Human Gene Therapy Products 
Incorporating Human Genome Editing 
 
Guidance for Industry 
 
 
 
 
 
 
 
Additional copies of this guidance are available from the Office of Communication, Outreach 
and Development (OCOD), 10903 New Hampshire Ave., Bldg. 71, Rm. 3128, Silver Spring, 
MD 20993-0002, or by calling 1-800-835-4709 or 240-402-8010, or email ocod@fda.hhs.gov, or 
from the Internet at https://www.fda.gov/vaccines-blood-biologics/guidance-compliance- 
regulatory-information-biologics. 
 
For questions on the content of this guidance, contact OCOD at the phone numbers or email 
address listed above. 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research 
January 2024   
Contains Nonbinding Recommendations 
Table of Contents 
I.  INTRODUCTION ..............................................................................................................1 
II.  BACKGROUND .................................................................................................................1 
III.  CONSIDERATIONS FOR PRODUCT DEVELOPMENT ...........................................2 
A.  General Considerations.......................................................................................... 2 
1.  Genome Editing methods .............................................................................2 
2.  Type and degree of genomic modification ...................................................3 
3.  Genome Editing Component Delivery Method ...........................................3 
B.  Chemistry, Manufacturing and Controls (CMC) Recommendations ...............4 
1.  Genome Editing Component Design............................................................5 
2.  Genome Editing Component Manufacture and Testing ...............................5 
3.  Drug Product Manufacture and Testing .......................................................7 
IV.  CONSIDERATIONS FOR NONCLINICAL STUDIES ................................................9 
A.  Product Evaluated in Nonclinical Studies ..........................................................11 
B.  Assessment of Activity .........................................................................................11 
C.  Assessment of Safety ............................................................................................12 
V.  CONSIDERATIONS FOR CLINICAL STUDIES .......................................................12 
A.  Study Population ..................................................................................................13 
B.  Dose and Dose Schedules .....................................................................................13 
C.  Treatment Plan .....................................................................................................14 
D.  Monitoring and Follow-Up ..................................................................................14 
1.  Assessment of Product-Related Adverse Events........................................14 
2.  Long Term Follow-Up ...............................................................................14 
E.  Study Endpoints ...................................................................................................15 
F.  Special Considerations for Research Involving Children .................................15 
VI.  COMMUNICATION WITH FDA .................................................................................16 
VII.  REFERENCES .................................................................................................................17 
APPENDIX ...................................................................................................................................19 
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Human Gene Therapy Products Incorporating Human Genome 
Editing 
Guidance for Industry 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or 
Agency) on this topic. It does not establish any rights for any person and is not binding on FDA 
or the public. You can use an alternative approach if it satisfies the requirements of the 
applicable statutes and regulations. To discuss an alternative approach, contact the FDA staff 
responsible for this guidance as listed on the title page. 
 
 
I.  INTRODUCTION 
 
In this guidance, we, FDA, are providing recommendations to sponsors developing human gene 
therapy1 products incorporating genome editing (GE) of human somatic cells. Specifically, this 
guidance provides recommendations regarding information that should be provided in an 
Investigational New Drug (IND) application in order to assess the safety and quality of the 
investigational GE product, as required in Title 21 of the Code of Federal Regulations 312.23 
(21 CFR 312.23). This includes information on product design, product manufacturing and 
testing, nonclinical safety assessment, and clinical trial design. 
In general, FDAs guidance documents, including this guidance, do not establish legally 
enforceable responsibilities.  Instead, guidances describe the Agencys current thinking on a 
topic and should be viewed only as recommendations, unless specific regulatory or statutory 
requirements are cited. The use of the word should in Agency guidance means that something 
is suggested or recommended, but not required. 
 
 
II.  BACKGROUND 
 
The level of interest in human GE as a scientific technology used in the treatment of human 
disease has increased substantially, and there has been rapid development of gene therapy 
products incorporating GE. While the potential of such products for the treatment of human 
 
1 Human gene therapy seeks to modify or manipulate the expression of a gene or to alter the biological properties of 
living cells for therapeutic use. FDA generally considers human gene therapy products to include all products that 
mediate their effects by transcription or translation of transferred genetic material, or by specifically altering host 
(human) genetic sequences. Some examples of gene therapy products include nucleic acids, genetically modified 
microorganisms (e.g., viruses, bacteria, fungi), engineered site-specific nucleases used for human genome editing, 
and ex vivo genetically modified human cells. Gene therapy products meet the definition of biological product in 
section 351(i) of the Public Health Service (PHS) Act (42 U.S.C. 262(i)) when such products are applicable to the 
prevention, treatment, or cure of a disease or condition of human beings. (See Federal Register Notice: Application 
of Current Statutory Authorities to Human Somatic Cell Therapy Products and Gene Therapy Products (58 FR 
53248, October 14, 1993), https://www.fda.gov/media/76647/download). 
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disease is clear, the potential risks are not as well understood. To assist in the translation of these 
products from the bench to clinical trials, this guidance includes recommendations for how to 
assess the safety and quality of these products and address the potential risks of these products. 
For the purpose of this guidance, human GE is a process by which DNA sequences are added, 
deleted, altered or replaced at specified location(s) in the genome of human somatic cells, ex 
vivo or in vivo, using nuclease-dependent or nuclease-independent GE technologies. Human 
gene therapy products incorporating GE are referred to as human GE products throughout this 
guidance. 
 
