Overview
This project systematically promotes the development of mRNA drug platforms through multiple parallel and interrelated engineering cycles: at the design level, we conceived a multi-level regulatory blueprint including target degradation, protein expression, UTR optimization, molecular switching, and targeted delivery systems; In the construction stage, we used plasmid vectors and in vitro transcription techniques to successfully construct various functional mRNA molecules and surface display extracellular vesicles. During the testing phase, we used various methods such as RT-qPCR, Western Blot, fluorescence detection, and nanoflow cytometry to rigorously evaluate the functional efficiency of each component. Finally, in the learning phase, we conduct in-depth analysis based on the experimental data of each cycle (such as efficient siRNA silencing, UTR sequence effect differences, leakage expression problems of switches, etc.) , and then drive the next round of more targeted optimization design (such as integrated MFE optimization) . UTR model, exploring switches with different CAG repeats, etc. and gradually transformed the initial concept into an integrated drug platform with increasingly perfect functions and more accurate regulation.
Drug Target Selection Cycle
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Design
Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that plays a complex dual role in liver physiology and pathology. In hepatocellular carcinoma (HCC) , TGF-β protein inhibits tumors in the early stages and strongly promotes their malignant progression in the late stages. Here, we select TGF-β protein as our protein degradation target. In order to verify that the protein expression of this target can be artificially regulated, we designed siRNAs targeting TGF-β protein mRNA. It is hoped that RNA interference technology can be used to silence TGF-β protein gene expression from the RNA level.
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We designed siRNAs targeting the 48th, 339th, and 642nd bases of TGF-β protein mRNA, respectively , the prepared siRNA was transfected into Hepg cell lines by lipid nanoparticles (LNPs) , and the gene silencing effect was tested after 48h of culture.
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Test
The cells were collected, RNA was extracted, and the mRNA expression level of TGF-β gene was detected by RT-qPCR. After cell lysis, Western blot experiments were performed to test the expression level of target genes from the protein content level.
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Based on the RT-qPCR experimental results, the degradation rate of the three siRNAs of TGF-β reached 70% or more, and at the same time Western Blot experimental grayscale analysis also showed that we successfully silenced the TGF-β gene. It was proved that the protein expression of this target can be artificially regulated, laying the foundation for our subsequent experimental design.
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Design
Based on the verification that the expression of TGF-β protein can be artificially regulated, we designed mRNA with a protein degradation module to degrade TGF-β (TGF-β degradation RNA) to reduce the expression of this gene at the protein level. The element consists of three modules, namely the Trim21 protein, the ligation peptide, and a short peptide that can specifically bind to TGF β proteins. The fusion protein composed of these three modules can specifically bind to TGF-β protein and reduce the content of TGF-β protein by mediating the ubiquitination degradation pathway. We plan to deliver this mRNA into cells to verify the effectiveness of our design by detecting changes in the content of TGF-β protein.
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We used the pUC57 plasmid as a vector to design expression plasmids capable of characterizing target RNA. For TGF-β degradation RNA plasmids, the target sequences include: short peptide + ligation peptide + trim21 sequence targeting TGF β protein. We also integrate the T7 promoter into plasmids for in vitro transcription of TGF-β degradation RNA.
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Test
After constructing the plasmid, we linearized the expression vector and performed T7 RNA polymerase-mediated RNA transcription in vitro, but during the experiment, we found that the RNA concentration of the transcript product was low, making it difficult to proceed to the next experiment.
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After reviewing the literature and researching multiple sources, we decided to replace the T7 promoter with a more reliable one, hoping to obtain a higher concentration of RNA products for subsequent validation experiments, while providing a guarantee for RNA transcription in other module experiments.
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Design
Further, we designed a protein regulatory system with both components, and the two regulatory elements are linked by P2A peptides. It is hoped that the expression of tumor suppressor gene-related proteins can be improved while degrading the proteins expressed by the original oncogene in hepatocellular carcinoma cells, thereby improving the anti-cancer effect of drugs.
In order to verify the feasibility of upregulating and downregulating two different target proteins at the same time, we plan to first introduce eGFP-expressing mRNAs into cells, and then target eGFP mRNA, siRNA and mRNA expressing Luciferase were delivered into cells, and the feasibility of this strategy was verified by detecting changes in fluorescence intensity before and after cell transfection.
