Can antiviral oligonucleotides better prepare us for the next pandemic?

Todd Stawicki, Global Marketing Manager for MS Biopharma at SCIEX, offers an in-depth analysis of oligonucleotides and their role in preparing for the next pandemic

Covid-19, the disease caused by SARS-CoV-2, is arguably the greatest public health threat in a generation. It has temporarily paralysed our just-in-time economy^1,2 and highlighted many of the fragilities in our social support mechanisms^1,3. As of this writing, more than 169 million people have been infected with the virus worldwide^4,5, with approximately 3.5 million ensuing deaths^4,5 globally. Even without having to personally experience the symptoms of the virus, Covid-19 has left an imprint on nearly every person on the planet. It will be a milestone event for every living person, and people will think about where they were “before Covid” and “after Covid”. Fortunately, we are on the cusp of globally reducing deaths and disease transmission with the accelerating use of several different and highly effective vaccines against SARS-CoV-2. While we cannot consider Covid-19 to be “in the rear view mirror”, we can start thinking about how we better prepare for the next potential global respiratory pandemic.

Pathology in the lungs

Covid-19 is considered first, and foremost, an infection of the lungs. The virus is spread from hosts to targets through oral or nasal aerosols, and there is now some evidence of airborne transmission⁶. The SARS-CoV-2 virus attaches with high affinity to the angiotensin converting enzyme 2 (ACE2) receptor in nasopharyngeal mucosa and alveolae⁷. Covid-19 progresses in three phases: 1) asymptomatic, 2) upper airway, and 3) acute respiratory disease syndrome (ARDS)⁸. Patients can be walking around yet be highly infectious as the virus rapidly replicates and infiltrates deeper into the pulmonary system. Clinical pulmonary symptoms can start to manifest in early infection, and progress to major signs and symptoms that are the result of a hyperinflammatory response that affects multiple organ systems⁹. This can manifest as severe hypoxia, pneumonia and fibrosis, resulting from the programmed cell death (apoptosis) of numerous alveolar cells due to a severe host immunologic (inflammatory) response to high loads of viral protein – the so-called cytokine storm¹⁰.

Emergence SARS viruses over time

SARS-CoV2 is the seventh member of the coronavirus subfamily to achieve transmission in humans^11,12. Of these, only three manifested as severe acute respiratory syndromes: SARS-CoV in 2002, MERS (Middle East Respiratory Syndrome) in 2012 and lastly SARS-CoV2 in 2019¹¹. These three SARS-related viruses belong to the betacoronavirus group or “genus”, with SARS-CoV and SARS-CoV-2 being the most closely related in the sarbecovirus lineage or “subgenus” (Lineage B) while MERS is of the merbecovirus subgenus (Lineage C). These SARS-type viruses shows varying levels of infectivity (R0 or “R-naught”) and mortality (see Table 1). While they do vary, one thing is clear, they are all quite high in infectivity and mortality. For this entire subfamily of viruses, there are large zoonotic reservoirs, including a wide variety of mammals such as rodents, bats, civets and pangolins among others^13,14. It is unlikely that SARS-CoV-2 will be the last SARS coronavirus to propagate in humans. Furthermore, even with mass vaccination programs, it is likely that SARS-CoV-2 will persist as a regularly found or “endemic” virus worldwide as there are likely to now be significant human reservoirs of the disease spread across the globe.

Limitations of existing therapies

Due to the efforts of researchers and physicians all over the world, several types of genetic vaccines have received emergency use authorisation (EUA) in multiple countries. To date, 1.5 billion doses of vaccine have been administered worldwide⁴, constituting 10% of the population^21,22. A variety of different vaccines have demonstrated high efficacy against SARS-CoV-2^23,27. While this is a tremendous development, vaccination is a completely prophylactic solution – it has no efficacy for people already infected or symptomatic. Several antibody cocktails against SARS-CoV-2 have also received EUA^28,29. Unfortunately, these monoclonal antibody cocktail therapies are currently not approved for the treatment of people who have already been hospitalised or require oxygen therapy because of Covid-19^28,29,30,31. So again, these treatments will not help those who have progressed to the severe stages of Covid-19. However, there are several small molecule antivirals in various stages of preclinical and clinical development, which are aimed at treating people who have already contracted Covid-19. Some of these include remdesivir, favipiravir, umifenovir, lopinavir, penciclovir, among others.

