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

Volker Wiebking , Ciaran M Lee , Nathalie Mostrel , Premanjali Lahiri , Rasmus Bak , Gang Bao , Maria Grazia Roncarolo , Alice Bertaina , Matthew H Porteus 
Source: Haematologica
Publication Date: (2021)
Issue: 106(3): 847-858
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit

T cells were activated with Dynabeads Human T cell Activator (Gibco) for 3 days and beads removed before electroporation. For gene editing, electroporation was performed as previously described1. HPLC-purified sgRNA with 2’-O-methyl-3’-phosphorothioate modifications at the three terminal nucleotides on both ends3 (Synthego) was complexed with high-fidelity spCas9 protein4 (IDT) at a molar ration of 2.5:1 (sgRNA : protein) and electroporated in buffer P3 (Lonza) into activated T cells using a 4D-Nucleofector (Lonza) in 16-cuvette strips. 1E6 activated T cells were used per electroporation using program EO-115. The cells were resuspended directly after electroporation in 80µl of complete T cell medium and then diluted to the target density. For gene targeting, cells were incubated within 15 minutes after electroporation with rAAV6 for transduction at a multiplicity of infection (MOI) of >5000 vg/cell unless otherwise stated. After the specified transduction time, the suspension was diluted with complete medium to reach thetarget cell concentration as indicated


Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of aß 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 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 aß 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 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 aß T-cell-derived, genome-edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of aß T-celldepleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.