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We designed siRNAs targeting eGFP fluorescent proteins (eGFP siRNAs) and those that can stably express eGFP/Luciferase, respectively mRNA (eGFP mRNA and Luc mRNA) . The eGFP mRNA was transfected into 293T cells first, and then the eGFP siRNA was combined with the Luc mRNA. The feasibility of this strategy was verified by detecting the fluorescence intensity of cells after 24h of transfection.
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Test
The validation experiment was performed using the 293T cell line, and the control group was transfected with only eGFP mRNA and Luc mRNA .The remaining groups were introduced with Luc RNA + eGFP RNA + eGFP siRNA respectively, and another was set up Blank control group. Fluorescence detection was performed after 48h of transfection.
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The experimental results showed that the green fluorescence intensity of the experimental group transfected with eGFP siRNA was significantly different from that of the control group without transfection of siRNA (p<0.0001) and was treated in the same experiment .There was no significant difference in fluorescence intensity between the two groups at 560nm wavelength (p>0.05) , indicating that we successfully expressed eGFP while using siRNA to reduce expression Luciferase proved the feasibility of up-regulating and down-regulating two different target proteins at the same time, laying the foundation for further experimental exploration.
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Design
In order to evaluate the application potential of the drug, firstly, we plan to use siRNA to simultaneously silence the expression of two different fluorescent proteins, and verify the feasibility of down-regulating the expression of multiple proteins at the same time by detecting fluorescence intensity. We expect this drug to complement as a molecularly targeted drug in combination therapy and to be used in combination with antibody drugs targeting key immune checkpoints (e.g., PD-1/PD-L1 axis) , as well as targeting tumor angiogenesis (eg. VEGF/VEGFR signaling pathway) to reverse the immunosuppressive microenvironment and inhibit tumor progression through a multi-mechanism combination, thereby improving the overall therapeutic effect of advanced liver cancer.
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Build
We designed siRNAs targeting eGFP fluorescent proteins/Luciferase (eGFP siRNA and Luc mRNA, respectively) and mRNAs that stably express eGFP/Luciferase (eGFP mRNA and Luc mRNA, respectively ) . The eGFP mRNA and Luc mRNA were transfected into the cells first, and then the eGFP siRNA and Luc siRNA were transfected Co-delivered into cells to verify the feasibility of the strategy by detecting changes in fluorescence intensity before and after cell transfection.
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Test
The 293T cell line was used for validation assays, and the control group was transfected with eGFP siRNA and Luc mRNA, and the experimental group was transfected with Luc RNA + eGFP RNA + eGFP siRNA +Luc siRNA, and a blank control group. Fluorescence detection was performed after 48h of transfection.
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The experimental results showed that the green fluorescence intensity of the experimental group transfected with eGFP siRNA and Luc siRNA was significantly different from that of the control group without transfected siRNA (p<0.0001) Under the same experimental treatment, there was a significant difference in fluorescence intensity between the two groups at 560nm wavelength (p<0.001) , indicating that we used it eGFP siRNA and Luc siRNA successfully reduced both eGFP and Luciferase proved the feasibility of our strategy of down-regulating the expression of multiple proteins at the same time, and laid the foundation for further experimental exploration.
UTR Optimization Loop
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Design
In order to obtain the manually optimized design of the 5’UTR sequence, we selected two models, UTRGAN and UTR-Insight, and built the basis based on the model characteristics and operation requirements. The computing environment of the PyTorch framework is configured with GPU acceleration resources to meet the computing power requirements of deep neural network training, and the version conflict problem of the two model dependencies is solved through virtual environment isolation – UTRGAN. The required WGAN-GP optimization module, transpose convolution implementation component, and UTR-Insight dependent UTR-LM pre-trained language model. The Conv-Former architecture-related tools are deployed in separate virtual environments to ensure that their functions are functioning properly.
Based on these two independent models, we generated the first UTR sequences. After UTR-Insight prediction and screening, 10 high MRL sequences were selected for the next experimental verification. We selected Luciferase as the reporter protein and designed an RNA capable of Luciferase expression (Luc-UTR RNA) . To evaluate the regulatory efficiency of different 5’UTR variants.
