How CRISPR-Cas9 Gene Editing May Improve the Effectiveness of Cellular Therapeutics in Patients With Cancer

A Conversation With Carl H. June, MD

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The edited T cells we infused in all three patients remained at stable levels for at least 9 months—compared with about 2 months in comparable CAR T-cell therapy studies.
— Carl H. June, MD

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The results from the first in-human phase I clinical trial in the United States evaluating CRISPR-Cas9–edited T cells in patients with advanced cancer has shown that the therapy is both feasible and safe, representing a big step forward in the potential of using gene editing to boost the natural ability of human T cells to recognize and attack cancer cells.1 The study, conducted by Carl H. June, MD, and his colleagues at the Abramson Cancer Center of the University of Pennsylvania, used clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene-editing technology to engineer T cells in three patients with refractory cancer. Two patients with advanced refractory multiple myeloma and one patient with metastatic sarcoma underwent the treatment.

The researchers removed T lymphocytes from the patients and used CRISPR-Cas9 to delete two genes encoding the endogenous T-cell receptor (TCR) alfa and beta chains from the patients’ T cells to reduce TCR mispairing and to enhance the expression of a synthetic cancer-specific TCR transgene, NY-ESO-1. A third gene encoding programmed cell death protein 1 (PD-1) was also removed to improve antitumor immunity. The autologous T cells were engineered by lentiviral transduction to express an HLA-A201 antigen, which is expressed in only a subset of patients. The patients received lymphodepleting chemotherapy with cyclophosphamide and fludarabine prior to the administration of a single infusion of the CRISPR-Cas9–engineered cells.

The phase I study was designed to investigate whether CRISPR-Cas9–engineered T-cell products are feasible and safe and not whether the therapy is effective against cancer. However, the study findings showed that the treatment provided limited benefit to the three patients. For example, the patients with myeloma initially experienced stable disease, and the patient with sarcoma had a 50% decrease in his abdominal mass—although other lesions in his body progressed—but all of the patients eventually experienced disease progression, and one of the patients with myeloma has died.

The ASCO Post talked with Dr. June, the -Richard W. Vague Professor in Immunotherapy in the Department of Pathology and Laboratory Medicine; Director of the Center for Cellular Immunotherapies at the Perelman School of Medicine; and Director of the Parker Institute for Cancer Immunotherapy at the University of Pennsylvania, about the results of his study and the potential of CRISPR-Cas9 gene editing in the treatment of cancer and other life-threatening diseases.

Enhancing the Body’s Immune System to Destroy Cancer Cells

Please talk about the results of your study and the potential of CRISPR-Cas9 gene editing to enhance the body’s immune system to destroy cancer cells.

We and others have shown that if you knock out the PD-1 gene with CRISPR so it cannot be expressed in the T cells, it would improve the function and persistence of engineered T cells and make them safer. We introduced a synthetic, cancer-specific, TCR transgene, NY-ESO-1, to recognize tumor cells, and then monitored the engineered T cells to determine whether the cells could persist after the CRISPR-Cas9 modifications.

We found the persistence of the T cells expressing the engineered endogenous TCR was much more durable than in previous clinical trials in which T cells were infused that retained expression of the endogenous TCR and the endogenous PD-1. Chromosomal translocations were observed in vitro during cell manufacturing, and they decreased over time after we infused the engineered cells into the patients.

Our study has shown that genome editing and CRISPR-Cas9 technology seem to live up to its promise of being able to target three genes at one time.
— Carl H. June, MD

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The edited T cells we infused in all three patients remained at stable levels for at least 9 months—compared with about 2 months in comparable chimeric antigen receptor (CAR) T-cell therapy studies—and there were no clinical toxicities associated with these engineered T cells. The length of time the modified T cells persisted in these patients suggests that immunogenicity is minimal under these conditions and demonstrated the feasibility of using CRISPR gene editing for cancer immunotherapy.

Changing the DNA of T Cells to Attack Cancer Cells

Although all three patients had an initial modest response to this therapy, eventually their cancers progressed. What did you learn from your study about the effectiveness of this treatment?

Our trial was designed to see whether it is safe and feasible to do multiplex CRISPR gene editing, which had never been done before. The trial wasn’t designed to see whether it would cure patients with cancer. If we were attempting to do that, we would have included dose escalation and given more infusions of the modified T cells. Instead, we gave the patients just a single dose and only one time. The endpoints of the study were to investigate the safety aspect of CRISPR gene editing and whether it is precise in changing the DNA of cells. We found out that it is very, very precise.

And, remember, CRISPR actually comes from bacteria, Staphylococcus aureus and Streptococcus pyogenes, and we have built up immunity to the proteins from these bacteria over time. We showed in our study that all of our patients had preexisting immune responses to Cas9, the bacterial protein most commonly used in CRISPR gene editing. So, one safety aspect of the study was to investigate whether Cas9 would cause side effects and whether it would trigger rejection of our engineered T cells. We found there were no safety side effects, and it did not trigger a rejection of the engineered T cells.