FDA evaluates human GE products using a science-based approach weighing the benefits and 
risks of each product. The benefit-risk profile for each product depends on the proposed 
indication and patient population, the extent and duration of therapeutic benefit achieved, and the 
availability of alternative therapeutic options. Some of the specific risks associated with GE 
approaches include off-target editing, unintended consequences of on-target editing, and the 
unknown long term effects of on- and off-target editing. 
 
Human GE is a rapidly evolving field and this guidance encompasses FDAs current thinking 
regarding the development of human GE products for clinical studies and licensure. As the field 
evolves, product design advances, and we gain information on the safety of human GE products, 
we may revise our recommendations to take into account such changes. 
 
 
III.  CONSIDERATIONS FOR PRODUCT DEVELOPMENT 
 
A.  General Considerations 
 
A GE technology may be composed of a single or multiple GE component(s). For the 
purpose of this guidance, a GE component is considered any material that is essential for 
the intended genomic modification, including those that may not appear in the final drug 
product. GE components may include, but are not limited to, the editor, DNA targeting 
elements (i.e., elements used to dictate the target DNA sequence, such as guide RNA) 
and a donor DNA template (i.e., DNA sequence provided to repair the target sequence), if 
applicable. When developing a human GE product, we recommend that sponsors 
consider:  1) the method by which the DNA sequence change will be achieved; 2) the 
type of genomic modification needed for the desired therapeutic effect; and 3) the 
delivery method of the human GE components. 
 
1.  Genome Editing methods 
 
GE can be achieved by either nuclease-dependent or nuclease-independent 
methods. Nuclease-dependent GE technologies introduce site-specific breaks in 
the DNA, which may result in modification of the DNA sequence at the editing 
site. Some examples of nuclease-dependent GE technologies include, but are not 
limited to, zinc finger nucleases (ZFNs), transcription activator-like effector 
nucleases (TALENs), modified-homing endonucleases (meganucleases), and 
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clustered regularly interspaced short palindromic repeat (CRISPR)-associated 
(Cas) nucleases. Nuclease-independent GE technologies can change a DNA 
sequence without cleaving the DNA. Examples of nuclease-independent GE 
technologies include, but are not limited to, some forms of base editing and 
synthetic triplex-forming peptide nucleic acids. When choosing a specific GE 
technology, consideration should be given to the nature of the desired editing 
outcome (e.g., gene inactivation, restoration, or introduction), the ability to 
specifically target the desired DNA sequence, and the ability to optimize the GE 
components to improve safety, efficiency, specificity, or stability. 
 
2.  Type and degree of genomic modification 
 
The type of genomic modification needed for the desired therapeutic effect is 
another important consideration. Many GE approaches rely on intrinsic DNA 
damage repair pathways to perform genomic modification. Commonly utilized 
DNA damage repair pathways include, but are not limited to, homology directed 
repair (HDR) and non-homologous end-joining (NHEJ). HDR utilizes a 
homologous DNA sequence to repair the DNA break. NHEJ repairs the DNA 
break by rejoining two ends of cleaved DNA without a homologous repair 
template. Both HDR and NHEJ can be used to therapeutically modify the 
genome (Ref. 1). However, it is important to note that NHEJ is relatively 
independent of the cell cycle, while HDR is most active during S/G2 phase. It is 
also important to keep in mind that, although these processes can be accurate, 
they can also result in DNA insertions or deletions (indels) with possible 
unanticipated consequences. DNA cleavage events, which could be caused by 
multiplex on-target editing or a combination of on- and/or off-target effects, can 
also lead to chromosomal rearrangements, including translocations. 
 
The degree of genome modification needed for the desired therapeutic effect (i.e., 
therapeutic editing threshold) may depend on the indication and the intended 
patient population. We recommend considering the therapeutic editing threshold 
(e.g., frequency of editing, number of cells edited) when developing a therapeutic 
product incorporating human GE. For some conditions, clinical data may be 
available to support a given therapeutic editing threshold. The potential efficacy 
of a human GE product will depend on its ability to achieve this therapeutic 
editing threshold. If clinical data supporting a therapeutic editing threshold are 
not available, we recommend sponsors provide a justification for the potential 
efficacy of the achievable editing threshold to initiate a clinical study. 
 
3.  Genome Editing Component Delivery Method 
 
When determining the optimal delivery method of the GE components into cells, 
it is important for sponsors to consider the advantages and limitations of each 
potential method (e.g., the amount of nucleic acid the delivery vector can contain, 
efficiency and specificity of targeted delivery, and GE component persistence and 
stability). For example, with regard to persistence of the GE components, the 
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longer the GE component (e.g., the nuclease) is functionally active, the greater the 
risk of unintended genomic modifications, specifically off-target editing and 
chromosomal rearrangements. Therefore, to limit the degree of potential off- 
target editing, the duration of GE component persistence should be minimized to 
the time needed to perform the desired genomic modification, to the extent 
possible, based on individual product characteristics. 
The optimal method for delivering the GE component(s) may depend on whether 
the product involves ex vivo or in vivo genomic modification. Ex vivo 
modifications are introduced into cells while the cells are outside the body. The 
modified cells are then administered to the patient. In vivo modifications result 
from administration of the GE components in their final formulation to the 
patient. Sponsors should consider whether ex vivo or in vivo genomic 
modification is best suited to their target indication and patient population. 
 