To date, these small molecule antiviral therapies have shown little or no efficacy in treating acute cases of Covid-19. Even remdesivir, which the FDA approved for use in hospitalised Covid-19 patients in 2020³², subsequently received a recommendation against its use by the World Health Organization only a few months later³³. The failure of these different strategies to adequately address more advanced cases of Covid-19 highlights the significant unmet medical need in treating acute cases of Covid.

Why oligos?

Part of the problem with small molecule antivirals is that there is not enough of a gap in mechanisms of action between the host and virus targets. Replication and transcriptional protein structures are some of the most conserved across all forms of life – which makes selective targeting with small molecules very difficult. For instance, remdesivir and other nucleoside analogs target RNA-dependent RNA polymerase (RdRp)³⁴, but have shown limited clinical efficacy and can have complicating side effects³⁵. Furthermore, viruses hijack native host cellular machinery to reproduce their viral genome and enzymatic and structural proteins. Targeting only the enzymes of infected host cells while excluding healthy cells could provide almost impossible for small molecules. Oligonucleotides represent a wholly different approach to antiviral treatment. Therapeutic oligonucleotides are fully synthetically produced short sequences of modified nucleotides. They can be either single-stranded antisense oligonucleotides (ASOs) or double-stranded small interfering RNAs (siRNAs). They can range anywhere from eight bases up to over 50 bases for a duplex siRNA. Depending on the type of therapeutic oligonucleotide, they have the ability to edit pre-mRNAs (pre-messenger RNAs) for alternative splice variants or destroy RNA (usually mRNA) via either an RNase H or RISC-dependent mechanism³⁶. What is critically important about any type of therapeutic oligonucleotide is that they have the ability to modulate diseases at the mRNA level. Where small molecules or protein therapeutics target at the protein level, oligos target at the genetic level. As oligonucleotides target RNAs for destruction, they represent the possibility to target RNA viruses like the SARS family at multiple levels. They could directly destroy viral genome RNA, as well as viral mRNAs that get expressed.

Genetic druggability of the SAR family

A recent publication found significant commonality in much of the sequence of many of the SARS coronaviruses³⁷. In an examination of 109 different genomes of the coronavirus subfamily, they found “moderate levels of protein sequence identity across all genera and included nsp3-10, nsp12-16 (RNA-dependent RNA polymerase, helicase, 3’-to-5’exonuclease, endoribonuclease, and 2’-O-ribose methyltransferase), and the structural proteins spike (S), membrane (M), and nucleocapsid (N)”³⁷. They also found regions of high sequence identity that are specific for members of the betacoronavirus Lineage B, of which SARS-CoV-2 is a member. Notable among these are the envelope protein and NSP10 (a putative replication cofactor). There are therefore many validated and novel genetic targets that could be broadly applicable as targets for therapeutic oligonucleotides against either the entire coronavirus family or the specific lineage of SARS-CoV-2.

Host targets

In addition to viral targets, there is the possibility to use therapeutic oligonucleotides to selectively modulate maladaptive host responses in Covid-19. Much of the mortality from late stage Covid-19 cases come from the cytokine storm response to viral proteins. Oligonucleotide cocktails could temporarily down-regulate pro-inflammatory cytokines such as interleukin-6 or interferon. Blocking certain host cell proteins and receptors could also slow Covid-19 progression. SARS-CoV-2 binds with high affinity to ACE2 receptors and its entry into the cell is facilitated by a transmembrane serine protease (TMPRSS2)^14,38. Blocking the expression of one, or both, of these proteins could reduce the viral spread³⁸.

