Metabolic Stability and Targeted Delivery of Oligonucleotides: Advancing RNA Therapeutics Beyond The Liver

Abstract

Oligonucleotides have emerged as a formidable new class of nucleic acid therapeutics. Fully modified oligonucleotides exhibit enhanced metabolic stability and display successful clinical applicability for targets formerly considered "undruggable". Accumulating studies show that conjugation to targeting modalities of stabilized oligonucleotides, especially small interfering RNAs (siRNAs), has enabled robust delivery to intended cells/tissues. However, the major challenge in the field has been the stability and targeted delivery of oligonucleotides (siRNAs and antisense oligonucleotides (ASOs)) to extrahepatic tissues. In this Perspective, we review chemistry innovations and emerging delivery approaches that have revolutionized oligonucleotide drug discovery and development. We explore findings from both academia and industry that highlight the potential of oligonucleotides for indications involving different extrahepatic organs─including skeletal muscles, brain, lungs, skin, heart, adipose tissue, and eyes. In all, continued advances in chemistry coupled with conjugation-based approaches or novel administration routes will further advance the delivery of oligonucleotides to extrahepatic tissues.

Figure 1

FDA-approved NATs. There are currently 24 approved NATs, which include siRNAs (6), ASOs (11), CpG oligonucleotides (2), mRNAs (3), and aptamers (2). Interestingly, 20 out of the 24 NATs were approved in the past decade, signifying a resurgence for this class of therapeutics. NAT: nucleic acid therapeutic; siRNA: small interfering RNA.

Figure 2

Chemical designs and FDA-approved delivery platforms for siRNAs. (Left) Commonly employed modifications include 2′ ribose modifications (2′-O-Me, 2′F) and backbone modification (PS, 5′VP). Glycol nucleic acid modification at position 7 on the antisense strand prevents seed region-mediated off-target toxicity. 5′ Morpholino modification at the sense strand mitigates loading of the sense strand into the RISC. (Middle) Evolution of chemical designs for siRNAs. Onpattro (patisiran) was the first approved siRNA therapeutic with minimal chemical modifications, whereas all other approved members in this class contain extensive chemical modifications. As displayed using future design chemistry, the mechanism of action of siRNA involves binding of the antisense strand to the RISC machinery to generate an antisense-RISC, which carries out catalytic degradation of multiple copies of the targeted mRNA. (Right) LNPs and trivalent GalNAc are the two approved platforms for delivery of siRNA to the liver. 2′F: 2′-fluoro; 2′O-Me: 2′ O-methyl; 5′VP: 5′-vinylphosphonate; GalNAc: N-acetylgalactosamine; PS: phosphorothioate; RISC: RNA-induced silencing complex; siRNA: small interfering RNA.

Figure 3

Mechanisms of action and chemical modifications of ASOs. (Left) ASO operates via two main mechanisms: mRNA degradation through endonuclease RNase H1, and mRNA modulation by steric blocking of the faulty exon. The RNase H1 mechanism is activated by the binding of a gapmer ASO to mature mRNA, ultimately resulting in the degradation of target mRNA. Steric-blocking ASOs bind to pre-mRNA containing an exon to form a heteroduplex. The ASO masks a portion of the exon, allowing for the formation of a shortened but functional mRNA. (Right) Depiction of the structures of approved ASO medicines Inotersen and Nusinersen. Inotersen is a gapmer ASO while Nusinersen is predominately made up of fully modified RNA. Typical chemical modifications to ASOs include backbone modifications (PS, TMO, or PMO), 2′ ribose modifications (2′O-MOE, LNA, 2′-cEt) or base modification (5-methyl-pyrimidine). 2′-cEt: 2′-O-(2-methoxyethyl) ethyl; 2′MOE: 2′O-methoxyethyl; gapmer (ASO with a central DNA gap flanked by modified RNA); LNA: locked nucleic acid; PMO: phosphorodiamidate morpholino oligonucleotide; PS: phosphorothioate; RNase H1: ribonuclease H1; TMO: thiomorpholino oligonucleotide.

Figure 4

Proposed mechanism of action of Avidity Biosciences’ investigational drug, AOC 1001, for myotonic dystrophy. (A) In patients with myotonic dystrophy, DMPK mRNA forms toxic (CUG)n repeats that result in the formation of a hairpin structure. Subsequently, MBLN—which is an essential splicing factor, is sequestered by these repeats, which leads to a splicing aberration. (B) AOC 1001 is generated by the conjugation of a TfR1 mAb to DMPK siRNA. Following systemic administration, AOC 1001 binds to TfR1 expressed on skeletal muscles. Further, AOC 1001 is internalized into endosomes, and the siRNA is released to cause DMPK silencing, which likely leads to the release of MBNL followed by the restoration of normal splicing machinery. In their phase 2 clinical trials, Avidity recently reported that this molecular correction (subserved by AOC 1001) is associated with an overall improved muscle performance. MBLN: muscle bind-like protein; TfR: transferrin receptor.