For ex vivo genome modification, GE components are commonly delivered into 
cells via transfection, transduction, electroporation, or other mechanical methods. 
The GE components may be delivered as DNA, RNA, protein, or 
ribonucleoprotein complexes (RNPs). If HDR is the repair pathway being 
utilized, the donor DNA template can be supplied as a plasmid, or using a viral 
vector, such as recombinant adeno-associated virus (AAV). The chosen GE 
component delivery method may depend on the ability of the cell type of interest 
to be efficiently electroporated or transduced by a vector and maintain acceptable 
levels of viability following electroporation, transfection, or transduction, for 
example. 
 
For in vivo genome modification, GE components may be delivered by viral 
vectors or nanoparticles. When choosing an in vivo delivery method, it is 
important to consider the ability of the delivery vector to target the cells/tissue of 
interest and minimize distribution to non-targeted tissue. Consideration should 
also be given to the ability to control expression of vector-delivered GE 
components (e.g., using tissue-specific promoters, small molecule inhibitors, etc.), 
if appropriate. Viral vectors may support sustained expression of GE component 
transgenes, and nanoparticles may allow the temporal delivery of GE components 
as DNA, RNA, or proteins. The potential for vector-mediated toxicity as well as 
pre-existing immunity to the GE components and vector should also be 
considered. The sponsor should select the appropriate delivery method based on 
the intended use. 
 
B.  Chemistry, Manufacturing and Controls (CMC) Recommendations 
 
This guidance is intended to address considerations specific to GE products and is not 
designed to be a stand-alone CMC guidance. The general CMC considerations for 
product manufacturing, testing and release of human GE products are the same as those 
previously described in FDAs guidance entitled Chemistry, Manufacturing, and Control 
(CMC) Information for Human Gene Therapy Investigational New Drug Applications 
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(INDs): Guidance for Industry, January 2020 (Ref. 2) (hereinafter referred to as the 
GT CMC Guidance). Additional recommendations specific to human GE products 
regarding design, manufacture, and testing of the GE components, as well as the drug 
product (DP), are described below. 
1.  Genome Editing Component Design 
Many platforms exist to design GE components, particularly the targeting 
elements. We recommend sponsors utilize design platforms that are most 
applicable to their genomic target and the type of intended genomic modification. 
A description of, and rationale for, the design and screening processes should be 
provided in the IND. The IND should also include the sequences of the GE 
components and/or expression constructs. 
 
We recommend sponsors optimize the GE components to reduce the potential for 
off-target genome modification, to the extent possible. Optimization can be 
performed on the editor or the targeting elements, depending on the GE 
technology being utilized. GE components, such as guide RNA, can also be 
optimized to inhibit degradation. The optimization strategy should be described 
in detail in the IND. 
 
2.  Genome Editing Component Manufacture and Testing 
 
GE components can be administered in vivo (e.g., using nanoparticles, plasmids, 
or viral vectors), or they can be used to modify cells ex vivo. When administered 
in vivo in the form of DNA, RNA and/or protein via nanoparticles, the GE 
components are considered the active pharmaceutical ingredients or drug 
substances. A GE component in its final formulation for in vivo administration is 
generally considered a DP. For example, when the GE components are expressed 
in vivo by directly administered plasmids or vectors, the plasmid or vector in its 
final formulation encoding the GE component is considered the DP. If used to 
modify cells ex vivo, the GE components are considered critical components for 
the manufacture of the final product because without these components, the 
resulting cell product would not have the same pharmacological activity. 
 
The recommendations in section B.2 of this guidance apply to GE components 
used routinely in the manufacture of each DP lot, either as drug substances or 
critical components. If the GE component is used only once, for example in the 
manufacture of a master cell bank (MCB) that is then used in routine DP 
manufacture, the extent of information and release testing for the GE components 
may be reduced but should still be sufficient to support the quality of the GE 
component and safety of the starting material (e.g., MCB). In these cases, we 
recommend discussing the information available for the GE components and the 
GE component control strategy with FDA early in product development. 
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Detailed descriptions of how each GE component is manufactured, purified and 
tested must be provided in the IND (21 CFR 312.23(a)(7)). We recommend a 
description of the manufacturing process and any in-process controls for each GE 
component include a flow diagram(s) and a detailed narrative. We recommend 
that sponsors provide lists of the raw materials/reagents used during these 
processes and representative certificates of analysis. Summaries of the following 
should also be provided in the IND for each GE component manufacturing site: 
  The quality control and quality assurance programs in place; 
  Procedures in place to ensure component tracking and segregation; 
  Procedures in place to prevent, detect and correct deficiencies in the 
manufacturing process; and 
  Procedures for shipping of the GE component from the component 
manufacturing site to the final product manufacturing site. 
 
