Zamas Lam Senior Vice President of Preclinical Development, QPS
John Kolman Vice President of Translational Medicine, QPS
Todd Stawicki Global Marketing Manager for MS Biopharma, SCIEX, a Danaher Operating Company
As the world continues to adapt to the changes required to minimize the spread of COVID-19, scientists around the globe are working to develop both vaccines and antiviral drugs. With new strains of the SARS-CoV-2 virus emerging, it may only be a matter of time until vaccine-resistant strains surface. Therefore, it is likely that we will need both approaches to successfully combat this disease and overcome the pandemic. To that end, an exciting new type of antiviral is being developed using genetic medicines – a modality hailed as the third major drug development platform (after small molecules and biologics). This builds on the revival we have seen in recent years of oligonucleotide (oligo) drugs.
Although the oligo modality for gene therapy was first conceived of 30 years ago it is only now being hailed as the third major drug-development platform after small molecules and biologics.1,2 Despite several setbacks, oligo-based medicines have finally delivered on their promise; providing novel disease-modifying treatments to patients with otherwise undruggable diseases.3 Improved chemistries, a better understanding of the basic biology of oligonucleotides, and more sophisticated delivery systems have led to the development of ten approved oligo drugs to date.1,3
Oligonucleotide therapeutics generally are modified short single-stranded or double-stranded DNA or RNA sequences, typically comprised of around 15–30 nucleotides. The first class of oligos developed were the antisense oligonucleotides (ASO), then aptamers, then small interfering RNA (siRNA), with microRNA (miRNA), deoxyribozymes (DNAzymes), and short DNA/RNA heteroduplex oligonucleotides (HDOs) distributed in the whole discovery and development spectrum. Each class has different modes of action but the first step for all these oligos is the recognition and binding of specific endogenous homologous or complementary sequences in the target DNA, messenger RNA (mRNA), other RNA, or protein.
ASOs are single-stranded oligos that induce blockage, cleavage or degradation of the target mRNA. The first ASO drug, Vitravene (fomivirsen), was approved in 1998 and also happens to be an antiviral. Vitravene works by binding to a specific sequence of cytomegalovirus (CMV) mRNA and stopping its translation to the protein that leads to cytomegalovirus retinitis. One of the more recently approved but controversial ASOs is Exondys 51 (eteplirsen). This specific ASO works by exon-skipping and specifically targets exon 51 of the mutated Duchene muscular dystrophy (DMD) gene. By ‘skipping’ exon 51, the downstream reading frame is shifted, which allows the production of a modified but still functional dystrophin protein. As there are many DMD gene mutations, Exondys 51 only works for patients who have a confirmed mutation amenable to the skipping of exon 51. Since 1998, eleven ASOs have been approved in the past 21 years.
Aptamers are single-stranded oligos or peptides with a threedimensional structure that non-covalently bind to specific targets, which can be small molecules, peptides or proteins. Macugen (pegaptanib), the only approved aptamer drug, was approved in 2004. Macugen is a DNA oligo modified with a long polyethylene glycol chain (PEGylation). It binds to and acts as a vascular endothelial growth factor (VEGF) antagonist for the treatment of wet aged-related macular degeneration (AMD).
Small interfering RNA was first described in 1998 by Fire and Mello, and they were awarded the Nobel Prize for Medicine a scant eight years later in 2006 – one of the fastest records for recognition by the Nobel committee.4 siRNA are double-stranded oligos that bind to and activate the RNA-induced silencing complex (RISC). The two strands in the RISC are decoupled, with the sense strand (or passenger strand) discarded while the RISC-associated antisense strand (or guide strand) is retained, so that it recognizes a specific target mRNA sequence and silences that specific gene. The first siRNA drug, Onpattro (patisiran), was approved in 2018. Onpattro works by silencing the transthyretin (TTR) gene mutation that causes the production of misfolded TTR protein monomers. These monomers aggregate and form amyloid structures, such as fibrils, which lead to polyneuropathy. The second siRNA drug, Givlaari (givosiran), was approved in 2019, for the treatment of acute hepatic porphyria. Applications for marketing approval have also been submitted to FDA last year for two additional siRNA drugs. This potentially brings the count of approved siRNA drugs up to four – an incredible achievement in the 22 years since the discovery of this class of biologic.
