CRISPR and allogenic CAR-T Cells is revolutionary for cancer treatments
I recently listened to “The Sheekey Science Show’s” conversation with Ryan Murray on the future on cancer therapies. The podcast was very intriguing, so I also read Ryan Murray’s preprint research article: Comprehensive genome editing confers ‘off-the-shelf’ CAR-T cells superior efficacy against solid tumors. Ryan Murray is a current PhD student at North Eastern University who has been using an innovative form of gene editing, called base editing, to engineer allogenic T-cells to more effectively target cancer cells.
The paper discusses a novel approach to genetically engineering off-the-shelf CAR-T cells in order to make more effective solid-tumor targets, by minimizing effects from biochemical and immunological inhibitors. In this article I am discussing/summarizing Murray’s really cool research, and I hope you enjoy. Here is the podcast, if you would like a thorough introduction:
T-Cells are a major part of the immune system, which target pathogens like viruses and bacteria. Today, they are intensely researched for therapeutic purposes in immuno-oncology, or fighting cancer. T Cells have an innate ability to target cancer cells, which is what CAR T cells are somewhat designed to mimic.
When there is a foreign or infected cell, natural T cells are designed to target that cell using there TCR (T cell receptor). However, cancer has many immuno-evasion mechanisms, that prevent interaction with T cells or suppress the immune response, making T cells ineffective. For example, cancer cells have many potential T cell targets (antigens) that are also expressed on healthy normal tissue. Thus, T cells may not recognize cancer cells as foreign or dangerous, therefore not initiating an immune response to destroy them.
Administrating Chimeric Antigen Receptor or CAR T Cells is a method to make T cells better recognize and precisely target cancer cells. This involves engineering specific extra antigens on T-Cells that help them evade immunosuppressive strategies that cancer cells employ. Modern research is trying to make CAR T cells more potent, persist in the patient longer (to eradicate tumors), more efficient, and target different types of cancers (i.e.. tumors and blood cancers).
The standard treatment method is to extract a patient’s T Cells, engineer it with chimeric antigen receptors in the lab, and administered back into the patient (Autologous CAR T cells). However these method is extremely lengthy, challenging, and cost ineffective to administer for each individual patient (especially when patients cannot tolerate the time needed to engineer their T cells and administer them back). As a result treatments are moving away from autologous methods and more towards allogenic CAR T cells (still in its development), where T cells are taken from a healthy donor, engineered, and administered into the patient. CAR T cells are essentially stored in the freezer and can be administered on demand (called “off the shelf”). There is no delay in the engineering process. This however requires more engineering in the lab to make sure that when it is administered into the patient, the body doesn’t see them as foreign (allorejection). The other alternative is in-vivo engineering (new and on the horizon), where T cells are engineered within the body. Treatments involve proteins, genetic engineering methods, etc. that can create the CAR T cells within the body. There is no risk of immune rejection, the need for removal of T cells, or lengthy times for treatment.
Engineered cell therapies — after years of fighting cancer using small molecules and antibodies, we’re entering the era of cell therapies against cancer. Using gene editing, we can engineer immune cells to kill cancer in a robust and specific way while avoiding many toxicities of traditional cancer therapies — CRISPR Therapeutics
There are many genetic engineering strategies to increase the potency of CAR T cells including gene editing. This article discusses the use of gene editing through CRISPR Cas 9 technology (specifically base editing) to make allogenic CAR T cells resistant to allorejection and biochemical/immunological inhibitory signaling (tricksters that trick T cells) within a tumor microenvironment (TME), making them have greater effectiveness against solid tumors. This research is very important to make CAR T cells an appropriate and effective treatment against all cancers.
Biological (high levels of adenosine in the TME as a product of high metabolism which can suppress immune response) and immunological signals (PD-L1 and TGF-β) inhibit CAR T cells, explaining their clinical failures associated with tumor related cancers (but they are very good at blood cancers for the same reason — cancer cells are dispersed equally). PD-1 is a protein on the TCR which acts as an “off switch” (helping keep T cells from attacking healthy tissue) when it attaches to PD-L1 (an antigen on normal and cancer cells). TGF-β is a key cytokine that suppresses T cell activation and function through direct and indirect cytokine pathways.
High levels of metabolism and tumor growth results in hypoxia or low oxygen levels in the TME. This expresses HIF-1α which impacts expression of various genes in the cell. For example it reduces ATP consumption, promotes anaerobic methods to produce ATP (fermentation), and creates an adenosine rich environment (causes immunosuppression in a TME). HIF-1α also increases the expression of PD-L1 and TGF-β that bind to PD-1 and TGF-βRI/RII to stop T cell activity (as discussed before).