Ability to target lungs

An obvious complication with very large molecules, such as therapeutic oligonucleotides, has been achieving systemic administration. While systemic administration of an antiviral would certainly be advantageous, the critical site of infection and disease progression of SARS viruses occurs in the air passages and lungs. Fortunately, efficacious administration with oligos has already been clinically illustrated with the ASO, eluforsen³⁹. Eluforsen was an investigational 35-mer oligonucleotide therapeutic in development by ProQR Therapeutics. Eluforsen was formulated to be delivered via nebulisation to lungs. Preclinical mouse studies demonstrated successful delivery of nebulised delivery to alveolar cells³⁹. A phase Ib clinical trial of cystic fibrosis patients with an F508del homozygous mutation, demonstrated the ability to deliver eluforsen to the lungs to achieve “clinically relevant improvements in respiratory symptoms”⁴⁰. Therefore, it is feasible that therapeutic oligonucleotides can be delivered to the site of SARS pathology, the lungs⁴¹.

Antiviral oligos in development

The table below illustrates the status of some of the antiviral oligonucleotides in development.

To date, there are a limited number of antiviral therapeutic oligonucleotides in development and even less so, specifically for Covid-19. Program terminations suggest difficulties in the development of this modality. This is not the first setback for therapeutic oligos, however. Oligonucleotides were first investigated clinically by the University of Nebraska Medical Center with an antisense oligonucleotide directed against p53 in the early 1990s⁴². Ironically enough, the first FDA-approved therapeutic oligonucleotide was an antiviral (fomiversen) against cytomegalovirus retinitis (CMV) in 1998⁴³. Then ensued a 15-year dry spell for the therapeutic modality and the next oligonucleotide therapeutic was not approved until 2013 with mipomersen for the treatment of homozygous familial hypercholesterolemia⁴⁴. The first siRNA drug was not approved until 2018 with patisiran for polyneuropathy of hereditary transthyretin amyloidosis (hATTR)⁴⁵. Now, there are 14 oligonucleotide-based drugs approved in the US^46-48 and several hundred more in clinical and preclinical development.

During this massive dry spell, much of the stability and delivery issues were worked out with new generations of oligonucleotide chemistry. The in vivo nuclease resistance of oligonucleotides has been dramatically improved with the use of many types of chemical modifications including 2’-ribose modifications including the additions of fluoro, O-methyl or methoxyethyl groups. The phosphodiester backbone has been replaced with phosphorothioate. Furthermore, novel types of backbone chemistries are being leveraged including locked nucleic acids (LNAs), morpholino residues or peptide backbones^49,50. Formulation has been dramatically improved with greater sophistication in the design and production of lipid nanoparticles. In addition, many siRNA and ASO molecules have been developed with the addition of covalent moieties to target specific tissues. For instance, patisiran from Alnylam has a triantennary n-acetylgalactosamine (GalNAc) group covalently linked to the 3’ end of the sense strand. The GalNAc group binds with high affinity to the asialoglycoprotein receptor (ASGPR) which is expressed at high levels on hepatocytes, thus ensuring effective delivery to the liver⁵¹. The significant improvements in the stability, formulation and targeting chemistry of oligonucleotides combined with their intrinsic ability to target with genetic specificity make it likely they can overcome setbacks as an antiviral modality.

These therapies will need to be flexible enough to treat a variety of coronaviruses while remaining highly selective in targeting the virus and not the host. Furthermore, these need to be developed quickly, requiring judicious decisions as early as possible in the drug development program. A key to faster and more confident decision-making throughout the drug development process is analytics. Insights obtained from mass spectrometry and capillary electrophoresis analyses can greatly inform critical decisions, from the development of manufacturing processes through to the analysis of biological samples from preclinical and clinical studies. To ensure that biopharma scientists have the most advanced and precise tools available, SCIEX works closely with customers and collaborators to develop innovative solutions that enable better and faster analytics at all stages of therapeutic oligonucleotide discovery and development.

There is clearly still an unmet need for effective treatment of patients with severe Covid-19. This need likely extends to future patients with acute respiratory disease arising from new SARS coronaviruses (or potential SARS-CoV-2 mutants), especially as new vaccines may be needed. We have made incredible strides incredibly quickly with many new types of genetic vaccines and passive immunity therapies. It is now time to complete our protection with the successful development of antiviral oligonucleotides.

About the author

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.