The information regarding manufacturing and testing of the GE component is 
needed even if the GE component is manufactured and/or tested by a contract 
manufacturer (Ref. 3) and may be incorporated into the IND by cross-reference if 
it is present in an existing IND or Master File (Ref. 4). For most Phase 1 clinical 
investigations, sponsors should follow the recommendations in FDAs Guidance 
for Industry: CGMP for Phase 1 Investigational Drugs for the manufacture of 
these components (Ref. 5).  However, for later Phase studies and for licensure, 
GE component manufacturing must comply with CGMP under section 
501(a)(2)(B) of the Federal Food, Drug, and Cosmetic Act (FD&C Act), with 
particular consideration for control of raw material/reagent quality, manufacturing 
process, and analytical methods. 
We recommend each GE component be tested appropriately. GE components 
should be evaluated for sterility, identity, purity and activity, in a phase 
appropriate manner. Additional testing, such as that for process residuals, should 
be included, depending on the manufacturing process. Descriptions of the 
analytical procedures utilized for GE component testing, including the sensitivity 
(e.g., limit of detection/quantitation) and specificity of the procedures, should be 
included in the IND. Sponsors should also outline any in-process testing 
performed to ensure the quality of the GE components, as appropriate. 
 
We recommend GE components be assessed for stability if being stored. Outlines 
of stability study protocols and any available stability data should be provided in 
the IND. Stability studies should be conducted on all applicable GE component 
presentations (e.g., lyophilized and reconstituted materials). Stability studies 
should include stability-indicating tests assessing critical product attributes, such 
as purity and activity, that may be affected during storage. 
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3.  Drug Product Manufacture and Testing 
An IND should contain a detailed description of the DP manufacturing process, 
and any in-process controls. We recommend this description include a flow 
diagram(s) as well as a detailed narrative. We recommend lists of the reagents 
used during manufacture and certificates of analysis be provided. Please note that 
for DP intended to be sterile, but that cannot be terminally sterilized, sponsors 
should provide descriptions of the procedures in place to assure aseptic 
processing. 
 
An IND should also contain a detailed description of the testing plan for the DP. 
To ensure that the DP meets acceptable limits for identity, strength,2 quality and 
purity as defined in 21 CFR 312.23(a)(7)(iv), the DP testing plan should 
incorporate evaluations that address any safety concerns introduced due to the 
manufacturing process or identified during nonclinical studies. For human GE 
products consisting of ex vivo-modified cells, DP testing should include 
determination of GE efficiency (e.g., the degree of editing at the on-target site) 
and may include an assessment of specificity (e.g., the degree of editing at off- 
target sites). The DP should also be tested for sterility. 
 
Sponsors should describe in detail the analytical procedures used for testing the 
DP. The descriptions should include the accuracy, precision, sensitivity, and 
specificity of the assay (as appropriate for the stage of development), as well as 
any controls and, if applicable, reference materials used to ensure proper assay 
performance. 
 
To help assure product safety, the DP specifications should be developed based 
on the starting materials, manufacturing process, desired final product attributes 
and nonclinical studies. As discussed, the DP may consist of GE components 
intended for in vivo administration or may be composed of ex vivo -modified 
cells. In the following sections, we provide recommendations pertaining 
specifically to each of these human GE DP types: 
 
i.  In vivo-administered Human Genome Editing Drug Products 
 
If the GE components will be expressed by a plasmid or viral vector 
that is administered to patients in vivo, the plasmid/vector in its final 
formulation is considered the DP and thus a complete description of 
plasmid/vector manufacturing and testing should be provided in the 
IND (Ref. 2). 
 
2 For purposes of this guidance, strength is the equivalent of potency. As defined in 21 CFR 600.3(s), the word 
potency is interpreted to mean the specific ability or capacity of the product, as indicated by appropriate laboratory 
tests or by adequately controlled clinical data obtained through the administration of the product in the manner 
intended, to effect a given result. During the IND stage, sponsors must submit data to assure the identity, quality, 
purity and strength (21 CFR 312.23(a)(7)(i)) as well as stability (21 CFR 312.23(a)(7)(ii)) of products used during 
all phases of clinical study. Biological products regulated under section 351 of the PHS Act must meet prescribed 
requirements of safety, purity and potency for BLA approval (21 CFR 601.2). 
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If the GE components will be administered using nanoparticles, a 
detailed description of the nanoparticle formulation, a description of 
the manufacture of the nanoparticle components, as well as the DP, 
should be provided in the IND. A description of the tests performed 
on each nanoparticle component as well as on the DP should also be 
provided. Please note that release testing of the DP should include 
assays to evaluate the efficiency of incorporation of each GE 
component into the nanoparticles. 
 
We recommend sponsors develop potency assays to measure multiple 
aspects of activity for in vivo human GE DPs. For early phase studies, 
potency assays evaluating the ability of the GE components to perform 
the desired genetic sequence modification may be adequate. However, 
for studies intended to provide primary evidence of effectiveness to 
support a marketing application, potency assays should include an 
assessment of the intended downstream biological modification (e.g., 
corrected cellular function). We recommend that, whenever possible, 
the potency assays be performed in the target cells or tissues (or a 
representative surrogate). We also recommend inclusion of such a 
potency assay in the DP stability studies. Additional information on 
the development of appropriate potency tests can be found in FDAs 
Guidance for Industry: Potency Tests for Cellular and Gene Therapy 
Products (Ref. 6). 
ii. Ex vivo-modified Human Genome Editing Drug Products 
When describing the manufacturing processes for ex vivo-modified 
human GE DPs, sponsors should clearly indicate the timing of the GE 
step within the overall manufacturing process. Descriptions of process 
controls and in-process testing should also be included for critical 
steps that may have significant impact on the efficiency or specificity 
of editing (e.g., RNP formation step in the case of CRISPR-mediated 
editing). Acceptance criteria or limits should be provided and 
justified. 
 
