In the previous investigation, we found that type 2 inflammatory response is common in the onset of asthma, so we chose two key roles: ILC-2 cells and TH2 cells. ILC-2 cells can be activated by TSLP secreted by lung epithelial cells, and can function as a accomplice in driving type 2 inflammation; TH2 cells produce many kinds of interleukins mediate inflammation, and GATA3 is the transcription factor for them. (Fig.1A) We designed siRNAs for TSLP and GATA3, hoping to knock down the expression of these two targets and inhibit type 2 inflammation.
Our project this year mainly involves two parts. The first part is to reprogram lung cells so that lung epithelial cells can produce some effective siRNA. Since almost every cell in the human body releases exosomes, this extracellular vesicle is a very common tool for intercellular communication. Therefore, the reprogrammed lung epithelial cells can secrete exosomes carrying effective siRNA and target our desire cells with high bioaffinity. After embedding the appropriate targeting peptide on the exosomal membrane, the exosomes are able to target epithelial cells or TH2 cells.
The second part is the uptake and function of exosomes. Through two targeting peptides: RGD and CKLF1-C19, RGD can help exosomes target epithelial cells and CKLF1-C19 can help target TH2 cells. Ingested siRNA will form a RISC complex to reduce the translation of TSLP in lung epithelial cells and GATA3 in TH2 cells, respectively.
So, we plan to deliver the minicircle DNA (mcDNA, a kind of DNA circuit) expressing these two siRNA and targeting peptides to the lung of mice using liposome by nasal inhalation. According to our hypothesis, the lung will uptake mcDNA and expresses corresponding siRNAs and targeted peptides. Through exosomes secreted by the lung itself and targeting peptide, these siRNA can reach targeted cells and have their therapeutic effects (Fig.1B).
1. Preparation of mcDNA
What work we have done
✓mcDNA induction and extraction
✓Verification the sequence of mcDNA
✓ Choosing the best liposome, LID
✓Choose the best ratio of DNA to liposome
1.1 Extraction of mcDNA
Based on our experimental design, we ordered the plasmids from GenScript.
We first selected strain ZYCY10P3S2T of E. coli as the chassis organism for mcDNA production. This particular E.coli strain has been reported to be very suitable for mcDNA production. After amplifying E. coli ZYCY10P3S2T, we transferred the plasmid into E. coli by heat shock and coated it on LB plates containing kanamycin. On the second day, a suitable colony was selected for amplification and preservation.
We inoculated the bacteria into a special mcDNA induction medium, cultured E.coli to an appropriate concentration, and induced by L-arabinose to allow E.coli to loop out the desired mcDNA. The procedure is shown at Fig.2. mcDNA was purified and extracted with plasmid extraction kit.
We then compared the mcDNA with the mother plasmid by agarose gel electrophoresis . The following figure (Fig.3) shows the electrophoresis results. The left four lanes are mother plasmid of GATA3-si-1, TSLP-si-1, TSLP-si-2, TSLP-si-3, and the right lane is mcDNA of TSLP-si-1. According to the right marker we can say that, comparing to the mother plasmid (around 7000b.p.), our mcDNA is incredible small, which is around 3000b.p.
2. Screening of liposomes
After completing plasmid construction and mcDNA extraction, we considered how to deliver our mcDNA to epithelial cells with both safety and efficiency. Therefore, we interviewd Professor Wu Jinhui from Institute of Drug R&D, Nanjing University. Professor Wu recommended that we select some cationic liposomes and liposomes with targeted peptides for experimental screening, and select the best liposome for treatment. Therefore, cationic liposome Lipofectamine 2000, DOTMA/Chol, Lipofectin and LID were initially selected for screening through literature searching.
We select pcDNA3.1-GFP as cargo in the liposome and use GFP protein expression as indicator for the efficiency of liposome transfection. We selected different kinds of liposomes and ratio of DNA : liposome for screening.
2.1 Screening of liposomes by efficiency
Four kinds of liposomes, namely Lipofectamine 2000, DOTMA/Chol, Lipofectin and LID, and three DNA: liposome ratio, 1:5, 2:5 and 4:5, were selected. The optimal liposome and DNA : liposome ratio were screened by the following experimental results.
