Crazy for CRISPR!

Author: Sara M. Nolte, 12/05/16

On November 15th, my social media pages exploded with posts and comments regarding the latest news about how the gene-editing ‘CRISPR-Cas9’ technology had been used in the first human patient.

The article, published by Nature, was entitled “CRISPR gene-editing tested in a person for the first time.” It described how a group of Chinese scientists administered CRISPR-Cas9-modified immune cells to an advanced lung cancer patient, as part of a clinical trial investigating the technology as a cancer immunotherapy. The writer speculates how this announcement may affect a similar trial commencing soon in the United States, and what the results of the trials could mean for the future of cancer treatment.

My friends continued the debate on social media. They raised some interesting questions, like what side effects might be expected, and the practicality of cell-based immunotherapies. Most were truly excited as they use CRISPR in their own research. A small group questioned how ‘gene-editing’ and ‘in a person’ might be misconstrued by many as suggesting that the scientists genetically engineered a person.

With many questions of my own, here I am again (it’s been a while!) to, hopefully, shed some light on the situation.

The trial

The Chinese clinical trial, led by oncologist Lu You, from Sichuan University’s West China Hospital, was originally announced by Nature in July 2016. The clinical trial – officially called “PD-1 Knockout Engineered T Cells for Metastatic Non-small Cell Lung Cancer” – is a Phase I Clinical Trial (see my blog and infographic on the clinical trial process), to primarily determine the safety of the genetically modified T cells.

The study is being performed in fifteen patients with stage IV (very advanced) non-small cell lung cancer that has continued to progress, despite all currently approved therapies. A patient’s own (autologous) T cells are collected from their blood, genetically altered ex vivo (in a lab) using the CRISPR-Cas9 technology to remove (knockout) the PD-1 gene. The modified cells are then infused back into the patient. Several different dose plans will be tested to determine dose-related safety and efficacy.



At this time, the treatment is going as planned – though there are still several patients to enter the trial, and several months of monitoring.

 The technology

The CRISPR-Cas9 system is a method of gene editing borrowed from bacteria. Bacteria originally used CRISPR-Cas9 as an immune defense against bacteriophages (bacteria-specific viruses).  The ‘CRISPR’ portion – Clustered Regularly Interspaced Short Palindromic Repeats – refers to a pattern of DNA sequences that can be recognized by the Cas9 endonuclease: an enzyme that cuts DNA. Using CRISPRs as a recognition sequence, Cas9 can cut the DNA in specific locations.

Scientists have modified the system to target specific genes. This is done by creating a ‘guide RNA’ (gRNA) that is unique to the gene of interest. The gRNA allows for CRISPRs to be inserted around the gene of interest in the target genome. Cas9 recognizes the CRISPRs as a ‘signal’ to cut the DNA around the gene of interest, removing it from the target genome. Andy Warner has a great illustrated commentary on CRISPR, or watch this video from the McGovern Institute for Brain Research at MIT.

In the case of this clinical trial, the researchers have designed a gRNA that is specific to PD-1, their gene of interest. The cancer patients’ T cells are manipulated with the CRISPR-Cas9 PD-1 construct, resulting in PD-1 being ‘edited’ out of the genome.

The target

PD-1 – Programmed cell Death protein 1 – is a receptor protein found on the surface of T cells. T cells are among the immune cells necessary to kill off invading pathogens (e.g. bacteria) or ‘unhealthy’ cells (e.g. tumour cells). PD-1 predominately acts as an ‘off’ switch for the T cell immune response.

When PD-1 binds one of its ligands, PD-L1 or PD-L2, a signal is sent into the T cell, telling it to shut down the production of immune defense molecules, to stop dividing, and to undergo apoptosis (cell death). This is a necessary regulatory pathway for normal immune responses: once the ‘threat’ to the body is gone, the immune response needs to be turned off.

But why have scientists decided to pursue PD-1 as a target for gene editing with CRISPR-Cas9? This is primarily for two reasons:

  1. Knocking out PD-1 essentially turns ‘off’ the immune response ‘off’ switch. This allows the T cells to stay ‘on,’ allowing them to attack tumour cells.
  1. Tumour cells, especially in non-small cell lung cancers, express PD-1 ligand PD-L1, allowing the tumour to turn off T cell-mediated attacks. By knocking out PD-1, T cells are no longer susceptible to ‘off’ signals from the tumour.

Essentially, the researchers are fine-tuning the patient’s immune response to better attack their cancer.

Why it could work

PD-1 has already been shown to be a useful target for cancer immunotherapies. For example, nivolumab and pembrolizumab are antibodies against PD-1, blocking it from interacting with PD-L1 on tumours. Both antibodies were approved by the Food and Drug Administration in 2015 for treatment of non-small cell lung cancers. The effectiveness of the therapies (improved survival and tumour regression), suggest that eliminating PD-1 signaling pathways will be successful.

A different gene-editing technology – zinc finger nucleases (ZFNs) – has successfully been used in patients with HIV. The ZFNs were used to alter the gene for the receptor that HIV uses to infect host cells, making the receptor dysfunctional.  Patients with this gene-editing treatment had lower levels of HIV in their blood, and higher numbers of immune cells – all good things! This success story suggests that CRISPR-Cas9, which is more specific in its gene-editing than ZNFs, can also be successful in human patients.

What are the potential problems?

While all this sounds amazing and cool, you’re probably sensing a ‘but…’ somewhere. And there are a few potential problems that may arise.

The first set of issues has to do with the logistics of the therapy. Each cycle of therapy needs twenty million per kilogram of PD-1 knockout T cells. So if you weigh 50kg, you will need one billion genetically modified T cells. This number is per cycle, and the experimental arms are looking at 2-4 cycles… As someone who has worked in cell biology, getting that number can be very hard to do! On top of this, it’s possible that after the initial treatment, patients may require additional rounds of therapy, which means… even more T cells are needed!

Perhaps more concerning are potential side effects of the proposed therapy. Studies have demonstrated that PD-1 knockout mice have a higher prevalence of autoimmune diseases, such as dilated cardiomyopathy and lupus.  At first glance, this seems highly alarming, but rest assured that in order to have moved to a Phase I clinical trial, pre-clinical animal studies would have demonstrated efficacy and minimal safety concerns. Please also bear in mind that CRISPR-Cas9 knockout of PD-1 in T cells in a Petri dish is very different than global knockout of PD-1 in a mouse.

With all that said…

I think we’re in for some exciting times in the next few years. There is certainly promising and encouraging data for CRISPR-Cas9 gene-edited PD-1 knockout T cells as a cancer immunotherapy, but there is also uncertainty. Only time and science will tell!


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Sara M. Nolte

Sara Nolte holds a Bachelor of Health Sciences and Masters of Science in Biochemistry & Biomedical Sciences from McMaster University. Her MSc research focused on developing of cancer stem model to study brain metastases from the lung. She then spent a year working on developing cell-based cancer immunotherapies. Throughout a highly productive graduate career, Sara became interested in scientific communication and education. She is now involved in developing undergraduate programs and courses in the health sciences at McMaster, and is looking for ways to improve scientific communication with the public. Outside of science, Sara enjoys participating in a variety of sports, and is a competitive Olympic weightlifter hoping to compete at the National level soon!
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