Multiple hurdles face developers of novel cell-based therapies, such as CAR-T, to successfully produce effective therapeutics. Following is a brief summary of some of the key challenges and how they are being addressed through the use of gene editing.  

If the patient’s own T cells are to be used, there must be enough healthy cells available for harvest and proliferation. However, the cancer a patient may be fighting, or the treatment they’ve received, may have damaged the cells or reduced their number. Even if this is not the case and it is possible to manufacture the modified cells to transfuse into the patient’s body, those cells then face a number of functional challenges. They may attack the patient’s cells if they’re derived from a different donor. They may even attack each other. Supposing those problems are avoided, the cancer afflicting the patient can only be fought effectively if the engineered immune cells can find the cancerous cells and actively attack them for a long enough period. They must do this even when the cancer may have created a toxic or soporific microenvironment around itself. Lastly, the attack must not cause undue harm to the rest of the patient’s body. All these risks must be mitigated, and researchers have homed in on specific genes to help accomplish the feat.  

Knocking out the TRAC, CIITA and B2M genes can mitigate the risk of graft versus host disease (GvHD).[1,2] Without functional copies of those genes, modified cells lose their endogenous T-cell receptor (TCR) and major histocompatibility complex (MHC) proteins, and thus won’t be triggered to attack healthy host cells. Researchers are searching for ways of avoiding GvHD to make it easier to use healthy donor (allogenic) cells rather than having to use potentially ailing and scarce patient-derived (autologous) cells. Allogenic CAR-T cells could also be ready and waiting as soon as a patient needs them. 

To address the need for sustained activity of the CAR-T cells being introduced, PD-1, CTLA-4, LAG-3, and TIM-3 are additional targets for disruption. T-cell exhaustion describes a decline in the the ability of the modified immune cells to stay alive and active as they attack the cancer. The causes of this fatigue are multifactorial and an area of active research. Investigators have reported reduced T-cell exhaustion by turning off these four genes.[1,2] 

Lastly, GM-CSF and IL-6 are two gene targets whose deactivation can improve the safety of CAR-T cell therapies. [1] The cytokines encoded in these genes entail risk of Cytokine Release Syndrome and neurotoxicity if their production becomes dysregulated.  

The ultimate goal of having pre-made, off-the-shelf modified immune cells is within sight, though still out of reach. Perhaps the day is not too far off when allogenic CAR-T cells will be available on demand through the modification of these and other genes. 


  1. Dimitri, A., Herbst, F. & Fraietta, J.A. Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing. Mol Cancer 21, 78 (2022). 
  2. Liu X, Zhang Y, Cheng C, Cheng AW, Zhang X, Li N, Xia C, Wei X, Liu X, Wang H. CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells. Cell Res. 2017 Jan;27(1):154-157. doi: 10.1038/cr.2016.142. Epub 2016 Dec 2. PMID: 27910851; PMCID: PMC5223227.