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We used the pUC57 plasmid as a vector to construct an expression vector for evaluating the efficiency of 5’UTR. A Luciferase coding sequence was inserted into the plasmid as a reporter gene, and the T7 promoter was integrated to enable in vitro transcriptional synthesis of Luc-UTR RNA. We designed 10 pairs of specific primers for the UTR sequence output of the model, integrated the designed sequence into the expression vector by PCR, and obtained the corresponding artificial 5'UTR and LUC-UTR RNA by in vitro transcription technology.
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The prepared mRNA was transfected into a 293T cell line by lipid nanoparticles (LNPs) , cultured for 24h and then lysed the cells, Luciferase substrate was added, and the fluorescence intensity was tested within 2h.
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Through the analysis of wet experimental data, we found that there is still a big gap between the regulatory ability of the 5’UTR generated by the existing model and the existing artificially synthesized high-expression 5’UTR sequences, generate the model for further improvement and optimization.
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Design
Based on the experimental results of cycle1, we have improved the generative model. The core constraint system that integrates the minimum free energy (MFE) prediction ability and systematically incorporates the key regulatory dimension of mRNA stability into the sequence design is achieved through deep coupling of the original model prediction link. Simultaneous prediction - the stability of the secondary structure can be quantified in real time during the sequence generation process, and the risks such as strong stem-ring structure caused by energy anomalies can be identified in advance, so as to solve the experimental problems caused by stability imbalance in the first batch of sequences. At the same time, we add a gradient optimization mechanism, based on the original generation logic of UTRGAN, and dynamically guide the parameter space generated by the sequence through the gradient optimization algorithm, hoping to improve the effect of the sequence.
We use the self-designed fusion model to optimize the design of the sequence, calculate the minimum free energy of the resulting sequences, and select the second batch of 15 sequences for wet experimental verification after considering MRL and MFE.
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To test the expression efficiency of the 5’UTR sequences we generated, while unifying our characterization criteria, data from different test batches can be compared. We followed the plasmid design in cycle1, designed 15 pairs of specific primers for the 15 UTR sequences output by the fusion model, and integrated the designed sequences into the expression vector by PCR. mRNA with the corresponding artificial 5’UTR was obtained by in vitro transcription technology.
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The prepared mRNA was transfected into a 293T cell line by lipid nanoparticles (LNPs) , cultured for 24h and then lysed the cells, Luciferase substrate was added, and the fluorescence intensity was tested within 2h.
Figure 11. Efficiency Characterization of the Second Set of 5'UTR Sequences Sequence numbers 11-24 represent 14 differently designed 5'UTR sequences. The PC group uses Pfizer's publicly available artificially optimized 5'UTR sequence, while NC serves as a blank control without transfection of RNA. Luciferase activity was measured and is presented as Relative Light Units (RLU).
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Learn
Compared to the reference sequence, we designed the sequence with low expression. We analyze the reasons for the results of the new round of experiments: (1) In the actual reproduction of the UTR-Insight model, we did not perform the key pre-training steps and directly used the randomly initialized transformer. The encoder is connected to the main training of the model. This omission led to the encoder not being able to effectively capture the hierarchical biological features of the sequence, ultimately resulting in the model's prediction accuracy failing to reach the level in the literature. (2) Due to too many changes to the code during the use of the model, the model effect is affected. (3) There is no reference for control, and only the sequence optimized by the model random design is used for numerical screening.
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Design
In response to the problems encountered in the first two loops, we further improved the model by retraining the model and designing an automated workflow to optimize the design of the sequence by running the two models in parallel. The MRL scores of UTR sequences with known high efficiency were calculated, and the sequences with the same level or higher scores were screened, and the above sequences were compared in terms of MFE and CG content.
We designed the third batch of sequences with the new model and selected 14 sequences for the next experimental verification. In order to further characterize our 5’UTR effect, we collected NCA-7d-5'UTR sequences and Albumin-5'UTR. The former is a highly expressed endogenous 5’UTR, and the latter is an albumin gene-derived 5’UTR, both of which are naturally occurring 5’UTR sequences in nature. It has been used in mRNA therapy research. We want to compare it with the regulation effect of our self-designed 5’UTR to better evaluate our optimization results.
