Nonclinical Testing of 
Individualized Antisense 
Oligonucleotide Drug Products for 
Severely Debilitating or 
Life-Threatening Diseases  
Guidance for Sponsor-Investigators 
 
DRAFT GUIDANCE
 
 
  This guidance document is being distributed for comment purposes only. 
 
Comments and suggestions regarding this draft document should be submitted within 60 days of 
publication in the Federal Register of the notice announcing the availability of the draft 
guidance.  Submit electronic comments to https://www.regulations.gov.  Submit written 
comments to the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 
Fishers Lane, Rm. 1061, Rockville, MD  20852.  All comments should be identified with the 
docket number listed in the notice of availability that publishes in the Federal Register. 
 
For questions regarding this draft document, contact (CDER) Ronald Wange at 301-796-1304.  
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
April 2021 
Pharmacology/Toxicology 
42059790dft.docx 
04/07/21   
Nonclinical Testing of 
Individualized Antisense 
Oligonucleotide Drug Products for 
Severely Debilitating or 
Life-Threatening Diseases  
Guidance for Sponsor-Investigators 
 
 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information 
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor 
Silver Spring, MD  20993-0002 
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov 
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
April 2021 
Pharmacology/Toxicology 
   
TABLE OF CONTENTS 
 
I.  INTRODUCTION ........................................................................................... 1 
II.  PROOF OF CONCEPT................................................................................... 2 
III.  IND-SUPPORTING SAFETY STUDIES........................................................ 2 
A.  Duration and Timing of General Toxicity Studies ................................................. 4 
B.  For the Clinical Phenotype of Slower Progression ................................................. 4 
IV.  FIH DOSE SELECTION ................................................................................ 4 
V.  DOSE ESCALATION ..................................................................................... 5 
VI.  FACTORS SUPPORTING ABBREVIATED NONCLINICAL 
ASSESSMENT APPROACH DESCRIBED IN THIS GUIDANCE ............... 5 
 
 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1  Nonclinical Testing of Individualized Antisense Oligonucleotide 
2  Drug Products for Severely Debilitating or 
3  Life-Threatening Diseases  
4  Guidance for Sponsor-Investigators1 
5   
6   
7   
8   
9  This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
10  Administration (FDA or Agency) on this topic.  It does not establish any rights for any person and is not 
11  binding on FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
12  applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff responsible 
13  for this guidance as listed on the title page. 
14   
15   
16   
17   
18  I.  INTRODUCTION 
19   
20  The purpose of this guidance is to describe the nonclinical information that FDA recommends to 
21  support an investigational new drug application (IND) for an antisense oligonucleotide being 
22  developed to treat a severely debilitating or life-threatening (SDLT) disease caused by a unique 
23  genetic variant where only a small number of individuals are prospectively identified (usually 
24  one or two).  The investigational antisense oligonucleotide should be from a well-characterized 
25  chemical class2 for which there is substantial nonclinical information and clinical experience that 
26  is publicly available or to which the sponsor-investigator (hereafter referred to as sponsor) has a 
27  right of reference. 
28   
29  This guidance is not intended to address nonclinical testing for commercial development of 
30  oligonucleotides. 
31   
32  The contents of this document do not have the force and effect of law and are not meant to bind 
33  the public in any way, unless specifically incorporated into a contract.  This document is 
34  intended only to provide clarity to the public regarding existing requirements under the law.  
35  FDA guidance documents, including this guidance, should be viewed only as recommendations, 
36  unless specific regulatory or statutory requirements are cited.  The use of the word should in 
37  Agency guidance means that something is suggested or recommended, but not required. 
38   
 
1 This guidance has been prepared by the Office of New Drugs in the Center for Drug Evaluation and Research at 
the Food and Drug Administration. 
 
