Structure-activity relationships and cellular mechanism of action of small molecules that enhance the delivery of oligonucleotides.

Rudolph L Juliano,Lindsey James,Mark Suto,Edward G Brown,Yamuna Ariyarathna,Chengqiong Mao,Xin Ming,Ling Wang,Francis Tavares

doi:10.1093/nar/gkx1320

Abstract

The pharmacological effects of antisense and siRNA oligonucleotides are hindered by the tendency of these molecules to become entrapped in endomembrane compartments thus failing to reach their targets in the cytosol or nucleus. We have previously used high throughput screening to identify small molecules that enhance the escape of oligonucleotides from intracellular membrane compartments and have termed such molecules OECs (oligonucleotide enhancing compounds). Here, we report on the structure–activity relationships of a family of OECs that are analogs of a hit that emerged from our original screen. These studies demonstrate key roles for the lipophilic aromatic groups, the tertiary nitrogen, and the carbamate moiety of the parent compound. We have also investigated the intracellular site of action of the OECs and have shown that activity is due to the release of oligonucleotides from intermediate endosomal compartments rather than from early endosomes or from highly acidic downstream compartments. At high concentrations of OECs toxicity occurs in a manner that is independent of caspases or of lysosomal cathepsins but instead involves increased plasma membrane permeability. Thus, in addition to describing specific characteristics of this family of OECs, the current study provides insights into basic mechanisms of oligonucleotide trafficking and their implications for oligonucleotide delivery.

Highlights

The precise gene regulating actions of siRNA, antisense oligonucleotides (ASOs), and splice switching oligonucleotides (SSOs) offer substantial promise for the therapy of cancer and other diseases [1,2,3]
The structure of UNC7938 is depicted in Figure 1A with the groups that were modified in this study indicated by highlighting
A wide variety of delivery technologies including many types of lipid and polymer nanoparticles, cell penetrating peptides, and ligand-oligonucleotide conjugates have been used in attempts to improve the pharmacological actions of oligonucleotides [47,48]

Summary

Introduction

The precise gene regulating actions of siRNA, antisense oligonucleotides (ASOs), and splice switching oligonucleotides (SSOs) offer substantial promise for the therapy of cancer and other diseases [1,2,3]. Use of oligonucleotides in therapy has been constrained by the inability to effectively deliver these relatively large and highly polar molecules [9,10]. An important aspect of the delivery problem concerns trapping of oligonucleotides in endosomal compartments limiting access to their intracellular sites of action in the cytosol or nucleus [11,12,13]. Discontinuities in the lipid bilayer can occasionally occur [17], allowing partial escape of vesicle contents [18], and permitting the pharmacological effects of oligonucleotides. Intracellular trafficking involves numerous proteins and lipids that regulate the formation, movement and coalescence of membrane bound vesicles [19,20]. Small organic molecules could potentially influence various aspects of intracellular trafficking and endomembrane stability, but few such molecules have been described far [21]

Methods

Results

Conclusion