Developing innovative antivirals for novel viruses

An exciting and innovative focus for antiviral therapy is the application of a radical new drug modality.¹ Oligonucleotide (oligo) therapeutics are modified short single- or double-stranded DNA or RNA sequences, typically comprising 15–30 nucleotides, which can interact with DNA, RNA or proteins to silence gene expression.²

Although the idea behind these therapeutics was first conceived 30 years ago, setbacks has since hindered the development of these drugs.³

Not so long ago, however, we saw the first oligo drug, Vitravene (fomivirsen), reach the market and clinic.^2,4 Since then, nine other drugs have been approved and this modality has now been hailed as the third major drug development platform after small molecules and biologics.^1,4

This new modality currently encompasses a variety of classes, including antisense oligonucleotides (ASOs), aptamers, small interfering RNAs (siRNAs), microRNAs (miRNAs), deoxyribozymes (DNAzymes) and short DNA/RNA heteroduplex oligonucleotides (HDOs).

Getting a head start with oligonucleotide antivirals

Fortunately, oligo drugs are already being developed to treat viral infections, giving us a head start in the race to develop a much-needed antiviral against COVID-19. Oligonucleotide companies such as Arbutus, Dicerna, Ionis, Janssen and Vir are developing ASO and siRNA antivirals for hepatitis B.

The hepatitis B virus (HBV) infects liver cells and hijacks their molecular machinery to replicate. Sometimes, during this process, a substantial quantity of foreign proteins is produced, which can trigger all sorts of cellular and immune responses with immediate and long-term effects.

So, HBV infection can cause chronic inflammation and increase the risk of developing liver cancer and other life-threatening diseases.

This multifaceted but unclear viral pathogenesis could be said to be comparable in a way with the more immediate “cytokine storm” disease mechanism of COVID-19 caused by the SARS-CoV-2 virus.⁵

Several HBV oligo drugs are being tested in clinical trials. A key component of these trials, as well as of their requisite preclinical studies and any production after marketing approval, is the bioanalysis of drug exposure relating to its biological activity (dose response).

This includes the performance of toxicology studies and establishing drug metabolism and pharmacokinetics (DMPK) profiles for each experimental medicine.

However, with new modalities, there are always unknowns. In recent years, oligos have been increasingly modified and developed, varying greatly from generation to generation and class to class in terms of synthesis, pharmacology, DMPK, safety, chemistry and size.²

The increasingly complex modifications and growing diversity — and their inability to be categorised as a small molecule or large biologic — present novel challenges for drug bioanalysis.⁶

New analytical solutions for a next-generation drug modality

This is why contract research organisations (CROs) such as QPS and advanced analytical instrument vendors such as SCIEX have had to develop better methods and tools to analyse oligo therapeutics — from discovery through development to final regulatory approval.

Multiple analytical platforms are employed to analyse oligo drugs; but, as with all new modalities, shared experience is limited regarding how best to analyse the different classes. Indeed, in our experience, different platforms are needed for different types of oligo, depending mainly on their size, chemistry and complexity.

The stability of the oligo can greatly affect its analytical parameters, as well as impacting efficacy and safety.

Because of the stability concern, it has been critical to understand their potential chemical degradants and biological metabolites.

Oligo metabolites usually arise from exonuclease activity and minor metabolites can be generated by the deamination of nucleic acids. Therefore, it is important to use a platform that can provide high analytical resolution.

Only a few methodologies are applicable for oligo bioanalysis, namely hybridisation enzyme-linked immunosorbent assay (hELISA), liquid chromatography (LC)-UV and hybridisation LC-fluorescence (hLC-FLD), all of which come with advantages and disadvantages.

The oldest and traditionally the most sensitive method is hELISA, which is able to detect concentrations in the picogram/mL range. But it either cannot separate the parent oligo from its metabolites or requires that individual hELISAs are developed for each particular metabolite.

