult CD34+ cells increased HbF levels; however these studies did not address the potential for targeting an individual let-7 family member to regulate HbF levels. We initially determined the expression levels of the let-7 miRNAs in purified cell populations sorted from human peripheral blood and let-7a was identified as a predominantly expressed species in reticulocytes. Therefore, we hypothesized that specifically targeting let-7a may be sufficient to regulate HbF. For this purpose, a lentiviral construct with the tough decoy (TuD) design to target let-7a was compared with empty vector controls. Transductions were performed in human CD34+ cells from five adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Down-regulation of let-7a was confirmed by Q-RT-PCR at day 14 (control: 1.4E+07 +/- 2.4E+06 copies/ng; let-7a-TuD: 1.6E+06 +/- 4.6E+05 copies/ng; p=0.0003). Expression levels of globin genes were evaluated with significant increases in gamma-globin mRNA expression levels after let-7a-TuD transduction (control: 1.2E+06 +/- 6.8E+05 copies/ng; let-7a-TuD: 1.1E+07 +/- 4.5E+06 copies/ng; p=0.004). Remarkably, HPLC analyses at day 21 demonstrated robust increases in HbF levels after let-7a-TuD transduction (control: 4.7 +/- 0.6%; let-7a-TuD: 38.2 +/- 3.8%; p=0.00003). Our findings identify let-7a as a predominantly expressed let-7 family member in human reticulocytes and that targeted reduction of let-7a in erythroblasts is sufficient to cause robust increases in HbF levels.

Scientific Focus Area: Cell Biology

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