References

1. United Nations Conference on Trade and Development. Impact of the COVID-19 pandemic on trade and development: transitioning to a new normal. UNCTAD. Accessed May 27, 2021. https://unctad.org/webflyer/impact-covid-19-pandemic-trade-and-development-transitioning-new-normal.
2. Rynne B, Reader G, Heading S. COVID-19 and the global economy. With uncertainty comes either paralysis or opportunity. KPMG. Accessed May 27, 2021. https://home.kpmg/xx/en/home/insights/2020/06/covid-19-and-the-global-economy.html
3. Organisation for Economic Co-operation and Development. OECD Policy Responses to Coronavirus (COVID-19). COVID-19: Protecting people and societies. Version 31 March 2020. OECD Accessed May 27, 2021. https://www.oecd.org/coronavirus/policy-responses/covid-19-protecting-people-and-societies-e5c9de1a/
4. World Health Organization. WHO Coronavirus (COVID-19) Dashboard. WHO. Accessed May 27, 2021. https://covid19.who.int/.
5. Coronavirus. COVID-19 coronavirus pandemic. Worldometer. Accessed May 27, 2021. https://www.worldometers.info/coronavirus/?utm_campaign=homeAdUOA?Si.
6. Greenhalgh T, Jiminez JL, Prather KA, Tufekci Z, Fisman D, Schooley R. Ten scientific reasons in support of airborne transmission of SARS-CoV-2. Lancet. 2021; 397: 1603 – 1605. DOI: 10.1016/S0140-6736(21)00869-2.
7. Matheson NJ, Lehner PJ. How does SARS-CoV-2 cause COVID-19? Science. 2020; 369: 510–511. DOI: 10.1126/science.abc6156.
8. Mason RJ. Pathogenesis of COVID-19 from a cell biology perspective. Eur Respir J. 2020; 55: 2000607. DOI: 10.1183/13993003.00607-2020.
9. Siddiqi HK, Mehra MR. COVID-19 illness in native and immunosuppressed states: A clinical-therapeutic staging proposal. J Heart Lung Transplant. 2020; 39: 405–407. DOI: 10.1016/j.healun.2020.03.012.
10. Shimizu M. Clinical features of cytokine storm syndrome. In: Cron RQ, Behrens EM, eds. Cytokine Storm Syndrome. Springer; 2019: 31 – 41. Accessed May 27, 2021. https://link.springer.com/book/10.1007/978-3-030-22094-5.
11. US Centers for Disease Control and Preventions. COVID-19. Human coronavirus types. CDC. Accessed May 27, 2021. https://www.cdc.gov/coronavirus/types.html.
12. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol. 2020; 5 :536–544. DOI: 1038/s41564-020-0695-z.
13. da Costa VG, Moreli ML, Saivish MV. The emergence of SARS, MERS and novel SARS-2 coronaviruses in the 21st century. Arch 2020; 165: 1517–1526. DOI: 10.1007/s00705-020-04628-0.
14. Rabi FA, Al Zoubi MS, Kasasbeh GA, Salameh DM, Al-Nasser AD. SARS-CoV-2 and coronavirus disease 2019: What we know so far. Pathogens. 2020; 9: 231. DOI: 10.3390/pathogens9030231.
15. Liu J, Wanli Xie W, WangY, et al. A comparative overview of COVID-19, MERS and SARS: Review article. Int J Surg. 2020; 81: 1–8. DOI: 10.1016/j.ijsu.2020.07.032.
16. Lu L, Zhong W, Bian Z, et al. A comparison of mortality-related risk factors of COVID-19, SARS, and MERS: A systematic review and meta-analysis. J Infect. 2020; 81: e18–e25. DOI: 10.1016/j.jinf.2020.07.002.
17. Chen Z, Boon SS, Wang MH, Chan RWY, Chan PKS. Genomic and evolutionary comparison between SARS-CoV-2 and other human coronaviruses. J Virol Methods. 2021; 289: 114032. DOI: 10.1016/j.jviromet.2020.114032.
18. US Centers for Disease Control and Preventions. About SARS. SARS Basics Fact Sheet. CDC. Accessed May 27, 2021. https://www.cdc.gov/sars/about/fs-sars.html.
