dna-helix-for-webOn June 21st, 2016, the United States National Institutes of Health (NIH) authorized the very first human clinical trial involving CRISPR gene editing. Led by scientists from the University of Pennsylvania (UPenn), the upcoming Phase I trial will test the safety of CRISPR-edited immune cells in eighteen patients with either late-stage blood immune cancer (myeloma), solid tumours in the joints (synovial sarcoma) or solid tumours in the skin (melanoma). The proposed procedure is similar to traditional immunotherapy in that it involves reinvigorating a cancer patient’s immune cells and injecting them back into the same person; however, this time, CRISPR will be used to fashion immune cells with an enhanced ability to destroy cancer. Financing this effort is the billionaire co-founder of Napster and Facebook’s first president, Sean Parker, representing a rare union of the biotechnology and information technology sectors.

Ending Cancer, Not with a Bang, but Three CRISPR Edits

The CRISPR-Cas technique, put simply, involves a system of molecular scissors that cuts genes in a targeted fashion and deletes or inserts new DNA at that site. This gene-editing tool is expected to transform gene therapy because it is cheaper, faster and more accurate compared to other gene manipulation methods. In the upcoming Phase I trial, CRISPR will be used to edit DNA in mature T cells—immune cells that target and kill infected, cancerous or otherwise abnormal cells. Since any modifications in these cells do not get passed down to future generations, this application has not had to face the same ethical objections as gene edits in embryonic stem cells, leading to its rapid trajectory to the clinic.

Led by UPenn oncologists, including immunotherapy leader Dr. Carl June, this trial involves using viral gene delivery as well as CRISPR gene editing to re-engineer a cancer patient’s T cells. Lentiviral vectors will deliver a receptor that is structurally similar to the T cell’s natural receptor into T cells collected from the patient. This T cell receptor (TCR) will recognize a molecule called NYESO-1, which is present predominantly on cancer cells. This will allow the T cells to identify and specifically destroy the patient’s tumour. In addition, two CRISPR edits will remove the pair of genes that make up the T cell’s natural receptor; this will eliminate confounding signals and reduce pairing of natural and inserted TCRs, resulting in increased tumour killing efficiency and decreased toxicity of the re-injected T cells. The third CRISPR edit will remove PD-1, an off-switch on T cells used by the tumour to protect itself from immune destruction. Although CRISPR is a great DNA deletion tool, it is less effective for gene overexpression than the well-established lentiviral system – part of why a combination of these tools is being used. Ultimately, these four gene manipulations will generate an enhanced T cell expected to annihilate both blood cancers and solid tumours.

This CRISPR-based form of personalized medicine brings together gene therapy, cell therapy and immunotherapy, and has the potential to be a definitive cure for cancer. If it proves effective, this therapy may augment or replace radiation and chemotherapy, which can lead to off-target killing of healthy cells and requires multiple doses for effective tumour clearance. This technology also stands to compete with tumour-infiltrating lymphocyte (TIL) therapy. Although therapeutic TILs respond to more than one cancer-associated molecule, they are susceptible to silencing by mechanisms like PD-1, at least when used as a stand-alone treatment. Ultimately, because the FDA simply requires that a therapy be safe and efficacious for approval, an unbiased comparison of the currently available options will be need to take place to determine whether CRISPR immunotherapy can make a real impact in the clinic.


[B]asic science research must continue to uncover the best gene targets for CRISPR-based therapies.”


Precautionary Measures

As this red-hot CRISPR-based therapy gets its first green light into the clinic, scientists must remain cautious of adverse outcomes. The foremost safety concern comes from the fact that these engineered T cells will be a mixed bag – that is, the T cells will contain one to four edits. As such, there is a risk that the endogenous TCR could remain intact in some of the edited populations, possibly producing self-reactive T cells. Although all sixteen potential combinations of edited T cell populations were found to be safe and efficacious in animals, their activity in humans remains unclear.

