Genome editing of donor-derived T-cells to generate allogenic chimeric antigen receptor-modified T cells: Optimizing αβ T cell-depleted haploidentical hematopoietic stem cell transplantation: optimizing aβ T-cell-depleted haploidentical hematopoietic stem cell transplantation

_A ^llogeneic therapy _{based on} for _the^{hematopoietic} high-risk _selective leukemias. _removal^{stem cell}_of In _aβ^{transplantation} children, _T cells _and graft _B^is_cells manipulation ^an_has^effective_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 aβ T 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 in-frame 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 receptor-positive 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 aβ T-cell-derived, genome-edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of aβ T-cell-depleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.