Chimeric antigen receptor T cells, otherwise known as CAR T cells, are a marvel of today’s molecular medicine, made possible by genome editing. In CAR T-cell therapy, the body’s own T cells can be engineered by introducing a CAR domain so that they recognize and destroy tumors. The therapy has proved extremely successful in many different trials, particularly for the treatment of blood cancers.
That being said, CAR T-cell therapy faces many challenges. For one, trials have not been very successful in the treatment of solid tumors, as it is more challenging for the CAR T cells to penetrate the tumor microenvironment and exhibit any kind of antitumor effect. There are other potential drawbacks for CAR T cells, such as antigen escape or off-target effects, as well as severe and sometimes life-threatening toxicity brought about by high levels of systemic cytokines.
However, research is not prepared to give up on such a promising therapy. There are many solutions to the problems facing CAR T-cell therapy. For example, T cells could be engineered to have more potent antitumor activity with transgenic cytokine expression, while being able to successfully navigate the tumor microenvironment. Toxic side effects can also, eventually, be engineered out of the T cells.
All these solutions have one thing in common: they require modification of the genome of the T-cell population. On the face of it, this is great news. We have CRISPR genome modification technology. Its originators received the Nobel Prize, and it has been hailed as the discovery of the century. Even if it doesn’t work, there are alternative genome modification technologies that could be used instead.
However, there is still a huge hurdle to overcome, and if we don’t find a solution, it could mark the end of the road for CAR T cells. The problem is that creating CAR T cells requires that T cells take up DNA, which is then used to modify the genome. In the future, to make better CAR T cells that can attack a wider range of cancers, we need even more DNA to perform more aggressive modification on T cells. The problem is that until now, when we try to modify T cells, many of them die.
When we expose T cells to high concentrations of double-stranded DNA, up to 80% of the population dies. This is problematic, as often the starting material for these modifications consists of T cells, which are rare and often difficult to harvest from sick patients. If 80% of the material from the patients is wasted, then it makes performing the genetic manipulations and administering the therapy all the more difficult, not to mention expensive.
If high DNA concentrations are killing the T cells, why not simply lower the concentration? The problem is that low concentrations are not effective enough. There is a DNA response in therapeutic cells with the sole purpose of removing foreign DNA. This is an important defense mechanism for the cells, but it makes inserting CAR constructs difficult and, for now, means that high DNA concentrations are necessary.
This problem could be a significant barrier for the scale-up of CAR T-cell therapies. As previously mentioned, CAR T cells are available in personalized therapies that use autologous T cells from patients. This is extremely costly and labor intensive and has to be repeated for every single treatment. A solution would be to produce universal CAR T cells from healthy allogeneic donors. This approach would be less costly and make CAR T cells more widely available. However, it would be difficult to make universal CAR T-cell treatments if T-cell genomes had to be extensively modified using current approaches.
One potential positive avenue of research is the use of specific cocktails of inhibitors to block the DNA sensing in the cell, which would prevent killing and increase the yield of transfections. This would mean that high concentrations of DNA would be unnecessary, and toxic effects would be significantly reduced.
Another important aspect to consider is the DNA itself. Ultimately, it is the properties of the DNA that cause it to be recognized by the cell, resulting in toxic effects. If these properties could be modified, then toxicity could be lowered.
There is an increasing body of evidence to show that single-stranded DNA is much less toxic to cells than double-stranded DNA. Single-stranded DNA also has a number of other documented benefits, such as eliminating off-target events during editing, which could cause unwanted mutations. It’s also less immunogenic, so there are fewer problems when working directly with patients.
Single-stranded DNA can also be easily modified to produce molecules with unique biological properties. Modified single-stranded DNA can reach cells more easily and avoid recognition by the DNA sensing mechanism. These improvements should lead to higher efficiency of transfection. They could also reduce the cost of development, which is an important consideration for clinical trials.
It is important to address the problems facing CAR T-cell manufacturing now to ensure that this life-changing therapy can impact more people and ultimately save lives. With manufacturing issues solved, it will be easier to address more long-term therapy options such as Tonic Signaling, in which low levels of CAR T cells are maintained in patients to be stimulated in the case of cancer relapse.
Solving the issues with DNA will make genome editing a far more precise and effective tool in CAR T-cell therapy. It will be much easier to produce more highly modified cell lines with unique properties tuned to individual use. As previously mentioned, it will be possible to introduce transgenic cytokines and additional targeting domains without reducing cell viability or raising the risk of off-target effects. CAR T cells have the potential to revolutionize cancer therapy, and in some ways, they are beginning to realize this potential. However, without the right tools to perform efficient genome editing on T cells, CAR T cells may be sidelined as a niche therapy with limited use.
Cosimo Ducani, PhD, is the co-founder and CEO of Moligo Technologies.