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To test the expression efficiency of the 5’UTR sequences we generated, while unifying our characterization criteria, data from different test batches can be compared. We followed the plasmid design in cycle1, designed 15 pairs of specific primers for the 15 UTR sequences output by the fusion model, and integrated the designed sequences into the expression vector by PCR. mRNA with the corresponding artificial 5’UTR was obtained by in vitro transcription technology.
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Test
The prepared mRNA was transfected into a 293T cell line by lipid nanoparticles (LNPs) , cultured for 24h and then lysed the cells, Luciferase substrate was added, and the fluorescence intensity was tested within 2h.
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According to the experimental data analysis, the design quality of the third batch of 5’UTR was improved compared with the previous two batches, and the regulatory effect of the 5’UTR sequence No. 37 was better than that of the two high expressions in nature 5’UTR, with application potential. So far, the iteration of our UTR optimization model has achieved certain results.
Switch Cycle
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Design
The Toehold switch is a modularly designed cis-acting riboswitch that functions to regulate the translation of downstream genes by sequence-specific binding to specific trigger RNAs. Its composition includes a switch element located in the untranslated region of the 5' end of the mRNA, which spontaneously folds into a hairpin secondary structure, thereby hiding the ribosome binding site and inhibiting translation initiation; When fully complementary trigger RNAs bind to this region through a strand replacement reaction, they cause switch conformational rearrangements, exposing ribosome binding sites that specifically activate protein expression of the gene of interest.
At the beginning of the project, we found a toehold switch sequence with microRNA as an aptamer that can be used in eukaryotic systems through literature research, and hoped to optimize it and apply it to our drug platform system to improve the cell selectivity of our drugs. For this sequence, we designed an mRNA (t-T-mRNA) with a toehold switch sequence and a Luciferase sequence. The trigger RNA in the switch is miR-21.
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In order to test the expression regulation efficiency of the toehold switch sequences collected by us, we used pUC57 plasmid as a carrier to construct an expression vector for evaluating the expression regulation efficiency of molecular switching. Here we use a toehold switch whose trigger RNA is miR-21. The Luciferase coding sequence is inserted into the plasmid as a reporter gene, and the T7 promoter is integrated for in vitro transcription and RNA synthesis. We designed a pair of specific primers for the artificially designed toehold sequence, and the switch sequence was integrated into the vector by PCR 5’ UTR behind the RNA sequence, mRNA with corresponding switch sequences (t-T mRNA) was obtained by in vitro transcription technology.
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Test
The prepared t-T RNA and miR-21 were transfected into a 293T cell line by lipid nanoparticles (LNPs) , and the cells were lysed after 24h of culture, and Luciferase substrate was added. The fluorescence intensity was tested within 2h.
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The experimental results showed that there was a significant difference in the fluorescence intensity measured at 560 nm between the control group transfected with t-T-mRNA and the experimental group transfected with t-T-mRNA and miR-21 at 560 nm (p<0.0001) , indicating that the toehold sequence has a regulatory effect. However, there is still room for optimization at the opening level, so we decided to try to generate and optimize some toehold sequences for subsequent experimental validation.
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Design
To solve the problem of low level of openness, we studied the special structure of the eukaryotic. Toehold sequence and other factors that affect its function, such as Kozak offset, toe pairing, GC content, etc. Two sequences were manually designed for dry experimental verification, and it was found that the characterization efficiency was too low and the results were not ideal.
Next, we took the Tau team's Triggate project in 2022 to try out switch designs using their platform, which we obtained 25. The Toehold switch sequence of the eukaryotic system was based on the next experimental verification.
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To test the expression efficiency of the Toehold switches we generated, and to harmonize our characterization criteria, data from different test batches can be compared. We followed the plasmid design in cycle1 and designed 25 pairs of specific primers for the 25 UTR sequences output by the triggate platform . For specific primers, the corresponding mRNAs are t-A mRNA to t-Z mRNA. The designed sequences were integrated into the expression vector by PCR, and mRNA with the corresponding artificial Toehold switch sequence was obtained by in vitro transcription technology.
