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

Abstract

Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of αβ T 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 that allogeneic hematopoietic stem cell transplantation can be used in. Leukemic relapse, however, remains a problem. T cells expressing chimeric antigen receptors can potently eliminate leukemia, including in the central nervous system. We hypothesized that by modifying donor αβ T cells to simultaneously express a CD19-specific chimeric antigen receptors and inactivating the T cell receptor by genome editing, 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 integrate a CD19-specific chimeric antigen receptor in-frame into the TRAC locus. Greater than 90% of cells lost TCR expression, while >75% expressed the CAR. The product was further purified to ultimately have less than 0.05% residual TCR+ cells. In vitro, the CAR 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 xenogeneic 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 αβ T cell-depleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.

How to Combine the Two Landmark Treatment Methods—Allogeneic Hematopoietic Stem Cell Transplantation and Chimeric Antigen Receptor T Cell Therapy Together to Cure High-Risk B Cell Acute Lymphoblastic Leukemia?

“…Recently, Wiebking et al designed an intriguing approach which combined both allo-HSCT and CAR-T cell therapy with complementary anti-leukemia mechanisms: the HLA-dependent activity of GVL effect and the HLA-independent mechanism of CAR-T cell (101). In this setting, a TCRab/CD19-depleted haplo-HSCT platform was employed, which was associated with very low transplantation-related mortality and GVHD incidence (102)(103)(104)(105).…”

Towards Clinical Implementation of Adeno-Associated Virus (AAV) Vectors for Cancer Gene Therapy: Current Status and Future Perspectives

“…Greater than 90% of cells lost TCR expression, while >75% expressed the CAR. These cells efficiently killed target cells in vitro and in a xenograft model in vivo, without off-target effects or induction of graft-versus host disease [ 173 ].…”

CRISPR/Cas9 for the treatment of haematological diseases: a journey from bacteria to the bedside

“…Very recently, ab T cells treated with CRISPR/ Cas9 editing and an AAV donor template resulted in> 75% expression of the CAR along with> 90% TCR loss. 88 These unprecedented rates of targeted integration of large gene cassettes bode well for improved HDR efficiencies in other Tcell settings but additional optimisation will be required before such impressive results are seen in long-term engrafting stem cells. Although the clinical trial of CRISPR/Cas9 editing of HSPCs for ablating CCR5 expression as a treatment for HIV showed promising results in one patient (NCT03164135 69 ), the numbers of modified cells achieved post-transplant were subtherapeutic, illustrating the ongoing challenges in this area.…”

“…In comparison, HDR rates reported in T cells to insert a CAR within the TRAC locus were more encouraging. Very recently, αβ T cells treated with CRISPR/Cas9 editing and an AAV donor template resulted in> 75% expression of the CAR along with> 90% TCR loss 88 . These unprecedented rates of targeted integration of large gene cassettes bode well for improved HDR efficiencies in other T‐cell settings but additional optimisation will be required before such impressive results are seen in long‐term engrafting stem cells.…”