Synthetic gene circuitry to refine CAR T therapy

Here we describe a novel way to control CAR T therapy through synthetic gene circuits. To learn more about controlling CAR T therapy, read our article on the use of antibody switches to control CAR T cell activation. Previous episodes also deal with the CAR T fundamentals and its applications for B cell cancers, multiple myeloma, lupus, multiple sclerosis and the heart, as well as new search on the suit CRISPR/CAR T therapy.

Although CAR T therapy represents one of the most impressive innovations in cancer care, the treatment can cause autoimmune-like side effects. Many have faced the dilemma of maximizing the therapy’s benefits while minimizing its side effects. A recent breakthrough published in Science holds great promise for improving control of CAR T therapy with the hope of eventually benefiting patients.

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CAR T Therapy Challenges

VSchimerical Antigen Rreceiver J cell therapy relies on the engineering of synthetic receptors on a patient’s immune cells to recognize and eliminate a programmed target. The treatment has been shown to be effective in targeting antigens, or biological markers, found on the surface of certain blood cancer cells. Although successful in this regard, the modified T cells cannot be controlled once injected back into the body. This factor, coupled with other hurdles such as its inability to target more than one antigen at a time, leaves room for improved treatment.

Possible solutions

There are several nascent research initiatives looking for possible solutions. One method of controlling CAR T therapy discussed previously is to create CAR T cells that bind to antibody switches. Researchers found that a single infusion of antibody switches could help alleviate the worst toxic side effects of CAR T therapy. Although this method has the potential to fight solid tumors by changing the antibody target, it did not not yet tested in animal or human models.

A recent study by Boston University scientists offers an alternative and versatile platform that could transform the implementation of CAR T therapy. The team looks to synthetic gene circuits for answers and is getting encouraging results.

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A new alternative: synthetic gene circuits

In a genetic circuit, the process of transforming an input into a desired output can be controlled by the use of a synthetic regulator (see Figure 1). The synthetic regular can, in theory, tailor a cell’s gene expression to produce a desired result. The researchers in this study designed such a circuit using synthetic proteins and small molecule switches.

To establish the circuit, the team relied on synthetic versions of proteins called transcription factors. Transcription factors can recognize certain DNA patterns and help convert that DNA into RNA, genetic instructions for protein synthesis. They take advantage of synthetic zinc fingers (SynZiFTR) especially because of its compact size and its human origin; Figure 2 illustrates the structure. Together, these two factors allow the protein to move efficiently through human cells while minimizing unwanted side effects. Additionally, multiple zinc fingers can be joined together to create a structure capable of recognizing potentially unique human DNA sequences in the genome, as shown in Figure 3.

This circuit must be controlled using a gene switch. The team experimented with three clinically approved small molecule inducers to accomplish this task. The unique combination of zinc fingers and small molecule inducers allowed genes to be turned on with the introduction of the inducer and turned off with its removal.

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Combining synthetic gene circuits with CAR T therapy

How do these synthetic gene circuits behave when used on CAR T cells in vitro and live? The team investigated this question in several stages using cellular and animal models.

The researchers first tested the efficiency of a gene circuit controlling the expression of chimeric antigen receptors. They found that in a xenograft liquid tumor model, expression of the altered receptor could indeed be controlled in a drug-dependent manner. Treatment could be enhanced using the same stimuli and secondary infusion of chimeric antigen receptors with a different antigen target, demonstrating the adaptable premise of the platform.

The platform also produced similar results when tested in blood tumor models in mice. Mice treated with a drug inducer or drug-enhancing cocktail could clear the tumor, while those without had a high tumor burden. This phase illustrated how genetic circuits can be influenced in a drug-dependent manner in living beings.

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One of the most fascinating findings of this study is the creation of a double-switched genetic circuit. The researchers engineered a genetic circuit that impacted cytokines known to influence cell proliferation. This cytokine circuit could successfully prime double-switched CAR T cells in mice with leukemia; a secondary signal to encourage CAR expression triggered their anti-tumor activity. The sequential and synergistic effect of the double-switch circuit can be seen in Figure 4. This duo reduced tumor burden in mice more successfully than untreated CAR T cells or CAR T cells induced with anti stimuli. – tumors only.

Looking forward to

Here, researchers show how synthetic gene circuits can enhance CAR T cell proliferation and anti-tumor activity in animal models. This promising platform could prove clinically viable once translated into humans. However, the implications of this study extend beyond the rapidly growing field of CAR T therapy. The underlying mechanism could be customized to improve other gene and cell therapies, or combined with other powerful technologies. such as CRISPR-Cas9 for more personalized results.

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