Identification of HLA-A, HLA-B and HLA-C triple homozygous and double homozygous donors: a path towards synthetic superdonor Advanced Therapeutic Medicinal Products

Daniel Naumovas^1,2Barbara Rojas¹Catalina M. Polanco¹Rita Čekauskienė³Victor Andrade⁴Inga Laurinaitytė²Artūras Jakubauskas³Laimonas Griskevicius³Ivan Nalvarte⁵Jose Inzunza^6,7Daiva Baltriukiene⁸Jonathan Arias^1,9*

• ¹Vilnius University Life Science Center EMBL partnership institute for gene editing technologies, Laboratory of nuclease enabled cell therapies, Vilnius, Lithuania
• ²Vilnius Santaros Klinikos Biobank, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania., Vilnius, Lithuania
• ³Department of Molecular Medicine; Hematology, Oncology and Transfusion Medicine Center, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania, Vilnius, Lithuania
• ⁴Division of Neurogenetics and Molecular Psychiatry, Department of Psychiatry and Psychotherapy, Medical Faculty, University of Cologne, 50937 Cologne, Germany., Colgone, Germany
• ⁵Karolinska Institutet, Department of Neurobiology, Care Sciences and Society, BioClinicum, Visionsgatan 4, Solna, Sweden, Stockholm, Sweden
• ⁶Karolinska Institutet Stem Cell Organoid (KISCO) facility, Department of Laboratory Medicine, Alfred Nobels allé 8, Huddinge, Sweden, Huddinge, Sweden
• ⁷Karolinska Institutet, Department of Laboratory Medicine, Alfred Nobels allé 8, Huddinge, Sweden
• ⁸Department of Biological Models, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania, Vilnius, Lithuania
• ⁹Karolinska Institutet. Karolinska Institutet Stem Cell Organoid (KISCO) facility, Department of Laboratory Medicine, Alfred Nobels allé 8, Huddinge, Sweden

Human induced pluripotent stem cells with broad immune compatibility are highly desirable for regenerative medicine applications. HLA class I homozygous cell sources are ideal for immune compatibility modeling. Here, we profile HLA-A, HLA-B, and HLA-C alleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% of HLA-A, 43.2% of HLA-B, and 59.2% of HLA-C, 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 HLA-A, HLA-B, and HLA-C. Compatibility modeling showed triple homozygous profiles match 60.5% of Lithuanians, 13.4% of British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disrupt HLA-A or HLA-B, while preserving HLA-C, producing edited profiles matching over 97.9% of Lithuanians, 95.7% of European-Americans, and 95.5% of British population. Finally, we established 15 fibroblast lines from triple homozygotes, as a bioresource for the derivation of human induced pluripotent stem cells, and for immune-compatibility studies.