Background
According to the WHO GLOBOCAN database, colorectal cancer (CRC) is the third most common cancer in men and the second most common cancer in women, with 1.8 million new cases and approximately 881,000 deaths worldwide in 2018 [1]. Tumors evolving from polyps are the leading cause of colorectal cancer [2,3]. Unfortunately, the 5-year survival rate for advanced-stage colorectal cancer is very low [4]. Colorectal cancer treatment options include minimally invasive surgery [5,6]; chemotherapy (of which 5-FU is the most commonly used agent) [5,6]; radiotherapy [5,6]; targeted therapy [7]; immunotherapy [8]; and others: Lonsurf ® (orally active antimetabolite) [9] and celecoxib (Celebrex®) (COX-2 inhibitor and NSAID) [8]. Despite the treatment of cancer, many results have been achieved, all these approaches have their challenges and limitations in the field of cancer therapy. Nonspecific toxicity to normal cells is one of the main challenges of conventional cancer therapy. In addition, some chemotherapeutic agents can lead to the formation of multidrug-resistant cells [10,11]. We have compiled a list of the various disadvantages of today's colorectal cancer treatment methods as shown in the table below, more details can visit implementation.
Since gene programming enables bacteria to perceive and respond to in situ physiological conditions, this method will change the existing mode of diagnosis and treatment of diseases. For programming hosts, E. coli strains are easier to handle, although many microbes are chassis organisms, thanks to their clear genetic background and mature tools for heterologous expression of large amounts of proteins. Among them, E. coli Nissle 1917 (EcN) has reported selective colonization and replication in solid tumors [12-15]. It has been proved that recombinant EcN has no effect on the migration, cloning and amplification in the autoimmune environment, or the induction or destruction of peripheral T cell tolerance [16,17].
On the other hand, a key consideration for the use of engineered bacteria in drugs is the need to limit the growth of microorganisms to the disease site to prevent off-target tissue damage and septic shock [18,19]. Therefore, it is possible to solve the challenge of transforming the next generation of microbial therapy by engineering gene circuits to limit the growth of bacteria in specific parts of the human body. So far, most bacterial treatments rely on the natural tendency of bacteria, which is defined as preferential growth in specific host tissues or microenvironments (such as gastrointestinal tract, skin and tumor). Although depending on the inherent growth preference of bacteria can sometimes control the localization of bacteria, many bacteria can grow outside their natural ecological positions – rapidly spread to unexpected positions, and lead to off-target effects.
Genetic engineering methods, such as metabolic undernutrition, dependence on synthetic amino acids and toxin / antitoxin systems, have been used to control bacterial growth [20-24]. Combining these technologies with environmental responsive biosensors can improve the containment of engineering bacteria and prevent accidental diffusion [20-24], but it is still a challenge to accurately locate tumor tissues. Therefore, sensing one or more different physiological characteristics through genetic circuit programming may enhance the tropism of engineered bacteria under predetermined conditions of organ ecological niches [25].
Design
Localization
In order to construct a bacterial biosensor system that can distinguish the unique organ environment, oxygen, pH and lactic acid are considered as unique indicators of colorectal cancer tumor tissue. Due to the Warburg effect, namely aerobic glycolysis, a large amount of lactic acid is released into the tumor microenvironment and leads to a decrease in pH [26,27]. This metabolic pathway enables rapid and direct energy acquisition [28] and weakens anti-tumor immune responses [29]. On the other hand, hypoxia is one of the extreme factors affecting the microenvironment of CRC [30]. Rapid proliferation of colorectal tumor leads to rapid depletion of oxygen supply without vascular supply, which is a common cause of hypoxic microenvironment in colorectal cancer.
Three operons were used to specifically recognize hypoxia, low pH and lactic acid. Hypoxia-sensing promoter (pPepT) is used to sensehypoxia. It is mainly regulated by transcription activator (FNR) [31]. In the absence of oxygen, FNR binds to the $[4Fe-4S]^{2+}$ cluster to generate homologous dimers with transcriptional activity. However, in the presence of oxygen, $[4Fe-4S]^{2+}$ clusters are degraded, resulting in the dissociation of FNR dimers into inactive monomers [32].
L-lactic acid induction is based on lldPRD operon [33]. The lactic acid sensing system consists of two parts. pLldP drives the expression of interested genes. LldR repressor binds to pLldP to inhibit the expression of reporter genes, unless bound to lactic acid [34,35]. The pH-sensitive promoter pCadC, regulated by membrane-tethered activator protein (CadC), exhibits higher activity in acidic media than in neutral pH media.
Stabilization
However, many challenges limit the clinical application of current biosensor systems, for example, the signal processing ability is limited, and it is unable to integrate multiple biomarkers for accurate diagnosis, and response time is not compatible with the diagnosis requiring rapid results. In order to enable living cells to perform complex signal processing operations, amplifying genetic switches and Boolean logic gates based on serine integrase (Bxb1, TP901) are used in the design of biosensor systems [36]. These genetic devices enable bacteria to perform reliable detection, multiplex logic and data storage of clinical biomarkers in human clinical samples [37,38] to meet the requirements of medical testing.
To be specific, amplifying genetic logic gates use the asymmetric transcription terminator as the reversible switch to control the RNA Pol flow between gate input and output. However, only when serine recombinase catalyze the unidirectional reversion of DNA in the corresponding recognition site can the status of signal output be changed.
Adhesion
Next, the tumor cell adhesion module was designed: HlpA from Streptococcus gallolyticus can combine with the HSPG (heparan sulfate glycoprotein) on tumor surface (especially with the upregulated syndecan 1 during carcinogenesis). HlpA has been shown to improve the penetration of engineered bacteria into colorectal tumor cells. In order to export HlpA-binding protein to the surface of EcN, shortened INP (ice nuclease protein) tag was fused into the N-end of HlpA.
Treatment
To give the engineered strains the therapeutic ability on tumor cells, Haemolysin E, CCL21 and CDD-iRGD (Bit1 fusion protein of cell death domain and tumor perforin) are used as therapeutic parts. Haemolysin E is coded by hlyE from E.coli and is testified to able to be used as a pore-forming antitumor toxin. CCL21 and CDD-iRGD are able to activate the immune response of host. The former recruits T-cells and dendritic cells while the latter triggers tumor cell apoptosis.
Lysis
In the end, bacteriophage lysis gene (phiX174 E) was put into the module, which can lead to bacteria lysis and death when expressed, resulting in the release of therapeutic factors stored inside the strain.
For safety reason, we determined to introduce an arabinose induced killing switch which Worldshaper-HZBIOX sent to us. Therefore, patients can take some arabinose first to allow the engineered bacteria to function, it is equivalent to an additional protective barrier The part’s sequence is based on the BBa_K2556051. But this part is still in the test phase, more details can visit partnership-Worldshaper-HZBIOX.
Capsule
Since the experimental results of the killing switch were not promising, for a second layer of assurance, we made microcapsules from sodium alginate and chitosan, also known as ACA. More details can visit hardware.
Result
We verified our engineered strains at the molecular and cellular levels, both for single parts and for overall efficiency. The experimental results show that our design is successful and feasible for future implementation, more details for this part can visit proof of concept. We also developed models for the analysis of three biosensors, more details can visit model.
For the future prospect, for the sake of increasing safety, we also intend to add killing switch and capsule, killing switch and Worldshaper-HZBIOX cooperation is still in progress. If the subsequent series of experiments are successful there is a good chance that our engineered microbes can enter the market, for this reason we have also asked the government departments in advance to understand the requirements for marketing our probiotics in China, more details can visit integrated human practice-government.
Reference
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