Delivery of Oligonucleotides: Efficiency with Lipid Conjugation and Clinical Outcome

Abstract

Oligonucleotides have shifted drug discovery into a new paradigm due to their ability to silence the genes and inhibit protein translation. Importantly, they can drug the un-druggable targets from the conventional small-molecule perspective. Unfortunately, poor cellular permeability and susceptibility to nuclease degradation remain as major hurdles for the development of oligonucleotide therapeutic agents. Studies of safe and effective delivery technique with lipid bioconjugates gains attention to resolve these issues. Our review article summarizes the physicochemical effect of well-studied hydrophobic moieties to enhance the cellular entry of oligonucleotides. The structural impacts of fatty acids, cholesterol, tocopherol, and squalene on cellular internalization and membrane penetration in vitro and in vivo were discussed first. The crucial assays for delivery evaluation within this section were analyzed sequentially. Next, we provided a few successful examples of lipid-conjugated oligonucleotides advanced into clinical studies for treating patients with different medical backgrounds. Finally, we pinpointed current limitations and outlooks in this research field along with opportunities to explore new modifications and efficacy studies.

Keywords: LNP; cholesterol; delivery; fatty acid; lipid conjugates; oligonucleotide; squalene; tocopherol.

Figure 1

Pros and cons comparison of oligonucleotide versus small molecule drugs. Some highlighted advantages of oligonucleotide would be inhibitory specificity, expedient manufacture, and low in drug–drug interaction. Disadvantages of cost, stability, and formulation remained.

Figure 2

An illustration of main oligonucleotide mechanism of action as divided into two main groups: (1) Occupancy-only and (2) occupancy-mediated degradation. In occupancy only, oligonucleotide would act as steric blocker or inhibitor preventing any upcoming interaction with precedent targets. Meanwhile, occupancy-mediate degradation activates cleavage the targeted RNA via RNase or AGO-2.

Figure 3

Four biological barriers preventing activity of therapeutic oligonucleotide. (A) Endo-lysosomal entrapment. ON required escape from late endosome before subjected to lysosomal degradation. (B) Exonuclease cleavage. Initiate hydrolysis of phosphodiester backbone, which deteriorate ON. (C) Reticuloendothelial system. Digestion of ON by macrophage can terminate its activity. (D) Endothelium lining. Blockage of transverse ON from vascular lumen to interstitial fluid.

Figure 4

Three generations of common nucleic acid modifications. First generation replaced phosphodiester (PO) backbone to phosphorothioate (PS) to enhance degradative resistance. Second generation includes modification of 2′ ribose into 2′-O-methyl (2′-OMe) and 2′-O-Methoxyethyl (2′-MOE), which are popular in gapmer synthesis. Third generation restricts conformational flexibility by introducing a methyl bridge between 2′-O and 4′ of ribose. Locked nucleic acid (LNA) and constraint ethyl (cET). Ribose moiety would be completely replaced with tricyclic DNA (tcDNA) or cyclohexene (CeDNA). Alternative modification was phosphorodiamidate morpholino oligomer (PMO). This neutral nucleic acid was described with an additional amine accompanied with phosphorodiamidate backbone.

Figure 5

Chemical structure of two most advanced LNP that assisted in delivery of 2 mRNA COVID vaccines. ALC-0315 and SM-102 contains similar structure with ethanolamine head (one is shorter than others). ALC-0315 has two branched degradable ester tail, while SM-102 only has one branched ester tail.

Figure 6

General molecular structure of conjugated oligonucleotides including: (1) synthetic oligonucleotide, (2) linkers, and (3) bioconjugates.