Universal Donor Megakaryocytes

点击翻译按钮获取中文摘要

 DESCRIPTION (provided by applicant): Cellular products derived from pluripotent stem cells (PSCs) are being developed as possible treatments for diseases affecting almost every organ system. Megakaryocytes (MKs) are an especially promising application, since they produce platelets that are needed in thrombocytopenic patients to prevent bleeding, and they can be irradiated to ensure safety. However, the clinical use of cells derived from PSCs is limited by the host's rejection of transplanted cells due to polymorphisms in the human leukocyte antigen (HLA) genes. This has led to proposed solutions such as the establishment of HLA-typed stem cell banks or the derivation of PSCs from every patient, which will require that many different cell lines be established, characterized and approved for clinical use. Here we propose instead to develop universal donor PSCs derived by HLA- engineering that can be used in any recipient, which will greatly reduce the time and expense required to advance cellular therapies to the clinic. Our goal is to derive universal donor MKs from these PSCs that could be given to patients and produce platelets that function in the presence of the anti-HLA antibodies commonly found in transfusion-refractory patients. We will use adeno-associated virus (AAV) gene targeting vectors to knock out the B2M and RFXANK genes in human PSCs, which are required for HLA class I and II gene expression respectively. In order to prevent the lysis of HLA-negative cells by host Natural Killer (NK) cells, we will also express single chain versions of the non-polymorphic HLA-E class Ib protein fused to B2M, which should bind to the inhibitory CD94/NKG2A receptor on NK cells. These HLA-engineered PSCs will be differentiated in vitro into MKs and cultured with human blood cells to demonstrate that they no longer elicit immune responses or are lysed by NK cells. This approach will also be verified in mouse transplantation experiments by deriving analogous universal donor mouse PSCs, expressing the murine homolog of HLA-E (Qa-1b), differentiating these cells into MKs, and infusing the MKs into isogenic and allogeneic mouse recipients. These mouse experiments will directly test the hypothesis that universal donor MKs can survive in allogeneic hosts and produce functional platelets. We will also infuse human universal donor MKs into immunodeficient mice, to study the platelets they produce in vivo and the effects of anti-HLA antibodies.