Release testing of ex vivo-modified human GE DPs should include 
evaluation of on-target editing efficiency and the total number (or 
frequency) of genome-edited cells. Additional characterization of the 
editing events occurring at the on-target site should also be performed. 
Assessments of off-target editing frequency, intrachromosomal and 
interchromosomal rearrangements, and residual GE components may 
also need to be included for release of the DP based on the outcomes 
of nonclinical studies. We also recommend that the number of edited 
cells or the frequency of GE be monitored during stability testing of ex 
vivo-modified human GE DP. 
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When establishing potency tests for ex vivo -modified human GE DP, 
we recommend assays be developed that measure the properties of the 
cells and the intended downstream biological modifications resulting 
from GE. For example, we recommend that potency assays for a 
genome edited CD34+ hematopoietic stem/progenitor cell product 
measure both the stem/progenitor cell activity and the biologically 
relevant outcome of the GE. For early phase studies, confirming the 
desired genetic sequence modification may be adequate to support 
potency of the DP. However, for studies intended to provide primary 
evidence of effectiveness to support a marketing application, potency 
assays should include an assessment of the intended downstream 
biological modification (e.g., corrected cellular function). We also 
recommend inclusion of such a potency assay in the DP stability 
studies. In some instances, surrogate potency tests may be acceptable; 
however, if proposing a surrogate potency assay, it is critical that the 
data provided supports a correlation between the output of the 
surrogate potency test and the biologically relevant outcome of the GE 
(Ref. 6). 
 
Please note that if the ex vivo-modified human GE DP is an allogeneic 
human cell product, where a product lot is meant to treat multiple 
patients, additional DP testing and establishment of acceptance criteria 
may be appropriate. For example, additional adventitious agent 
testing, stringent acceptance criteria for the number of potentially 
alloreactive lymphocytes and absence of aberrant growth (i.e., if the 
DP is an allogeneic T cell product) should be included in lot release 
testing. Additional information on allogeneic products, including 
donor eligibility and testing recommendations for cell banks 
originating from allogeneic cells or tissues, are discussed in the GT 
CMC Guidance (Ref. 3) 
 
Additional in-process, lot release, and characterization testing may be 
needed for more complex products (e.g., products incorporating 
multiple rounds of genome editing or the creation of multiple cell 
banks). Also, the timing and type of testing may depend on when the 
GE process is performed in manufacturing. For example, if a genome 
edited MCB is used to produce the DP without additional GE steps, 
some testing may be able to be performed on the MCB. 
 
 
IV.  CONSIDERATIONS FOR NONCLINICAL STUDIES 
 
The overall objectives of a nonclinical program for a human GE product are generally the same 
as those described for gene therapy products in FDAs Guidance for Industry: Preclinical 
Assessment of Investigational Cellular and Gene Therapy Products (Ref. 7) (Preclinical 
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Assessment Guidance). These objectives include: 1) identification of a pharmacologically- 
active dose level range; 2) recommendations for an initial clinical dose level, dose -escalation 
scheme, and dosing regimen; 3) establishment of feasibility and reasonable safety of the 
proposed clinical route of administration (ROA); 4) support for the target patient population; 
and 5) identification of potential toxicities and physiologic parameters that help guide clinical 
monitoring and risk mitigation plans. More details for these general considerations in 
nonclinical studies are available in the above noted guidance (Ref. 7). The following general 
elements should be incorporated into the nonclinical development program for a human GE 
product: 
  Nonclinical proof-of-concept (POC) studies should be conducted to establish feasibility 
and support the scientific rationale for administration of the investigational human GE 
product in a clinical trial. 
 
  The use of in vitro models (examples include, but are not limited to, cultured 
o
cells, tissues, explants, organoids, etc.) should be considered for evaluating the 
activity of a human GE product in the target cell type(s) for genomic 
modification. 
 
  The animal species and/or models selected for in vivo studies should demonstrate 
o
a biological response to the human GE product (see section IV.A of this guidance 
for further discussion). Given the differences in the genomic sequences between 
humans and animals, analysis of the biological activity may be done in a species- 
specific context (e.g., using a surrogate product), as appropriate. 
 
  Nonclinical safety studies should be designed to identify potential risks associated with 
administration of the GE product. Toxicities may be related to the human GE product, 
delivery method, and/or modification of the genomic structure. 
 
  The safety assessment should include identification and characterization of on- 
o
and off-target editing, chromosomal abnormalities, and their biological 
consequences. 
 