The current fifteen approved oligo drugs have all been used to treat orphan indications and rare diseases. However, concerted effort has been made to migrate over to the ‘bigger’ indications that transform oligos from a curiosity to a major drug modality. Alnylam’s siRNA, Leqvio (inclisiran), which targets proprotein convertase subtilisin/ kexin type 9 (PCSK9) for the treatment of hypercholesterolemia has been hailed as the first oligo drug to have ‘large’ patient trials.
Both ASOs and siRNAs are now being developed to combat infectious diseases, such as hepatitis B – caused by the hepatitis B virus (HBV). HBV is a highly ‘successful’ virus as it causes what is known as a ‘silent’ disease. It is estimated that 2 billion people worldwide have been infected with HBV, which is a quarter of the world population. Each minute, two people die from HBV and related complications.5 Although HBV vaccines are available, the annual infection rate is 30 million people, an infected pregnant mother can pass on HBV to the baby during delivery. HBV is a highly successful virus, HBV is highly species-specific and tissue-specific. Humans are the only known natural hosts and hepatocytes, which is the main cell in the liver, is the only targeted cells. HBV infect hepatocytes and hijack their molecular machinery to replicate. Although HBV kills the host hepatocytes during replication, the liver is a very robust organ which can retain its function with only 20% of viable cells. However, during infection a substantial amount of foreign viral proteins are synthesized, which can trigger all sorts of cellular response and immune response with immediate and long-term implications. In simple terms, this can cause chronic inflammation, the potential of developing hepatic cancer, and other life-threatening diseases. In a way, this multifaceted but unclear viral pathogenesis can be comparable to the more immediate “cytokine storm” disease mechanism of COVID-19 caused by the SARS-CoV-2 virus.6
New Drug Modalities Bring New Mechanisms of Action and Analytical Challenges
Oligonucleotide companies, such as Arbutus, Dicerna, Ionis, Janssen, and Vir are developing ASO and siRNA antivirals for HBV. A potential ASO antiviral is Ionis’ IONIS-HBVRx and potential siRNA antivirals include Arbutus’ AB-729, Dicerna’s RG6346 (formerly DCR-HBVS), Janssen’s JNJ-3989 (formerly ARO-HBV), Vir and Alnylam’s VIR-2218 (also known as AL-HBV02), all of which employ a mechanism that interferes at the molecular level of RNA to disrupt the expression of HBV proteins and HBV surface antigens. By knocking down the genes in the most conservative region of the viral genomic, and the subsequent viral surface antigen, it is postulated that the host immune cells will be able to function properly and target HBV for removal. These experimental RNA interference (RNAi) oligo drugs are all being evaluated in clinical trial programs. A key part of such evaluations, as well as of their requisite preclinical studies and of all stages of development and production, is the bioanalysis of drug exposure relating to its biological activity (dose-response). This includes performing toxicology studies and establishing drug metabolism and pharmacokinetics (DMPK) profiles for each experimental drug.
Although bioanalysis is standard within drug development, the novelty of the oligo modality presents new analytical challenges. Early hurdles in the development of oligo drugs included their inability to enter cells because of their chemical properties – namely their hydrophilicity and high molecular weights. Naked and unmodified oligos were also easily degraded by exonuclease and endonuclease enzymes in the body and could be rapidly cleared and excreted by the liver and kidneys. Therefore, direct modification of the oligos was needed, as well as advanced delivery systems.7 The modality now encompasses a growing variety of classes, with different sizes, chemistries, levels of complexity, and delivery. For example, most people now recognize the current fourth-generation ASO is very different from the first generation ASO 30 years ago in the context of synthesis, pharmacology, DMPK, and safety, and thus cannot be grouped together as another ASO. With each generation, ASOs become more stable to the nuclease enzymes and have a better safety profile and are less toxic. In a way, chemical modification, molecular targeting, and drug delivery run in parallel to the indication. For example, Vitravene was administrated via intravitreal injection which circumvented stability and potential off-target toxicity, while Leqvio will be a ‘normal’ pill projected to be taken every three months. The increasing complexity and diversity of oligo drugs, and their lack of a ready categorization as a small molecule or large biotherapeutics, means that contract research organizations (CROs) and advanced analytical instrument vendors have had to develop better methods and tools for analyzing this new modality from discovery through development to final approval.
