FDA Issues New Guidance: Nonclinical Safety Assessment Of Oligonucleotide-Based Therapeutics

By Peter H. Calcott, Ph.D., FRSC, president and CEO, Calcott Consulting LLC

FDA (CDER) issued a draft guidance in November 2024, Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics (ONT), to support clinical development and marketing of these products.¹ As is customary, industry has 60 days to assess it and reply with feedback for consideration. This guidance should be considered with the also recently issued guidance by EMA² on the development and manufacturing of oligonucleotide-based therapies to see that the two, although covering slightly different topics, are extremely complementary.

CDER points out that this guidance offers recommendations specific to these molecules. It describes how these molecules are similar to other small molecules but that there are areas unique to this class of molecule. The guidance is specific to single stranded or double stranded ONTs, created synthetically or via biological processes. They can be native or modified backbones or nucleoside structures. Examples of included oligonucleotides are antisense, small interfering RNA, microRNA, transfer RNA, decoys, and aptamers. Immune stimulatory oligonucleotides (e.g., CpG motifs acting via Toll-like receptors) are excluded, as are CBER-regulated products (e.g., DNA/RNA vaccines, virally delivered ONTs, messenger RNA and RNA used for gene editing). This is identical to the situation in the EMA guidance. Conjugated forms of products made from applicable ONTs are included. For ONTs intended to treat cancer, sponsors should consult International Council for Harmonisation (ICH) guidances for industry S9 (March 2010)³ and S9 Questions and Answers (June 2018)⁴ regarding the nonclinical studies recommended to support development of those products. Principles outlined in this guidance may also be relevant to the design and conduct of studies recommended when assessing the nonclinical safety of an ONT for the treatment of cancer.

As would be expected the guidance is divided into the classic safety considerations that all pharmaceuticals must address. It includes sections on mode of activity and impact on safety, an assessment related to structural considerations and non-structural implications. The general areas of pharmacokinetics and general and specific safety considerations are also addressed. Some of the specific considerations include genotoxicity, reproductive and developmental toxicity, carcinogenicity, local tolerance, immunotoxicity, and photosafety. In addition, the impact of formulation, targeting moiety, and impurities/degradants (oligo related and other impurities) are considered, as is impact on juvenile animals.

1. Modes Of Activity

This class of molecule has multiple modes of action to achieve pharmacological action. While these molecules do have an affinity for specific sequences in the DNA, they can cause many effects, such as expressing a gene or silencing it. Further, some molecules can interact with the precursor mRNA to elicit an action to modulate genes. Other ONTs (aptamers) can bind to proteins to elicit a pharmacological action. There may be others not fully appreciated at the moment. Because of this diversity of actions, there is no simple formula specifying what needs to be done to assure safety.

2. Safety Assessment

Safety assessments must take into consideration on- and off-target effects (i.e., intended and unintended effects). The on-targeted effects can be assessed in a target species where the molecule is effective. That is to look for exaggerated effects. If no specific species is available, it is permissible to adjust the molecule to target the species. The guidance goes into more specific detail on the questions to be posed and addressed.

Off-target effects are caused by the binding of the ONTs to regions of the genome, mRNA, or proteins not contemplated. This is caused by similarities in the structure of these regions. Points to consider for sequence-dependent off-target assessments are:

• Because there are multiple types of products, each with their own MoA, the criteria for defining high-risk off-target binding and sensitivity to mismatches are expected to differ for each ONT type. Appropriate criteria should be provided and justified for the applicable type of ONT drug product.
• All elements of the ONT drug product and its predicted metabolites that are available for
• off-target hybridization should be assessed, including both the sense and the antisense
• strands, overlapping ends, etc.
• Potential hybridization to the transcriptome and nuclear and mitochondrial genome should be addressed.
• The in silico methods used, including the criteria for defining potential off-target binding, should be justified.
• The importance of any potential off-target hybridization identified in silico should be further investigated in in vitro assays (e.g., cell-based analysis, RNA-seq). Scientific justification for the assays selected should be provided.
• Information from human genetic diseases and animal models (e.g., mouse knockouts) can be used as part of a weight of evidence (WoE) assessment to understand potential consequences of off-target effects.
• Relevance of toxicology animal species for assessing off-target effects should be considered by determining whether the human off-target site is conserved in the animal.
• Additional factors, such as temporal and cell-type-specific expression, pharmacokinetic (PK) properties, and hybridization-dependent binding efficiency, may be considered in the overall risk assessment of potential off-target effects.