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The obtained t-C mRNA, t-E mRNA, t-I mRNA, t-K mRNA will be prepared and cycle1 prepared t-T-mRNA and miR-21 transfected to 293T by lipid nanoparticles (LNPs) , respectively. In the cell line, the cells were lysed after 24h of culture, Luciferase substrate was added, and the fluorescence intensity was tested within 2h.
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The experimental results show that the fluorescence expression of the self-designed toehold switch is greatly increased compared with the sequence in cycle1, but the leakage expression is higher at the same time, and the regulatory effect is poor. Switch generation models while exploring other feasible molecular switch designs.
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Design
We set our sights on other types of RNA molecular switches. After investigation, we found an RNA-ON switch based on exCAG sequences. This switch consists of a miR-21 binding region and a repressive sequence, located downstream of the mRNA polyA tail. The repression region consists of repeated CAG sequences that silence the ORF region by inducing RNA gelation. When the concentration of miR-21 in the cell is high, its complementary region pairs with miR-21 and recruits the RISC complex to cleave the inhibitory sequence, thereby degelating the RNA and making the ORF normal expression for cell selectivity.
Based on this principle, we designed an mRNA (CAG30 mRNA) with the mCherry sequence as the reporter gene. The posterior insertion of the polyA tail of RNA adds a miR-21 binding sequence and a 30x repeated CAG sequence as an inhibitory sequence. The trigger RNA switched in this mRNA is miR-21.
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In order to test the expression regulation efficiency of the exCAG switch sequence we designed, we designed and constructed an expression vector using the pUC57 plasmid as a carrier. The mCherry coding sequence was inserted into the plasmid as a reporter, and the miR-21 binding sequence was added sequentially after the polyA tail, and 30× Repeated CAG sequences and integration of the T7 promoter upstream of the 5’UTR for in vitro transcriptional synthesis. CAG30 mRNA with corresponding switch sequences was obtained by in vitro transcription.
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Test
The prepared CAG30 mRNA and miR-21 were transfected into a 293T cell line by lipid nanoparticles (LNPs) and cultured for 24 h and then by flow cytometry B610 channel detects fluorescence signals.
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The experimental results showed that the experimental group transfected with CAG30 mRNA and miR-21 inhibitor was the same as that transfected with CAG30 mRNA and miR-21 mimic. There was no significant difference in the fluorescence intensity of mCherry detected by B610 channel by flow cytometry (p>0.05). The switch design and control effect is not good, and there is still room for optimization. Therefore, we decided to explore the optimal number of replicates of exCAG in the inhibition sequence to increase the switch-induced fold and the level of the open state.
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Design
In order to further optimize the switch design, increase the induction factor and open state level, and reduce the impact on normal cells, and explore the lowest CAG repeats, we designed two sets of switches with the number of CAG repeats of 3× and 10× for further experimental verification. We speculate that the optimal repeat may be between 3× and 30×, and hope to further narrow the range of sequence screening through this experiment, and finally find the optimal sequence through engineering cycle iteration.
We designed two mRNAs with mCherry sequences as reporters (CAG3 mRNA and CAG10 mRNA) , and miR-21 was added to the posterior insertion of the polyA tail of both RNAs. The sequences were combined, and two exCAG sequences with 3× and 10× repeats were inserted as inhibitory sequences, respectively. The trigger RNA switched in this mRNA is miR-21.
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To test the expression efficiency of the molecular switches we designed, while unifying our characterization criteria, data from different test batches can be compared. We followed the plasmid design in cycle3 while designing specific primers to modify the CAG repeat number in the inhibition sequence to 3× and 10×, CAG3 mRNA and CAG10 mRNA with corresponding switch sequences were obtained by in vitro transcription.
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Test
The prepared CAG3 mRNA and CAG10 mRNA were compared with the CAG30 mRNA prepared in cycle1 and miR-21 by lipid nanoparticles (LNP) into 293T cell lines, and fluorescence signals were detected in the B610 channel by flow cytometry after 24h of culture.
