Team:NEFU China/Design
Overview
We want to develop a steerable in vivo tumor therapeutic system carried by E. coli Nissle 1917. The followings are our core design modules of the system:
• Tumor microenvironment sensor module
• Uric acid regulation module
• Targeting and self-regulation module
• Plasmid protection module
As we know, at physiological conditions, malignant cells can stack together to constitute a tumor, and the tight arrangement of tumor cells creates a unique tumor microenvironment. Tumor micro-environment is characterized by hypoxia and high level of lactic acid that are two inducers of our tumor micro-environment sensor module. We aim to sense those two parameters to activate the expression and secretion of a mutant form of tumor necrosis factor-α (mTNF-α) with enhanced activity of killing tumor cells.
As our project is based on the bacterium therapy in vivo, we wanted to choose a bacterial carrier that can survive in human tissues without causing intolerable inflammatory response. To prevent these bacteria from adversely affecting normal tissues, we used the cytosine deaminase (CD) as a part of our targeting and self-regulation module, which can convert 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) to kill the bacteria. We also made bacteria express ftnA-M, a mutant form of the ferritin gene, which could efficiently accumulate iron ions in bacteria, and thus can help bacteria gather on tumor sites with the help of a magnetic field.
When tumor cells lysing, purines will be released into the inner environment of human body and finally be converted to uric acid, elevating the risk of acute uric acid nephropathy. This usually happens as a part of The Acute Tumor Lysis Syndrome in cancer patients undergoing chemotherapies or radiation therapies for the treatment of malignancies with rapid cell turnover. Current therapeutic approaches of cancers frequently ignore the effects of these syndromes on patients, which needs substantial cost and time to cure. In our therapeutic system, we aim to solve this problem by adding a uric acid regulation module containing uricase to catalyze the oxidation of uric acid to 5-hydroxyisourate. Besides, we will also use this module to regulate the rates of tumor cell lysis and consequently reduce the risk of acute tumor lysis syndrome.
Figure 1. The schematic representation of our cancer therapeutic system. The system contains 3 main modules, including the Tumor microenvironment sensor module, the Uric acid regulation module, and the Targeting and self-regulation module. The Tumor micro-environment sensor module will sense tumor microenvironment and product agents. The Uric acid regulation module can oxidize uric acid to reduce the risk of acute uric acid nephropathy. The Targeting and self-regulation module can help bacteria survive in tumor and regulate the production speed of the anticancer agent.
Tumor microenvironment sensor module
To ensure our therapeutic system can work only when reaching tumor cells, we chose hypoxia and high lactic acid levels, characteristics of tumor microenvironment, as the detection indexes of our sensor module. We utilized a hypoxia-inducible promoter, PfnrF8 to restrict gene expression specifically in the hypoxic environment of tumors. The transcriptional activation promoted by the PfnrF8 needs a specific transcription factor called FNR, which can bind to oxygen. When oxygen level is at a normal range in the internal environment of human body, the transcription factor FNR will associate with oxygen, leading to its impeded binding to the PfnrF8 promoter, and reduce the transcription of its downstream gene (Figure 2, a, c). However, in hypoxic environment of tumors, FNR will disassociate with oxygen and activate its downstream gene [1].
Lactic acid concentration is another detection index of our sensor module. The lldPRD operon (previously named as lct) is responsible for aerobic L-lactate metabolism. It consists of three genes that form a single transcriptional unit inducible by growth in L-lactate. The three genes lldD, lldP and lldR encode a dehydrogenase, a permease and a regulatory protein, respectively. We chose lldR and lldP to build our lactic acid response unit. In this circuit, lldR and lldP are downstream of the Anderson promoter, and are always expressed in the bacteria in culture medium. LldR protein can bind to the operators O1 and O2 located on each side of the PfnrF8. When lactic acid level is low, two lldR molecules will individually bind to the O1 and O2 sites and form a tetramer to make DNA strand form a hairpin structure, which can turn off the expression of the downstream gene (Figure 2, a, b). When lactic acid level reaches a certain point, lldR will be released from the O1 and O2 operators, and the DNA hairpin will be resolved, leading to activated transcription of the downstream gene. We also used lldP to increase the sensitivity of our system to lactic acid alteration, so that our system can sense the specific lactic acid concentration in tumor microenvironment [2].