These should be carefully addressed and considered. If these cannot be addressed in the animal model, they should be examined and considered during the clinical program.

The guidance also describes off-target hybridization-independent effects. This covers unanticipated effects not ascribed to the specific hybridization of the molecule with an unintended molecule but, rather, generalized effects caused by other effects. ICH M3 (R2) Questions and Answers (February 2013)⁵ is referenced. These are difficult to predict because of the breadth of potential interactions; often, empirical assessments are the only way to go.

3. Pharmacology

Primary pharmacology studies should investigate the mode of action and/or effects of an ONT in relation to its desired therapeutic target. These studies may also provide information about the duration of effect and can contribute to dose selection for both nonclinical and clinical studies. Some ONTs may be highly specific for human targets and consequently show no pharmacological activity in nonhuman species. In these cases, pharmacology information may be obtained by using a test species-active surrogate oligonucleotide. In vitro studies using human cells or genetically modified animals expressing the human target or in which the target is knocked out also can provide information about potential biological effects from the ONT. Not every typical study used to assess classical small molecules may be necessary for ONTs. However, if effects are seen that are unexpected in animal and eventually human studies, these issues may need to be revisited. Even if not anticipated, a generalized safety pharmacology should address all critical systems to assure safety, preferably with a relevant species. When a drug is to be delivered to a specific organ or system, that target should be studied at depth. The guidance recommends ICH S7B (October 2005), E14 and S7B Questions and Answers (August 2022).⁶

4. Pharmacokinetics

As for other investigational drugs, the nonclinical absorption, distribution, metabolism, and excretion parameters of an ONT should be understood to inform the safety assessment to support clinical trials.⁵ This should be conducted in a pharmacologically relevant species. In vitro plasma protein binding data for animals and humans should be evaluated before initiating human clinical trials. For well-characterized ONTs, class data may be used, if justified. Metabolites of ONTs are not considered necessary to be studied. However, if novel chemistry is used, then it should be addressed. If a conjugated ONT is used, then the conjugants must be studied. Other pharmacokinetic parameters and issues are discussed.

5. General Toxicity Studies

Species, Design, Duration, And Timing

Toxicity studies of ONTs should generally be conducted in two species: one rodent and one nonrodent. Generally, at least one species should be pharmacologically relevant. In cases where a surrogate molecule is used, animal studies should typically include some groups treated with the clinical candidate and an additional group (or groups) treated with the surrogate. The design of toxicity studies for ONTs, including endpoints assessed, are generally similar to those for small molecule drugs. Criteria for high doses and durations used for ONTs in toxicity studies are similar to those described in ICH M3(R2).⁵ Appropriate dosing frequency and duration of the toxicity study should consider aspects of drug distribution and half-life, as well as the clinical dosing regimen. The half-life of ONTs can be shorter in animals than humans. Other factors and parameters are well discussed.

First In Human

The first-in-human dose in healthy volunteers should be selected using the general principles outlined in the guidance for industry (July 2005).⁷ In general, a reasonable starting dose can be defined as one that is expected to result in no more than one-tenth of the exposure observed at the no observed adverse effect level in the most relevant (or more sensitive) species and is typically estimated by calculating a human equivalent dose of the animal dose normalized to body surface area. For riskier molecules, further reduction in primary dose may be considered.

6. Genotoxicity Studies

Genotoxicity testing of ONTs composed exclusively of natural nucleic acids is not necessary. ONTs containing non-native nucleic acids, backbone structures, or other structures, such as conjugates or linkers, should be assessed for genotoxic potential. However, if they do not fit fully into that class, the tests in ICH guidance for industry S2(R1) (June 2012)⁸ are relevant.

