Small Activating RNA-Mediated Induction of <i>HBG</i> Via Liposome Delivery for <i>In Vivo</i> Treatment of Sickle Cell Disease and Beta-Thalassemia

Small activating RNAs (saRNAs) are short, double-stranded RNA molecules that leverage an evolutionarily conserved mechanism to selectively induce the transcription of target genes. While they share similarities with RNA interference, such as the requirement for Argonaute proteins, saRNAs exhibit distinct kinetics and mechanisms of action. Specifically, saRNAs function within the nucleus, promoting the recruitment of the RNA-induced transcriptional activation (RITA) complex which leads to changes in epigenetic markers associated with gene activation. The clinical validation of saRNA-mediated RNA activation has been demonstrated for the transcription factor C/EBPα using intravenous in vivo delivery via the liposome NOV340 (PMID 34407972, NCT02716012, NCT04105335). Hemoglobin (Hb) is a tetramer composed of two alpha globin chains and two beta globin chains. The beta globin gene locus includes embryonic (epsilon, ε, HBE), fetal (gamma, g, HBG), and adult (beta, b, HBB) forms of beta globin chains. Sickle cell disease (SCD) is caused by a mutation in the HBB gene, producing haemoglobin S (Hb S), which causes red blood cells to deform under hypoxic conditions leading to painful vaso-occlusive episodes, anaemia, and organ damage. Beta-thalassemia, also caused by mutations in the HBB gene, reduces the production of beta globin chain, resulting in low of Hb levels and severe anaemia. Both conditions cause significant morbidity and mortality. Clinical studies have shown that the expression of fetal gamma globin (Hb F) can compensate for the dysfunctional HbS, providing a validated therapeutic approach for managing beta-hemoglobinopathies in SCD patients. Recent drug development efforts have focused on reactivating HBG using small molecules and CRISPR technology. However, small molecules have not achieved transformational levels of Hb F induction, and ex vivo gene editing approaches, while highly effective, pose a high treatment burden and challenging patient accessibility. In this study, we identify saRNAs that induce g-globin from the cell's HBG locus by upregulating endogenous transcription and translation. We designed saRNA sequences targeting the HBG1/2 promoter region using our proprietary algorithm and established an erythroid-derived progenitor cell model using bone marrow CD34 cells isolated from human donors. Our results demonstrate dose-dependent and durable upregulation of g-globin mRNA and protein, with expression observed in a pan-cellular manner. Previously, we demonstrated that packaging saRNAs into the clinically validated NOV340 liposomal formulation enables highly efficient targeting of erythroid progenitor cells (ErPs) in the bone marrow of non-human primates. This in vivo system achieves delivery to approximately 60% of CFU-E and Pro-E cells. Notably, equivalent in vivo delivery efficiency was observed in peripheral blood monocytes, where the activity of another NOV340 formulation (MTL-CEBPA) was previously reported in clinical studies. To predict the performance of the saRNA / liposome formulation in SCD patients, we conducted a quantitative systems pharmacology analysis. The model predicts a peak in HbF induction after three months of treatment with at least 2.5 fold increase in % Hb F levels with biweekly or less frequent dosing. Collectively, these studies establish that in vivo-delivered saRNA oligonucleotides hold promise for best-in-class in vivo HbF induction, with the potential to achieve protective levels of HbF in a significant proportion of patients. This approach offers significant improvements in safety, tolerability, and accessibility over current gene editing and small molecules therapies in development.