  In vivo nonclinical safety studies for a human GE product (or surrogate product) 
o
should incorporate elements of the planned clinical trial (e.g., dose level range, 
ROA, delivery device, dosing schedule, study endpoints, concomitant therapies, 
etc.), to the extent feasible. Study designs should be sufficiently comprehensive 
to permit identification, characterization, and quantification of potential local and 
systemic toxicities, their onset (i.e., acute or delayed) and resolution, and the 
effect of dose level on these findings. 
  Assessment of biodistribution should be conducted to characterize the distribution, 
persistence, and clearance of the GE product, any expressed GE components in vivo, 
editing activity in target and non-target tissues, and the potential for inadvertent germline 
modification. These evaluations may be conducted independently or in conjunction with 
POC and/or safety studies. 
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Specific recommendations for the assessment of activity and safety of a human GE product are 
as follows: 
A.  Product Evaluated in Nonclinical Studies 
  The intended clinical GE product should be evaluated in the definitive POC and 
safety studies, as feasible. 
  Due to differences in the genomic sequences between animals and humans, POC 
and/or safety studies may warrant the use of a surrogate GE product (e.g., 
substitution of the human elements including GE components, promoter(s), and 
transgene(s) with the respective species-specific elements in the GE product) in 
situations where administration of the investigational human GE product would 
not be informative. We recommend sponsors provide scientific justification for 
the administration of a surrogate GE product, and establish the biological 
relevance of the surrogate compared to the human GE product.3 
  For ex vivo-modified human GE products, the clinical cell source/type should be 
used for the definitive nonclinical studies. Scientific justification should be 
provided if an alternative cell source/type is used in any studies. 
  Each human GE product lot evaluated in the nonclinical studies should be 
characterized according to appropriate specifications, consistent with the stage of 
product development. Retention of adequate samples from each nonclinical lot is 
recommended in case future reanalysis is warranted. 
B.  Assessment of Activity 
 
Pharmacology studies conducted to assess the activity profile of a human GE product 
can address important considerations such as the following: 
  Specificity and efficiency of editing in target and non-target cells; 
 
  Functionality of the corrected or expressed gene product (e.g., protein, RNA), if 
applicable; 
  Editing efficiency required to achieve the desired biological activity or 
therapeutic effect; and 
  Durability of the genomic modification and resulting biological response 
 
3 The nonclinical program for any investigational product should be individualized with respect to scope, 
complexity, and overall design. We support the principles of the 3Rs, to reduce, refine, and replace animal use in 
testing when feasible. Proposals, with justification for any potential alternative approaches (e.g., in vitro or in silico 
testing), should be submitted during early communication meetings with FDA. We will consider if such an 
alternative method could be used in place of an animal test method. 
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C.  Assessment of Safety 
Comprehensive safety studies should be conducted to characterize the risks of a human 
GE product. These studies can include the following: 
  Identification of on- and off-target editing events, including the type, frequency, 
and location. 
 
  Multiple methods (e.g., in silico, biochemical, cellular-based assays) that 
o
include a genome-wide analysis are recommended to reduce bias in 
identification of potential off-target sites. When possible, the analysis 
should be performed with the relevant human cell type(s) obtained from 
multiple donors. For in vivo GE products, the analysis should also 
include the major cell types in which editing events are detected. 
  Verification of off-target sites should be conducted using methods with 
o
adequate sensitivity to detect low frequency events. For ex vivo GE 
products, the final clinical product obtained from multiple donors should 
be evaluated. For in vivo GE products, the analysis should also include 
the major cell types in which editing events are detected. 
 
  Appropriate controls should be included to confirm the quality of the 
o
assay and to assure interpretability of the results and its suitability for the 
intended use. 
 
  Assessment of genomic integrity, including chromosomal abnormalities, 
insertions or deletions, and potential oncogenicity or insertional mutagenesis. 
This may include assessment for clonal expansion and/or unregulated 
proliferation of edited cells. 
  Evaluation of the biological consequences associated with on- and off-target 
editing, including, but not limited to, viability and function of the edited cells 
(e.g., differentiation capacity of progenitor cells). 
  Assessment of immunogenicity of the GE components and expressed 
transgene(s). 
 
V.  CONSIDERATIONS FOR CLINICAL STUDIES 
 
We recommend that clinical development programs of human GE products address both the risks 
associated with the gene therapy product itself as well as the additional risks associated with the 
GE, including off-target editing and unintended consequences of on-target editing, which may be 
unknown at the time of product administration. Clinical trial design should include an 
appropriately-defined patient population, an efficient and safe approach to product 
administration (including data-based dosing, dose schedule, and treatment plan), adequate safety 
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monitoring, and appropriate safety and efficacy endpoints. Additionally, long term follow-up is 
recommended for clinical trial subjects receiving human GE products for evaluation of clinical 
safety. In general, the overall study design, assessment of adverse events (AEs), and subject 
follow-up plans should be well described in the IND. The overall considerations for clinical trial 
design for GE products are similar to those outlined for other cellular and gene therapy products 
(Ref. 8) and are briefly described in section V.A-F of this guidance. 
A.  Study Population 
Selecting the appropriate study population ensures maximum benefit, while minimizing 
the potential risk to subjects. We recommend the choice of study population be well 
supported based on the product MOA and study rationale, along with balancing the 
potential risks of the product. Human GE products may have significant risks and an 
uncertain potential for benefits. Therefore, first-in-human trials involving such products 
generally should enroll only subjects for whom no other treatment options are available 
or justified. Factors to consider in determining the study population include: 
  The MOA of the product in the context of a specific disease; 
  The anticipated duration and magnitude of therapeutic benefit; 
  The availability, safety, tolerability, and effectiveness of alternative 
therapeutic options for the patient population; 
  Subjects with severe or advanced disease may be more willing to accept the 
potential risks of an investigational human GE product. However, these 
subjects may be predisposed to experiencing more AEs or be receiving 
concomitant treatments, which could make the safety or effectiveness data 
difficult to interpret. Therefore, in some instances, subjects with less 
advanced or more moderate disease may be appropriate for inclusion in first- 
in-human clinical studies. 
B.  Dose and Dose Schedules 
 
