Cholesterol-conjugated miR-29b induces fetal haemoglobin expression via γ-globin promoter demethylation in the Townes mouse model for sickle cell anaemia.

Cholesterol-conjugated miRNA has emerged as a viable system for preventing small molecule degradation in plasma with low toxicity and high delivery efficiency. We previously established that miR-29b induces fetal haemoglobin (HbF) in Townes sickle cell anaemia (SCA) mice using a continuous infusion mini-osmotic pump. Herein, we expanded the scope of our work to determine the ability of intermittent subcutaneous cholesterol-conjugated miR-29b (Chol-miR-29b) to induce HbF expression in SCA mice. Blood was collected for automated complete blood counts with differential, reticulocyte count and γ-globin and β(S)-globin mRNA levels quantified by reverse transcription-quantitative PCR; flow cytometry measured the percentage of HbF-positive cells (F-cells). Methylation assays determined 5-methylcytosine levels, and RNA sequencing changes in gene expression for different treatment conditions. SCA mice receiving Chol-miR-29b exhibited normal behaviour and weight gain without peripheral blood count toxicity. The γ-globin mRNA levels significantly increased, and F-cell percentages were enhanced after Chol-miR-29b treatment. Subsequently, spleen tissue confirmed decreased DNMT3A protein and 5-methylcytosine levels in the γ-globin gene promoter. RNA-seq analysis supports oncogene silencing and tumour suppressor gene activation by Chol-miR-29b. These data demonstrate that a daily subcutaneous dose of Chol-miR-29b induces HbF expression, highlighting its potential for clinical development in SCA.