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8 Tips for Successfully Developing Oligonucleotide Therapies

By Eva Gil Berglund and Nathalie H Gosselin

Oligonucleotide therapeutics are a validated class of drugs that can modulate a multitude of genetic targets with gene silencing to prevent expression of encoded “disease-related” proteins. Developing drugs in this class is challenging. That’s why we’ve put together this list of 8 clinical pharmacology and pharmacokinetic considerations that you should know for these drugs:

Oligonucleotide therapeutics: What are they and how to they work?

1: There are two types of oligonucleotide therapeutics:1
  • antisense oligonucleotides (ASOs) bind a target RNA through Watson-Crick base pairing to cause gene silencing.
  • double-stranded short interfering RNA (siRNA):  a small RNA duplex associates with the RNA-induced silencing complex (RISC), one strand (the passenger strand) is lost, and the remaining strand (the guide strand) cooperates with RISC to bind complementary RNA, degrade it, and silence the gene. For example, patisiran’s (ONPATTRO) lipid nanoparticles protect its siRNA from degradation by endo- and exo-nucleases in the circulatory system and improves delivery to the target site of action, the liver.
Figure 1 adapted from Knockdown of lncRNAs: Exploring RNAi and antisense oligo methods [Digital image]. (n.d.). Retrieved January 27, 2021, from

Regulatory Clinical Pharmacology: Considerations for oligonucleotides

2: Do I need to consider possible drug-drug interactions (DDIs)

Oligonucleotides have showed potential effects on drug metabolizing enzymes and transporters in vitro.2-4 Oligonucleotides could also theoretically impact the expression of enzymes and transporters though influencing regulatory pathways. Does this mean that oligonucleotides could have clinical effects on the disposition of other drugs? It has been debated whether effects in vivo can really be obtained and whether oligonucleotides can enter hepatocytes, enterocytes, and kidney cells. However, oligonucleotides may be designed to enter hepatocytes and, in this case, their presence at the usual interaction sites can be assumed.5 For older oligonucleotides such as eteplirsen and ataluren, the half-lives are short, and thus a possible DDI perpetrator effect observed in vitro could, depending on administration frequency, be very transient in vivo. More recently developed, stabilized oligonucleotides have a more sustained exposure that could cause clinically relevant DDIs.

3: Are in vitro DDI studies a regulatory requirement?

Due to scientific knowledge gaps, there is presently no FDA or EU regulatory guidance describing which studies are expected for new investigational oligonucleotide drugs when submitting a marketing application. In 2019, the FDA published a request for public comments on the characterization of the effects of hepatic and renal impairment, DDIs, and immunogenicity and cardiac safety of these drugs.6  Rogers et al. has very recently published an FDA review of data for 21 oligonucleotides submitted as NDAs or INDs between 2012 and 2018.7 Seven in vivo DDI studies were performed and in one case, the study was considered positive.

As perpetrator effects by oligonucleotides could not be excluded, it is reasonable to believe that regulators have been recommending in vitro DDI perpetrator studies in obtained scientific advice. All the FDA registered drugs in the paper had in vitro data on perpetrator effects on enzymes and transporters. Based on the FDA Clinical Pharmacology review reports, this was also the case for the post-2018 approved viltolarsen (VILTEPSO) and golodirsen (VYONDIS 53).

4: What considerations do we need to make regarding patients with renal and/or hepatic impairment?

Reduced hepatic or renal function may not cause increased exposure for many of the presently available oligonucleotides. There are, however, exceptions. In the recent FDA paper, only one drug in the dataset, the exon skipping eteplirsen, indicated for Duchenne muscular dystrophy was identified as having marked renal excretion, and renal impairment effected exposure. Moderate renal impairment caused a mean 2.4-fold increase in eteplirsen exposure.8  Interestingly, renal extraction is the major elimination pathway of the post-2018 approved viltolarsen and golodirsen for the same indication, showing that this should not be excluded as a major elimination mechanism. The effect of renal impairment on golodirsen exposure was somewhat lower than observed for eteplirsen, i.e. 2-fold in severe renal impairment.

The need for investigating in vitro DDI perpetrator effects as well as the role of renal excretion of oligonucleotide elimination and effect of renal impairment should be considered when planning clinical pharmacology development programs of oligonucleotides. We hope that the major regulatory agencies will update their guidelines with information on the present study requirements. Such recommendations could then be further updated when more information is available. This year the FDA is planning to publish a draft guidance on “Clinical Recommendations to Support IND Submissions for Individualized Antisense Oligonucleotide Drug Products for Severely Debilitating or Life-Threatening Diseases”. We are looking forward to this guidance and hope that it also provides advice on the clinical pharmacology space.

