Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of αβT cells and B cells has been shown to reduce the risk of acute and chronic graft- versus -host disease, thus allowing the use of haploidentical donors which expands the population of recipients in whom allogeneic hematopoietic stem cell transplantation can be used. Leukemic relapse, however, remains a challenge. T cells expressing chimeric antigen receptors can potently eliminate leukemia, including those in the central nervous system. We hypothesized that by engineering the donor αβT cells that are removed from the graft by genome editing to express a CD19-specific chimeric antigen receptor, while simultaneously inactivating the T-cell receptor, we could create a therapy that enhances the anti-leukemic efficacy of the stem cell transplant without increasing the risk of graft- versus -host disease. Using genome editing with Cas9 ribonucleoprotein and adeno-associated virus serotype 6, we integrated a CD19-specific chimeric antigen receptor inframe into the TRAC locus. More than 90% of cells lost T-cell receptor expression, while &75% expressed the chimeric antigen receptor. The initial product was further purified with less than 0.05% T-cell receptorpositive cells remaining. In vitro , the chimeric antigen receptor T cells efficiently eliminated target cells and produced high cytokine levels when challenged with CD19 ~(+) leukemia cells. In vivo , the gene-modified T cells eliminated leukemia without causing graft- versus -host disease in a xenograft model. Gene editing was highly specific with no evidence of off-target effects. These data support the concept that the addition of αβ T-cell-derived, genome-edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of αβT-celldepleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft- versus -host disease.
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