Let's play a game of word association. I'll go first.
…What words spring to mind? CRISPR? Medicine? Genetic disorders? Cancer? Gene therapy?
What about green tea? Unlikely, I imagine.
But in a new study published today in Science Advances, researchers from East China Normal University have created an elegant system for activating genetically edited cells using green tea.1
Realizing the promise of cell therapies
Engineered cell therapies, deemed the "next frontier" in modern medicine, contain specific cellular material that triggers a desired effect in vitro or in vivo. Such therapies are in development in laboratories across the globe for an array of different conditions, including acute myocardial infarction (heart attack), brain cancer, breast cancer, diabetes and liver diseases. They offer a novel avenue of therapeutics for patients suffering from diseases for which treatment options are limited.
For their efficacious and safe use in the clinic, scientists need to be able to regulate the activity of these cells in vivo. Essentially, they require a "remote control". This has proven a major barrier for the delivery of cell therapies to patients. Initial work in this field has adopted antibiotics such as doxycycline or tetracycline as remote-control triggers for gene expression in the cells. However, regular use of antibiotics may result in antibiotic resistance and other adverse side effects.
So, what alternatives exist?
Haifeng Ye, Professor at East China Normal University, says "Ideal trigger molecules for clinical biomedical applications would be natural, non-toxic, highly soluble, inexpensive, and perhaps even beneficial to health."
Previous studies have reported that remote control switches can be activated through the use of food or cosmetic preservatives, vanillic acid, benzoate and phloretin for example. These molecules do not naturally occur in food however, and the safety implications of their long-term use is not well known.
A green solution?
Nothing beats a good cup of tea. It is the second most popular beverage on the planet (following water) and can be found in the household cupboards of 80% of Americans. Tea is available in a variety of forms, including but not limited to black tea, oolong tea, white tea and green tea. A plethora of research studies have documented the numerous health benefits of green tea consumption, including anticarcinogenic, anti-inflammatory, antimicrobial, and antioxidant effects.
The components of green tea most heavily researched with regards to health are the polyphenols, of which the most pertinent are flavonoids, and the most pertinent flavonoids are the catechins.2
Post green-tea consumption, the tea catechins and phenolic acids undergo metabolic processing to form the antioxidant protocatechuic acid (PCA). In their latest study, Ye and team have utilized this antioxidant as a "remote control" for activating gene switches in cells. "PCA is a major tea catechin compound produced by humans following green tea consumption that has powerful antioxidant activity. Therefore, in this study, we showed the use of protocatechuic acid (we call it PCA), a metabolite after tea drinking, as a trigger molecule," Ye told Technology Networks.
PCA-inducible gene switches
In the study, the scientists engineered PCA-inducible gene switches in mammalian cells. Initially, they explored the potential for using PCA to monitor cell-based long-term therapies in vivo by integrating the genetic switch into HEK-293 cells and found that the cell line demonstrated reversible and tunable induction kinetics, which the authors regard as "excellent switching performance". This was characterized by negligible basal expression and nonsaturating increases in the transgene output over the course of a 15-day trial.
Next, they microencapsulated and implanted the HEK cells into mice. Ye tells us, "The alginate-poly (L-lysine)-alginate-based encapsulation technology was used in our study for cell therapy. This clinically validated implant technology enables the free diffusion of metabolites, nutrients and proteins of lower molecular weights (<72 kDa) across the biocompatible capsule membrane while shielding their cellular content from physical contact with the host’s immune system. The implant technology has been successfully validated in human clinical trials and the performance of the material is continuously improved for clinical applications."
The researchers found that, regardless of delivery method (intraperitoneal, oral intake from water, or oral intake from concentrated green tea), PCA could control the secretion of a reporter protein, SEAP, in a dose-dependent manner.
Making CRISPR more – crisp?
