Delivery of Oligonucleotide Therapeutics: Chemical Modifications, Lipid Nanoparticles, and Extracellular Vesicles

Abstract

Oligonucleotides (ONs) comprise a rapidly growing class of therapeutics. In recent years, the list of FDA-approved ON therapies has rapidly expanded. ONs are small (15-30 bp) nucleotide-based therapeutics which are capable of targeting DNA and RNA as well as other biomolecules. ONs can be subdivided into several classes based on their chemical modifications and on the mechanisms of their target interactions. Historically, the largest hindrance to the widespread usage of ON therapeutics has been their inability to effectively internalize into cells and escape from endosomes to reach their molecular targets in the cytosol or nucleus. While cell uptake has been improved, "endosomal escape" remains a significant problem. There are a range of approaches to overcome this, and in this review, we focus on three: altering the chemical structure of the ONs, formulating synthetic, lipid-based nanoparticles to encapsulate the ONs, or biologically loading the ONs into extracellular vesicles. This review provides a background to the design and mode of action of existing FDA-approved ONs. It presents the most common ON classifications and chemical modifications from a fundamental scientific perspective and provides a roadmap of the cellular uptake pathways by which ONs are trafficked. Finally, this review delves into each of the above-mentioned approaches to ON delivery, highlighting the scientific principles behind each and covering recent advances.

Keywords: RNA therapeutics; cellular uptake; endosomal escape; extracellular vesicles; intracellular trafficking; lipid nanoparticles; oligonucleotide; oligonucleotide delivery.

Figure 1

Commonly used types of ONs and their target species. Five types of ONs are discussed in this review. Gapmers target mRNA with high affinity thanks to LNA base pairs. The unmodified nucleotides in the central region allow RNase H1 to bind, degrading the mRNA. Splice-switching ONs target splice sites of pre-mRNA, preventing the splicing machinery from forming and altering the resultant mRNA. Aptamers have a 3D structure which mimics the ligands of the proteins they target with high specificity and affinity. The guide strand of double-stranded siRNA guides the RISC to the target mRNA, leading to RISC-mediated degradation. miRNA is activated by cleavage by Dicer, where it binds to mRNA preventing the formation of RNP complexes and ultimately destabilizing the mRNA. Abbreviations: SSO, splice switching ON; siRNA, short inhibiting RNA; RISC, RNA-induced-silencing complex; miRNA, micro-RNA; mRNP, mRNA–protein complex. Figure created in BioRender.

Figure 2

Common chemical modifications for RNA ONs. The three sites for common modifications of RNA ONs include the nucleobase, the phosphate backbone, and the carbohydrate sugar. Advantageous characteristics of modifications are listed for each site, and chemical modifications which are utilized FDA-approved ONs are listed for each. The 5-carbon of the nucleobase and the 2′-carbon of the carbohydrate are annotated with their relevant location number. Abbreviations: PS, phosphorothioate; PMO, phosphorodiamidate morpholino oligomer; 2′-OMe, 2′-O-methyl; 2′-O-MOE, 2′-O-methoxyethyl; 2′-F, 2′-fluoro; LNA, locked nucleic acid. Figure created in BioRender.

Figure 3

Endocytic uptake and endosomal escape of ONs. The major identified internalization routes of ONs are clathrin-dependent endocytosis, clathrin-independent endocytosis, and macropinocytosis. ON is then trafficked sequentially to the early endosome and sequentially to the late endosome, where it is trafficked to the lysosome or to the multivesicular bodies and exocytosed. Late endosome membrane remodeling and transition to MVB or lysosome have been indicated as likely points of endosomal escape. Commonly used endosomal markers are shown. Abbreviations: Rab, Ras-associated protein; EEA1, early endosome antigen 1; LBPA, lysobisphosphatidic acid; LAMP1, lysosomal-associated membrane protein 1. Figure created in BioRender.

Figure 4

RNA-loading approaches for EVs. EV cargo-loading can be broadly classified as passive endogenous loading, active endogenous loading, or exogenous loading. Endogenous pathways involve transfection or transduction of genetic material into the EV-producing cells. In passive endogenous loading, an RNA overexpression construct leads to stochastic EV loading of an abundantly transcribed RNA. In active endogenous loading, an additional construct comprised of an EV marker protein and an RBD capture the target RNA and shuttle it to EVs during their biogenesis. Exogenous loading occurs after EVs have been isolated and involve physical or chemical techniques to insert RNA into the EV lumen. Abbreviations: RBD, RNA-binding domain; MVB, multivesicular body. Figure created in BioRender.