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CRISPR Gene Editing Puts Researchers Closer to an “Off-the-Shelf” CAR T-Cell Product for ALL

December 30, 2021

Last year marked the first U.S. Food and Drug Administration approvals of autologous T-cell immunotherapies for the treatment of cancer, including acute lymphocytic leukemia (ALL) and non-Hodgkin lymphoma (NHL). However, several challenges have limited the clinical development of chimeric antigen receptor (CAR) T-cell therapies against T-cell malignancies, including difficulties harvesting non-malignant autologous T cells from affected patients. Allogeneic, off-the-shelf CAR T-cell products would overcome some of these limitations, but the shared expression of target antigens between effector cells and malignant cells can lead to "self-killing" of CAR T cells.

Researchers at the Washington University School of Medicine in St. Louis have developed a new CAR T-cell strategy using the gene-editing technology CRISPR/Cas9 to engineer "fratricide-resistant" CAR T cells. Lead author Matthew L. Cooper, PhD, and colleagues published their results in Leukemia.1 The preclinical data from this mouse model demonstrates the feasibility of an allogeneic strategy to treat T-cell malignancies.

"Cancerous T cells and healthy T cells have exactly the same protein – CD7 – on their surfaces, so if we program T cells to target CD7, they would attack both the cancerous cells and each other, thus undermining this approach," senior author John F. DiPersio, MD, PhD, explained.2 "To prevent this T-cell fratricide, we used CRISPR/Cas9 gene editing to remove CD7 from the CAR T cells, so they no longer carry the target." CD7 was also selected because this antigen could be deleted in T cells without affecting immune function, the investigators noted.

The researchers generated the CD7-CAR product, UCART7, using CRISPR/Cas9 gene editing and T-ALL cells collected from patients being treated at their institution. Then, they tested this approach in mice xenografted with either T-ALL cell lines or primary cells from human T cell ALLs.  The xenografted mice were randomized to receive either UCART7 or UCART19, an allogeneic T cell product directed against CD19 (expressed on B-cells, not T cells) that served as a negative control. Mice received CAR T-cell doses at 2×106/kg.

Following transduction of T cells, the investigators observed significantly fewer UCART7 cells than UCART19 cells. UCART7 cells were biased toward a CD4 phenotype, compared with UCART19. Mice receiving UCART7 had significantly prolonged survival and reduced tumor burden, compared with mice receiving UCART19 (p=0.0003).

Also, mice treated with the gene-edited T cells targeted to CD7 survived 65 days, compared with 31 days for those that received CD19 targeted cells (p=0.0003).

The investigators then tested the capacity of UCART7 to kill primary T-ALL in vivo without inducing an alloreactive graft-versus-leukemia effect or xenogeneic graft-versus-host disease (GVHD). T-ALL blasts were absent in peripheral blood of mice receiving UCART7, compared with those receiving UCART19 (p<0.0001). UCART7 recipients also had normal-sized spleens, while those receiving UCART19 exhibited splenomegaly.

"These UCART7 cells efficiently kill human T-ALL cell lines and patient-derived primary T-ALL in vitro and in vivo without resulting in xenogeneic [GVHD]," the researchers wrote of their findings, indicating that CD7 is a candidate for gene editing of CAR T cells. "Should rejection occur, UCART7 would still provide a viable bridge to transplantation, which many feel is the primary benefit of [CAR T-cell therapy]."

"[An] additional benefit of this approach is that a patient could receive this therapy much more quickly after diagnosis," said Dr. Cooper. "We wouldn't need to harvest the patient's own T cells and then modify them, which takes time. We also wouldn't have to find a matched donor. We could collect T cells from any healthy donor and have the gene-edited T cells ready in advance."

The study is limited by its use of mice and will need to be validated in a human model. Studies are underway to assess the viability of scaling UCART7 for clinical trials.

The authors report no financial conflicts.


  1. Cooper ML, Choi J, Staser K, et al. An "off-the-shelf" fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies. Leukemia. 2018 February 20. [Epub ahead of print]
  2. Washington University School of Medicine in St. Louis press release, March 5, 2018.

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