19. World Health Organization – Regional Office for the Eastern Mediterranean. Middle East respiratory syndrome. MERS situation update, December 2020. WHO. Accessed May 27, 2021. http://www.emro.who.int/health-topics/mers-cov/mers-outbreaks.html.
20. The New York Times. Coronavirus World Map: Tracking the Global Outbreak. The New York Times. Updated May 27, 2021. Accessed May 27, 2021. https://www.nytimes.com/interactive/2021/world/covid-cases.html.
21. Our World in Data. Statistics and Research. Coronavirus (COVID-19) vaccinations. Oxford Martin School, University of Oxford. Accessed May 27, 2021. https://ourworldindata.org/covid-vaccinations.
22. Mathieu, E., Ritchie, H., Ortiz-Ospina, E. et al. A global database of COVID-19 vaccinations. Nat Hum Behav. 2021. DOI: 10.1038/s41562-021-01122-8.
23. World Health Organization – Africa. What is COVID-19 vaccine efficacy? WHO. February 26, 2021. Accessed May 27, 2021. https://www.afro.who.int/news/what-covid-19-vaccine-efficacy.
24. Baden LR, El Sahly HM, Essink B, et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med. 2021; 384: 403–416. DOI: 10.1056/NEJMoa2035389.
25. Polack FP, Thomas SJ, Kitchin N, et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med. 2020; 383: 2603–2615. DOI: 10.1056/NEJMoa2034577.
26. Voysey M, Clemens SA, Madhi SA, et al. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet. 2021; 397: 99–111. DOI: 10.1016/S0140-6736(20)32661-1.
27. Sadoff J, Gray G, Vandebosch A, et al. Safety and efficacy of single-dose Ad26.COV2.S vaccine against Covid-19. N Engl J Med. 2021. DOI: 10.1056/NEJMoa2101544.
28. US Food and Drug Administration. Coronavirus (COVID-19) Update: FDA Authorizes Monoclonal Antibodies for Treatment of COVID-19. FDA News Release. February 9, 2021. Accessed May 27, 2021. https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-monoclonal-antibodies-treatment-covid-19-0.
29. US Food and Drug Administration. Coronavirus (COVID-19) Update: FDA Authorizes Monoclonal Antibodies for Treatment of COVID-19. FDA News Release. November 21, 2020. Accessed May 27, 2021. https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-monoclonal-antibodies-treatment-covid-19.
30. Gottlieb RL, Nirula A, Chen P, et al. Effect of bamlanivimab as monotherapy or in combination with etesevimab on viral load in patients with mild to mderate COVID-19: A randomized clinical trial. JAMA. 2021; 325: 632–644. DOI: 10.1001/jama.2021.0202.
31. Parkins K. Regeneron’s antibody cocktail helps prevent and treat Covid-19in Phase III studies. Clinical Trials Arena. News. April 13, 2021. Accessed May 27, 2021. https://www.clinicaltrialsarena.com/news/regenerons-antibody-cocktail-regen-cov-helps-prevent-and-treat-covid-19-in-phase-3-studies/.
32. US Food and Drug Administration. FDA approves first treatment for COVID-19. FDA News Release. October 22, 2020. Accessed May 27, 2021. https://www.fda.gov/news-events/press-announcements/fda-approves-first-treatment-covid-19.
33. World Health Organization. WHO recommends against the use of remdesivir in COVID-19 patients. WHO Newsroom Feature Stories. November 20, 2020. Accessed May 27, 2021. https://www.who.int/news-room/feature-stories/detail/who-recommends-against-the-use-of-remdesivir-in-covid-19-patients.
34. Dyer O. Covid-19: Remdesivir has little or no impact on survival, WHO trial shows. BMJ 2020; 371: m4057. DOI: https://doi.org/10.1136/bmj.m4057.