The previous success of Phase II trials using lentivirus to insert the NY-ESO-1 TCR provides some assurance of its safety; however, its side effects when used alongside these CRISPR edits remain unknown. One potential consequence of using CRISPR alone is the off-target editing of genes, which could be fatal if abnormal T cells are generated. To address this issue, June’s group plans to measure the frequency of off-target modified T cells to evaluate safety for each patient before and after injection. Another concern is the persistence of the bacterial Cas proteins, which, compounded by the persistence of viral proteins and DNA, could lead to the rejection of modified cells. While suicide genes would be one way to deal with these leftover proteins, they are not included as part of the editing strategy in this trial, perhaps to reduce gene manipulation. Instead, cases of toxicity will be managed as needed with immunosuppressants and the re-administration of standard cancer therapy.

Finally, should this therapy make it through the Phase I trial, it will still need at least two more rounds of successful clinical trials before it is approved by the FDA and implemented in the clinic. Efficacy issues like outgrowth of tumour cells without NYESO-1, limited receptor function in some patients, relapses, poor survival of injected T cells, and silencing by mechanisms other than PD-1 may arise.


The Model TCR

screen-shot-2016-09-18-at-4-18-41-pm-pngDr. June’s research team, in addition to testing many blood cell gene therapies for HIV and cancer, led one of the first Chimeric Antigen Receptor (CAR) T cell trials against the blood cancer Chronic Lymphoid Leukemia in 2011. Here, a CAR consisting of a domain directly recognizing CD19 expressed on leukemic cells, without requiring the self-protein context needed by natural TCRs, was introduced using lentivirus. The CAR also included chimeric (from a different source) T cell activation regions to turn on T cell defences without the need for other stimulatory signals. From 2011 to 2015, June and other investigators conducted many trials using lentiviral vectors to deliver relevant CARs to patients with many different cancers. Other teams used the TALEN gene-editing tool to insert the CAR. CAR T cells were able to eliminate cancerous cells in most patients, with mild to severe symptoms caused by tumour death. In other patients however, a severe attack of healthy tissues was observed, presumably the result of uncontrolled CAR-driven immune activation. An FDA evaluation of these Phase II trials is pending and will determine whether CAR-T therapy is viable in the long-term.

For now, perhaps due to the lack of Phase II data using a NYESO-1 CAR alone or due to the expectation of fewer adverse effects using the traditional TCR in the absence of PD-1, the current proposal does not use the CAR approach. This restricts its clinical impact because, unlike its CAR counterpart, this TCR can only detect NYESO-1 in a limited cohort of patients. However, pharmaceutical companies may be interested in uniting the CAR and CRISPR technologies. After establishing CAR-T trials, Novartis bought Intellia Therapeutics earlier this year, a start-up that develops CRISPR methods to insert CARs into T cells and make gene edits in blood stem cells. Although Novartis’ plans have not been released, we can likely expect more CAR-T and CRISPR monotherapy trials in the near future.


The CRISPR Runaway

Within a month of UPenn’s NIH approval, Sichuan University’s Dr. You Lu also received approval to use CRISPR to remove PD-1 in T cells from lung cancer patients. Canada too is not far off in using CRISPR to address clinical problems, with Health Canada and NIH policies being similarly permissive to this robust form of gene editing. U of T affiliated scientist Dr. Ronald Cohn is exploring the use of CRISPR to treat Duchene muscular dystrophy. But CRISPR’s potential is not limited to direct clinical applications. In the past, Brazil has authorized the release of genetically altered mosquitoes to control Dengue fever and is now considering using CRISPR to curb the Zika virus outbreak.

The biomedical community remains enthusiastic and is working hard to realize CRISPR’s clinical promise. It is, however, important to keep in mind that without an astute understanding of gene function in health and disease, CRISPR is merely a tool. For CRISPR to reach its full utility in the clinic, basic science research must continue to uncover the best gene targets for CRISPR-based therapies.


  1. Brower, V. The CAR T-cell raceThe Scientist 2015.
  2. Porter, DL et al. Chimeric Antigen Receptor – Modified T cells in chronic lymphoid leukemiaThe New England Journal of Medicine 2011; 365: 725-733.
  3. Rapoport, AP et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myelomaNature Medicine 2015; 21: 914-921.
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Priya Makhijani

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