Identification of HLA-A, HLA-B, and HLA-C triple homozygous and double homozygous donors: a path toward synthetic superdonor advanced therapeutic medicinal products

Human-induced pluripotent stem cells with broad immune compatibility are highly desirable for regenerative medicine applications. Human leukocyte antigen (HLA) class I homozygous cell sources are ideal for immune compatibility modeling. Here, we profile <i>HLA-A</i>, <i>HLA-B</i>, and <i>HLA-C</i> alleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% of <i>HLA-A</i>, 43.2% of <i>HLA-B</i>, and 59.2% of <i>HLA-C</i>, with HLA-A*02:01:01, HLA-B*07:02:01, and HLA-C*07:02:01 being the most common. Lithuanian allele frequencies closely resemble those of European-American and British populations. We identified 153 double homozygotes and 51 triple homozygotes for <i>HLA-A</i>, <i>HLA-B</i>, and <i>HLA-C</i>. Compatibility modeling showed that triple homozygous profiles match 60.5% of Lithuanians, 13.4% of the British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disrupt <i>HLA-A</i> or <i>HLA-B</i> while preserving <i>HLA-C</i>, producing edited profiles matching over 97.9% of Lithuanians, 95.7% of European-Americans, and 95.5% of the British population. Finally, we established 15 fibroblast lines from triple homozygotes as a bioresource for the derivation of human-induced pluripotent stem cells and immune compatibility studies.