7. Reproductive And Developmental Toxicity Studies

The assessment of developmental and reproductive toxicity (DART) for ONTs should be conducted in accordance with the ICH guidance for industry S5(R3) (May 2021).⁹ Typically, this assessment relies on animal studies that assess effects on fertility and early embryonic development, embryo/fetal development (EFD), and pre- and postnatal development (PPND) endpoints. Fertility and early embryonic development and PPND studies are routinely conducted in rodents, whereas two species, typically rodents and rabbits, are used in EFD studies. Class data that have been obtained may be relevant to help design studies. If no pharmacologically relevant species is available, a surrogate molecule can be used if characterized adequately. Accumulated data from the same class of molecules can be used to argue for no additional studies.

ICH M3(R2) should be consulted regarding the timing of submission of DART study reports as it relates to supporting enrollment into clinical trials of women of childbearing potential or pregnant women as well as submission of the marketing application.⁵ For ONTs intended to treat cancer, SDLT hematologic disorders, or rare SDLT indications, the relevant guidance should be consulted.

8. Carcinogenicity Studies

Sponsors should follow ICH guideline S1A (March 1996)¹⁰ with regard to determining whether a carcinogenicity assessment is warranted for a particular development program. As ONTs are generally developed to treat chronic conditions, a carcinogenicity assessment will be expected for most programs. If the ONT is from a well-characterized category, there may be some flexibility.

Additionally, as described in the ICH guidance for industry S1B(R1) Addendum to S1B (November 2022),¹¹ the use of a WoE approach for concluding that a rat carcinogenicity study would not add value to the carcinogenicity assessment is also available to those ONT programs with data that can address all the factors described in that guidance. Specific dosing strategies are described in detail for various types of molecules.

It is recognized that there are scenarios under which an in vivo assessment of the carcinogenicity of the pharmacological activity of a surrogate oligonucleotide may not be warranted, including when the pathway being perturbed is well understood and is known to contribute to tumorigenesis, the surrogate oligonucleotide would affect wild type rodents in a manner that does not recapitulate the intended pharmacology (e.g., for some splice-modulating ONTs that target pre-mRNA to alter the nucleic acids that are included in the mature mRNA, thereby affecting reading frame), or the target does not exist in rodents.

There is the potential for human-specific, off-target hybridization-dependent activity that could affect carcinogenic risk (e.g., the ONT or a metabolite affects the activity of an unintended target with a known role in carcinogenesis). This type of hazard should be addressed through a WoE assessment, clinical monitoring, and appropriate communication of potential risks via informed consent and product labeling, as animal studies are likely to be uninformative in assessing this risk.

9. Local Tolerance

Local tolerance can generally be assessed as part of single- or repeated-dose toxicity studies. The local toxicity of ONTs with complex vehicles should be assessed by testing the entire clinical formulation in toxicity studies, if feasible.

10. Immunotoxicity

The assessment of immunotoxicity of ONTs should follow ICH and FDA guidance on this topic.¹² Although ONTs may engage the immune system, stand-alone, dedicated immunotoxicity assessments are not typically warranted. However, assessment of ONT-induced effects on the immune system (e.g., cytokines, complement) in general toxicity studies can provide useful context for understanding observations seen in these studies.

11. Photosafety

The ICH guidance for industry S10 (January 2015) should be consulted for recommendations on photosafety assessment. If ultraviolet-visible light absorption of an ONT is determined to be solely due to natural nucleic acid bases, additional photosafety assessment is not warranted.

12. Other Toxicity Studies

Formulation, Targeting Moiety

The ONT should be tested in the presence of the formulation. For novel excipients, preliminary toxicology studies can be done alone for these moieties. See guidance for industry (May 2005)¹³ for further assistance. Any conjugating agent should be assessed, but general data collected separately can be considered. Advice from FDA is encouraged.

Impurities/Degradants

As for other synthetically derived drug products, impurity levels in ONT drug products should be adequately controlled and demonstrated to be present at levels that do not raise safety concerns. Impurities derived from ONTs can be classified into two broad categories: oligonucleotide-related substances and other impurities (e.g., organic small molecule impurities, residual solvents, elemental impurities). Historical data may be relevant and used as appropriate.

Oligo-related impurities may be difficult to separate and quantify, so the approach is to control the manufacturing process to assure consistency of the product and test that material. Other impurities associated with the manufacturing process should be considered like other impurities in small molecular weight pharmaceutical products.¹⁴

Juvenile Animal Studies

When evaluating the need for juvenile animal studies for ONTs being developed for pediatric indications, sponsors should follow the recommendations provided in the ICH guidance for industry S11 (May 2021).¹⁵

13. Notes

• Where possible, do not use non-human primates.
• Considerations should be made on dosing levels, frequency, and other factors.

