GMP compliant CRISPR/Cas9 gene editing of B2 microglobulin in cord blood derived megakaryocytes and platelets for patients with refractory thrombocytopenia

Abstract

Abstract Introduction: A reliable and sustainable supply of platelets remains a critical challenge in managing thrombocytopenic patients, particularly those undergoing chemotherapy, hematopoietic stem cell transplantation, or surgical procedures. Over 2 million platelet units are transfused annually in the United States alone. However, platelet transfusion is often complicated by platelet transfusion refractoriness (PTR), characterized by an inadequate post-transfusion platelet count increment. PTR occurs in up to 30% of chronically transfused patients and is frequently caused by alloimmunization to HLA class I antigens. Alloimmune PTR presents a major clinical hurdle, as HLA-matched donor platelets are logistically difficult to source and limit timely access to effective transfusion therapy. The reliance on donor-derived apheresis platelets is further constrained by the short shelf life of platelets and limited donor availability. To address the shortage, we developed a novel strategy for the large-scale ex vivo generation of HLA class I-deficient GMP-grade megakaryocytes (MKs) and platelets from CD34⁺ cord blood (CB) cells. Methods: We have developed a GMP-compliant method to produce unedited, expanded MKs and platelets in vitro, using a multi-phase co-culture system of CB-derived CD34⁺ cells and CB-MSCs. The culture system is supplemented with dynamically titrated cytokines to simulate the bone marrow microenvironment across distinct developmental stages. Additional components include a caspase-3 inhibitor, a histone deacetylase inhibitor, and a ROCK inhibitor to enhance MK viability and platelet production. CD34⁺ cells, sourced from the MDACC Cord Blood Bank, and CB-MSCs, from the MSC Bank, are expanded and differentiated over five sequential culture phases. Platelet release is induced via shear force in a closed-loop bioreactor, and the final MK-platelet product is harvested on day 24. The product is phenotyped, assessed for in vitro aggregation, and infused into irradiated NSG mice for functional evaluation.. To overcome platelet transfusion refractoriness (PTR), we then targeted HLA class I expression on MKs. On day 3 of culture, CRISPR/Cas9 gene editing is performed using a single-guide RNA targeting exon 1 of B2 microglobulin M (B2M), delivered via the Lonza 4D-Nucleofector system. Editing conditions were optimized to minimize off-target effects while achieving robust B2M disruption. Results: After 24 days, the unedited cultures yielded ~50 × 10⁶ mature polyploid MKs and ~0.5 × 10¹⁰ platelets. Upon infusion of 4.2 × 10⁶ MKs and 0.5 × 10⁸ platelets into sublethally irradiated thrombocytopenic NSG mice, human CD41⁺/CD61⁺ platelets were detected in circulation within 1 hour. In vivo–generated platelets appeared by day 4 and peaked at day 11, with persistence in circulation through day 21, indicating sustained thrombopoietic activity. B2M knockout was then successfully integrated into the culture protocol. Flow cytometry confirmed effective downregulation of HLA class I, with only ~15% of cells expressing residual HLA-I. In vitro functional assays showed thrombin-induced aggregation of platelets, confirming functional competency. Importantly, in vitro cytotoxicity assays showed that the B2M knockout cells were resistant to allogeneic T cell–mediated killing, supporting the immune evasion potential of this product in alloimmunized patients. Conclusions: We have established a scalable, GMP-compliant platform for generating MKs and platelets from cord blood. The combined infusion of MKs and platelets enables both immediate and sustained platelet reconstitution. To address platelet transfusion refractoriness (PTR), we incorporated CRISPR/Cas9-mediated knockout of HLA class I, generating universal, donor-independent platelets with immune-evasive properties. This platform offers a renewable and potentially off-the-shelf source of platelets. Clinical trials are planned to evaluate the safety and efficacy of this approach.