We compared the transfection efficiency of LID（Lipofectin : peptide=1：0.76）,DOTMA / Chol, Lipofectamine 2000 and Lipofectin encapsulated with different concentrations of DNA into mouse lung epithelial cell line 16HBE cells (Fig.4A)
The fluorescence of two random pictures in the same well cells with the same exposure time (1/2.5s) and the same sensitivity were captured, and the fluorescence intensity was analyzed by ImageJ. The results showed that the transfection efficiency was the best when LID was used as liposome to wrap DNA and the best DNA : liposome ratio is 4:5 (Fig.4B)
2.2 Screening of liposomes by safety
We used CCK8 Assay to screen liposome in the view of safety. After the optimal DNA : liposome ratio was determined, we transfected mouse airway epithelial cell line 16HBE cells with four liposomes and their corresponding optimal DNA : liposome ratio. The cargo DNA we used was still pcDNA3.1-GFP. 8 hours after transfection, we diluted the cells to 6000 cells/well and seeded into 96-well plates. The cells were further cultured with 2% DMEM. CCK8 reagent was added at 12, 24, 36 and 48h to detect the cell growth. The experimental results (Fig.5) showed that there was no obvious toxicity for our selected liposome LID compared to the mock group, but significantly better than the commonly-used liposome, Lipofectamine 2000.
3. Verification for our targeting peptides
What work we have done
✓The fusion proteins were constructed by computer
✓The function of fusion proteins were verified by protein-protein docking
3.1 Construction of fusion protein model
In order to demonstrate that our targeting peptides, CKLF1C19-Lamp2b and RGD-Lamp2b, can locate on exosome surface, we constructed our proteins structure using homology modeling with the help of I-TASSER. Further, we conduct alignments with our targeting peptides to the natural Lamp2b protein (Fig.6). The alignment results showed that our fusion proteins were almost the same as the natural Lamp2b regarding to the transmembrane domain, which promised that our targeting peptides can successfully anchored on the exosome memberane.
3.2 Protein-Protein docking between fusion proteins and receptor proteins
To further illustrate our targeting peptides features, CLUS PRO software was used to simulate the protein-protein docking process. Both the two targeting peptides results showed the feasibility of targeting affinity (Fig.7). For detail, please go to Model
3.3 Supplementary material on targeting ability
In order to verify the targeting ability of our Lamp2b fusion protein in self-assembled siRNA delivery system, someone in our lab performed wet experiments as shown below (data not published). In this experiment, exosomes with or without targeted peptide GE11-lamp2b were co-cultured with desired cells. siRNA content in intracellular was measured to reflect the difference in the ability of targeted peptides to target cells. The results clearly showed that the amount of siRNA in cells co-cultured with targeted peptide GE11-lamp2b on exosomes was higher.
4. Proof-of-Concept in vitro
What work we have done
✓Verification and screen of GATA3-siRNAs
✓Verification and screen of TSLP-siRNAs
✓Verification of that our siRNAs can be encapsulated by exosomes and knock down target mRNAs
✓Verification of the safety of our mcDNAs
4.1 Verification of efficiency of GATA3-siRNAs
We designed the mcDNA-CKLF1C19-Lamp2b-GATA3-siRNA to express GATA3-siRNA, hoping that GATA3-siRNA would knock down the GATA3 mRNA and thus downregulating GATA3 protein in the mouse T lymphoma cell line EL-4 cells. For the GATA3-siRNA, we designed 3 different sequence named GATA3-siRNA-1, GATA3-siRNA-2 and GATA3-siRNA-3. Through in vitro screening, we hoped to find the best siRNA sequence and would apply it in animal experiments afterwards.
4.1.1 GATA3-siRNA can be expressed in HEK293T cells
We transfected HEK293T cells with mcDNA-GATA3-siRNA-1/2/3. Through RT-qPCR, a significant amount of GATA3-siRNAs were detected in HEK293T cells, while no siRNA expression were detected in the negative control (NC) group transfected with cassette expressing scramble sequence (Fig.8).This result indicates that the design of our siRNA expressing cassettes could correctly express our desired siRNAs.
4.1.2 GATA3-siRNAs can knock down GATA3 mRNA
To evaluate the therapeutic effect of our designed mcDNA-GATA3-siRNAs，we electrotransfected EL-4 cells with mcDNA-GATA3-siRNA-1/2/3. 6h after the transfection, we used TPA and cAMP to induce the expression of GATA3 in EL-4 cells. 24h after our transfection, we harvested cells and extracted mRNA in order to detect the expression of GATA3 mRNA through RT-qPCR. The result (Fig.9) showed that GATA3-siRNA-3 can significantly downregulate the GATA3 mRNA in EL-4 cells.
4.1.3 GATA3-siRNAs can knock down GATA3 protein
To further evaluate the therapeutic effect of our designed mcDNA-GATA3-siRNAs, we electrotransfected EL-4 cells with mcDNA-GATA3-siRNA-1/2/3. 6h after the transfection, we used TPA and cAMP to induce the expression of GATA3 in EL-4 cells. 36h after our transfection, we harvested cells and extracted protein in order to detect the expression of GATA3 protein through Western Blot. The result is showed in Fig.10A; through quantification of protein expression by ImageJ, we plotted the relative expression of GATA3 protein in Fig.10B. From the result, we can say that GATA3-siRNA-3 can significantly downregulate the GATA3 protein in EL-4 cells, which correspond to the mRNA level.
