Construction of High Coverage Japanese Dental Pulp Cell Stock with Selective Genome Editing in the HLA Allele

Abstract

This study was conducted to investigate selective gene modification of human dental pulp cells using Zinc Finger Nucleases (ZFNs), targeting the HLA-A*02 allele. We analyzed the HLA haplotypes of 177 dental pulp donors at Gifu University, creating a list of frequently occurring haplotypes. The coverage rate of each haplotype was calculated, and the cumulative coverage rates in the Japanese population was estimated as being more than 80%.

Next, the design of ZFNs for specifically targeting the exon 3 region of HLA-A*02:01:01:01 was executed, focusing on the unique amino acid sequences encoded in this exon as compared with the allele counterpart, HLA-A*33:03:01:01. Electroporation was employed to introduce plasmids encoding ZFNs into the dental pulp cells under optimized voltage and pulse conditions. Post-electroporation, the cells were cultured to assess the cell viability and plasmid transfection efficiency, evaluated through fluorescence microscopy and flow cytometry, respectively. The study also included the use of anti-HLA-A*02 antibodies for staining and analysis via flow cytometry, ensuring the specificity of the ZFN action.

The results demonstrated successful targeting of the HLA-A*02 allele with no effect on the HLA-A*33 allele, indicating the potential for creating HLA-homozygous cell lines. This advancement is significant for developing cell banks for transplantation, as it addresses the challenge of HLA compatibility in regenerative medicine. The findings suggest that the ZFN technology can be effectively utilized to generate genetically modified dental pulp cells, paving the way for personalized cell therapies and improved outcomes in tissue engineering.

In conclusion, this research highlights the feasibility of using ZFNs for precise gene editing in human dental pulp cells, with implications for enhancing the efficacy of regenerative medicine approaches. Future studies should focus on the long-term stability of the modifications and the functional implications in clinical applications.