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The experimental results showed that the control group transfected with CAG10 mRNA and the experimental group transfected with CAG10 mRNA and miR-21 mimic were detected by flow cytometry using B610 channels. There was no significant difference in mCherry fluorescence intensity (p>0.05). However, the control group transfected with CAG3 mRNA and the experimental group transfected with CAG3 mRNA and miR-21 mimic showed a significant difference in fluorescence intensity (p<0.01). Similarly, the control group transfected with CAG30 mRNA and the experimental group transfected with CAG30 mRNA and miR-21 mimic also showed a significant difference in fluorescence intensity (p<0.001). Based on the experimental results, we guessed that the better CAG repeat number might be around or above 30. As for the regulatory effect of CAG3 mRNA, it may be caused by other mechanisms, which needs to be further explored.
Expression Vector Cycle
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Design
We expect to give mRNA drug delivery vectors tumor-specific targeting capabilities through engineering design. Based on the literature review, we selected GPC3 protein, a member of the glycosaminoglycan family, which is highly expressed in 70–80% of patients with hepatocellular carcinoma, especially in poorly differentiated tumors, as a potential delivery recognition molecule , it is proposed to confer the specific recognition ability of HCC cells by conjugating GPC3 antibodies on the surface of lipid nanoparticles (LNPs) .
The specific strategy consists of three steps: (i) presentation of GPC3 antibodies using the Lpp-OmpA display system in E. coli BL21; (ii) Preparation of extracellular vesicles (EVS) that present GPC3 antibodies on the surface; (iii) It was heterozygated with LNPs to prepare LNP-EV heterozygous vectors carrying GPC3 antibodies on the surface.
As a confirmatory experiment, we first constructed an Lpp-OmpA-eGFP protein presentation system to present eGFP protein on the surface of Escherichia coli, and then prepared EVSThe protein expression level was also detected to verify the feasibility of the design idea.
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We used the pET-28a (+) plasmid as a vector to construct an expression vector for the presentation of eGFP on the surface of Escherichia coli in BL21. The plasmid contains a sequence encoding the Lpp-OmpA-eGFP fusion protein, which integrates lactose operons into the expression vector to regulate protein expression. At the same time, 6× His tags were added to the end of the fusion protein for Western Blot experimental characterization.
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The plasmid was transformed into the BL21 strain. The bacteria were cultured in a 100 ml system until the OD600 reached 0.5. Protein expression was then induced by adding 50 μl of IPTG, followed by overnight incubation at 18°C. EVs were prepared by ultrasonic centrifugation after fragmentation of cells, and the fluorescence intensity of EVs was detected by nanoflow cytometry to characterize the number of eGFP surface presentations.
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Through the nanoflow cytometry characterization results, we can indicate that the target protein has been presented on the surface of EVs, which basically verifies the feasibility of our Lpp-OmpA display system and can be used for the next characterization experiment.
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Design
In cycle1, we basically validated the feasibility of our Lpp-OmpA demonstration system, and further we conjugated a single-stranded antibody of the GPC3 protein to the OmpA protein. The C-terminus of the EVS was prepared to surface display GPC3 single-stranded antibodies for subsequent heterozygous vector preparation.
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We used the pET-28a (+) plasmid as a vector to construct an expression vector for the presentation of GPC3 single-stranded antibodies on the surface of E. coli. The plasmid contains a sequence encoding the Lpp-OmpA-GPC3 single-stranded antibody fusion protein, integrating lactose operons into the expression vector to regulate protein expression, and adding 6xHis tags to the fusion protein end for Western blots experimental characterization.
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Test
The expression plasmid was transformed into BL21 strain. The bacteria were cultured in a 100 ml system until the OD 600 reached 0.5. Protein expression was then induced by adding 50 μl of IPTG, followed by overnight incubation at 18°C. EVS were prepared by ultrasonic centrifugation after crushing cells, and then Western blot assay was performed to detect the expression of target protein.
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In this cycle, we performed Western Blot experimental characterization of EVs displaying eGFP and GPC3 single-stranded antibodies on the surface. From the development results, we successfully presented these two proteins on the surface of EV s, which further proved the effectiveness of our protein presentation system design and laid the foundation for our subsequent LNP-EVS hybridization experiments.
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