Knockout of the Beta-2 Microglobulin Gene in Adipose Tissue-Derived Cells Using CRISPR/CAS9 System for the Generation of Universal HLA Class I Platelets

Platelet refractoriness is a recurring problem in the treatment of thrombocytopenic patients, hindering the restoration of normal circulating platelet levels after platelet transfusion therapy. The primary cause is the incompatibility between the transfused platelets' HLA Class I antigens and the patient's antibodies. The production of platelets in vitro from genetically edited stem cells may become a future source of HLA Class I universal platelets that could help mitigate this problem. Adipose tissue-derived mesenchymal stem/stromal cells line (ASCL), generated from the dedifferentiation of adipocytes using the ceiling culture method, can be obtained in abundance and differentiated into megakaryocytes and platelets under suitable conditions. HLA Class I antigens are fixed on the surface of human cells through the binding to Beta-2 Microglobulin (B2M), expressed by the B2M gene. In this study, we performed gene editing on ASCL cells to obtain cell populations without HLA Class I antigens by knocking out the B2M gene using the CRISPR/Cas9 tool. Three guide RNAs (sgRNA) were designed and synthesized specifically to target DNA breaks in exons 1 or 2 of the B2M gene. The different sgRNAs were cloned into the pL-CRISPR.EFS.GFP plasmid, which was then sequenced by the Sanger automatic sequencing method for confirmation. Each cloned plasmid, along with two others (second generation) for lentiviral particle assembly, was transfected into HEK 293T cells, and each resulting lentiviral variety was collected and stored at -80°C until use. Different batches of each produced lentivirus variety were pooled, and a small sample was used for K562 cell transductions to estimate lentiviral production by titration, quantified by detecting green fluorescent protein (GFP) via flow cytometry. HEK 293T cells were also used for initial transduction tests and knockout efficiency using each of the three lentiviral varieties, either individually or in combination. The lentivirus carrying the cloned plasmid with the sgRNA guide#2 (LV-CRISPR-B2M-G2) was the most effective in reducing HLA Class I levels on the surface of HEK 293T cells, compared to the control (LV-CRISPR-B2M-EMPTY), and was then chosen for further study. Transductions of ASCL cells using the lentivirus LV-CRISPR-B2M-G2, with an MOI of approximately 13.5, resulted in viable, GFP-positive cell populations lacking B2M protein and HLA Class I antigens on their surface. The culture of transduced ASCL cells proliferated in vitro for more than 42 days, with at least 90% remaining negative for B2M and HLA Class I. Transduced and non-transduced ASCL cells were induced to megakaryocytic and platelet differentiation in vitro for 12 days, and the resulting cells were characterized for the expression of specific lineage markers and ploidy. The ASCL cells transduced with LV-CRISPR-B2M-G2 generated megakaryocyte and platelets populations with reduced expression of B2M and HLA Class I. The functionality of the platelets was verified through the augmented expression of p-selectin (CD62p) after stimulation with thrombin. The efficiency achieved in the transduction and knockout of the B2M gene using CRISPR/Cas9 technology, along with the significant reduction in HLA Class I antigen levels detected on the surface of ASCLs, megakaryocytes and platelets, demonstrates the potential of this approach for generating functional platelets lacking HLA Class I antigens. This suggests that these modified cells may be stored and later induced to differentiate into HLA Class I negative platelets, offering a promising strategy for developing alternative platelet sources for transfusions in refractory patients.