35. Jiang Y, Yin W, Xu HE. RNA-dependent RNA polymerase: Structure, mechanism, and drug discovery for COVID-19. Biochem Biophys Res Commun. 2021; 538: 47–53. DOI: 10.1016/j.bbrc.2020.08.116.
36. Fusetti F. Bioanalysis of therapeutic oligonucleotides by HRMS. Technology Networks. Webinar. March 16, 2021. Accessed May 27, 2021. https://www.technologynetworks.com/biopharma/webinars/bioanalysis-of-therapeutic-oligonucleotides-by-hrms-345118.
37. Chan AP, Choi Y, Schork NJ. Genomic and evolutionary comparison between SARS-CoV-2 and other human coronaviruses. Version 1. bioRxiv. 2020 Jul 6. DOI: 10.1101/2020.07.06.190207.
38. Uludağ H, Parent K, Aliabadi HM, Haddadi A. Prospects for RNAi therapy of COVID-19. Front Bioeng Biotechnol. 2020; 8: 916. DOI: 10.3389/fbioe.2020.00916.
39. Beumer W, Swildens J, Leal T, et al. Evaluation of eluforsen, a novel RNA oligonucleotide for restoration of CFTR function in in vitro and murine models of p.Phe508del cystic fibrosis. PLoS One. 2019; 14: e0219182. DOI: 10.1371/journal.pone.0219182.
40. Drevinek P, Pressler T, Cipolli M, et al. Antisense oligonucleotide eluforsen is safe and improves respiratory symptoms in F508DEL cystic fibrosis. J Cyst Fibros. 2020; 19: 99–107. DOI: 10.1016/j.jcf.2019.05.014.
41. Verma NV, Fazil MHUT, Duggan SP, Kelleher D. Combination Therapy Using Inhalable GapmeR and Recombinant ACE2 for COVID-19. Front Mol Biosci. 2020; 7: 197. DOI: 10.3389/fmolb.2020.00197.
42. Bayever E, Iversen PL, Bishop MR, et al. Systemic administration of a phosphorothioate oligonucleotide with a sequence complementary to p53 for acute myelogenous leukemia and myelodysplastic syndrome: initial results of a phase I trial. Antisense Res Dev. Winter 1993; 3: 383–390. DOI: 10.1089/ard.1993.3.383.
43. US Food and Drug Administration. Approval letter – reference: NDA 20-961. August 26, 1998. Accessed May 27, 2021. https://www.accessdata.fda.gov/drugsatfda_docs/nda/98/20961_Vitravene_Approv.pdf.
44. US Food and Drug Administration. Approval package for application number: 203568Orig1s000. January 29, 2013. Accessed May 27, 2021. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2013/203568Orig1s000Approv.pdf.
45. US Food and Drug Administration. Approval package for application number: 210922Orig1s000. August 10, 2018. Accessed May 27, 2021. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/210922Orig1s000Approv.pdf.
46. Xiong H, Veedu RN, Diermeier SD. Recent Advances in Oligonucleotide Therapeutics in Oncology. Int J Mol Sci. 2021; 22: 3295. DOI: 10.3390/ijms22073295.
47. US Food and Drug Administration. FDA Approves First Drug to Treat Rare Metabolic Disorder. FDA News Release. November 23, 2020. Accessed May 27, 2021. https://www.fda.gov/news-events/press-announcements/fda-approves-first-drug-treat-rare-metabolic-disorder#:~:text=Approval%20is%20for%20primary%20hyperoxaluria,and%20loss%20of%20kidney%20function&text=Today%2C%20the%20U.S.%20Food%20and,)%2C%20a%20rare%20genetic%20disorder.
48. US Food and Drug Administration. HEPLISAV-B. FDA. Accessed May 27, 2021. https://www.fda.gov/vaccines-blood-biologics/vaccines/heplisav-b#:~:text=Indications%3A,years%20of%20age%20and%20older.
49. Roberts TC, Langer R, Wood MJA. Advances in oligonucleotide drug delivery. c–94. DOI: 10.1038/s41573-020-0075-7.
50. Springer AD, Dowdy SF. GalNAc-siRNA conjugates: Leading the way for delivery of RNAi therapeutics. Nucleic Acid Ther. 2018; 28: 109–118. DOI: 10.1089/nat.2018.0736.