Pharmacologic activation of HbF can interrupt the most proximal events in the pathophysiological cascade of sickle cell disease (SCD), and is therefore a preferred approach to multi-organ disease modification. Its clinical potential, however, has not been fulfilled. We hypothesize that removal of epigenetic activation marks at the HBG locus is an early and necessary step in the fetal to adult globin switch, and hence, inhibiting specific chromatin-modifying enzymes that remove these activating epigenetic marks (e.g. LSD1 or DNMT1) will efficiently prevent HBG repression. In SA1, we determine if there is an obligatory sequence in the placement of epigenetic marks at HBG during the maturational globin switch. Precisely staged erythroid progenitor cells are analyzed by ChIP. The interdependence of epigenetic marks is analyzed by use of drugs and siRNA knockdowns targeting the enzymes responsible for catalyzing these modifications. In SA2 we will explore the possibility that either of two recently discovered non-cytotoxic inhibitors of LSD1 could lead to more efficacious human SCD medications. One of these compounds, tranylcypromine (TC), is already FDA approved. The other compound, a natural product belonging to the chromen-4-one class and potent non-cytotoxic LSD1 inhibitor, will be improved with further SAR and medicinal chemistry prior to evaluation both in vitro and in murine models of SCD, to generate critical pre-clinical in vivo proof of principle. Thus we already have three candidate epigenetic drugs (the two LSD1 inhibitors and the DNMT1-depleting drug, decitabine) to evaluate and ask whether some combination of these compounds can synergistically induce HbF, enabling individual medication dosages to be kept at levels well clear of possible toxicity. Ideally, candidate drugs would be targeted to the erythroid compartment, to further enhance therapeutic index. This need is addressed in SA3, using a highly innovative, versatile method that connects any developed drug to an EPOR targeting peptide by way of gold nanocluster intermediaries and chemistry that does not impede the function of either, and which facilitates in situ release of drug by thiol biomolecules at target cells. In SA4 we will explore in the baboon model individual and combinatorial drug efficacy and safety, providing dose and schedule guidance for formal toxicology and formulation studies and definitive progress to clinical trials. While many pre-clinical animal models inadequately predict clinical utility, the baboon model has reproducibly facilitated clinical translation of novel therapies for SCD. By using highly innovative methods and rational concepts to develop novel drug combinations and delivery systems that can maximize therapeutic index, this project strives to fulfill the potential of HbF activation therapy. (End of Abstract)