Allele-Selective Genome Editing of the Human Leukocyte Antigen Locus in Human Dental Pulp Cells Using Zinc Finger Nucleases.

Human dental pulp cells (DPCs) hold promise for cell-based therapies, but their allogeneic use is limited by human leukocyte antigen (HLA) incompatibility. Utilizing HLA haplotype-homozygous (HHH) donors can improve compatibility, yet sourcing donors for diverse haplotypes remains challenging. Generating pseudo-HHH cells by disrupting a specific HLA allele via gene editing offers a potential solution. This study aimed to establish zinc finger nuclease (ZFN)-mediated gene editing for allele-specific disruption of HLA-A in DPCs. We designed ZFNs to target the HLA-A*02:01 allele in DP144 DPCs (HLA-A*02:01/A*33:03, homozygous at HLA-B, -C, -DR). Following ZFN transfection and fluorescence-activated cell sorting (FACS)-mediated cell enrichment, allele-specific modifications were assessed using targeted deep sequencing, whole-genome sequencing (WGS) for off-target analysis including structural variants (SVs), and next-generation sequencing (NGS)-based HLA typing. Results demonstrated efficient (>96% indels) and specific disruption of the targeted HLA-A*02:01 allele, with minimal modification of the HLA-A*33:03 allele. Whole-genome sequencing revealed overall genomic stability, although two minor deletion-type SVs were detected in non-coding regions. Importantly, NGS HLA typing confirmed the integrity of the non-targeted HLA-A allele and other key HLA loci. These findings demonstrate the feasibility of using ZFNs to generate pseudo-HHH DPCs by allele-specific HLA knockout, providing a potential strategy to expand the donor pool for DPC-based therapies. However, careful assessment of genomic integrity is crucial for future clinical translation.