Delivery of oligonucleotide-based therapeutics: challenges and opportunities

Abstract

Nucleic acid-based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid-based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid-based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide-based therapeutics.

Keywords: RNA therapeutics; delivery; oligonucleotides; preclinical models; safety.

Figure 1. Oligonucleotide chemistries

Commonly used nucleic acid chemistries. The often used phophorothioate (PS) backbone replaces the natural phosphodiester (PO). Modifications to the ribose at the 2ʹ‐O position of RNA and 2ʹ‐position of DNA include the 2ʹ‐O‐methyl (2ʹ‐OMe), 2ʹ‐O‐methoxy‐ethyl (2ʹ‐MOE) and 2ʹ‐fluoro (2ʹ‐F) are the most commonly used. Conformationally constrained DNA analogues, locked nucleic acid (LNA), constrained 2′‐O‐ethyl (cEt) and tricyclo‐DNA (tcDNA), provide greater binding affinity. LNA and cEt are constrained by a methyl bridged from the 2′‐O and 4′ position of the ribose. tcDNA introduces of an ethylene bridge with a cyclopropane ring between the 3′ and 5′ carbon positions of ribose. Alternative chemistries include changes in the nucleobase, e.g. phosphorodiamidate morpholino oligomers (PMO) and peptide nucleic acid (PNA).

Figure 2. Mechanisms and location of action for oligonucleotides

Representative mechanisms of action and intracellular localisation for (1) gapmer and mRNA degradation, (2) aptamer, (3) nuclear steric blockage for splice switching, (4) blockage the assembly of RNA‐binding factors, (5) TLR activation of innate immunity, (6) miRNA and antagomir, steric block, translational upregulation, (7) agomir, translational inhibition, and (8) siRNA, RISC, RNAi silencing ONs.

Figure 3. Delivery of oligonucleotides to the brain and eye

(A) ONs are prevented from passive diffusion into the central nervous system (CNS) by the vascular BBB. (B) ONs without a delivery reagent require direct administration into the brain or spinal cord. The most frequently used CNS administration route in humans is intrathecal (IT) administration, where ONs are administered into the subarachnoid space of the spinal cord to pass the pia mater and enter the parenchyma. This results in an immediate high ON concentration in the cerebral spinal fluid, meaning that a lower dose can be used, which reduces side effects. Also, the BBB prevents transport of ONs into the peripheral circulation resulting in long‐lasting high ON concentrations. (C) The eye is a contained and immune‐privileged organ of the CNS that allows local delivery. ONs are effective and well tolerated when administered directly by intravitreal injection. Subretinal delivery is also possible, but the treated area will be reduced. (D) Certain macromolecules can cross the vascular barriers via receptor‐mediated endocytosis after systemic administration (Pardridge, 2007). The transferrin transport pathway has been exploited in several rodent studies to carry ONs into the brain parenchyma (Lee et al, 2002; Kozlu et al, 2014). Systemically delivered ONs covalently conjugated to arginine‐rich CPPs have been shown to cross the BBB in mice (Du et al, 2011) and have been used for ON delivery in mouse models of SMA (Hammond et al, 2016). Several studies have shown exosome‐mediated delivery of small RNAs across the vascular barriers into the CNS (Alvarez‐Erviti et al, 2011; Yang et al, 2017). (E) Drugs dosed by intranasal administration can be transported into the brain along the olfactory, trigeminal nerve and rostral migratory stream (Curtis et al, 2007).

Figure 4. Delivery technologies for oligonucleotides

Delivery technologies used to improve the ADMET properties of ONs, including chemical conjugates (left) and nanoparticulate carriers (right). Polymers, cell‐penetrating peptides (CPPs) and lipids represent examples of molecules used for covalent conjugation to ONs for passive targeting, whereas covalent conjugation of ONs to antibodies, receptor ligands and aptamers are applied for active targeting. Drug conjugates display a defined stoichiometry. CPP conjugation is only compatible with uncharged ONs, e.g. PMOs and PNAs, whereas lipids and GalNAc are compatible with all types of ONs. Nanoparticulate carriers can be used to encapsulate negatively charged ONs and can be based on lipids, e.g. lipid nanoparticles (LNPs) and exosomes, polymers, e.g. dendrimers, poly(lactide‐co‐glycolic acid) (PLGA) and polyphosphazenes, and peptides, or on hybrid systems composed of several different types of compounds. The complexity of these systems poses new challenges in the development with respect to cost, manufacturability, safety, quality assurance and quality control.

Figure 5. ASO mediated toxicities

Schematic representation of the most common ON‐mediated toxicities, which are mainly classified as hybridisation‐dependent (Watson–Crick hybridisation) or hybridisation‐independent effects (tissue accumulation, proinflammatory mechanisms and/ or protein binding effects). Some of them are strictly class specific (sequence independent), while others can be influenced by the sequence (sequence specific).