GUIDE-seq and NGS-based off-target analysis support genome editing safety assessment for engineered Treg therapy

GentiBio recently presented genome editing safety data during the “On- and Off-Target Method Development” session st the 2026 ASGCT Meeting, highlighting the extensive off-target characterization strategy used to support development of GNTI-122, an engineered regulatory T cell (Treg) therapy for type 1 diabetes (T1D).

The presentation is particularly timely given the recent FDA draft guidance, “Safety Assessment of Genome Editing in Human Gene Therapy Products Using Next-Generation Sequencing,” which emphasizes comprehensive NGS-based evaluation of genome editing outcomes, including off-target activity assessments, on-target characterization, and broader genomic integrity studies.

GNTI-122 is manufactured using CRISPR/Cas9-mediated dual genome editing with AAV-delivered transgene insertion at two genomic loci. Because multiplex genome editing can introduce unintended modifications, comprehensive assessment of off-target activity is increasingly viewed as a critical component of regulatory safety packages.

GUIDE-seq enabled comprehensive empirical off-target site identification

To evaluate editing specificity, GentiBio implemented a multi-layered off-target assessment strategy combining computational prediction methods with empirical GUIDE-seq analysis supported by Avance Biosciences.

Initial off-target nomination used in silico methods incorporating guide-target sequence homology, predicted cleavage probability, and human genetic variants capable of creating potential off-target sites not represented in the reference genome.

Importantly, these predictions were complemented by GUIDE-seq (Genome-wide Unbiased Identification of Double-stranded Breaks Enabled by Sequencing) performed by Avance Bioscience using T cells obtained from six T1D donors.

GUIDE-seq provides an unbiased genome-wide approach for identifying CRISPR-induced double-strand breaks and is increasingly recognized as a powerful orthogonal method for empirical off-target discovery. The assay pipeline demonstrated sensitivity to detect rare editing events below 0.1% indel frequency, supporting detection of low-frequency genome modifications relevant to clinical safety evaluation.

Alignment with emerging FDA expectations for off-target analysis

The FDA draft guidance “Safety Assessment of Genome Editing in Human Gene Therapy Products Using Next-Generation Sequencing” places significant emphasis on comprehensive assessment of off-target activity as part of genome editing safety evaluations.

Among the key recommendations highlighted by FDA are:

• Comprehensive off-target site identification using orthogonal approaches
• Integration of computational predictions with empirical methods for off-target discovery
• Application of NGS-based assays to quantify editing outcomes at nominated sites
• Use of bioinformatics workflows to support off-target characterization and risk assessment

The off-target analysis strategy presented for GNTI-122 closely aligns with these emerging expectations by combining in silico prediction methods with GUIDE-seq empirical site nomination to create a comprehensive list of potential off-target loci.

Growing importance of GUIDE-seq in genome editing programs

As genome-edited cell and gene therapies continue advancing into the clinic, unbiased methods such as GUIDE-seq are becoming increasingly important for building comprehensive safety packages.

Empirical off-target discovery approaches provide an important complement to computational prediction methods and help establish the analytical evidence needed to support regulatory submissions, particularly as expectations evolve toward broader NGS-based genome safety assessments.

The GNTI-122 program illustrates how integrated off-target characterization strategies can support development of complex multiplex-edited therapies while addressing emerging regulatory expectations for genome editing safety.

References

• FDA Draft Guidance – Safety Assessment of Genome Editing in Human Gene Therapy Products Using Next-Generation Sequencing
• Oliver B, Crevier T, Hernandez S, de Jesus T, Uenishi G, Stuart A, del Rio Espinola A, Lugena A, Berry R, Chen T, Christin B, Wickham T, Patel C, Repic M. (2026) Comprehensive genotoxicity evaluation of GNTI-122, an engineered Treg therapy for type 1 diabetes. Abstract 413, presented at the ASGCT Annual Meeting 2026, Boston, MA, USA. [abstract].