Adopting safe and effective product delivery methods is important for minimizing any 
potential AEs related to product delivery to target tissues. Both the delivery and the 
proposed dose schedules should be supported by comprehensive nonclinical data and, 
where available, guided by previous clinical experience from similar products, including 
cellular or gene therapy products that may or may not have been genome edited. 
Additional aspects of dose and regimen for clinical trials evaluating human GE products 
are similar to those for other cellular and gene therapy products and can be found in 
section IV.D of FDAs Considerations for the Design of Early-Phase Clinical Trials of 
Cellular and Gene Therapy Products; Guidance for Industry (Ref. 8). 
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C.  Treatment Plan 
We recommend that any risk(s) anticipated in association with the GE product be 
mitigated by staggered subject enrollment, with a specified time interval between product 
administration to sequential subjects within and between cohorts. For first-in-human 
studies, the staggering interval should be of sufficient duration to detect acute and 
subacute AEs prior to treating additional subjects at the same dose, or prior to increasing 
the dose in subjects treated subsequently. The staggering interval should also take into 
account the expected duration of activity of the human GE product. 
 
Selection of study cohort size depends on the size of the proposed patient population and 
the amount of acceptable risk in that study population for the GE product. In addition, 
other considerations, such as assessments of tolerability, feasibility, and pharmacologic 
activity may influence choice of cohort size. Additional cohort size considerations are 
outlined in section IV.E.2 of FDAs Considerations for the Design of Early-Phase 
Clinical Trials of Cellular and Gene Therapy Products; Guidance for Industry (Ref. 8). 
 
D.  Monitoring and Follow-Up 
 
1.  Assessment of Product-Related Adverse Events 
 
A thorough safety monitoring strategy, with a well-defined toxicity grading 
system, and a toxicity management plan is crucial for clinical trials evaluating 
human GE products. Specific consideration should be given for adequate 
monitoring of any off-target editing and adequate assessment of the outcomes of 
off-target editing and unintended consequences of on-target editing as anticipated 
from nonclinical studies. Additional monitoring should capture AEs related to 
aberrant cellular and chromosomal changes, immunogenicity, and tumorigenicity. 
The product-related adverse event monitoring plan and toxicity grading and 
management strategy should be described in the clinical protocol. 
 
Applicable reporting requirements outlined in 21 CFR 312.32 for adverse 
experiences associated with the use of the human GE product must be followed. 
Additional information concerning good clinical practice can be found in FDAs 
E6(R2) Good Clinical Practice: Integrated Addendum to ICH E6(R1); Guidance 
for Industry (Ref. 9). 
 
2.  Long Term Follow-Up 
 
Prior to enrolling subjects in a clinical study evaluating a human GE product, they 
should be asked to provide voluntary, informed consent to long term follow-up 
(LTFU). As discussed, the long-term safety and therapeutic effects of intended 
on-target editing, as well as off-target editing and unintended editing at the on- 
target loci may be unknown at the time of GE product administration. Therefore, 
we recommend that sponsors conduct LTFU for up to 15 years after product 
administration, as outlined in FDAs Long Term Follow-Up After Administration 
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of Human Gene Therapy; Guidance for Industry (Ref. 10).  FDA also 
recommends that a plan be provided for follow-up, including funding, in the event 
the sponsor ceases to operate or decides to inactivate, transfer, or withdraw the 
IND before completion of the long term follow up. 
E.  Study Endpoints 
We recommend that study endpoints be based on the proposed indication. For efficacy 
studies, the primary endpoint should also reflect a clinically meaningful effect of the GE 
product on how patients feel, function, or survive. The experience gained from early- 
phase clinical studies can help guide the selection of a primary endpoint for late-phase 
studies. Further information may be obtained from FDAs Guidance for Industry: 
Providing Clinical Evidence of Effectiveness for Human Drugs and Biological Products 
(Ref. 11). Additionally, accelerated approval may be an appropriate pathway for 
approval of a human GE product intended to treat a serious or life-threatening disease or 
condition, where there is a lack of available alternative treatments. Under accelerated 
approval, a surrogate endpoint or marker (e.g., a laboratory measurement) that is 
reasonably likely to predict clinical benefit will need to be selected; alternatively, an 
intermediate clinical endpoint that that is reasonably likely to predict an effect on 
irreversible morbidity or mortality or other clinical benefit will need to be selected.4 
Approval under this pathway may be subject to a requirement to conduct an appropriate 
postapproval study or studies to verify and described the predicted effect.5 FDA is 
supportive of the use of accelerated approval for GE products and encourages sponsors to 
discuss the potential eligibility of a GE product for such program, including the proposed 
surrogate endpoints or intermediate clinical endpoints, early in development of the 
clinical trial. FDAs Guidance for Industry: Expedited Programs for Serious Conditions 
 Drugs and Biologics (Ref. 12). 
 
