FDA drafts guidance on using next-generation sequencing to assess gene therapy safety

The US Food and Drug Administration (FDA) has released draft guidance on how sponsors can utilize next-generation sequencing (NGS) methods in nonclinical studies to assess the safety risks associated with gene therapy products. This guidance aims to support the submission of investigational new drug applications (INDs) and biologics license applications (BLAs).

The guidance was developed by the Office of Therapeutic Products (OTP) within the Center for Biologics Evaluation and Research (CBER). The document is meant to build on a previous guidance issued in January 2024, which addresses the nonclinical, clinical, and chemistry, manufacturing, and controls (CMC) considerations for human gene therapy products that incorporate human genome editing. (RELATED: FDA: Genome editing therapies may use accelerated approval therapy, Regulatory Focus 30 January 2024)

The guidance notes that the clinical development program for genome edited (GE) products “should address both the risks associated with the gene therapy product itself as well as the additional risks associated with GE, including off-target editing and unintended changes to the genome for therapies targeting genetic diseases, including individualized therapies.” 

“Genome editing holds extraordinary promise for treating previously incurable genetic diseases, and today’s announcement represents the FDA's forward approach to drive innovation and advance the development of genome editing therapies. This guidance provides sponsors with clear, scientifically-grounded recommendations for evaluating off-target editing risks using state-of-the-art sequencing technologies. We are serious about moving this ball forward,” said FDA Commissioner Marty Makary.

The guidance explains that there are various gene editing techniques available for editing cells either outside the body (ex vivo) or inside the body (in vivo). These techniques can be applied to cells sourced from either patients or healthy donors. Additionally, gene editing can be performed in vivo using messenger RNA (mRNA) or DNA-based vectors. However, in both cases, gene editing at unintended sites or chromosomal translocations poses potential safety risks, as such edits may negatively impact normal cell function.

To reduce these risks, FDA advises sponsors in the guidance to conduct a complete analysis to evaluate the risks of off-target editing and its effects on the chromosomal integrity of gene editing products.

Companies should use “an appropriate sequencing strategy that meets the goals of the on-target and off-target edit site analysis method being implemented.”

For example, in assessing the editing of off-target sites that result in changes to a short stretch of DNA, a sequencing strategy using short-read sequencing-based methods may be best. However, evaluating longer stretches of sequence may require implementing a sequencing strategy that uses long-read sequencing-based methods.

The guidance notes that “sequencing should be performed using an adequate amount of the input material (genomic DNA, cell number, or RNA, as applicable) at a depth that allows detection of potential off-target editing events occurring at frequencies or edit rates that are usually lower than the on-target edit rate.  Sponsors should 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.”

FDA recommends that for GE techniques known to cause DNA double-strand breaks, as well as medium to large insertions or deletions that could affect the genomic integrity of cells after editing, sponsors should perform a “sensitive and quantitative” assessment of genomic integrity in edited cells using NGS.

The guidance states that in these situations, “the sponsor may consider choosing targeted sequencing and/or primer-based sequencing or other genome-wide NGS-based methods to detect low frequency translocation events and report chromosomal translocation rate(s) between on-target genomic site(s) where double strand break events occurred during GE.”

The deadline to comment is 15 June. Submit comments to www.regulations.gov. Refer to docket number FDA-2026-D-1255.

Draft guidance