Non-genotoxic transplantation and in vivo selection through epitope editing

Key Points:

• Researchers developed bidirectional Sleeping Beauty transposon plasmids encoding codon-optimized KIT cDNA or its mutated variants, combined with fluorescent and puromycin resistance reporters, to enable stable gene integration and selection in human and mouse cell lines.
• A degenerate codon library targeting the KIT extracellular domain was created and analyzed via flow cytometry and next-generation sequencing to identify mutations affecting antibody binding, facilitating epitope mapping and therapeutic antibody characterization.
• Base editing and prime editing techniques were optimized in human K562 cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), with editing efficiencies assessed by flow cytometry and Sanger sequencing, enabling precise genetic modifications.
• Extensive in vitro and in vivo assays, including antibody-mediated selection, erythroid differentiation, colony-forming unit assays, and xenotransplantation into immunodeficient mice, were performed to evaluate the functional impact of KIT editing and therapeutic antibody treatments.
• Comprehensive genomic and transcriptomic analyses, including CHANGE-seq, rhAmpSeq, RNA-seq, barcode-based clonal tracking, and off-target assessments, were conducted to characterize editing specificity, clonal dynamics, gene expression changes, and potential chromosomal rearrangements, ensuring safety and efficacy of gene editing approaches.