4.2 Verification of efficiency of TSLP-siRNAs
Likewise, we designed the mcDNA-RGD-Lamp2b-TSLP-siRNA to express TSLP-siRNA, hoping TSLP-siRNA would knock down the TSLP mRNA and thus downregulating TSLP protein in the mouse lung adenocarcinoma cell line LA-4 cells. For the TSLP-siRNA, we also designed 3 different sequence named TSLP-siRNA-1, TSLP-siRNA-2 and TSLP-siRNA-3. Through in vitro screening, we hoped to find the best siRNA sequence and would apply it in animal experiments afterwards.
4.2.1 TSLP siRNAs can be expressed in HEK293T cells
We transfected HEK293T cells with mcDNA-TSLP-siRNA-1/2/3. Through RT-qPCR, a significant amount of TSLP-siRNAs were detected in HEK293T cells, while no siRNA expression were detected in the negative control (NC) group transfected with cassette expressing scramble sequence (Fig.11).This result indicated that the design of our siRNA expressing cassettes could correctly express our desired siRNAs.
4.2.2 TSLP-siRNAs can knock down TSLP mRNA
To evaluate the therapeutic effect of our designed mcDNA-TSLP-siRNAs，we transfected LA-4 cells with mcDNA-TSLP-siRNA-1/2/3. 6h after the transfection, we used TPA to induce the expression of TSLP in LA-4 cells. 24h after our transfection, we harvested cells and extracted mRNA in order to detect the expression of TSLP mRNA through RT-qPCR. The result (Fig.12) showed that TSLP-siRNA-2 can significantly downregulate the TSLP mRNA in LA-4 cells.
4.2.3 TSLP siRNA carried by exosomes interferes with the expression of TSLP mRNA in target cells
Further, we tried to find out whether TSLP-siRNAs can be encapsulated by the natural exosomes, and eventually play its role in knocking down the TSLP mRNA in LA-4 cells. Similarly, if we can prove that TSLP-siRNAs can be encapsulated by exosomes and targeted into LA-4 cells by targeting peptides on exosome membrane, so can GATA3-siRNAs.
We harvested exosomes from HEK293T cell medium after transfected with mcDNA-TSLP-siRNA-2 , and co-incubated these exosomes with LA-4 cells (10^6 cells per well, 6h after inducement). After 36h co-incubation, LA-4 cells were harvested for total RNA extraction and subsequent RT-qPCR of TSLP mRNA. The result (Fig.13) shows that through co-incubation, the exosomes from HEK293T, which has expressed TSLP-siRNA-2, can encapsulate TSLP-siRNA-2, and that help TSLP-siRNA-2 "enter" the LA-4 cells to degrade the expression of TSLP mRNA.
4.3 Verification of safety on our mcDNAs and Plasmids
After our screening for the best siRNA sequence for GATA3-siRNA and TSLP-siRNA, we wanted to prove that the vector we use, namely mcDNA, is safe to be applied on animal or human, and that mcDNA outperform its mother plasmid. We used CCK8 Assay to verify the safety issue. We transfected HEK293T cell with mcDNA-TSLP-siRNA-2 and its mother plasmid pMC-TSLP-siRNA-2, using liposome lipofectamine 2000 with DNA; liposome ratio of 4:5. 8h after our transfection, when all the DNA was believed to had been uptaken by HEK293T cells, we diluted cells to 6000 cells/well and seeded into 96-well plates. The cells were further cultured with 2% DMEM. CCK8 reagent was added at 12, 24, 36 and 48h to detect the cytotoxicity effect on cells. CCK8 Assay result (Fig.14) shows that there is no obvious cytotoxicity effect of mcDNA on cells, and that the mcDNA overall has fewer cytotoxicity than its mother plasmid.
4.4 Verification of long-acting feature on mcDNAs and Plasmids
In order to verify the advantage of mcDNA compared with plasmid in duration, we transfected HEK293T cells with equal molar mcDNA and plasmid at the same time, and DNA circuits in cells were extracted at 24h, 48h and 72h repectively. RT-qPCR was used for relative quantitative analysis of the decay changes in both. As shown in supplementary figure 2 below. According to the results, we found that mcDNA was significantly better than plasmid in the overall trend of decay over time, which is exactly what we expected from the beginning. However, it's a pity that we didn't set longer time to characterize mcDNA and plasmids over a longer period of time. But someone else in our lab has demonstrated in vivo that mcDNA can be expressed in vivo for significant longer periods than plasmids. Details can be found at Fig.2 in our Design.
