Industry wants more clarity on NGS for assessing gene therapy safety
The pharmaceutical industry has largely endorsed the US Food and Drug Administration's (FDA) guidance on next-generation sequencing (NGS) for assessing the safety of human gene therapy products. However, they have requested clarification on several points, including off-target data, nomination methods, and additional information on how to assess these products following any manufacturing changes.
These comments were made in response to FDA’s draft guidance on NGS-based methods used in nonclinical studies to support initiating clinical trials of investigational human genome editing (GE) products. The guidance was released in April 2026 and the agency received more than 30 comments by the deadline. (RELATED: FDA drafts guidance on using next-generation sequencing to assess gene therapy safety, Regulatory Focus 14 April 2026)
The guidance received support from various pharmaceutical industry groups, including the Biotechnology Innovation Organization (BIO), the Pharmaceutical Research and Manufacturers of America (PhRMA), and Eli Lilly.
PhRMA said that it “supports FDA’s continued efforts to advance a scientifically rigorous and risk-based framework for the development of genome editing therapies. The Draft Guidance provides valuable recommendations regarding the application of next-generation sequencing (NGS) technologies to evaluate potential off-target editing and genome integrity risks associated with genome editing products. The guidance also appropriately recognizes the diversity of genome editing modalities and the importance of tailoring analytical approaches to product-specific characteristics.”
BIO wrote that it “appreciates FDA’s forward‑looking draft guidance as it represents a pointed advancement for the field and provides much‑needed direction around NGS‑based safety assessments for genome editing products that supplements the January 2024 final genome editing guidance. We strongly support the clarity provided on sequencing modality selection, bioinformatics transparency, variant‑aware analysis, and the risk‑based approach to chromosomal integrity. The guidance appropriately expands scope to include epigenome and transcriptome editors and provides a necessary roadmap across pre-IND, IND, and BLA stages.”
Lilly also said that it “applauds this Draft Guidance and views it as an important step toward the kind of predictable, transparent, and science-based regulation that the genetic medicines field needs. We appreciate the Agency grounding their analytical expectations in the mechanism of action of the editor rather than applying uniform requirements across fundamentally different editing chemistries.”
More clarity on off-target editing data
BIO and Lilly stated that more information is needed regarding the off-target editing data of in vivo products. Off-target editing refers to unintended and accidental modifications to the genome that occur when gene-editing tools, such as CRISPR-Cas9, bind to and cut DNA at incorrect sites—specifically, those with similar sequences to the intended target.
The guidance states that “for the assessment of off-target editing activity of in vivo products, we recommend that sponsors use appropriate cell types that are representative of the drug product’s intended target tissue in vivo and use editing parameters that results in the intended level of editing rates at the on-target site. However, if appropriate cell type(s) are unavailable, difficult to obtain, or if the cells are not amenable to in vitro studies, then sponsors may consider using alternative methods or cell samples to perform studies assessing off-target editing activity and provide scientific justification to support their strategy.”
“This language is insufficiently clear regarding what level of in vivo exposure to non-target tissues warrants inclusion of representative non-target cell types in off-target editing analyses. For in vivo products with engineered tissue tropism and minimal off-target tissue exposure at therapeutic doses, the current ambiguity may drive sponsors to perform in vitro analyses in non-target cell types that have low translational relevance to human safety,” BIO wrote.
Lilly concurred. The company stated that “the current language is ambiguous on when off-target analyses can be limited to the intended target cell type. For in vivo products that concentrate editing in a single tissue (e.g., LNP delivery to hepatocytes), in vitro off-target studies in non-target cell types at saturating concentrations have low translational relevance when biodistribution shows minimal in vivo editor exposure in those tissues. Explicit recognition of this principle would right-size analytical investments without compromising safety.”
Nomination methods
BIO and Lilly suggested that FDA provide more clarity on the strategies, or nomination methods, that should be used to deliver and edit the genetic material to treat diseases. The guidance recommends three potential approaches for off-target site nomination: in silico methods, biochemical assays, and cell-based assays.
BIO states that “while the guidance lists in silico, biochemical, and cell‑based assays as available nomination approaches, some may interpret this as requiring all three. For modalities such as base editors and nickase‑based systems, biochemical assays often provide superior analytical sensitivity. Explicitly stating that modality‑appropriate combinations are acceptable would harmonize expectations and prevent unnecessary testing.”
“Explicit clarity is desired to acknowledge that, while multiple approaches are recommended, all three approaches are not always required,” Lilly wrote.
More data on CMC
The Alliance for Regenerative Medicine (ARM) and PhRMA requested more information on assessing these products after manufacturing changes.
The guidance states that “additional off-target editing analysis may be needed during product development if major changes to the manufacturing process(es) impacts either the editor activity and/or editing rate(s) at on-target site(s) that may potentially impact the off-target edit site profile of the drug product.”
ARM responded that the guidance “appropriately recognizes that manufacturing process changes may warrant additional off-target analyses during product development. However, the guidance would benefit from greater clarity regarding the scope of analyses expected following manufacturing changes, particularly to support late stage CMC lifecycle planning and BLA preparation.”
The guidance “identifies manufacturing process changes as a trigger for additional off-target analysis but provides limited detail on what constitutes an adequate bridging study at the BLA stage. Many products will undergo scale-up between clinical supply and commercial manufacturing, and sponsors would benefit from further clarity, such as guiding principles for late-stage CMC development and planning,” PhRMA said.
More time for meetings
PhRMA also stated that the FDA should ensure they allocate time to meet with sponsors of these products through INTERACT meetings.
The guidance states that “sponsors are encouraged to explore meeting options to discuss their strategies assessing off-target editing activity in an INTERACT or pre-IND setting…. The sponsor may provide a brief description of their off-target analysis plan in these submissions.”
“PhRMA appreciates FDA's encouragement of sponsors to engage through INTERACT and pre-IND meetings to discuss off-target study plans, and we agree that this type of early dialogue is valuable for aligning on study design expectations. However, sponsors have reported challenges obtaining timely opportunities to discuss genome editing-specific questions through existing meeting pathways. PhRMA encourages FDA to ensure that INTERACT and other early engagement mechanisms remain accessible for novel genome editing programs.”
