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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE276352 | GEO | 2025/06/18

REPOSITORIES: GEO

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Project description:Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation . In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations. Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable. Keywords: Microarrays, PAXgene, globin reduction, whole blood, PBMC There are 10 samples for each of PBMC, PAX and PAX globin-reduced, where 5 samples come from sickle-cell patients and 5 from healthy controls.

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

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