5. Proof-of-Concept in vivo
What work we have done
✓Verification of our DNA circuit therapeutic effect on target genes
✓Verification of our DNA circuit therapeutic effect on type 2 inflammation
✓Verification of our DNA circuit therapeutic effect on asthma symptom
✓Verification of nasal inhalation can take effect with precision and efficiency
After completing the above experiments, we had proved that our idea was safe and effective in vitro, so we wanted to carry out experiments in mice in order to further prove our concept.
In brief, our animal experiments consist of two part. The first is to treat asthma mice with mcDNA mixture of mcDNA-GATA3-siRNA-3 and mcDNA-TSLP-siRNA-2,which both had been proved to be the optimal to knock down the target genes through in vitro screening, to see their therapeutic effects on asthma mice. We compared mcDNA group with positive control group and mock group in our selected targets' expression in mRNA and protein level, expression in type-2-inflammation-related IgE level, and tissue sections, which eventually prove that our therapy is promising in treating asthma. The second part of our animal experiment is the verification for safety. We wanted to prove that nasal inhalation of DNA circuit would take effect specifically on our desired tissue with precision and efficiency.
5.1 Our mcDNA can effectively reverse asthma criteria in mice
5.1.1 Asthma mice modeling and treatment strategy
6-8 week female BALB/c mice were intraperitoneally injected with 50mg OVA with 2mg aluminum hydroxide in 200ul PBS on day 0,14,28 for sensitization; and on days 42-44, 50mg OVA in 30ul PBS will be given by nasal inhalation for challenge.
For treatment, we divided asthma mice into mcDNA group and Positive Control (PC) group. For the mcDNA group, we treated the mice in day 30-40 with mcDNA, which is equal mass mixture of mcDNA-GATA3-siRNA-3 and mcDNA-TSLP-siRNA-2 (Fig.15); the mcDNA was mixed with liposome LID with DNA : liposome ratio of 4:5, and administrated into mice by nasal inhalation. Total amount of mcDNA which was administrated into a mouse is 5mg/kg and there was a gap day between two treatment. For the Positive Control group, we treated them with equal volume of PBS through nasal inhalation the same time mcDNA group got treated. Additionally, we use equal amount of unmodeled mice as mock group, which were treated with PBS whenever other two group got treated for asthma modeling or treatment.
5.1.2 GATA3 and TSLP mRNA were downregulated after mcDNA treatment
At day 45 we harvested the lung of mice and extracted the total RNA in lung tissues. Through RT-qPCR analysis, as shown in fig.16, we were confident that the mcDNA mixture of mcDNA-GATA3-siRNA-3 and mcDNA-TSLP-siRNA-2 could successfully downregulated our target genes with high efficiency.
5.1.3 GATA3 and TSLP protein were inhibited after mcDNA treatment
We also extracted total protein in mice lung tissue in order to analyze protein expression of GATA3 and TSLP using Western Blot. The result shows that both GATA3 and TSLP protein expression are inhibited after our mcDNA treatment.
5.1.4 Type 2 inflammation was suppressed after mcDNA treatment
We harvested serum of mice when sacrificing the mice in day 45. We tested IgE expression in mice serum, which is typical in type 2 inflammation, using ELISA, and the result (Fig. 18) shows that there is a significant difference between mock and positive control, which proves that asthma mice has sever type 2 inflammation; while those treated with mcDNA has downregulated IgE expression in serum, which proves that our therapy can successfully inhibit type 2 inflammation in asthma mice through inhibiting expression of GATA3 and TSLP.
5.1.5 Mice lung tissue sections indicated asthma criteria was reversed after mcDNA treatment
Paraffin-embedded tissue sections and H&E staining were performed on mice lung tissues after sacrificing. The results are shown in Fig. 19. The bronchial smooth muscle indicated by the red arrow was significantly thickened in the Positive Control group but not in the mock group. The smooth muscle thickening phenotype in the mcDNA group was successfully reversed. Similarly, metaplasia of airway epithelial cells into goblet cells was observed in the asthma group, as is indicated by blue arrow, but not in the mock group or the mcDNA group. The result of tissue sections indicated that the asthma mice had been successfully cured by our mcDNA treatment.
5.2 Our administration strategy is precise and effective
To verify our therapy is safe and tissue specific, we conduct experiments on eGFP mice, which express eGFP green fluorescent protein in almost every somatic cells. We administrated mcDNA-eGFP-siRNA using the same protocol as we did on asthma mice. We mixed mcDNA with liposome LID with DNA : liposome ratio of 4:5 through nasal inhalation in 10 days with interval of 1day (5 times in total). The total mass of mcDNA we administrated was 5mg/kg. The result of tissue section (Fig. 20) reveals that our administration strategy can successfully target the lung tissue with precision and efficiency.
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