Our project is mainly divided into 3 parts, namely GATA4 fusion protein, liposome carrier and red blood cell "hitchhiker" transportation. We aim to design a part which is synthesised to a fusion protein including the function of suppressing effect of GATA4 transcription factor, and the disagnosis usage of GFP fluoroscence, aiding the target of noticing expression of Therefore, we designed a T7-promoter-based gene expression plasmid carrying GFP coding region When expressed in E. coli BL21 and DH5a cells, this plasmid is expected to produce GFP proteins, which indicates the existance of Hepatic Stellate Cells

In our project, genetic modified fusion protein serves 2 functions: supressing the activation of HSC[1], and diagnosis of chronic liver disease at a very primary stage, which usually no specific syptoms and actions are shown. The T7 promotor we added in our modified DNA triggers the expressing of GFP fluoroscence. This part is designed with T7 promoter following with histags and ribosome binding sites.

Figure 1: Overview of our plasmid

Figure 2: Structure of our fusion protein design

[2]When the Nobel Prize in chemistry was awarded for the discovery of green fluorescent protein (GFP) in 2008, the Nobel Committee called GFP a guiding star for biochemistry enabling processes that were previously invisible, such as cancer cells spreading, to be strikingly visible. GFP and its red, blue, yellow, and orange cousins have revolutionized bio-medical research and enabled every major disease, both cause and effect, to “light-up” in the laboratory for researchers to visualize, understand and eventually conquer them. This also applies in our dedicated project but instead of cancer tumour cells, GFP acts as a signalling device for the pedominantly release aSMA receptors from the liver fibrosis site[3]. When liver fibrosis inducing realease of excessive aSMA receptors is detected, the expression started and GFP is expressed as a signal. In the future, by the advent of GATA4 acting as a "detector", and expressing GFP as a signalling device, it's believed that this fusion protein-driven liposome carrier drug could bring us to a far destination, not only as a quick test to chronic liver disease, but as a beacon of hope in curing chronic liver disease in instant.

Myofibroblasts are absent from normal tissues. During normal tissue repair, transient activation of myofibroblasts contributes to restoration of integrity of the tissue by forming a mechanical scar that is usually dissolved when the tissue is repaired. At this stage myofibroblasts are cleared by apoptosis or by inactivation.(1,2) In contrast, persistent myofibroblast activation causes accumulation and contraction of collagenous extracellular matrix (ECM), a condition called “fibrosis.”[3] By the possible solution of deactivating the activated HSC, our discovery and modified gene shed light on the future of chronic liver disease therapy. Proven that GATA4 transciption factor express effectively accelerate the process of deactivating aHSC, we connected GATA4 isoform and GFP fluoroscence in the well designed plasmid, offering a synthesis of fusion protein for a groundbreaking solution, hasten the progress of liver disease therapy in a breakneck speed. According to the research conducted in 2021[1], Gata4 overexpression reduces liver fibrosis in vivo, and is proved by a solid-grouned, evidence based series of experiments.

To test this hypothesis, [1]Universidad Pablo de Olavide researchers evaluated the effect of Gata4 overexpression on liver fibrosis regression in vivo. To this end, mice were treated with CCl4 for 4 weeks to induce liver fibrosis, followed by tail vein injection of Gata4-overexpressing adenovirus (Ad-GATA4) or Ad-GFP as a control. Liver tissues were collected 1 week after adenovirus infection, i.e., 9 days after the last CCl4 injection.(Figure 3)

Figure 3: [4] Figure 3.A-C

Histological evaluation using Sirius red revealed diminished fibrillar collagen deposition in the livers of mice injected with Ad-GATA4 compared with both control mice (no infection with adenoviruses) and mice injected with Ad-GFP . Decreased CD45 immunostaining indicated reduced liver inflammation in mice injected with Ad-GATA4 . Of note, injection of Ad-GATA4 in wild-type mice did not have any effect on collagen accumulation or liver inflammation. The repected team evaluated HSC activation in CCl4-treated mice by α-SMA immunohistochemistry. Livers of CCl4-treated mice injected with Ad-GATA4 showed a decreased number of α-SMA–positive areas compared with both control mice (no infection with adenoviruses) and mice injected with Ad-GFP. Quantification of Col1A1 and α-Sma mRNA levels confirmed a reduction of fibrosis and HSC activation in Gata4-overexpressing mice (Figure 4).

Figure 4: [4] Figure 3.T-V

Thus, Gata4 overexpression promotes liver fibrosis regression in vivo by deactivating HSCs.

