Team:SYSU-CHINA/Results
Results
We wonder if our system works as expected and has the potential to be applied to real-world scenarios. So we demonstrate the feasibility of this project through verification of each independent system, serial effect verification and modeling analysis.
What had we achieved?
1. Sensor: Identified tumor miRNA profile through miRNA sensor
2. Switch: Verified the response sensitivity of the system switch
3. Carrier: Reorganized and packaged the virus
4. Killer: Characterized the efficacy of genes that turn on the virus to kill tumors
5. The system works normally after being connected in series
6. Predicated and improved the working condition of oncolytic virus in tumor treatment by modeling
Differentiation of miRNA profiles using miRNA logic gates
L7Ae K-turn system
We engineered the L7Ae protein as an intermediate inhibitor in the miRNA sensor design. (Figure. 2a) By testing we found that it does not have obvious cytotoxicity (Figure.2 c,d)
Figure 2This picture shows the construction and characterization of L7AeO. a. According to the 3D structural analysis of the protein, the Thr on the α-helix interacting with the substrate is mutated to Pro by overlap PCR. b. 3D structure of L7Ae combined with ligand from PDB c.L7Ae/L7AeO cytotoxicity results microscopy d.Semi-quantitative determination of cytotoxicity by detecting cell reducing ability by MTS staining.
Then we quantitatively and qualitatively characterize the working ability of this system. The result shows that the mutant was weakened as expected but still functions in high concentration. Meanwhile, the 1000ng/ml L7Ae plasmid can depress the translation of mRNA with an efficiency of 84%. (Figure.3a) At the same time, we determined the relationship between the number of K-turn structures on mRNA and the inhibition efficiency. Only two Kt can achieve significant inhibition effect, and the inhibition efficiency of each Kt structure is obtained by data conversion for modeling. (Figure. 3b)
Figure 3 This figure shows the quantitative and qualitative characterization of the L7Ae/L7AeO-Kturn system. a. Determination of inhibition efficiency of LA7e/L7AeO and K-turn at different concentrations.(Blue：L7AeO；Black：L7Ae；Green：control plasmid pcDNA). b. The effect of different K-turn numbers on L7Ae/L7AeO, only 2 Kt-turn can achieve greater suppression efficiency.
miRNA target
Based on our analysis, we designed three different miRNA targets. We inserted it into the following miRNA detection plasmid (Figure 4a) to verify its independent ability to work and detect the expression of the corresponding miRNA in different HEK293. To verify whether the system causes tandem, we constructed three different plasmid contains 3 miRNA targets independently (miR-592, The detection plasmids of miR-663b and miR885-5p) were tested under different miRNA induction. After the plasmid is transcribed into the cell, the endogenous miRNA binds to the target on the mKate mRNA, blocking the expression of mKate, which cause a change in the value of mKate/EBFP that can be read by flow cytometry. The detection plasmids were tested under different miRNA induction.
By this method, we can compare the expression of the endogenous corresponding miRNA with the Positive Control which does not carry the miRNA target. At the same time, we transferred miR-592 mimetic, miR-885-5p and miR-663b inhibitors into corresponding cells to detect the regulation of the corresponding miRNA target by miR concentration, thus verifying the rationality and feasibility of single miRNA target design.
In the flow cytometry analysis (Fig. 4b, 5), we refer to the group with the lower affected group of mKate as the positive group, and the group with the larger influence of mKate as the negative group. We can identify a group of cells that are not obvious in the negative group (hexagonal circled part) in the positive group, and there is a significant difference in the ratio of cells between the positive group and the negative group in the interval of mKate/EGFP<0.4. Thus, we believe that miRNA targets can respond to changes in miRNA concentration.
Figure 4 Flow cytometry of miRNA detection plasmids. a. The detection plasmid is mainly composed of two fluorescent expression systems. The target of the target miRNA target is inserted into the 3'UTR sequence of mKate. If the miRNA target can work normally, the fluorescence ratio of mKate/EBFP can be changed. b. In the results, the positive group has a common cell population, which shows that all three miRNA targets work properly. At the same time, the concentration of endogenous miRNA corresponding to HEK293 can be displayed.
Figure 5 Density analysis of positive and unique cell populations revealed that the positive group had a deviation from the negative expression of mKate in the negative group
miRNA sensor composite system work
We constructed a plasmid with a complete miRNA sensor and verified the effects of the entire system at different miRNA concentrations. First, we regulated the concentration of miRNAs in HEK293 through three different miRNA mimics and inhibitors. miRNA-592 mimetic to shut down L7Ae translation, miRNA-663b inhibitor and miR-885-5p inhibitor to block EGFP mRNA degradation. That is to say, when three miRNA mimics are simultaneously transferred, the entire system reaches the maximum working efficiency to output EGFP. The results showed that EGFP was slightly upregulated when either miRNA mimic/inhibitor combination was input, with the combination of miR-663b inhibitor + miR-885-5p inhibitor being the least efficient. This may be due to the fact that the targets of these two miRNAs are on the same mRNA, and the two miRNAs will have a competing effect. In addition, when three miRNA mimics/inhibitors were simultaneously input, we observed a significant difference in fluorescence brightness, which means that our miRNA sensor can work normally only under the expression of a specific miRNA. This undoubtedly verifies its specificity.