Advanced Analytical Solutions to Support New Drug Development
Multiple analytical platforms are currently being used to analyze oligo drugs but as with all new modalities, there is limited shared experience regarding how best to analyze the different classes. Indeed, in our experience, different platforms are needed for different types of oligo, depending on their level of complexity, chemistry and most importantly, their size. With the stability concern, it is important to understand potential chemical degradants and biological metabolites. With the new generation of oligo chemistry, oligo metabolites are usually formed from exonucleases, give N 1, N 2, N 3, … metabolites, where N is the original length of the oligo, and the 1 or 2 or 3, is the individual nucleotides. Minor metabolites form from deamination of the nucleic acids. Therefore, it is important to use a platform that is able to provide high analytical resolution.
Due to previous analytical instrumentation limitations, and the belief that oligo metabolites either have similar pharmacologic activity as their parent drug or are rapidly degraded by nucleases, in vivo metabolism and potential toxicity has been regarded as less of a concern. However, with the newer generation of oligos, the metabolites are more stable and therefore there is a renewed focus on understanding the metabolism of the oligo, as well as the exposure to it, early on in the development process.
There are only a few methodologies for the bioanalysis of oligos, such as hybridization enzyme-linked immunosorbent assay (hELISA), liquid chromatography (LC)-UV, hybridization LCfluorescence (hLC-FLD), all of which are attendant with advantages and disadvantages. The oldest and traditionally the most sensitive method is hELISA. Hybridization ELISA has sensitivity down to the picogram/mL, but either cannot separate the parent oligo from the oligo metabolites or requires that individual hELIZAs are developed for each individual metabolite.
To ensure we were able to resolve the parent oligo, main metabolites and other minor metabolites with sufficient sensitivity and precision, we experimented with different platforms in parallel and consequently decided on high-resolution mass spectrometry (HRMS). This is not the only platform we employ for siRNA analysis, as we now recognize the advantages, limitations and suitability of various platforms for analyzing different types of oligo drugs. As such, we apply platforms ranging from hELISA to liquid chromatography (LC)-UV in the analysis of small oligos such as ASOs, miRNAs, and aptamers. We also use the same platforms to analyze PEGylated oligos. For large oligos, we usually use quantitative polymerase chain reaction (qPCR) assays.
Oligonucleotides Are Good Candidates for Fast-Development Antivirals
Oligonucleotide drugs have several key advantages over small molecule and biologic drugs, in that they may now be quicker and cheaper to develop and more easily adapted for personalized medicine. Unlike small-molecule drugs, in which a change in chemical structure almost certainly changes the drug’s pharmacokinetics, oligo drugs can be altered through their base nucleic acid sequence to hit different disease targets without impacting their biological mechanism of action.7 Unlike biologics that are generated using living organisms, which can be challenging to control, oligo drugs are specifically synthesized in a relatively straightforward and easily controllable manner. Moreover, because oligos work at the molecular level, the virus can be targeted precisely 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 in our understanding of disease mechanisms at the molecular level, oligo drugs have emerged as the next modality in antivirals, ideal for providing antiviral medicines quickly in response to new viral outbreaks.
The oligo HBV antivirals being developed work through RNAi to disrupt the disease mechanism. The viral hijack of the molecular machinery of the cell could be counteracted by RNAi to prevent the virus from replicating, and thus reduce the magnitude of the human immune response required to clear the virus from the body. In this way, oligo antivirals may deliver a cure for chronic hepatitis B, a currently incurable disease. Similarly, oligo antivirals may be developed for other diseases, like COVID-19, especially as they may be faster to develop than small molecule drugs or biologics. Indeed, in less than a year, Alnylam and Vir have already developed a candidate RNAi COVID-19 antiviral, known as both VIR-2703 and ALN-COV.8 Arrowhead Pharmaceuticals are also developing an RNAi-based antiviral called ARO-COV.9 It is likely that we will need both antivirals and vaccines to tackle COVID-19 effectively. With 234 vaccine and 316 antiviral candidates in development for COVID-19, and clinical trials such as the multinational Solidarity trial for COVID-19 treatments underway, it is hoped that we will soon have a medicine for fighting this pandemic disease.10–12
References
- Juliano RL. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 2016; 44: 6518–48. doi: 10.1093/nar/gkw236.
- Offord, C. Oligonucleotide Therapeutics Near Approval. The Scientist. Nov 30, 2016. Accessed Aug 2020. https://www.the-scientist.com/bio-business/oligonucleotidetherapeutics-near-approval-32446.
- Roberts TC, Langer R, Wood MJA. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov. 2020; 11: 1–22. doi: 10.1038/s41573-020-0075-7.