2 Examples of well-characterized antisense chemical classes, based on prior FDA experience, include single-
stranded phosphorothioate or mixed phosphorothioate/phosphodiester with or without 2-methoxyethyl substituted 
oligonucleotides (by systemic or intrathecal route), and phosphorodiamidate morpholino oligonucleotides (by 
systemic route). 
1  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
39   
40  II.  PROOF OF CONCEPT 
41   
42  Given that the administration of an investigational antisense oligonucleotide covered under this 
43  guidance will be to a small number of individuals with an SDLT disease, the nonclinical safety 
44  package recommended to support first-in-human (FIH) exposure is generally less extensive than 
45  what is typically recommended for development of antisense oligonucleotide products intended 
46  for broader use or use in less severe clinical circumstances.3  To offset a greater assumption of 
47  risk due to more limited data, it is important that sponsors provide convincing in vitro and/or in 
48  vivo proof of concept (POC) data as part of any pre-investigational new drug (pIND) meeting 
49  package or the original investigational new drug (IND) submission (if no pre-IND meeting was 
50  requested) for investigational antisense oligonucleotides covered under this guidance.  These 
51  data are important to support the potential for benefit for both adult and pediatric subjects. 
52   
53   
54  III.  IND-SUPPORTING SAFETY STUDIES 
55   
56  Sponsors should include the following nonclinical safety studies in their IND submission: 
57   
58    Hybridization-dependent off-target assessment: Basic Local Alignment Search Tool 
59  (BLAST) and other appropriate in silico and/or in vitro assessments of possible off-target 
60  binding. 
61   
62    Safety pharmacology: For systemically administered investigational antisense 
63  oligonucleotides, FDA recommends evaluating effects in the core safety pharmacology 
64  batterycardiovascular, central nervous, and respiratory systems.4  These endpoints may 
65  be assessed in the general toxicity study discussed below, if conducted in a rigorous 
66  manner (ICH S7A).  If a pharmacologically relevant species is available, sponsors should 
67  use that species. 
68   
69    In vitro human ether-a-go-go-related gene (hERG) testing is generally not warranted. 
70   
71    For products delivered directly to the central nervous system (e.g., intrathecally), the 
72  safety pharmacology assessment may be limited to central nervous system endpoints.  
 
3 FDA regulations provide flexibility in applying regulatory standards because of the many types and intended uses 
of drugs.  FDA exercise[s] its scientific judgment in determining the kind and quantity of data a sponsor is 
required to provide for individual drug development programs.  See, for example, 21 CFR 314.105(c).  This 
flexibility extends from the early stages of development to the design of adequate and well-controlled studies 
required to demonstrate effectiveness to support marketing approval and to establish safety data needed for the 
intended use.  For further information on this topic, see the draft guidance for industry Rare Diseases: Common 
Issues in Drug Development (January 2019).  When final, this guidance will represent the FDAs current thinking on 
this topic.  For the most recent version of a guidance, check the FDA guidance web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
 
4 See the ICH guidance for industry S7A Safety Pharmacology Studies for Human Pharmaceuticals (July 2001).  We 
update guidances periodically.  To make sure you have the most recent version of a guidance, check the FDA 
guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
2  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
73  FDA recommends that these endpoints be assessed following initiation of dosing and 
74  again toward the end of the study. 
75   
76    If the route of administration does not result in significant systemic or central nervous 
77  system exposures (e.g., intravitreal administration), safety pharmacology studies are 
78  generally not warranted. 
79   
80    Genotoxicity: Genotoxicity assessment is generally not warranted. 
81   
82    General toxicity 
83   
84    In combination with the results from the POC and safety pharmacology assessments, 
85  a single, adequately designed, good laboratory practicecompliant general toxicity 
86  study can support FIH dosing.  The toxicity study can be conducted in a rodent or 
87  nonrodent species.5  Sponsors should provide scientific justification for the species 
88  selected.  If a pharmacologically relevant species is available, sponsors should use 
89  that species. 
90   
91    The study should assess a standard battery of toxicological endpoints, including 
92  clinical observations, body weight, food consumption, clinical pathology, 
93  toxicokinetic analysis, and histopathology of a comprehensive panel of tissues. 
94   
95    The route of administration used in animal studies should be the same as the intended 
96  clinical route.  Sponsors should provide justification if an alternative route is 
97  proposed for the toxicity study. 
98   
99    To the extent feasible, the drug formulation used in animal studies should be 
100  comparable to the clinical formulation. 
101   
102    The dosing regimen (i.e., dose levels and frequency of dosing) should provide 
103  adequate coverage for the expected clinical exposure with regard to both starting dose 
104  and maximum anticipated dose.  To allow for the greatest flexibility in clinical dose 
105  selection, it is preferable for the high dose to be a maximally tolerated or maximum 
106  feasible dose.  Sponsors should justify selecting an alternative basis for high-dose 
107  selection.6 
108   
109    To ensure that the toxicity study can meet its objectives, FDA recommends that 
110  sponsors submit a draft protocol of the toxicity study to FDA for review and feedback 
111  before initiating a study. 
 
5 We support the principles of the 3Rs, to reduce, refine, and replace animal use in testing when feasible.  We 
encourage sponsors to consult with us if they wish to use a nonanimal testing method they believe is suitable, 
adequate, validated, and feasible.  We will consider if such an alternative method is adequate to meet the regulatory 
need. 
 
6 See the ICH guidance for industry M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials 
and Marketing Authorization for Pharmaceuticals (January 2010). 
 