To ensure we could sufficiently resolve parent oligos and major and minor metabolites, we experimented with different platforms in parallel and consequently decided on high resolution mass spectrometry (HRMS) using a SCIEX quadrupole time-of-flight (QTOF) 6600 instrument.

We mainly used HRMS to analyse siRNAs, but it is not the only platform we employ. We now recognise the advantages, limitations and suitability of various platforms to analyse different types of oligo drugs and apply platforms ranging from hELISA to liquid chromatography (LC)-UV in the analysis of small oligos such as ASOs, miRNAs and aptamers.

We also used the same platforms to analyse PEGylated oligos. For large oligos, we usually use quantitative polymerase chain reaction (qPCR) assays.

Oligonucleotides primed for rapid development

Another advantage of oligo drugs, especially in response to pandemic infectious diseases, are that they may now be quicker and cheaper to develop and more easily adapted for personalised medicine than small molecule and biologic drugs.

Although the efficacy and safety of small molecule drugs are nearly always affected by changes in their chemical structure, oligo drugs can be altered through their base nucleic acid sequence to hit different disease targets without impacting their biological mechanism of action.²

And whereas biologics require living organisms for their production, which can be challenging to control, oligo drugs are precisely synthesised in a relatively straightforward and easily controllable manner.

Moreover, because oligos work at the molecular level, viruses can be precision targeted as the viral DNA or RNA can be fully sequenced quickly and that sequence monitored for any mutations.

These advantages, combined with rapid advances in gene sequencing and our knowledge of molecular disease mechanisms, mean that oligo drugs have emerged as the next modality in antivirals and an ideal way to provide antiviral medicines quickly in response to new viral outbreaks.

For example, the HBV oligo antivirals under development all employ an RNAi mechanism of action to combat the disease, thus disrupting the viral hijack of the molecular machinery of the cell to replicate itself. This disruption could also reduce the magnitude of the human immune response required to clear the HBV from the body.

Therefore, oligo antivirals may well deliver a cure for hepatis B … even for chronic infections that are currently incurable.

Similarly, oligo antivirals may be developed for other diseases, such as COVID-19, especially as they may be faster to develop than small molecule drugs or biologics. Indeed, oligo companies developing HBV antivirals are also working on oligo antivirals against COVID-19.

Indeed, in less than a year, Alnylam and Vir already have a lead candidate called VIR-2703 (also known as ALN-COV) and Arrowhead Pharmaceuticals have one called ARO-COV, both acting through RNAi.^7,8

There are currently 316 antiviral and 234 vaccine candidates in development for COVID-19, and with clinical trials such as the multinational Solidarity Trial for COVID-19 treatments under way, it is hoped that we will soon have a medicine to fight this pandemic disease.^9–11

References

1. www.the-scientist.com/bio-business/oligonucleotide-therapeutics-near-approval-32446.
2. X. Lu and K. Zhang, “PEGylation of Therapeutic Oligonucleotides: From Linear to Highly Branched PEG Architectures,” Nano. Res. 11, 5519–5534 (2018).
3. R.L. Juliano, “The Delivery of Therapeutic Oligonucleotides,” Nucleic Acids Res. 44, 6518–6548 (2016).
4. T.C. Roberts, R. Langer and M.J.A. Wood, “Advances in Oligonucleotide Drug Delivery,” Nat. Rev. Drug Discov. 11, 1–22 (2020).
5. https://arrowheadpharma.com/pipeline.
6. www.pharmatimes.com/magazine/2019/june_2019/drugging_the_undruggables.
7. https://investors.alnylam.com/press-release?id=24796.
8. https://arrowheadpharma.com/pipeline.
9. https://vac-lshtm.shinyapps.io/ncov_vaccine_landscape.
10. https://airtable.com/shrSAi6t5WFwqo3GM/tblEzPQS5fnc0FHYR/viwJJuMA1ioIgAcPs?blocks=hide.
11. www.who.int/emergencies/diseases/novel-coronavirus-2019/global-research-on-novel-coronavirus-2019-ncov/solidarity-clinical-trial-for-covid-19-treatments.