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.