F.  Special Considerations for Research Involving Children 
 
When possible, clinical studies should enroll individuals who can understand and consent 
to the study procedures and risks. For clinical investigations involving children, 
associated with greater than minimal risk, a reviewing Institutional Review Board must 
find, among other things, that these risks are justified by the anticipated direct clinical 
benefit to the children (21 CFR 50.52). Such prospect of direct benefit should be 
evidence-based (e.g., from adult humans or appropriate animal models). Therefore, it is 
important to enroll at least an initial cohort of adult subjects, whenever feasible, to obtain 
preliminary data on safety and feasibility, bioactivity, and preliminary efficacy to support 
enrollment of children. If enrollment of children is justified based on the benefit-risk 
assessment, then an effort should be made to enroll adolescents prior to enrollment of 
younger children and infants, as appropriate for the specific disease of interest. See the 
draft guidance entitled Ethical Considerations for Clinical Investigations of Medical 
Products Involving Children; Draft Guidance for Industry, Sponsors, and IRBs, 
 
 
4 See FD&C Act section 506(c)(1)(A). 
5 See FD&C Act section 506(c)(2)(A). 
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September 2022 (Ref. 13) for additional recommendations on including pediatric subjects 
in cell and gene therapy trials. 
 
VI.  COMMUNICATION WITH FDA 
We recommend sponsors of human GE products communicate with the Office of Therapeutic 
Products (OTP) in the Center for Biologics Evaluation and Research (CBER) early in product 
development, before submission of an IND, to discuss the product-specific considerations for 
transitioning these products to the clinical phase of product development. There are different 
meeting types that can be used for such discussions, depending on the stage of product 
development and the issues to be considered. These include pre-IND meetings prior to 
submission of the IND (Ref. 14), and INitial Targeted Engagement for Regulatory Advice on 
CBER producTs (INTERACT) meetings, which can be used earlier in development to discuss 
issues such as nonclinical development or manufacturing, so that sponsors can obtain non- 
binding regulatory advice.6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6 For additional information about INTERACT meetings with OTP, please see https://www.fda.gov/vaccines-blood- 
biologics/cellular-gene-therapy-products/otp-interact-meeting. 
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VII.  REFERENCES 
1.  Cox D.B., et al. Therapeutic genome editing: prospects and challenges. Nature Medicine, 
2015. 21(2):121-131. 
2.  Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy 
Investigational New Drug Applications (INDs); Guidance for Industry, January 2020, 
https://www.fda.gov/media/113760/download. 
3.  Contract Manufacturing Arrangements for Drugs: Quality Agreements; Guidance for 
Industry, November 2016, https://www.fda.gov/media/86193/download. 
 
4.  Drug Master Files: Draft Guidance for Industry, October 2019, 
https://www.fda.gov/media/131861/download.* 
 
5.  Guidance for Industry: CGMP for Phase 1 Investigational Drugs, July 2008, 
https://www.fda.gov/media/70975/download. 
 
6.  Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products, January 
2011, https://www.fda.gov/media/79856/download. 
 
7.  Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene 
Therapy Products, November 2013, https://www.fda.gov/media/87564/download. 
 
8.  Considerations for the Design of Early-Phase Clinical Trials of Cellular and Gene 
Therapy Products; Guidance for Industry, June 2015, 
https://www.fda.gov/media/106369/download. 
 
9.  E6(R2) Good Clinical Practice: Integrated Addendum to ICH E6(R1); Guidance for 
Industry, May 2018, https://www.fda.gov/media/93884/download. 
 
10.  Long Term Follow-Up After Administration of Human Gene Therapy Products; 
Guidance for Industry, January 2020, https://www.fda.gov/media/113768/download. 
 
11.  Guidance for Industry: Providing Clinical Evidence of Effectiveness for Human Drugs 
and Biological Products, May 1998, https://www.fda.gov/media/71655/download. 
 
12.  Guidance for Industry: Expedited Programs for Serious Conditions  Drugs and 
Biologics, May 2014, https://www.fda.gov/media/86377/download. 
 
13.  Ethical Considerations for Clinical Investigations of Medical Products Involving 
Children; Draft Guidance for Industry, Sponsors, and IRBs, September 2022, 
https://www.fda.gov/media/161740/download.* 
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14.  Draft Guidance for Industry: Formal Meetings Between the FDA and Sponsors or 
Applicants, December 2017. https://www.fda.gov/media/109951/download.* 
 
* When finalized, this guidance will represent FDAs current thinking on this topic. 
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APPENDIX 
Abbreviations and Acronyms 
 
Term  Description 
AAV  Adeno-Associated Virus 
AE  Adverse Event 
Cas  CRISPR-associated 
CBER  Center for Biologics Evaluation and Research 
CGMP  Current Good Manufacturing Practice 
CMC  Chemistry, Manufacturing and Controls 
CRISPR  Clustered Regularly Interspaced Short Palindromic Repeat 
DNA  Deoxyribonucleic Acid 
DP  Drug Product 
FDA  Food and Drug Administration 
GE  Genome Editing 
HDR  Homology Directed Repair 
ICH  International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 
IND  Investigational New Drug 
Indels  Insertions or Deletions 
INTERACT  INitial Targeted Engagement for Regulatory Advice on CBER producTs 
LTFU  Long Term Follow-Up 
MOA  Mechanism of Action 
NHEJ  Non-Homologous End-Joining 
OTP  Office of Therapeutic Products 
PHS  Public Health Service 
POC  Proof-of-Concept 
RNA  Ribonucleic Acid 
RNP  Ribonucleoprotein Complex 
ROA  Route of Administration 
TALEN  Transcription Activator-Like Effector Nuclease 
ZFN  Zinc Finger Nuclease 
 
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