3  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
112   
113  A.  Duration and Timing of General Toxicity Studies 
114   
115  In the context of an investigational antisense oligonucleotide covered by this guidance, a single 
116  3-month toxicity study is considered adequate to assess safety for initiating human dosing, dose-
117  escalation, and chronic treatment. 
118   
119  For the clinical phenotype of rapid progression to death or rapid progression to substantial 
120  irreversible morbidity (e.g., within 1 year): 
121   
122    The IND submission should include at least 2 weeks of in-life data generated from an 
123  ongoing 3-month toxicity study. 
124   
125    Interim data should be provided periodically (e.g., monthly).  Sponsors must 
126  expeditiously report to the FDA findings suggesting a significant risk to the study 
127  participant(s) (21 CFR 312.32(c)(1)(iii)).  The institutional review board should 
128  likewise also be promptly informed of any such finding(s).7 
129   
130    A complete draft 3-month study report should be submitted as soon as completed.  
131  Sponsors should submit the final study report within 120 days of submitting the draft 
132  report. 
133   
134    Submission of the full study report should support continued dosing and dose 
135  escalation, assuming the data continue to support a conclusion of reasonable safety. 
136   
137  B.  For the Clinical Phenotype of Slower Progression 
138   
139    A completed 3-month toxicity study report should be submitted with the initial IND. 
140   
141   
142  IV.  FIH DOSE SELECTION 
143   
144  The primary goal of selecting the starting dose is to identify a dose that is expected to have 
145  pharmacologic effects and is reasonably safe, and it should be scientifically justified based on the 
146  totality of available data. 
147   
148  Sponsors should clearly describe and justify the method used for selecting the starting dose, 
149  including the basis for calculating safety margins between doses tested in animals and the dose 
150  or doses selected for administration in a human.  For local administration, sponsors should take 
151  into consideration organ weight, volume, or other measures as appropriate for interspecies dose 
152  scaling.  And for intrathecal administration, sponsors should calculate interspecies dose 
153  comparisons based on a dose normalized to cerebrospinal fluid volume. 
154   
 
7 For additional guidance on safety reporting, see the guidance for industry and investigators Safety Reporting 
Requirements for INDs and BA/BE Studies (December 2012). 
4  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
155   
156  V.  DOSE ESCALATION 
157   
158  When a steep dose-response or an exposure-response for severe toxicity is observed in 
159  nonclinical toxicity studies, or when no preceding marker of severe toxicity is available, 
160  sponsors should consider smaller than usual dose increments (e.g., fractional increments rather 
161  than dose doubling) for clinical dosing. 
162   
163  For investigational antisense oligonucleotides within the scope of this guidance, the highest dose 
164  or exposure assessed in the nonclinical studies does not necessarily limit the highest dose that 
165  can be evaluated in humans, depending on the available nonclinical and clinical information and 
166  the participants clinical situation. 
167   
168   
169  VI.  FACTORS SUPPORTING ABBREVIATED NONCLINICAL ASSESSMENT 
170  APPROACH DESCRIBED IN THIS GUIDANCE 
171   
172  The nonclinical safety package described here differs from that generally recommended for non-
173  SDLT diseases, for treatment modalities other than antisense oligonucleotides, and for SDLT 
174  diseases with larger patient populations.  FDA considers the nonclinical safety package 
175  recommended in this guidance acceptable to support INDs for investigational antisense 
176  oligonucleotides within the scope of this guidance, in part because of existing experience with 
177  antisense oligonucleotides and the ability to anticipate and manage some of the potential adverse 
178  effects.  However, the probability of identifying toxicity nonclinically may be reduced in 
179  comparison to standard nonclinical safety testing, and the potential for clinically significant 
180  adverse effects may therefore be increased.  With appropriate disclosures in the informed 
181  consent, this increased risk is considered acceptable to FDA at this time in the context of an 
182  investigational antisense oligonucleotide covered by this guidance. 
183   
184  Expansion of this approach to other oligonucleotide chemistries or mechanisms of action (e.g., 
185  siRNA), or to other treatment modalities (e.g., individualized biologics) should be supported by a 
186  nonclinical approach that provides a similar understanding of the chemistry and mechanism of 
187  action sufficient to allow for safe FIH dose selection, potential dose escalation, and an ability to 
188  predict the likely adverse effects that could occur, and how these can be clinically monitored.  
189  This will be considered by FDA on a case-by-case basis. 
190   
191  Expansion to a larger population or an intent to commercialize a treatment would typically 
192  warrant additional studies (e.g., longer duration general toxicity studies).8 
 
8 See ICH guidances for industry S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals 
(May 2012), S9 Nonclinical Evaluation for Anticancer Pharmaceuticals (March 2010), and ICH M3(R2). 
5