Advances in the Design of (Nano)Formulations for Delivery of Antisense Oligonucleotides and Small Interfering RNA: Focus on the Central Nervous System

Abstract

RNA-based therapeutics have emerged as one of the most powerful therapeutic options used for the modulation of gene/protein expression and gene editing with the potential to treat neurodegenerative diseases. However, the delivery of nucleic acids to the central nervous system (CNS), in particular by the systemic route, remains a major hurdle. This review will focus on the strategies for systemic delivery of therapeutic nucleic acids designed to overcome these barriers. Pathways and mechanisms of transport across the blood-brain barrier which could be exploited for delivery are described, focusing in particular on smaller nucleic acids including antisense oligonucleotides (ASOs) and small interfering RNA (siRNA). Approaches used to enhance delivery including chemical modifications, nanocarrier systems, and target selection (cell-specific delivery) are critically analyzed. Learnings achieved from a comparison of the successes and failures reported for CNS delivery of ASOs versus siRNA will help identify opportunities for a wider range of nucleic acids and accelerate the clinical translation of these innovative therapies.

Keywords: antisense oligonucleotide; blood−brain barrier; neurological diseases; small interfering RNA; systemic delivery.

Figure 1

RNAi-based therapeutic main approaches: single-stranded antisense oligonucleotides (ASOs) and double-stranded small interfering RNA (siRNA). ASOs: Once bound to the target mRNA, ASOs can form an RNA–DNA hybrid that becomes a substrate for RNase H, which results in mRNA degradation. siRNA: Double-stranded RNA (dsRNA) is processed by Dicer into siRNA. The guide RNA strand is incorporated into the RNA-induced silencing complex (RISC) and Argonaute 2 (Ago2). Ago2 cleaves the passenger strand, and siRNA/RISC complex then binds the complementary sequence of the target mRNA resulting in the degradation of the target transcript.

Figure 2

Physiological barriers to systemic delivery of RNA-based therapeutics. After systemic administration, the therapeutic nucleic acid must avoid interaction with bloodstream components, renal excretion, and uptake by phagocytes of the reticuloendothelial system (RES). Once at the targeted tissue, it must be internalized into the cell and escape from the endosome before degradation.

Figure 3

Schematic representation of the blood–brain barrier and the main transport routes for permeation and transport across the endothelium. (1) Small lipid-soluble agents can passively diffuse through the lipid bilayer. (2) Only small water-soluble molecules can diffuse through the intercellular spaces between endothelial cells. (3) The endothelium contains carriers for glucose, amino acids, nucleosides, purine bases, choline, and other substances. (4) Cationic molecules such as albumin and other plasma proteins are taken up by adsorptive-mediated transcytosis, which is consecutive of the endocytosis/exocytosis event. (5) Ligands such as insulin, transferrin, cholesterol-containing particles, and most other protein hormones are taken up by specific receptor-mediated transcytosis. Once across the BBB, the compounds must diffuse toward the disease site and be taken up by the diseased cells. TJ, tight junction; AJ, adherens junction.

Figure 4

Main strategies for central nervous system delivery of antisense oligonucleotides (ASOs) and small interfering RNA (siRNA). Unformulated/naked and chemical modifications predominate for ASOs, while conjugates and nanocarrier systems have been more widely studied for siRNA.

Figure 5

Schematic representation of a lipid–polymer hybrid nanoparticles (NPs). The NPs comprise a polymeric core containing a payload (siRNA or ASOs) surrounded by a lipid shell. Note that an additional outer PEG layer can be added and conjugated with targeting moieties such as aptamers, peptides, antibodies, and lipophilic derivatives (e.g., cholesterol). PEG, polyethylene glycol.

Figure 6

Schematic illustration of the 3D structure of cyclodextrin (CD). The CD comprises glucose units linked by α-1,4-glycosidic bonds and has a hydrophobic central cavity and a hydrophilic outer surface.