The take-home message from this guidance is that the industry should rely on the intelligent use of science, pragmatism and FDA guidances and ICH documents, even though the latter documents were not written specifically for this class of molecules. While the use of pharmacologically relevant species is preferred, sometimes it is impossible. In these cases, it may be possible to substitute a surrogate molecule, but care should be taken in interpreting the data. Data obtained from similar molecules can be useful but must be critically assessed.

[Editor’s note: The public comment deadline is January 14, 2025.]

References

1. FDA guidance, Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics Guidance for Industry, https://www.fda.gov/media/183496/download
2. EMA guidance on Development and Manufacturing of oligonucleotides https://www.ema.europa.eu/en/documents/scientific-guideline/draft-guideline-development-manufacture-oligonucleotides_en.pdf
3. ICH S9 Nonclinical Evaluation for Anticancer Pharmaceuticals (March 2010)^. www.ich.org
4. ICH S9 Nonclinical Evaluation for Anticancer Pharmaceuticals Questions and Answers (June 2018) (ICH S9 Q&A) www.ich.org
5. ICH M3 (R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals, Questions and 159 Answers (R2) (February 2013) www.ich.org
6. ICH S7B Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals (October 2005) and E14 and S7B Clinical and Nonclinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential--Questions and Answers (August 2022). www.ich.org
7. Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (July 2005) https://www.fda.gov/regulatory-information/search-fda-guidance-documents/estimating-maximum-safe-starting-dose-initial-clinical-trials-therapeutics-adult-healthy-volunteers
8. ICH guidance for industry S2(R1) 354 Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use 355 (June 2012). www.ich.org
9. ICH guidance for industry S5(R3) Detection of Reproductive and Developmental Toxicity for Human Pharmaceuticals (May 2021) www.ich.org
10.  ICH guideline S1A The Need for Long-term Rodent Carcinogenicity Studies of Pharmaceuticals (March 1996) www.ich.org
11. ICH guidance for industry S1B(R1) Addendum to S1B Testing for Carcinogenicity of Pharmaceuticals (November 2022) www.ich.org
12. ICH guidance for industry S8 Immunotoxicity Studies for Human Pharmaceuticals (April 2006) www.ich.org and the guidance for industry Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals (June 2023). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/nonclinical-evaluation-immunotoxic-potential-pharmaceuticals  
13. Guidance for industry Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients (May 2005) https://www.fda.gov/regulatory-information/search-fda-guidance-documents/nonclinical-studies-safety-evaluation-pharmaceutical-excipients
14. ICH Q3A(R2) and Q3B(R2) Impurities in New Drug Products (August 2006), and M7(R2) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk (July 2023). Residual solvents should be assessed with reference to the ICH 593 guidance for industry Q3C(R8) Impurities: Guidance for Residual Solvents. (December 2021). Elemental impurities should be assessed with reference to the ICH guidance for industry Q3D(R2) Elemental Impurities (September 2022). www.ich.org
15. ICH guidance for industry S11 Nonclinical Safety Testing in Support of Development of Pediatric Pharmaceuticals (May 2021).

[Editor’s note: You may also be interested in the author’s summary/analysis of the recent EMA guidance on oligonucleotides.]

About The Author:

Peter H. Calcott, D.Phil., is president and CEO of Calcott Consulting LLC, which delivers solutions to pharmaceutical and biotechnology companies in the areas of corporate strategy, supply chain, quality, clinical development, regulatory affairs, corporate compliance, and enterprise e-solutions. He has also served as an expert witness. He also teaches at the University of California, Berkeley in the biotechnology and pharmaceutics postgraduate programs. Previously, he was executive VP at PDL BioPharma, chief quality officer at Chiron and Immunex Corporations, and director of quality assurance for SmithKline Beecham and for Bayer. He has also held positions in R&D, regulatory affairs, process development, and manufacturing at other major pharmaceutical companies. He has successfully licensed products in the biologics, drugs, and device sectors on all six continents. Calcott holds a doctorate in microbial physiology and biochemistry from the University of Sussex in England. He has been a consultant for more than 20 years to government, industry, and academia.