CRISPR gene-editing shows promise in revolutionizing personalized medicine. A notable key issue with CRISPR, however, is the "off target" effects that limit its specificity. In this study, the scientists used the PCA-responsive cells to perform more targeted CRISPR gene editing: "By applying newly-designed fusion-protein-based PCA-controlled gene switches to Pol III promoters, we created trigger-inducible expression systems for gRNAs to program PCA-mediated CRISPR/Cas9-activity," says Ye.
Exploring diabetes treatment with PCA-induced cell therapy
Ye and colleagues next tested the potential of the PCA remote control system for treating experimental diabetes using a mouse model. Using the switch, they engineered two different cell lines: one that enabled PCA-inducible expression of the reporter protein SEAP and insulin, and the other producing a short variant of human glucagon-like peptide 1 and SEAP. Implantation of these cells into mouse models of type 1 diabetes and type 2 diabetes mellitus resulted in restored homeostatic fasting blood glucose concentrations and glucose tolerance upon PCA injection.
Recognizing that the translation of research findings from mouse models to humans in the clinic can be problematic, the scientists then decided to explore the PCA remote control switch efficacy in non-human primate models of diabetes. In parallel to the treatment efficacy observed in the type 2 diabetic mice, daily oral administration of PCA rapidly increased the expression of glucagon-like peptide 1 and restored glucose homeostasis in diabetic monkeys.
In terms of safety, blood biochemical analyses related to inflammatory responses found that white blood cell count, lymphocytes, monocytes, eosinophils, and basophils, did not increase at any point during the treatment when compared with pre-treatment.
The study findings certainly excite the authors, "Although there have not yet been preclinical studies for the application of engineered cell–based therapies in humans, this first-in-monkey study demonstrates the feasibility of safely and successfully scaling up a treatment strategy by controlling microencapsulated engineered cells to release therapeutic outputs from animals such as mice to larger NHPs. Therefore, this study substantiates the medical utility of concepts developed in synthetic biology," they note in the discussion of the paper.
How much tea is too much tea?
Hypothetically, if this therapy was to reach the clinic, I ponder over the possibility of an individual consuming "too much" green tea, and how this might impact the therapy. Ye is quick to inform me that this would not be an issue, "Only custom prepared concentrated green tea can activated the implanted designer cells. The normal green tea drinks cannot activate the implanted cells because of low concentration," he says.
The future looks green
The study is comprehensive, assessing the PCA "switch" in a variety of cell lines and mammalian models with a variety of control measures in place.
Thus, in which direction will this research go next? I ask Ye, who tells me, " We will next focus on solving the following limitations:
(1) The PCAON-switch was stably integrated into [the] genome by a "Sleeping Beauty" transposon system. Due to a random integration, unwanted insertional mutagenesis might occur. We will next consider using gene editing tools, such as CRISPR, to enable facile and permanent integration of the switch into the targeted genomic sequences in human cells without insertional mutagenesis;
(2) The chassis of the HEK-293 cells are easily handled, transfected, and compatible to the PCAON-switch. For translational applications, they must also be safe (no side effects) in humans. Hence, we will test the therapeutic efficiency of the PCAON-switch in autologous parental cells from patients’ own mesenchymal stem cells, which may provide immunocompatible and noncarcinogenic autologous or allogeneic cell sources;
(3) The lifespan of the designer cells inside the alginate microcapsules is an imperative issue. To realize long-term cell therapy, we will further improve the encapsulation technology."
Haifeng Ye, Professor at East China Normal University, was speaking with Molly Campbell, Science Writer, Technology Networks.
1. A green tea–triggered genetic control system for treating diabetes in mice and monkeys," by J. Yin; L. Yang; K. Dong; J. Jiang; S. Xue; Y. Xu; X. Wang; H. Ye at East China Normal University in Shanghai, China; L. Mou; Y. Lu at First Affiliated Hospital of Shenzhen University in Shenzhen, China.
2. Reygaert. 2018. Green Tea Catechins: Their Use in Treating and Preventing Infectious Diseases. Biomed Research International. doi: 10.1155/2018/9105261.