When both sensor parts are activated, a downstream anticancer protein, mTNF-α, will be expressed (Figure 2, d). It is a mutant form of the tumor necrosis factor-α (TNF-α), which can eliminate tumor cells without damaging normal tissues[3].
Figure 2. (a) In normal tissue, there are relatively high levels of oxygen and low levels of lactic acid. At this condition, two lldR molecules will form a homo-tetramer, and the binding of the transcription factor FNR to the PfnrF8 promoter will be impeded, leading to suppression of the mTNF-α gene. (b) When oxygen level of inner-environment rises while the lactic acid level is still low, FNR can bind to the PfnrF8 promoter, but lldR will still form a homo-tetramer and TNF-α will not be expressed. (c) When lactic acid level of inner-environment goes down while the oxygen level is still high, lldR will be released from the O2 site, but the PfnrF8 promoter can hardly associate with the transcription factor FNR and TNF-α will not be expressed. (d) In tumor micro-environment, with low levels of oxygen and high levels of lactic acid, lldR will be released from the O2 site and the transcription factor can bind to the PfnrF8 promoter, leading to TNF-α expression.
Uric acid regulation module
With the accumulation of tumor cell lysates, uric acid levels will rapidly increase and activate the uric acid regulation module. HucR is a protein that can bind to the Huc promoter and repress its mediated transcription of the downstream gene at low uric acid levels (Figure 3, a). When uric acid level increases to a threshold value, HucR will no longer bind to the Huc promoter, and transcription of downstream gene will be turned on (Figure 3, b) [4].
Uricase and lacI reside downstream of the Huc promoter. When the engineered bacteria are attracted to tumor sites and uric acid level is lower than the threshold value, HucR will bind to the Huc promoter and repress the transcription of uricase and lacI. With a large amount of tumor cells being killed, uric acid level will increase. When it reaches the threshold value, HucR will disassociate with the Huc promoter, and consequently the transcription of uricase and lacI will be activated. The produced uricase will catalyze the oxidation of uric acid to 5-hydroxyisourate, which is unharmful to human body and can be discharged from patients by excretion. The produced LacI will bind to the lacO site upstream of mTNF-α to repress its transcription and prevent tumor cells from being lyzed rapidly, which will reduce the risk of acute tumor lysis syndrome (Figure 3, b).
YgfU is a uric acid transporter of E. coli [5]. In our design, its coding sequence will be inserted downstream of the Anderson promoter, a constitutive promoter, to increase the sensitivity of the bacteria to uric acid.
Figure 3. (a) When the engineered bacteria reach in the tumors, the uric acid level at tumor microenvironment is low, and HucR will bind to the Huc promoter leading to repression of its downstream gene. (b) As tumor cells are rapidly killed, uric acid level of inner-environment will increase. This will lead to the release of HucR from the Huc promoter and consequent activation of uricase and lacI genes.
Targeting and self-regulation module
We used ftnA-M, a mutant form of the ferritin with enhanced capability of enriching ferrous ion, to attract bacteria to the tumor sites. Ferritin is a ubiquitous class of proteins present in living organisms and plays a crucial role in iron homeostasis. Ferritins form shells composed of 24 monomers in each shell, which create an inner cavity to store iron in a hydrated amorphous form of iron oxide, like mineral ferrihydrite. FtnA-M can enhance the magnetism and biomineralization capability of the engineered bacteria. [6] In our system, the ftnA-M coding sequence is driven by the BAD promoter (pBAD), which can be induced by arabinose. Before bacteria is intravenously injected, they will be cultured in medium supplemented with arabinose and iron ions. FtnA-M will be expressed to form shells with enriched iron ions. After bacteria are intravenously injected into mice, we will use a magnetic field to cover the tumor regions to attract bacteria to the tumor sites(Figure 4, a, b).
To eliminate bacteria that may stay in normal tissues, we will use 5-fluorouracil (5-FU) to kill the bacteria. In vivo, 5-fluorouracil is converted to the active metabolite 5-fluoroxyuridine monophosphate (F-UMP). Through replacing uracil, F-UMP incorporates into RNA and inhibits RNA synthesis, thereby inhibiting cell growth. As an anticancer drug, the presence of 5-FU will only promote tumor elimination. 5-FU interferes with DNA synthesis by blocking thymidylate synthase in converting deoxyribose uridine acid to thymidylate. 5-FU can be produced from 5-fluorocytosine (5-FC) under catalysis of cytosine deaminase (CD) [7]. Because 5-FC does not adversely affect animals or the engineered bacteria, we will feed mice with 5-FC that will be converted to 5-FU by expressed CD to eliminate the bacteria.
