By Shirin Bamezai
Since the beginning of the post-genomic era, one of the areas of research that has been consistently pushing the frontiers of medicine is gene and engineered-cell therapy. Gene and cell therapy aim to treat diseases by introducing genetic material in cells that either repairs or enhances their genome in order to produce therapeutic effects. In the last decade, the field has had some clinical success in the treatment of monogenic disorders, such as spinal muscular atrophy, haemophilia A and B, with great advances being made in the treatment of cancer as well. However, broader adoption of such therapeutics is challenging due to limitations in the control over the strength, timing and cellular context of the therapeutic effect, which causes safety concerns. For example, in clinical trials of engineered T cells, several fatal adverse events were reported. These events included neurotoxicity related to excessive activation of the said cells (Lim et al.), a result of the therapy relying on the over-expression of transgenes as most existing gene therapies do. Synthetic biology provides the tools required to overcome this hurdle, enabling the design of programmable gene and engineered-cell therapies with far-reaching applications.
In the field of synthetic biology, new synthetic biological systems are built through the marriage of traditional engineering concepts (such as abstraction, modularity, feedback control) and design rules specific to biology. This translates into researchers evaluating aspects of biological systems through the lens of engineering principles, enabling manipulation of the former. Modularity, for example, is a principle that allows engineers to design complex systems through the combination of simpler units (inputs and outputs). In the context of cell biology, this means viewing biological systems as a hierarchical connection of many simpler units, the simplest functional unit being molecular interactions (Kitada, 2018). Thus transcription can be classified as a module with two inputs (a DNA molecule and a transcription factor) and one output (an RNA molecule). This is of course an abstraction that neglects important details, however the simplicity gained enables bioengineers to more easily build complex synthetic systems via a composition of modules that interact with endogenous cell processes. The behaviour of the system is then assessed as a series of logic operations. The end result is a programable control over cell behaviour, that provides bioengineers with the ability to program and install personalised genetically encoded therapeutic programs.
Currently, synthetic biologists have developed three strategies to achieve precise control over the expression of gene and engineered cell therapies. The first is a simple switch, the implementation of a Boolean AND gate. A synthetic gene circuit is designed such that its activity is modulated externally by a small molecule drug administered to a patient. In engineering terms, the therapeutic gene-product (output) is produced when both DNA AND the small molecule drug are present (inputs). The application of this control mechanism has been tested for the treatment of Parkinson’s (PD), a neurodegenerative disease that is not monogenic. In a rat model of PD, the synthetic steroid hormone mifepristone was used to activate the production of GDNF, triggering a neuroprotective treatment (Agustín‐Pavón and Isalan, 2014). With further research, the switch model can be tailored such that the small molecule chosen also directly treats disease symptoms. The benefits of this strategy lie in the ability it gives the clinician to control the timing and intensity of therapeutic functions, resulting in personalised treatment.
Similar in principle to the first strategy, the second one employs the use of an AND gate for conditional gene expression. However, instead of a synthetic switch molecule, the gene circuit is designed to switch on when intracellular and extracellular disease biomarkers are sensed, enabling therapeutic activities to be spatially restricted to diseased cells and tissues. This type of gene circuit significantly increases the safety and efficacy of gene and engineered-cell therapies as cellular environment specificity increases the potency of treatment and reduces off-target activation (Kitad, 2018). A key area of exploration for the application of such strategy is CAR T cell-based immunotherapy. The antigen targeted by CAR is rarely expressed exclusively on tumour cells, hence T cell activation is at times detected in tissues other than the tumour, causing adverse effects to be observed in clinical trials. An approach to increase the specificity of the engineered CAR T cells, is to install a more complex logic circuit engineering the cell system to recognise two independent antigens. Roybal et al. achieved this creating a system composed of several biological modules including a synthetic Notch receptor, relying on a particular AND gate circuit. Essentially, the detection of the first antigen would trigger the release of a transactivator, the presence of which would activate DNA encoding the CAR, which once produced binds to the second antigen activating the T cell. Such greatly specific activation may serve as the foundation of safer therapeutics.
Finally, the third strategy is designed around a different engineering principle: feedback control loops. Gene circuits are built to modulate activity levels of therapies adaptively in response to disease biomarkers, via feedback loops. Aimed at disorders that result in disrupted homeostasis, this system would enable therapeutic functions to be activated solely at the right time and intensity. Prosthetic gene circuity has been developed to regulate insulin, thyroid hormones, blood pressure, pH and glucose, to name a few. Beyond metabolic disorders, feedback-regulation strategy is being explored for the treatment of chronic diseases that occasionally flare up but for which the use of prophylactics is not recommended. Schukur et al. were able to implement this technology to address psoriasis, with successful results in mice.
After more than two decades since the first clinical trial, the gateway to new potentials in gene and engineered-cell therapy unlocked by synthetic biology will propel innovative personalised treatment into the clinic. From the exploration and design of new modules, to the construction of more and more complex logic circuits, bioengineers are poised to develop safer and more effective treatment options for disease, genetic and otherwise.
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