We reported a phase I study in 2014 using a predecessor to the Cas9-engineered nucleases, the zinc-finger nuclease (ZFN), to modify the CCR5 gene, a major co-receptor for HIV, in the treatment of patients with HIV/AIDS.2 In that study, we used the ZFN technology to modify the CD4 T cells in the patients to mimic the CCR5-delta-32 mutation, which provides a natural resistance to the virus.

We found that the single infusion of the autologous CD4 T cells, in which the CCR5 receptor had been rendered dysfunctional by ZFNs targeting the CCR5 gene, was generally safe. We also learned that the long-term persistence of the CCR5-modified CD4 T cells in these patients suggested the cells were not rendered immunogenic as a result of the CCR5 disruption.

We worried whether gene editing with Cas9 was potentially immunogenic or toxic compared with the baseline low level of adverse events we observed in our previous clinical trial targeting NY-ESO-1 with transgenic TCRs. However, it turned out not to be a problem with one infusion of the CRISPR-Cas9–engineered T cells, so this is good to know for the field of gene editing. Now, that doesn’t imply that multiple infusions may not be a problem, but at least we know that one infusion is safe.

Providing Patients With Precision Medicine

Why is this CRISPR-Cas9–engineered T-cell therapy active in only a subset of patients with the HLA-A201 antigen?

It is a problem with TCRs. The CRISPR-edited T cells are not active on their own like CAR T cells, which are based on antibodies, and the same antibody that works in one patient would also work in another. The CRISPR-edited T cells used a TCR that requires the presence of the HLA-A201 antigen, which is expressed in only a subset of patients. That meant we had to screen patients before enrollment into the study to make sure their tumors were a match for this approach. When we use CAR T-cell therapy in patients, it’s more straightforward, because we don’t have to do any prior testing and every patient receives the same CAR T cells.

Combining Technologies to Boost Immunotherapy Effectiveness

The CRISPR-edited cells in your study showed that the therapy is safe and feasible in patients with cancer. Could this therapy potentially be used to boost the effectiveness of CAR T-cell therapy in more patients with cancer?

Yes. We proved that about 90% of patients with acute lymphocytic leukemia achieve a complete remission with CAR T-cell therapy, so the therapy is very effective. However, in patients with refractory lymphomas, the cure rate is only about 50%, so 50% of patients do not experience a long-term remission or a cure. Combining CRISPR gene-editing technology with CAR T-cell therapy can raise the remission or cure rate in these patients by another 40%, so eventually we’ll get closer to a 90% remission or cure rate in patients with refractory lymphomas.

Future of CRISPR-Cas9 Technology in Cancer and Beyond

What is next in your research in CRISPR gene-editing technology and its potential in cancer immunotherapy?

In our current study, we proved its safety and feasibility in patients with highly advanced cancers. For example, one of the patients with myeloma in our study had had eight lines of chemotherapy and three bone marrow transplants, and her immune system was really shot. So, we proved that CRISPR-edited immune cell therapy was feasible even in patients with highly advanced refractory disease. Next, we want to show its feasibility in patients with less-advanced cancers. Instead of using just CRISPR-Cas9 technology, we want to combine it with CAR T-cell therapy, so we don’t have the problem of enrolling only patients with specific HLA antigens.

Also, since our manufacturing process for the CRISPR-Cas9–engineered T-cell products was submitted to the U.S. Food and Drug Administration and approved in 2016, reagents in this field have improved, and we are able to make better forms of Cas9. In our next clinical trial, we are incorporating newer technologies instead of the basic vanilla form we had in this first pilot trial, so we should see improved results not just in terms of safety and feasibility, but in treatment effectiveness as well.

The bottom line is there has been a lot of hype about genome editing and CRISPR-Cas9 technology. Our study has shown that this therapy seems to live up to its promise of being able to target three genes at one time, and that knowledge will open up all kinds of progress in the field of immunotherapy, not just in the treatment of cancer. For example, there has been a report from an unpublished study that CRISPR-edited cells were used to correct a gene mutation in a patient with sickle cell disease.3

We are going to see amazing results in the treatment of a variety of diseases with this type of genome editing. The field is just at the beginning of learning how to harness this technology to treat cancer and other life-threatening diseases. 

DISCLOSURE: Dr. June has sponsored research grants from Novartis, receives royalties from Novartis for intellectual property licensed by the University of Pennsylvania to Novartis, and is a scientific founder of Tmunity Therapeutics.


1. Stadtmauer EA, Fraietta JA, Davis MM, et al: CRISPR-engineered T cells in patients with refractory cancer. Science. February 28, 2020 (early release online).

2. Tebas P, Stein D, Tang WW, et al: Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 370:901-910, 2014.

3. Stein R: Gene-edited ‘supercells’ make progress in fight against sickle cell disease. NPR, November 19, 2019. Available at Accessed April 3, 2020.