In our circuit, the expression of CD is regulated by the ECF20 promoter, which can recruit ECF20 σ-Factor to activate the transcription of its downstream gene. ECF20 σ-Factor is a member of the extra-cytoplasmic function (ECF) σ-Factors group and the binding of ECF σ-Factors to the ECF promoters can be impeded by their anti-factors [8, 9]. In our design, the anti-ECF20 factor is localized downstream of the promoter PfnrF8 and O2 site, and its expression is regulated by the tumor microenvironment sensor system.
When the engineered bacteria are intravenously injected into mice, they will be carried by blood to everywhere in the body. As they reach normal tissues, lldR will bind to the O1 and O2 sites. With the dimerization of two IIdR molecules, the promoter PfnrF8 will be inactivated and thus the transcription of anti-ECF20 factor will not be initiated. Meanwhile, ECF20 σ-Factor will bind to the ECF20 promoter and activate CD expression (Figure 4, c). We will feed the mice with 5-FC that will be converted to 5-FU to kill the bacteria in normal tissue. When the bacteria reach tumor tissues, lldR tetramer will collapse and the promoter PfnrF8 will be activated, leading to the transcription of anti-ECF20 factor. Anti-ECF20 factor will block the major RNA Polymerase binding determinants of ECF20 and prevent ECF-20 from associating with the core RNA Polymerase. As a result, transcription of CD will be impeded, so the bacteria can survive and exert its anticancer activity in the tumor microenvironment (Figure 4, f). Once tumors are cleared, the levels of oxygen and lactic acid will increase. In this case, lldR will release from O2 site and the promoter PfnrF8 will be activated to promote transcription. As a result, anti-ECF20 factor will not be expressed in bacteria and thus ECF20 σ-Factor will bind to the ECF20 promoter. This will activate CD transcription to produce 5-FU that kills the bacteria (Figure 4, c).
Figure 4. (a) In normal situation, bacteria transformed with ftnA will spread randomly all over the body. (b) When tumors are exposed to a magnetic field, bacteria will be attracted to the tumor sites. (c) In normal tissue, expression of anti-ECF20 factor will be turned off and ECF-20 will activate the expression of CD to kill the bacteria. (d) When oxygen level of inner-environment increases while the lactic acid level is still low, anti-ECF20 factor will not be expressed and thus the ECF-20 promoter will express CD to kill the bacteria. (e) When lactic acid level of inner-environment goes down while the oxygen level is still high, anti-ECF20 factor will still not be expressed and ECF-20 will still express CD to kill the bacteria. (f) In tumor micro-environment, anti-ECF20 factor will be expressed and bind to the ECF-20 promoter. Consequently, CD expression will be turned off, so that bacteria can function as a tumor oncologist at tumor sites.
Plasmid protection module
To prevent loss of the plasmids after being injected into the mice, we designed a plasmid protection module. We want to block the expression of a housekeeping gene of E. coli, and insert its coding sequence into one of our vectors. As a result, the bacteria can only survive in the experimental mice when harboring this vector. In our design, we used the genes alr and dadX as an example of our plasmid protection module (Fig. 5). These two genes are coding for alanine racemase, which are involved in the D-alanine biosynthesis of E. coli. If the two genes are knocked out at the same time, the E. coli will be not able to synthase the D-alanine and will not survive. Once one of the two genes is activated, the bacteria will survive[10]. We knocked out both the alr and dadX gene in the genome of E. coli. Once we insert the coding sequence of the alr or dadX gene into our vector, the bacteria will only survive with this vector. Therefore, the bacteria will not lose this vector.
Figure 5.The plasmid protection module. Knock out the genes alr and dadX in the genome of E. coli and insert one of them into our vector.
References
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[10] Bugg, T.D. and C.T. Walsh, Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance. Nat Prod Rep, 1992. 9(3): p. 199-215.
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