Cholesterol-Modified MIR29B Induces Fetal Hemoglobin via targeting DNMT3A in Townes Sickle Cell Mice

MicroRNA (miRNA) molecules hold promise as potential therapeutics capable of silencing target gene expression through RNA interference. The delivery of miRNAs without degradation has posed a significant challenge to their clinical use. However, cholesterol-conjugated miRNA has emerged as a promising delivery approach thanks to its low toxicity and high efficiency. We previously showed that MIR29B by 28-day continuous infusion using subcutaneous mini osmotic pump delivery, MIR29B induces HbF. The present study delves into evaluating intermittent delivery of a cholesterol-conjugate of MIR29B (Chol-MIR29B), to induce fetal hemoglobin (HbF) in Townes sickle cell mice. Therefore, we conducted additional in vivo studies to test intermittent dosing of Chol-MIR29B in Townes sickle cell mice. Blood was processed for complete blood count with differential and reticulocytes at weeks 0, 2, and 4, and γ-globin gene reactivation was determined by RT-qPCR. The percentage of HbF-positive cells was measured by flow cytometry. RNA seq was performed on the peripheral blood of mice treated with MIR29B. Mice treated with Chol-MIR29B exhibited normal behavior and weight gain. While there was no direct effect of Chol-MIR29B on γ-globin reactivation, delivery of Chol-MIR29B after 4 weeks of treatment significantly increased the percentage of HbF positive cells by up to 15-fold. Additionally, there were no toxic effects on complete blood counts and differential or activation of oncogenes as determined by RNA seq, supporting the safety of Chol-MIR29B while inducing HbF expression. Our results provide the first evidence of the ability of Chol-MIR29B to induce HbF in sickle cell disease transgenic mice.