To get a better idea of CRISPR, check out my comprehensive article discussing all the basics of the technology:
Base editors are helpful as they can make small changes in the genome without causing double stranded breaks. Double stranded breaks are often fatal to a cell as it shatters the integrity of DNA. It can potentially cause cellular senescence, the activation of the p53 gene (which tells cells to commit suicide), cancer, translocations in DNA, and more.
To knock out a gene, base editors can be administered to disrupt either the transcription of a gene or translation to stop the production of a protein. This can be done by targeting the start site of a gene or specific regions of intron/exon splice junctions (leads to non-sense mediated decay). To increase the range of target sites for base editors (often restrictive), they can be fused with a CRISPR-Cas nuclease (CRISPR systems have a very ubiquitous PAM motif). The researchers at Ryan Murray’s lab used a multiplex adenine base editor fused with a dead CRISPR-Cas12b nuclease to knock out 6 genes in CAR T cells that are associated with various immunosuppressive mechanisms.
- Stealth edits: B2M & CIITA for allorejection and CD3E for graft-versus-host disease (autoimmune response)
- ADORA2A for biochemical inhibitors (adenosine)
- PDCD1 (PD-L1) and TGFBR2 (TGF-β) for immunological inhibitors
Combined, a 6-plex gene edited CAR T cell called as Stealth-TKO CAR T cells is created. These variant CAR T Cells are “off the shelf”, can avoid autoimmune diseases, and are effective against solid tumors. In mouse models Stealth-TKO CAR T cells caused improved elimination of tumor cells.
CRISPR-based systems are widely used and powerful because they can be easily reprogrammed to target different sites in the genome. This new system is another way to make precise changes in human cells, complementing the genome editing tools we already have. — Feng Zhang
Main Research Findings
- Adenosine resistant CAR T cells resist immunosuppression caused by the accumulation of adenosine in TME
- Multiplex base editing of adenosine resistant CAR T cells make them resistant to other immunosuppressive methods.
- Stealth TKO CAR T cells resists allorejection and has potent anti-tumor activity
The researchers engineered adenosine resistant CAR T cells by removing the adenosine receptor A₂ₐ encoded by ADORA2A. They screened for sgRNA-base editor complexes by electroporating an adenine base editor or a cytosine base editor with various corresponding sgRNAs. By doing so they identified, an sgRNA-adenine base editor complex that targeted intron-exon junctions achieved the highest genome editing efficiency (88%). They found out that knocking out ADORA2A stopped A₂ₐ function, by observing no change in the level of pCREB with response to 1-chloroadenosine compared to a control group. Finally, they put adenosine resistant CAR T cells with lung cancer cell lines in the lab. After adding adenosine, they found out that the CAR T cell action was unaffected (there was still high levels of cytokine secretion and removal of tumor spheroids). On the other hand, untreated allogenic CAR T cells had their cytokine production suppressed when treated with adenosine. A similar experiment was conducted (in vivo) in mice with tumors and the same conclusion was achieved.
TMEs have adenosine but also many other immunosuppressive methods. So, adenosine resistant CAR T cells alone may not completely eliminate cancerous cells. The researchers mapped gene activity in the TME (digital spatial transcriptomics) and used flow cytometry and found out many more immunosuppressive methods and found that: in hypoxic areas there were higher levels of TGF-β and tumor cell surfaces have high levels of PD-L1. Through in vitro experiments they found out that adenosine resistant CAR T cells were sensitive to these alternative immunosuppressive methods. Adenosine resistant CAR T cells secreted amounts of cytokines inversely proportional to the amount of PD-L1 inhibition and amount of TGF-β in the TME.
The researchers then tried to create CAR T cells that were also resistant to TGF-β and PD-L1 inhibitory methods. They used the previous base editing complex type to target the PDCD1 gene that encodes the PD-1 receptor, resisting suppression of cytokines. They screened various gene editing complexes and identified a Cas12b-sgRNA complex to target TGFBR2 gene that encodes TGF-β receptor. These three gene edits (including adenosine) was named TKO (triple knock out). Slealth edits were also conducted and through various in vivo and in vitro experiments the 6 edits showed increased effectiveness against solid tumors.
Gene editing is constantly evolving, and when paired with CAR T cells, it can be revolutionary in cancer treatments. The future of the 🌎 is CRISPR 🧬!
Before you go…
You’ve learned about what Slealth TKO CAR T cells are, how they were discovered, and its efficiency. I simplified the main research findings (subjective based on my understanding) of Ryan Murray’s paper. You can read it for yourself here: https://www.biorxiv.org/content/10.1101/2023.06.13.544871v1.full
If you enjoyed reading through this article feel free to connect with me on LinkedIn