Figure 6 Different miRNA combinations control the output of miRNA sensors
Verify the response sensitivity of the system switch
Quantitative detection of relationship between Dox concentration and fluorescence induction
The result shows that only 270ng/ml DOX can induce half of the gene expression(Figure 7), which means a low dose of DOX can achieve the desired effect without considering the side effect of over-dose.
Figure 7 The effect of Dox concentration on induction efficiency, fitting half of the induction value
Effect of rtTA3 controlled under different promoters on Tet-on system
We also constructed two plasmids which contains the rtTA3 controlled by different promoters. One promoter is pCMV, the other one is hEF1α. The result shows that under the regulation of pCMV, the variance of expression is smaller. While, hEF1α has lower background and higher expression in a high concentration of DOX.(Figure.8)
Figure 8 Different promoters control the effect of rtTA3 on Tet-on system
Recombined and packaged the virus
We used the Adeasy system to insert our circuit into the Ad5 virus missing E1 and E3. First, we constructed the shuttle plasmid pShuttle-AdmiT in vitro to carry our circuit. Then transfer it with pAdeasy into E.coli BJ5183 through electroporation. These two plasmids were recombined in E. coli to obtain the Ad5-type adenovirus genome plasmid carrying our circuit. Figure b is a demonstration of a recombinant plasmid, 1 lane represents a successful recombinant plasmid, lane 2 represents pShuttle, and lane 3 represents a plasmid that has not been successfully recombined.
We then introduced the plasmid into HEK293 cells and packaged the virus. The image of Day3-8 is shown in the figure. It can be seen that significant CPE appeared on the third day after transfection, and a large number of suspended cells were produced on the eighth day. We harvested the virus suspension on day 8 and stored at -80 ° C.
Figure 9 Results of packaging recombinant adenovirus. a) a plasmid profile of our recombinant adenovirus b) The supercoiled clone in Line 1 is recombinant because it migrate at positions larger than the 10-kb marker and its shuttle vector. However line 3 is not. c-f) Use recombinants to transfect the packaging cell line(HEK293), and the CPE showed since 72h.p.t(hours post transfection).
Determination of virus titer
After harvesting the virus, we determined the TCID50 of the recombinant virus. The results showed that the TCID50 of the virus was 1x10-3.6/ml (Table 1). According to our modeling results, such titers can effectively kill tumors and avoid “escape” effects.
Table 1 The result of TDIC50 assay
Killer: Characterized the efficacy of genes that turn on the virus to kill tumors
We verified that E1A can restore the virus's self-replication ability by transfection experiments in HeLa cells, and test the killing effect of E1A induced by different concentrations of Dox. This shows that our design in Killer is feasible, we are verifying the impact of the E1B55K, which may be displayed on our posters and presentations. We found that E1A can induce the additive killing of adenovirus in HeLa cells, while the virus lacking E1A cannot increase in cells.
It can be seen from the cell microscopic (Figure.10) examination that the number of cell deaths increases with increasing E1A concentration. The detection of cell activity on day 4 also supported this result, indicating that our killer gene is effective. (Figure.11) When Dox is 0, E1A also causes apoptosis, which may be caused by pTRE leakage expression. At the same time, since virus infection enhances cell respiration, MTS detects mitochondrial activity, and the virus group data has partial deviation.
Figure 10 Microscopic examination after four days of virus infection
Figure 11 Detection of mitochondrial activity by MTS staining for detection of viral effects
The system works normally after being connected in series
Based on the above data, we verified the ability of each system to work independently. Now we will test different systems in series to prove that our circuit design is feasible.
Tet-on system regulates the initiation of miRNA sensor
We placed the miRNA sensor downstream of the pTRE promoter to detect if Tet on could work compatibly with it. miRNA-592 mimics were transported to switch off the miRNA sensor, while miRNA-885-5p inhibitor were transported to switch on the miRNA sensor. Experimental result shows that the two systems are well compatible.
Figure 12 Expression combination of miRNA sensor and Tet on
Tet-on system regulates the Ad5 to kill cancer cell
We co-transfected the adenoviral genome with the E1A gene located downstream of pTRE into HeLa cells. The cells were induced by different concentrations of Dox, and it was found that with the increase of Dox concentration, the cell survival of HeLa cell decreased gradually after 4 days of transfection. This shows that our adenoviral vector system, killer gene and Tet-on system are compatible.
Figure 13 Tet-on system control virus to kill cancer cell
Conclusion
The above data can prove that our design is reasonable and feasible, and through the improvement brought by modeling, this system can be predictably applied to the tumor environment in different situations for killing.