- Hopkin M. RNAi Scoops Medical Nobel – Gene Silencers Get Something to Shout About. Nature. News Oct 2, 2006. Accessed Sep 2020. https://www.nature.com/ news/2006/061002/full/061002-2.html.
- Hepatitis B Foundation. Hepatitis B Facts and Figures. Accessed Sep 2020. https://www. hepb.org/what-is-hepatitis-b/what-is-hepb/facts-and-figures/.
- Costela-Ruiz VJ, Illescas-Montes R, Puerta-Puerta JM, Ruiz C, Melguizo-Rodríguez L. SARSCoV-2 infection: The role of cytokines in COVID-19 disease. Cytokine Growth Factor Rev. 2020; 54; 62–75. doi: 10.1016/j.cytogfr.2020.06.001.
- Lu X, Zhang K. PEGylation of therapeutic oligonucletides: From linear to highly branched PEG architectures. Nano Res. 2018; 11: 5519–34. doi: 10.1007/s12274-018-2131-8.
- Alnylam Pharmaceuticals. Vir and Alnylam Identify RNAi Therapeutic Development Candidate, VIR-2703 (ALN-COV), Targeting SARS-CoV-2 for the Treatment of COVID-19. Press release: May 04, 2020. Accessed Aug 2020. https://investors.alnylam.com/pressrelease?id=24796.
- Arrowhead Pharmaceuticals. Pipeline. Accessed Sep 2020). https://arrowheadpharma.com/ pipeline/.
- Vaccine Centre, London School of Hygiene and Tropical Medicine. COVID-19 vaccine tracker. Accessed Aug 2020. https://vac-lshtm.shinyapps.io/ncov_vaccine_landscape/.
- Milken Institute. COVID-19 Tracker. Accessed Aug 2020. https://airtable.com/ shrSAi6t5WFwqo3GM/tblEzPQS5fnc0FHYR/viwJJuMA1ioIgAcPs?blocks=hide.
- World Health Organization (WHO). “Solidarity” clinical trial for COVID-19 treatments. Accessed Aug 2020. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/globalresearch-on-novel-coronavirus-2019-ncov/solidarity-clinical-trial-for-covid-19-treatments.
Zamas Lam, PhD, holds the position of Senior Vice President and Global Head of Bioanalytical for QPS (DE, USA), a global contract research organization providing discovery, preclinical and clinical drug development services since 1995. Lam is one of the world’s few high-resolution mass spectrometrists by training and by trade, with a passion for biologics mass spectrometry and gene therapy. Within QPS, he is an integral part of the preclinical and bioanalysis team supporting a first in the industry – the world’s largest gene therapy trial with patients numbering more than a million. Lam is a regularly published thought leader, with his expert insights appearing in publications such as Bioanalysis, Anal Chem, J Biol Chem, Biochemistry and Carbohydrate Research.
John Kolman, PhD, holds the position of Vice President and Global Head of Translational Medicine for QPS (DE, USA), a global contract research organization providing discovery, preclinical and clinical drug development services since 1995. Over 20 years in the pharmaceutical R&D industry, having held multiple leadership roles. He is also well known throughout the industry for his scientific leadership, as evidenced by recent speaking engagements at fora such as the World PGx (Pharmacogenomics) Summit (San Francisco), the International Conference on Biomarkers and Clinical Research (Philadelphia), and the PDA/ FDA (Parenteral Drug Association/Food and Drug Administration) Advanced Technologies for Virus Detection in Biologicals Conference (Bethesda). John received his Ph.D. in Molecular Biophysics and Biochemistry from Yale University and his published works have appeared in such noteworthy periodicals as Science and Biologicals, and are pending with Applied Microbiology and Biotechnology, the Journal of Pharmaceutical Science and Technology, BioProcessing Journal, and in the PDA Press. His experience in biomarkers, biologics, pharmacogenomics, genetics, and bioinformatics has earned him recognition as an expert in these fields.
Todd Stawicki is a Global Marketing Manager for MS Biopharma at SCIEX, a Danaher operating company and a global leader in the accurate and precise quantification of molecules. He has over 15 years of experience as a research scientist at several pharmaceutical and biopharmaceutical companies and as an LC-MS applications scientist at SCIEX. In his role as a marketing manager, he is passionate about empowering customers with the most innovative and effective LC-MS and CE-MS methods available to achieve their scientific goals.
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