Single-cell DNA sequencing provides a comprehensive assessment of genomic editing outcomes of gene-corrected CD4 T-cells for treating Hyper IgM1 supporting the start of clinical trial

Hyper-IgM 1 (HIGM1) is an X-linked combined immunodeficiency that is caused by a CD40LG mutation that impairs communication between active CD4+ T cells and effector immune cells. This results in an average lifespan of individuals with HIGM1 to be 25 years on average. Therefore, a one-size-fits-all, long-range corrective gene editing strategy is a viable therapeutic option for this cohort of individuals.

Safety requirements of gene corrected cell and gene therapies are currently under definition by regulatory agencies, and precise characterization of gene editing outcomes at the single-cell level play an important role in IND submissions. To help cure individuals with HIGM1 and inform regulatory agencies, Daniele Canarutto, M.D., Ph.D. and team developed a scalable, GMP-compliant gene therapy that utilizes CRISPR/Cas9, a corrective homology directed repair (HDR) template coupled to a selector, and an integrase-defective lentiviral vectors (IDLV) delivery system.

In this live session, Daniele will show how the adoption of a single-cell DNA sequencing (scDNA-seq) platform, coupled with their gene editing protocol and expected gene editing outcomes, enabled the rigorous validation of their gene therapy for HIGM1. The team successfully completed their preclinical safety evaluation using a custom scDNA-seq amplicon panel covering the X-chromosome, nuclease target, and donor template for characterization of gene editing outcomes.

Results of their preclinical safety evaluation using the custom scDNA-seq assays demonstrated how unsupervised clustering distinguishes precise HDR from on-target concatemers. Crucially, Daniele will show how using scDNA-seq assays confirmed the mutual exclusivity of donor integration and wild-type loci validating targeted integration and ruling out large deletions or off-targets to support their first-in-human trials.