UC Irvine

Endolysosomal trafficking determines the ultimate destination of molecules that enter cells and therefore has great impact on multiple cellular processes. Our lab has established that bioactive lipids called sphingolipids can rewire endolysosomal trafficking to result in accumulation of endocytosed material in pre-lysosomal compartments. In this thesis research, the impact of disrupted endolysosomal trafficking caused by sphingolipids on the activity of oligonucleotide therapeutics will be discussed. Therapeutic oligonucleotides include large RNA molecules such as mRNA vaccines and those that target RNA such as single-stranded antisense oligonucleotides (ASOs) or the double-stranded small interfering RNAs (siRNAs). ASOs base pair with a target RNA to elicit RNaseH-dependent degradation, inhibition of translation, or changes to splicing. siRNAs can produce prolonged target RNA silencing after being loaded into Ago2 to form the RNA-induced silencing complex (RISC). Given that all of these oligonucleotide therapeutic platforms require cytoplasmic entry for activity, inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. This thesis will discuss the development and characterization of a well-tolerated and orally bioavailable synthetic sphingolipid analog called SH-BC-893 that increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts multiple endolysosomal trafficking steps to improve the activity of oligonucleotide therapeutics in extrahepatic tissues.