Silencing the gene expression: ASOs & siRNA therapeutics

September 30th, 2020

SILENCING THE GENE EXPRESSION: ASOs AND siRNA THERAPEUTICS

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) are the two most widely used strategies for silencing (specifically reducing) gene expression, with several therapies already approved or in development. ASOs and siRNA mechanistically rely on Watson-Crick base-pairing, which means they recognize specific target oligonucleotide sequences.

ASOs are synthetic, single-stranded oligonucleotides with a typical length of 16 to 21 bp. The use of oligonucleotides as therapeutic agents has elicited a great deal of interest in the last decades. However, they have poor pharmacokinetic properties. Oligonucleotides are decomposed rapidly in biological systems by nuclease cleavage and then are excreted in the urine. These limitations affect their direct use in antisense therapeutics. Therefore, it has been crucial to find modifications, which strongly improve stability of ASOs in vivo and to make ASOs druggable. Modification of the phosphate linkage has been the first successful strategy for antisense drug developments. Then modifications on the base and on the sugar moiety have been investigated to increase their binding affinity and specificity, as well as their cellular uptake (Figure 1). 

ASOs act on the DNA/RNA level and confer gene silencing in different ways: 1) formation of DNA-RNA hetero-duplexes and subsequent degradation of the target mRNA (most common mechanism, mediated by RNase H), 2) inhibition of translation by a steric blockade of the ribosome and 3) prevention of assembly of the translation machinery by interaction with the 5′-terminus of the target mRNA.

In addition to gene silencing, certain ASOs function as regulators of RNA splicing and are of therapeutic interest to restore the function or expression of a protein involved in diseases. A prominent example is the application of splice-switching ASOs for the treatment of Duchenne muscular dystrophy (DMD), overcoming a frameshift mutation and thus restoring the expression of a functional, yet truncated, dystrophin protein.

siRNAs inhibit mRNA translation, employing the RNA interference (RNAi) pathway, and have potential for the cure of virus-transmitted and genetic diseases including cancer. siRNAs initially occur as RNA duplexes, but only one strand (guide strand) induces gene silencing, while the other (passenger strand) is enzymatically degraded.

Gene silencing takes place after binding of the guide strand to a complementary mRNA sequence and subsequent cleavage of the latter, employing the RNA-induced silencing complex (RISC). Synthetic siRNA duplexes are typically 19 to 22 bp in length, which is sufficient for function, but short enough to avoid most of the interferon response triggered by duplexes larger than 30 bp in length.

While ASOs continue to be used for gene silencing, the robustness of siRNAs and the relative ease of identifying active siRNAs have increased their clinical relevance. One of the challenges remains for example delivery, since ASOs as well as siRNAs must cross biological membranes to exert their activity.

S. Karaki et al., Antisense oligonucleotides, a novel developing targeting therapy. IntechOpen, (2019)

J.K. Watts and D.R. Corey, Silencing disease genes in the laboratory and the clinic. J. Pathol., 226(2), 365-79 (2012)

A. Deiters, Small molecule modifiers of the microRNA and RNA interference pathway. AAPS J, 12(1), 51-60 (2010)

A. Khvorova and J. K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility, Nat. Biotechnol., 238-248 (2017)

 

AFTER TWO DECADES OF RESEARCH: NEW OLIGONUCLEOTIDE THERAPIES ON THE HORIZON

Oligonucleotide therapeutics are a novel class of drug products comprised of strings of synthetic nucleotides. The potential of oligonucleotides as therapeutic drugs has generated hundreds of clinical trials. Currently, there are over 260 companies and organizations sponsoring clinical trials for oligonucleotide drug candidates.

The top five study sponsors of ongoing clinical trials of oligonucleotides include Ionis Pharmaceuticals, Alnylam Pharmaceuticals, Astra Zeneca, Biogen and Arrowhead Pharmaceuticals. Ionis is developing antisense oligonucleotides such as IONIS-AGT-LRX for the treatment of hypertension and IONIS-PKK-LRX for hereditary angioedema. Alnylam Pharmaceuticals is developing RNAi therapeutics such as lumasiran for primary hyperoxaluria type 1 and vutrisiran for the treatment of transthyretin-mediated (ATTR) amyloidosis. Meanwhile, Astra Zeneca has several oligonucleotide drug candidates in Phase I and Phase II studies such as AZD-8601 for Congestive Heart Failure and AZD-2693 for the treatment of Non-Alcoholic Steatohepatitis (NASH). In addition, Biogen has four antisense oligonucleotides currently in clinical studies for the central nervous system therapeutic area including nusinersen, tofersen sodium, BIIB078 and BIIB-094. Furthermore, Arrowhead Pharmaceuticals is leveraging RNAi technologies in several therapeutic areas including developing ARO-AAT for the treatment of liver disease associated with alpha-1 antitrypsin deficiency and JNJ-3989 for chronic hepatitis B infection.

In total, there are currently over 280 ongoing clinical trials of oligonucleotide drug candidates. The breakdown by clinical phase is shown in Figure 1.

Figure 1 – Number of Ongoing Clinical Trials by Phase. For more information on clinical phase definitions, please compare for example reference 3.

 The top therapeutic areas among the ongoing clinical trials is overwhelmingly oncology with 116 studies. Other top therapeutic areas include central nervous system, metabolic disorders, genetic disorders and infectious disease. The top 10 therapeutic areas for the current ongoing clinical trials are shown in Figure 2. The top indications for clinical trials of oligonucleotides include neurology, solid tumors, gastrointestinal tract cancer, skin cancer and blood cancer.

Figure 2 – Ongoing Clinical Trials by Top 10 Therapeutic Areas.

 With the recent approvals in 2020 and 2019 for NS Pharma’s Viltepso®, Sarepta Therapeutics’s Vyondys 53®, Alnylam Pharmaceuticals’s Givlaari®, Akcea Therapeutics’s Waylivra® and a loaded pipeline of clinical phase drug candidates, oligonucleotide therapeutics are on the edge of delivering on the promises of two decades of research and development efforts.

 References

1) Clinicaltrials.gov (2019)

2) GlobalData (2020)

3) National Cancer lnstitute Dictionary