Description
Depression medications pose burdens to the patients
Depression is a very common but serious mental disorder in modern society. Patients may suffer from the persistence of sad mood, anxiety, irritability, difficulty concentrating, loss of interest and even develop aches or pains without clear physical cause. Based on the latest researches, around 24.6% teenagers and 18.5% university students were diagnosed in 2019. [1]
Nowadays, the treatments for depression can be divided into three categories: medications, psychotherapies and brain stimulation therapies such as electroconvulsive therapy. Depression could be persistent and relapsed, which severely influences the everyday life of patients. To have it under control, systematic and standardized treatment with continuous maintenance of drug dosage is highly recommended in clinical practice.
However, prolonged medication intake has many drawbacks. For critical patients that require life-long medication, long-term drug use will bring them a double burden both economically and psychologically, and may cause drug abuse or drug resistance, which is not conducive to long-term and sustainable treatment. Meanwhile, for mild and moderate patients, long-term use of antidepressants may also do more harm than good [2]. Thus, innovations in antidepressants are of urgent requirements.
Drug delivery by engineered probiotics
After we learned the dilemma that most depression patients are facing, a question naturally occurs: Is there any way to make taking pills less bothering and easier to persist? Will it be much better if we don’t need to “take in” these pills anymore, when the drugs are automatically and periodically produced “inside” the body? This is when the intestinal microorganism enters our view. Recently, more and more research have shown the importance “Brain-gut axis” plays in various kinds of disease mechanisms, including the regulation of the central nervous system [3]. It is also found that the gut microbiota is remarkably different in depression patients, who shows severe intestinal dysbiosis. It is demonstrated that some original probiotic strains of Bifidobacterium longum (B. longum) have an intrinsic ability to treat depression. So we plan to use this strain as our final chassis.
SAMe: a natural antidepressant
We explored the possibility to engineer probiotics to deliver antidepressants in the gut. S-adenosyl-L-methionine (SAMe, S-adenosyl methionine) is a natural metabolite in all eukaryotes, which is sold in the United States as a dietary supplement. Abnormal levels of SAMe in the body have been reported in depression, and some studies have proven its beneficial effect as a natural antidepressant [4]. Unlike other usual antidepressants, side effects of SAMe are uncommon, and when they do occur, they are usually minor problems. So we choose it as our antidepressant molecule to relieve depression symptoms. SAMe is synthesized from ATP and methionine by SAMe synthetase. We found a SAMe synthetase Sam2, which doesn’t have a feedback inhibition phenomenon, from Saccharomyces cerevisiae (S. cerevisiae) to produce SAMe [5]. As for the secretion of SAMe out of bacteria, we also found a passive transporter Pet8p in S. cerevisiae.
Oscillator-mediated periodical production
Although SAMe is generally considered safe, we still wanted
to mimic normal drug uptake patterns, in which the drug was taken at intervals
rather than continuously. Therefore, we introduced an oscillator module to achieve
periodic SAMe secretion. Oscillator is composed of three transcriptional repressors
whose expressions are under the control of the last transcriptional repressors.
Another advantage of periodic administration is that it can also restrict the
possible side effects of drugs to a relatively short period of time, and can serve
as a method to maintain the blood drug concentration for treatment effect. Because
if the engineered bacteria are continuously administered, the total amount of drugs
secreted by the engineered bacteria is closely correlated to the cell density and
metabolic level of the engineered bacteria, which may be difficult to monitor and
control; if the drug secretion continues to be too low, the therapeutic effect will
not be achieved; if the drug secretion continues to be too high, the patient will
keep suffering from side effects. Therefore, it is beneficial to use oscillators to
achieve periodic and efficient drug secretion after a one-time intake of our
engineered probiotic.
From the various published and brainstormed oscillator
systems, we decided to use
the REPRESSILATOR [6]. This oscillating system contains three proteins, namely,
lambda Cl, TetR and LacI. These three proteins are all transcriptional repressors
that can inhibit the expression from their corresponding promoters, and these three
proteins are expressed under the drive of these three promoters. This enables the
three protein contents to change periodically respectively. We used this oscillator
to drive the expression of SAMe synthetase, so as to achieve the periodic change of
SAMe synthetase concentration, and further to achieve the periodic change of SAMe
production.
Directed evolution for a less sensitive LacI to function in the gut
The oscillator contains LacI protein as one of its repressors, However, the LacI will be influenced by the concentration of lactose. Since lactose commonly appeares in our daily diet, and of course in our intestine, it’s vital to improve this element to tolerate the interference of intestinal lactose if we want to keep a stable SAMe production cycle. Thus, we decided to develop a LacI protein that could tolerate high-level lactose without losing repression on the promoter. The detailed directed evolution scheme for LacI will be discussed in the Design page.
Suicide switches for biosafety
Considering we are designing engineered bacteria that will be used in human gut, the safety of our engineered bacteria should be stressed. On the one hand, our patients should be able to remove the bacteria actively as their wish. On the other hand, the bacteria should be prevented from leaking into environment. Thus, we designed a parallel safety switch to solve these problems. A temperature-sensitive circuit kills the engineered bacteria when it exits from the human digestive tract and senses low temperature, and a chemical-induced killing switch allows the bacteria to be eliminated when no longer needed. Holin, a protein that makes holes on cell membranes to cause the death of bacteria, is the downstream executor of the double kill switch.
Taken together, our project aims to develop a novel drug-delivery system that brings better treatment options to people suffering from depression. The SAMe production module will make the production of SAMe possible in the probiotic bacteria, the Oscillator module with improved LacI will update the toolbox for expression regulation, and the two-factor Suicide switches will also provide new tools to control the existence of bacteria.
Reference
[1] Fu, X., Zhang, K., & Chen, X. (2021). Blue Book of Mental Health: Report on
National Mental Health Development in China (2019-2020). Social Sciences Academic
Press (CHINA).
[2] Horowitz M, Wilcock M. Newer generation antidepressants and withdrawal effects:
reconsidering the role of antidepressants and helping patients to stop. Drug Ther
Bull. 2022 Jan;60(1):7-12. doi: 10.1136/dtb.2020.000080. Epub 2021 Dec 20. PMID:
34930807.
[3] Savignac HM, Kiely B, Dinan TG, Cryan JF. Bifidobacteria exert strain-specific
effects on stress-related behavior and physiology in BALB/c mice. Neurogastroenterol
Motil. 2014;26(11):1615-1627. doi:10.1111/nmo.12427
[4] Galizia I, Oldani L, Macritchie K, Amari E, Dougall D, Jones TN, Lam RW, Massei
GJ, Yatham LN, Young AH. S-adenosyl methionine (SAMe) for depression in adults.
Cochrane Database of Systematic Reviews 2016, Issue 10. Art. No.: CD011286
[5] Chu J, Qian J, Zhuang Y, Zhang S, Li Y. Progress in the research of
S-adenosyl-L-methionine production. Appl Microbiol Biotechnol. 2013 Jan;97(1):41-9.
doi: 10.1007/s00253-012-4536-8. Epub 2012 Nov 8. PMID: 23135229.
[6] Elowitz, M B, and S Leibler. “A synthetic oscillatory network of transcriptional
regulators.” Nature vol. 403,6767 (2000): 335-8. doi:10.1038/35002125