Gene Switch: A Novel Platform for Switching Genes On and Off

The notion that genes might be turned on and off was discovered several decades ago when studies revealed that E. coli bacteria, as well as lambda bacteriophage, can adapt to the alterations in the composition of their nutrient medium. Gene switches are sites on genes where regulatory molecules can bind to trigger transcription process, leading to expression of a particular gene. Further, the expression of any gene is dependent on the rate at which it is transcribed into mRNA and translated into proteins. There are various regulatory proteins or transcription factors that are responsible for affecting the transcription rate. Gene switches can be regulatory proteins or specific DNA sequences that act to either switch on or off the expression of a gene.

Basic Components of Gene Switch

Gene switches are composed of noncoding DNA sequences and transcription factors. These proteins recognize specific DNA sequences known as enhancers within a cell nucleus and determine whether the gene will be switched on or off.  Further, the transcription factors contain two molecular domains, the DNA binding domain and the activation domain. The DNA binding domain recognizes and binds to specific DNA sequences and the activation domain recruits transcription complexes to initiate transcription. Moreover, some transcription factors contain additional domains, such as ligand binding domain in order to interact with chemical signals.

 

Mechanism of Gene Switch

During the transcription process, the promoter region, which is located near the upstream end of each gene, binds to transcription factor, which is a specific type of protein. It is worth noting that the transcription factor is responsible for recruiting RNA polymerase to bind to the gene and produce messenger RNA, which is then translated into the protein. An additional level of genetic control is provided by gene switches that are located upstream of the promoter region. These genetic switches assist transcription factors in binding to the promoter region. Each gene has multiple gene switches that can be activated at any given point of time in order to allow the expression of a particular gene.  Figure below presents an overview of the mechanism of action of gene switch.

Type of Gene Regulation

Based on the type of transcription factor (activators / repressors), gene regulation has been categorized into positive and negative gene regulation. The two types of gene regulation have been discussed below:

• Positive Gene Regulation: Activator is a protein molecule that helps to initiate a positive gene regulation. When activator binds to the operon, it either speeds up or permits gene expression.
• Negative Gene Regulation: Repressor is a protein molecule that initiates negative regulation. When the repressor binds to the operon, it either slows or stops the gene expression.

Applications of Gene Switch

The design of gene switch platforms with distinct properties exemplifies the promise of synthetic biology in the creation of more advanced cell-based therapies. The design of gene switch platforms with distinct properties exemplifies the promise of synthetic biology in the creation of more advanced cell-based therapies. The applications of gene switch platforms can be broadly classified into the following two categories:

• Endogenous Gene Regulation
• Exogenous Gene Regulation

1. Endogenous Gene Regulation

The ability to regulate endogenous genes has various applications in biological research and gene therapy development. Several diseases are caused by sub-optimal gene expression or through uncoordinated or unsynchronized production of a gene product. Regaining control over the endogenous gene locus could be an effective approach for the treatment of such diseases. Additionally, regulation of endogenous gene expression can be a valuable tool for understanding fundamental biological pathways. Gene switches allow researchers to switch a gene of interest on or off, at a desired time point to study its effects.

• Exogenous Gene Regulation

Uncontrolled overexpression of exogenous genes often results in counter productivity when they are introduced into a host. This is true for any genetic products or byproducts of the metabolic pathways that are toxic to the host. Overproduction of such products results in imbalance and abnormalities in various metabolic processes of the body. Therefore, gene switches are used as an external inducible production mechanism in order to control expression of genes. The primary applications of gene switches have been described below:

• Management of Therapy-related Toxicities: Cell-based therapeutics employing engineered cells, including CAR-Ts and TCR-based therapies, have demonstrated promising results in clinical trials for the treatment of various indications. However, despite several advantages, engineered T-cell therapies are prone to several adverse events, which has limited the widespread use of such effective therapeutic modalities. Therefore, engineered T-cell responses must be regulated to prevent severe side effects. The safety of aforementioned cell-based therapies can be improved by incorporating a reversible ON or OFF safety, gene switch. Currently, several studies are underway to evaluate small-molecule-based safety switches for various cell-based interventions. Various small molecules, including rimiducid, FITC, folate, rapamycin, dasatinib and proteolysis-targeting chimera (PROTAC) compounds, are being investigated to design such safety switches.

• Regulation of Gene Expression: Another important application of gene switches is controlling the expression of transgene in various gene therapies without the requirement of additional protein components. For instance, studies have demonstrated that gene switches incorporated in gene therapies being developed for the treatment of anemia can regulate the production of hormone erythropoietin.

• Cell Proliferation: Gene switches are used for enhancing the effector cell proliferation and lifespan extension of activated cell therapies leading to durable patient responses to therapeutics. Gene switches, such as GoCAR technology of Bellicum Therapeutics, can enhance CAR-T cells proliferation and functional persistence by resisting exhaustion and driving production of immunomodulatory cytokines, thereby overpowering the inhibitory signals from the tumor microenvironment.

• Other Applications: Numerous cellular phenotypes are inaccessible under the control of a single gene locus due to its vastness and complexity. One of the most promising contributions of synthetic biology is the development of biological devices, such as gene switches, that can exploit regulation of metabolic pathways. Transcription regulators that respond to metabolites and RNA switches are used to alter the metabolic pathways. The same concept can be employed to discover novel drug targets and therapeutics for the treatment of various disorders.

Challenges Associated with Gene Switch

Gene switches have been used widely in order to increase efficacy and reduce the side effects posed by various gene therapies. However, activation of gene switch in vivo requires administration of small molecules that can cause unwanted side effects in patients. Therefore, although a gene switch may induce programmed cell death in culture cells to mitigate adverse events caused by engineered cell therapies, it could be considered insufficient / dangerous for clinical practice. Additionally, the genetically modified cell therapies might transform into uncontrollable mutant cells when the switch is inactivated. Moreover, when the genetically engineered cells die, they can release a significant amount of genetically altered DNA that might either integrate into host cells or trigger harmful anti-DNA immune reactions in the host body.[9] Consequently, the focus of researchers has shifted towards the development of regulatory switches that exhibit high efficacy and do not cause any adverse effects.

Concluding Remarks

Transcription regulators or gene switches controlled by small molecules have been proven to be an effective tool in many areas of synthetic biology. In developmental biology, signaling pathways can be altered by temporal suppression of genes within the pathways. Further, the advancement of synthetic biology has increased the demand for orthogonal gene switches that assist in the development of complex gene circuits, in order to increase the efficacy of gene therapies and reduce their associated side effects. In addition, there is a growing interest in exploring the potential of gene switches to regulate complex phenotypes of mammalian cells, including remodeling of complicated interconnected genetic network. The rising interest in gene regulation by genetic switches is supported by its potential to manufacture challenging yet highly effective cell therapies by reducing their side effects. Given the rising demand for cell and gene therapies and growing concerns associated with their safety, stakeholders in this industry are developing a number of safety switch / gene switch systems with an aim to gain control over infused cell products.

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