GATA4 reverts the active phenotype of HSCs by modulating the expression of fibrogenic and antifibrogenic genes.To confirm the cell-autonomous role of GATA4 in HSC deactivation and to get insight into the underlying molecular mechanisms, the team turned to cell culture using LX2 cells, a human HSC cell line that recapitulates many features of the aHSC phenotype. Due to safety consideration and equipment limitations, our team can't conduct cell lysis test on our own. Instead, this citation allows us to solidate our genetic engineering approach on GATA4, in the name of DIMSTAR treatment. The team devised a system that allows robust Gata4 activation in LX2 cells using Ad-GATA4 or Ad-GFP as a control. Adenovirus-mediated overexpression of Gata4 caused a marked reduction in laminin immunoreactivity, which further decreased as a multiplicity of infection (MOI) increased (Figure 5.1).

Figure 5.1: [4] Figure 4.B and C

At a MOI of 100, nearly 98% of LX2 cells were transduced. Transduction of LX2 with Ad-GFP at a MOI of 100 did not affect laminin expression (Figure 5.2).

Figure 5.2: [4] Figure 4.A

Next, microarray analyses were performed to analyze changes in gene expression in Gata4-overexpressing LX2 cells compared with GFP-overexpressing LX2 cells (Figure 5.3).

Figure 5.3: [4] Figure 4.D

Among the most downregulated genes were profibrogenic genes such as ECM components (ACTA2, LAMA1, and COLlA1), metalloprotease inhibitors (TIMP1), TGF-β receptors (TGFβR1 and TGFβR2) and PDGF receptors (PDGFRA and PDGRFB), TLR4, and IL6. Conversely, an increase in the expression of known antifibrogenic factors, such as STAT1, SMAD7, and the transcription factor TCF21, was observed. Quantitative RT-PCR and Western blot analysis confirmed the changes in expression in these fibrosis-related genes (Figure 5.4, 5.5).

Figure 5.4: [4] Figure 4.E

Figure 5.5: [4] Figure 4.G

Interestingly, these genes showed the reverse expression pattern in the liver tissue of embryos lacking Gata4 specifically in HSCs (10), confirming that GATA4 regulates the expression of multiple genes involved in liver fibrosis (Figure 5.6).

Figure 5.6: [4] Figure 4.H and I

There is still a problem left unfixed in our solution to chronic liver disease: how to, in practicular, inject the drug into our body. Acknowledging the fact that aged red blood cell is metabolised in the liver, which is the functioning site of our GATA4-producing fusion protein, into iron groups,(Figure 7) and then reused for production of new haemogoblin, we finally come to a perfect solution for our treatment's transport method: the "Hitchhiker".

Figure 7: Aged red blood cell metabolism

But what makes it perfectly applicable, is the fact that the cell responsible for red blood cell phagocytosis, is Kupffer cell, which also serves as the HSC secreter. With the advent of red blood cell, not only can we bring our liposome drug to the liver, but also effortlessly targetting the aHSC, where ECM is formed to cause liver fibrosis.

According to the research conducted by Department of Biosciences and Bioengineering, Indian Institute of Technology in 2022[6], it's proven that Bio-inspired solutions have come into limelight in pursuit of improved circulation, to facilitate release of drugs at the targeted sites, and preventing clearance of drugs from the human system. In the last two decades, there is a rapid increase in the research and development of RBC-based drug delivery systems(Figure 8)

Figure 8: [6]Figure 1

Additionally, the use of RBC membranes as a camouflaging coating on synthetic carriers like polymeric nanoparticles have been extensively developed in quest for improved circulation half-life and stability. These systems provide combined benefits of a natural coating along with a synthetic core, leading to controlled drug release (Figure 9). In this way, we've overcome the transportation problem of our project, and took a big step towards developing a complete, functionable drug.

Figure 9: [6]Figure 2

First hindrance appears during the development and conceptalisation stage. GATA4 is a advanced and newly discovered chemical, even generally saying in the medical industry. Furthermore, we are developing in a genetic engineering approach make the research on GATA4 gene extremely difficult. We mis-added the GATA4 transcription factor applicable in mice to our plasmid and for no doubt, does not work. Thanks to the joint endeavour of fellow students and teachers, we sucessfully design our first model of GATA4 fusion protein, but using Gibson Assembly method instead of GGA. With the advent of our fellow members, this approach of GATA4 treatment by genetic engineered medication, which has never been done, finally comes from blueprint to a semi-product. After countless experiments, one of our gels from gel electrophoresis showcased our designed DNA. The DNA was then extracted from the gel using the Monarch gel extraction kit. The transformed BL21 glowed under UV light, indicating the presence of GFP and hence our protein, since both proteins are linked together. We then tried to separate the desired proteins from the rest using SDS-page, which was unfortunately unsuccessful. With our failed experiments on hand, we repeated the entire experiment again from scratch, ensuring that every single step is handled with utmost care and precision. At first, the newly transformed BL21 bacteria showed fluorescence. However, after extraction of protein by cell lysis, and protein purification, extract did not show observable fluorescence. We performed SDS-page to confirm the presence of our desired protein, which was successful