Pharmacokinetic considerations

5: The pharmacokinetics of oligonucleotides drugs is governed by the type of modification, their conjugates for ASOs, and their carriers for siRNAs.
  • ASO. For example, ASOs that contain a phosphorothioate backbone are extensively bound to plasma proteins (≥ 85%) and mainly to albumin across all species including humans.9 This high degree of binding to human plasma proteins is expected to reduce urinary excretion and renal clearance of the drugs due to glomerular filtration. The binding affinity to albumin is relatively low, allowing uptake into tissues.
  • siRNA drugs are less charged or less bound to plasma proteins (e.g., siRNAs or morpholino nucleotide oligomers), and they are cleared rapidly from blood because of either nuclease metabolism in blood or excretion in urine. For example, < 2.1% of patisiran is bound to plasma proteins, and the terminal elimination half-life of patisiran is only 3 days (ONPATTRO).
6: How can ASGPR affect siRNA delivery?

Givosiran and inclisiran, two siRNA therapeutics, are conjugated to triantennary N– acetylgalactosamine carbohydrates (GalNAc) which bind to asialoglycoprotein receptors (ASGPR) on hepatocytes. Based on a pre-clinical model, even with a reduction of 50% in ASGPR levels, siRNAs-GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels.10

7: The time course for PK sampling of siRNAs can be shorter than for ASOs, but time course for PD sampling can be longer.

Total elimination half-lives (t1/2) of siRNA are shorter than t1/2 of ASO, due to the distribution of ASO in tissue and slower clearance from tissues.11 However, although siRNA therapeutics often have short elimination half-lives (up to 5 days), the dosing frequency intervals are up to 6 months, whereas the dosing frequency is weekly or monthly for ASO drugs.

8: There is often a temporal disconnect between the plasma PK of oligonucleotides, and their prolonged PD effects.

Choosing the dosing interval and making dosing adjustments are mainly guided by the length of drug effect and PD covariates, not the duration of drug exposure. Although inclisiran is eliminated after about 48 hours, a 6-month dosing interval was used in the Phase 3 studies.

Understanding of pharmacokinetics of oligonucleotides will help you to adequately plan your clinical trial data collection to facilitate population PK/PD modeling activities and support your dose recommendations.

To learn more about best practices for developing these complex biologic drugs, please register for this upcoming webinar:


1. Watts JK, Corey DR. Silencing disease genes in the laboratory and the clinic. J Pathol. 2012;226(2):365-379. doi: 10.1002/path.2993 [doi].

2. Kazmi F, Sensenhauser C, Greway T. Characterization of the in vitro inhibitory potential of the oligonucleotide imetelstat on human cytochrome P450 enzymes with predictions of in vivo drug-drug interactions. Drug Metab Dispos. 2019;47(1):9-14. doi: 10.1124/dmd.118.084103 [doi].

3. Kazmi F, Yerino P, McCoy C, Parkinson A, Buckley DB, Ogilvie BW. An assessment of the in vitro inhibition of cytochrome P450 enzymes, UDP-glucuronosyltransferases, and transporters by phosphodiester- or phosphorothioate-linked oligonucleotides. Drug Metab Dispos. 2018;46(8):1066-1074. doi: 10.1124/dmd.118.081729 [doi].

4. Ramsden D, Wu JT, Zerler B, et al. In vitro drug-drug interaction evaluation of GalNAc conjugated siRNAs against CYP450 enzymes and transporters. Drug Metab Dispos. 2019;47(10):1183-1194. doi: 10.1124/dmd.119.087098 [doi].

5. Gao S, Chen J, Dong L, Ding Z, Yang YH, Zhang J. Targeting delivery of oligonucleotide and plasmid DNA to hepatocyte via galactosylated chitosan vector. Eur J Pharm Biopharm. 2005;60(3):327-334. doi: S0939-6411(05)00103-7 [pii].

6. Evaluating the clinical pharmacology of oligonucleotide therapeutics; establishment of a public docket; request for information and comments. Federal Register. 2019;84(152):38634-38636.

7. Rogers H, Adeniyi O, Ramamoorthy A, Bailey S, Pacanowski M. Clinical pharmacology studies supporting oligonucleotide therapy development: An assessment of therapies approved and in development between 2012-2018. Clin Transl Sci. 2020. doi: 10.1111/cts.12945 [doi].

8. Exondys CHMP public assessment report (EPAR).  2018.

9. Geary RS. Antisense oligonucleotide pharmacokinetics and metabolism. Expert Opin Drug Metab Toxicol. 2009;5(4):381-391. doi: 10.1517/17425250902877680 [doi].

10. Willoughby JLS, Chan A, Sehgal A, et al. Evaluation of GalNAc-siRNA conjugate activity in pre-clinical animal models with reduced asialoglycoprotein receptor expression. Molecular therapy: the journal of the American Society of Gene Therapy. 2018;26:105-114.; doi: 10.1016/j.ymthe.2017.08.019.

11. Yin W, Rogge M. Targeting RNA: A transformative therapeutic strategy. Clin Transl Sci. 2019;12(2):98-112. doi: 10.1111/cts.12624 [doi].

About the authors

By: Eva Berglund
By: Nathalie Gosselin

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