ABSTRACT Acute myeloid leukemia (AML) is one of the most prevalent hematological malignancies. Unfortunately, due to a high relapse rate from chemotherapy, disease-free survival (DFS) remains at 40%. Greater than 80% of AMLs express high levels of the CD33 marker on their cell surface. Alternative therapeutic approaches, including Antibody-Drug Conjugate (ADC), anti-CD33 Mylotarg (gemtuzumab ozogamicin), and Radionuclide Antibody-Conjugates (RACs), have shown some promising results. However, both of these antibody therapeutic agents have significant side effects and suffer from indiscriminant killing of normal cells expressing CD33 as well as from killing normal cells due to non-specific tissue absorption and drug shedding. Unfortunately, AML also does not respond to anti-PD1 and PD-1L check-point inhibitors. Due to chromosomal translocations, the MLL gene fuses to many other genes, including the AF9 gene, resulting in MLL-fusion oncogenes that drive AML growth and survival. Expression of MLL- AF9 driver oncogene in AML has a particularly poor clinical outcome. Consequently, there is a great need to develop novel precision medicine therapeutics that selectively targets and kills only AML tumor cells based on their oncogenic genetic translocations, while sparing normal CD33 positive cells. RNA Interference (RNAi) responses have great potential to target MLL-AF9 and other MLL translocation fusion genes. Unfortunately, despite its promising therapeutic features, due to their requisite negatively charged phosphate backbone, siRNAs have no ability to enter cells and require a delivery agent. To tackle the RNAi delivery problem, we pioneered development of a next-generation RNAi trigger, called RiboNucleic Neutral (siRNN) prodrugs. siRNNs represent a “Prodrug” approach where the negative charge is directly neutralized by a bioreversible phosphotriester chemical group that is selectively cleaved off in the cytoplasm, but not outside of cells. The rate-limiting RNAi delivery step is escape from the endosome into the cytoplasm. To enhance endosomal escape, we will synthesize a next-generation endosomal escape domain (EED). We will target delivery of MLL-AF9 siRNN RNAi triggers to AML cells by conjugation to anti-CD33 antibodies, called Antibody-RNAi Conjugates (ARCs). The goal of this high risk/high gain technology development proposal is treat AML by generating precision medicine anti-CD33 Antibody-RNAi Conjugates (ARCs) that selectively target the MLL-AF9 fusion oncogene and kill AML cells based on their mutant genetics, but unlike ADCs and RACs, have no effect on CD33-positive normal cells.