Understanding the FDA’s latest guidance on safety assessment of genome editing therapies using next-generation sequencing

The U.S. Food and Drug Administration’s (FDA’s) Center of Biologics evaluation and Research (CBER) has recently released an updated draft guidance providing guidelines on the use of next-generation sequencing (NGS) for safety assessment of genome editing (GE) in human gene therapies¹.  

What are the new FDA recommendations 

The initial draft guidance on genome editing therapies covered broader areas of development for gene editing therapies². The current draft guidance specifically focuses on NGS as a technique and tool to assess safety in gene editing applications and outlines the agency’s expectations related to NGS assay planning, development and data analysis. The current guidance encompasses considerations for some newer classes of gene editing, epigenetic and RNA editing products, which were not included in the 2024 guidance. 

GE at off-target (i.e., unintended) site(s) and/or chromosomal translocations are a potential safety risk since such edits can be deleterious to normal cell function. Therefore, an adequate assessment to evaluate off-target editing risk and impact on chromosomal integrity of human GE products is crucial to minimize unintended changes in the target cells or tissues. In a wide range of GE products, computational and/or NGS-based methods are used to assess off-target editing activity. The current guidance provides FDA’s recommendations pertaining to NGS-based studies assessing off-target editing activity, NGS-based chromosomal integrity assessment studies, on-target edit site assessments, sequencing methods, considerations for sample selection, and reporting the findings to FDA. 

The guidance specifies that all the required studies on non-clinical off-target editing and chromosomal translocation should be performed based on the recommendations made in this guidance and completed prior to the submission of an original IND application. Moreover, the guidance outlines the FDA’s NGS-specific expectations at different stages of sponsor interactions before IND submission, i.e., the INTERACT and pre-IND meetings. This approach will be particularly helpful to sponsors as it provides a specific framework of FDA expectations related to NGS as an assay for safety assessment.  

Next-generation sequencing 

Next-generation sequencing (NGS) refers to more recent DNA and RNA sequencing technologies that offer benefits over previous methods. The method allows for high-throughput, massively parallel, deep sequencing of nucleic acid fragments, often used to identify genetic variation associated with diseases or other biological phenomena. NGS is subdivided into four main steps: 

1. Isolation and purification of nucleic acid 
2. Preparation of an NGS library 
3. Sequencing 
4. Data analysis 

For assessing editing at on- and off-target sites that result in changes to a short stretch of DNA (e.g., ≤50-bp), the agency recommends a sequencing strategy utilizing short-read sequencing-based methods may be adequate. However, evaluation of longer stretches of sequence for the presence of large insertions or large deletions may require implementing a sequencing strategy that uses long-read sequencing-based methods that can detect such changes. 

In the current guidance, sponsors are encouraged to provide data generated in-house, such as representative data from engineering NGS runs, and/or relevant information from peer-reviewed publications, to support the adequacy and the sensitivity of sequencing depth and to support their strategy to detect low frequency off-target editing events. 

A brief description of each step of sequence analysis including information on acceptance criteria applied at each step (as applicable) while processing the sequencing data. The acceptance criteria information should include information on sequencing quality acceptance criteria, sequencing depth acceptance criteria, and alignment metric acceptance criteria. However, whether acceptance criteria need to be set early in development is not clear from the draft guidance 

Samples used for on- and off-target site analysis should be edited with appropriate parameter(s) so that the resulting on-target editing rates are comparable to the rates proposed in the final drug product. 

Ex-vivo products 

If the GE strategy is aimed at correcting a mutation in patient cells, then the agency recommends that sponsors use patient-derived samples or normal cells that are engineered to harbor the mutation. This approach will facilitate reporting of editing rates at the on-target site. Importantly, the sponsor may use healthy donor samples where appropriate and provide scientific justification to support the adequacy of the approach. If the sponsor chooses a cell type that is different from the cell type they intend to edit, they should provide scientific justification to support the adequacy of their sample selection strategy. 

Human genetic variation 

The draft guidance points out that nucleotide variations, which are an inherent in the human genome, can reduce mismatches between target sequence and an off-target genomic edit site making, which could allow the sequence recognition domain of a gene editor, thereby increasing off-target risk. Specific considerations for in silico off-target analysis methods to adequately account for human genetic variation are discussed, including: selection of a human genetic variation database with variant information from healthy and patient populations, with justification for the specific database based on disease prevalence in the intended population and knowledge of disease-causing variations; and an in silico off-target nomination strategy for diseases with well-characterized prevalence that uses population stratification to select variants. 

Meetings with the FDA 

The guidance provides expected minimum information for INTERACT meetings as well as pre-IND stages of development. This is an important inclusion as it provides sponsors with a clear framework of the FDA’s expectations related to NGS at INTERACT meetings and pre-IND stages - to help sponsors prepare for their IND submission. For example, information on the editor used in the assay and the mechanism of action are expected at the INTEARCT meeting stage, while sequencing strategies with specifics about data quality, depth and alignment, along with samples used for each analysis, are expected at the pre-IND stage. 

The FDA has previously issued specific guidance on the design, development and analytical validation of NGS for whole exome human DNA sequencing (WES) or targeted human DNA sequencing intended to aid in the diagnosis of symptomatic individuals with suspected germline diseases or other conditions.³ 

Next steps 

For sponsors developing GE products, Parexel's regulatory experts provide deep insights into how to apply the FDA’s guidance to ensure agency alignment with your safety assessment approach and maximize approval success. Our team brings extensive CMC, nonclinical and clinical experience in cell and gene therapy assessments, enabling the translation of complex regulatory expectations into actionable development strategies that strengthen regulatory submissions and build compelling, science-driven narratives.   

Please contact us; we are always available for a conversation.  

Start a conversation

References 

1. FDA Guidance for Industry, Safety Assessment of Genome Editing in Human Gene Therapy Products Using Next-Generation Sequencing, April 2026 
2. FDA Guidance for Industry, Human Gene Therapy Products Incorporating Human Genome Editing, January 2024 
3. Considerations for Design, Development, and Analytical Validation of Next Generation Sequencing (NGS) - Based In Vitro Diagnostics (IVDs) Intended to Aid in the Diagnosis of Suspected Germline Diseases | FDA, April 2018 

Return to Insights Center