But we soon encounter another large obstacle, namely failing Golden Gate Assemblies due to the non-compatability of iGEM standard overhang of Type IIS enzyme and our design of 4-units overhang after the cleavage. Later, we fixed this by re-designing the cut sites.

Deciding to use TFH(Thin-Filmed-Hydration) liposome as our carrier at an early stage, we soon realise that the diameter of liposomes produced is not smaller than 10nm as our expectation. Since we have to insert the fusion protein into the liposome coat. [5]Liposomes are self-assembled (phospho)lipid-based drug vesicles that form a bilayer (uni-lamellar) and/or a concentric series of multiple bilayers (multilamellar) enclosing a central aqueous compartment. The size of liposomes ranges from 30 nm to the micrometer scale, with the phospholipidbilayer being 4–5 nm thick.

We control the size of liposomes by Thermal treatment, and hence reduce the diameter of each liposome to under 10nm, eventually aid the entry of GFP fusion protein in to the liposome carrier. (refer to Results page for further analysis). Also, we sucessfully coded the plasmid design and run PCR and Golden Gate Assembly to validate our work. (refer to results page for proofs). In addition, the backbone we used before the standards of iGEM release is not optimal for our promotor to work on and hence reduce the expressing efficiency of GATA4 GFP composite gene, we then switched up to the pUC-AmpRv1 backbone as the bedrock of our fusion protein, facilitating the process.

Figure 6.a: original design	Figure 6.b: enhanced design

The recombinant plasmid is then introduced into DH5α for further replication and storage of our plasmid. DH5α was then incubated at 37°C in agar plates. After several hours of incubation, 4 colonies of each agar plate were picked and incubated separately in LB broth. After more hours of incubation, DNA from transformed bacteria was extracted using Monarch PCR & DNA Cleanup Kit Protocol. Using the extracted DNA, gel electrophoresis was performed to confirm the size of our recombinant plasmid.

Although regrettably due to time limitations, we can't conduct the experiment to test out the combination efficiency of our fusion protein and red blood cell, also the percentage of GATA4 reaching the aHSC to function, this bald, yet grounded approach of us is guaranteed to be shedding light on curing chronic liver disease.

DIMSTAR

is the future of chronic liver disease cure.

Citations

[1]: Arroyo N, Villamayor L, Díaz I, Carmona R, Ramos-Rodríguez M, Muñoz-Chápuli R, Pasquali L, Toscano MG, Martín F, Cano DA, Rojas A. GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells. JCI Insight. 2021 Dec 8;6(23):e150059. doi: 10.1172/jci.insight.150059. PMID: 34699385; PMCID: PMC8675192.

[2]: Hoffman RM. Application of GFP imaging in cancer. Lab Invest. 2015 Apr;95(4):432-52. doi: 10.1038/labinvest.2014.154. Epub 2015 Feb 16. PMID: 25686095; PMCID: PMC4383682.

[3]: Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; PMCID: PMC5476301.

[4]: Kisseleva T. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology. 2017 Mar;65(3):1039-1043. doi: 10.1002/hep.28948. Epub 2017 Jan 11. PMID: 27859502; MCID: PMC5476301. Figure 4

[5]: Liu P, Chen G, Zhang J. A Review of Liposomes as a Drug Delivery System: Current Status of Approved Products, Regulatory Environments, and Future Perspectives. Molecules. 2022 Feb 17;27(4):1372. doi: 10.3390/molecules27041372. PMID: 35209162; PMCID: PMC8879473.

[6]: Vincy A, Mazumder S, Amrita, Banerjee I, Hwang KC, Vankayala R. Recent Progress in Red Blood Cells-Derived Particles as Novel Bioinspired Drug Delivery Systems: Challenges and Strategies for Clinical Translation. Front Chem. 2022 Apr 27;10:905256. doi: 10.3389/fchem.2022.905256. PMID: 35572105; PMCID: PMC9092017.