Proposed Implementation

When people have signs or symptoms that suggest they might have a myelodysplastic syndrome (MDS), they will likely need tests to look at their blood and bone marrow cells to see if they have MDS, such as complete blood count (CBC), bone marrow tests (aspiration and biopsy), blood samples, flow cytometry and immunocytochemistry, karyotype analysis and so on. However, these tests are not only expensive and inconvenient, but also rely on the experienced blood pathologists who can make an exact diagnosis with examining a sample of blood and bone marrow cells, therefore, the risk of incidence of MDS being underestimated.

Therefore, we hope we can develop a sensitive and simple diagnosis for MDS patients, which everyone could access to it. We constantly referred back to our human practice research, which influenced all the way from construct design to the marketing plan, to guide the implementation of our project.

Who are our proposed end users?

Considering the clinical phenotype of MDS is nonspecific, there is still a great challenge to arrive at the appropriate diagnosis. A large number of studies indicated dysregulation of RNA splicing played an important role in the pathogenesis of MDS. We found that there are many RNA splicing-related genes, such as SF3B1, U2AF2, and SRSF2, which have mutations in around 50% of MDS patients. Among these genes, SF3B1 is the most frequently mutated RNA splicing factor in MDS. Notably, SF3B1 mutations tend to be associated with a good prognosis in MDS. Thus, we develop several splicing sensors to diagnose MDS patients. Our project will provide some evaluation for the prognosis of MDS patients.

When the project was launched, it still require specific experimental training to lay some groundwork before we could go into the lab, such as the use of pipettes, virus packaging and plasmid transfection. We strongly recommend the professional people to do the procedure, like laboratory doctors. Indeed, all the experiments must be done following the standardized rules to make sure the accuracy of the project. Hence, the user should be someone with basic lab experiences.

How do we envision others using our project?

The disease of MDS is not incurable, the difficult part is the diagnosis. At this time, there are no widely recommended tests to screen for myelodysplastic syndromes (MDS). Screening is testing for cancer in people without any symptoms. MDS is sometimes found when a person sees a doctor because of signs or symptoms they are having. These signs and symptoms often do not show up in the early stages of MDS. Fortunately, our project can detect it in early stages, also, it's convenient and cheap for patients. Thus, MDS that is found early can be treated right away.

We envision our whole process of MDS detection being simple for patients. Also, our product requires the basic equipment so that clinics or community hospitals in the undeveloped places also have access to the apparatuses for detection. In other words, patient no longer need to travel all the way to the hospitals in the city center. This could reduce the inconveniency of testing in big hospitals, also save time, save money and reduce costs. In addition, when people do the physical exam as the process gets simplified to be implemented, thus allowing more people to get their detection earlier.

How would we implement our project in the real world?

For the implementation of our detection method, we primarily use cell lines and cell transfection to test the functions for splicing reporters. To apply our project in the real world, we extract primary bone marrow cells from patients, and the transfection of primary bone marrow cells using conventional cell transfection reagents was less efficient, resulting in a decrease in detection efficiency. According to the literatures[3], we found that Adeno-Associated Virus Type 2(AAV Type-2)can infect primary bone marrow cells. Hence, we assume that combine splicing reporters (MAP3K7-LUC or ZNF91-LUC) and a plasmid to construct a recombinant AAV virus. After collecting the virus supernatant and concentrating the virus, the virus was directly used to infect primary bone marrow cells. We collect infected primary bone marrow cells, to lysis and collect protein samples for luciferase or fluorescence tests. Thus, we can directly and conveniently get the result, and therefore help to diagnosis MDS.

Fig.1. RNA splicing reporter using AAV infection. Firstly, we transfect the MAP3K7-LUC /ZNF91-LUC and two packaged plasmids into the cell of 293T, then collect AAV-type2 virus supernatant samples 48 hours later. Secondly, we extract samples from patients' bone marrow cells, and use virus supernatant samples to infect bone marrow cells. Thirdly, after 48-72 hours infection, we start to lysis the cells and collect proteins. Finally, we add substrate buffer, detect the fluorescence values and get the corresponding sample results.

In addition, we can also use the cell-free system. We take primary bone marrow cell samples from patients, extract cells, and add splicing reporters (MAP3K7-LUC or ZNF91-LUC) into a kit of cell-free system to mimic the in vivo cellular environment, so the process of transcription and translation can be accomplished, directly from plasmid form to protein in vitro. Then, we perform the luciferase or fluorescence detection of the protein. As well as the virus infection system, it is very convenient and rapidly to get the e results of the test sample. Consequently, we can assess the patient's condition of MDS disease. We tried to test this system in the lab by using cell lysis from cells. More details were listed in Proof of Concept.

Fig 2. RNA splicing reporter in Cell-free system.Firstly, we draw the patient's bone marrow samples, extract bone marrow cells, purified and add MAP3K7-LUC /ZNF91-LUC with cell-free system reagents. Then we incubated in a metal bath at 30°C for 90 minutes to obtain reverse transcription protein samples. Finally, we add substrate buffer, detect the fluorescence values and get the corresponding sample results.

The safety aspects we would need to consider

When we did our experiment, we must accept pre-lab safety training in accordance with the lab safety protocols. Since we were working with E. coli, we were required to wear lab coats, gloves and masks before entering the laboratory, and when we were done with E.coli, we would sterilize the culture by reasonable ways, like high temperature or high pressure and dispose all the waste properly. We were also equipped with quick first kits, such as eye washers to protect ourselves if anything unexpected happened.

Meanwhile, we also perform cell culture and virus packaging, in which we do not apply any toxic reagents, and the AAV virus we choose is also low-risk [4]. But when applying to the real world, our product is relatively economical. There was no acid release or any significant dangers that we need to be aware of, and the cell-free systems we used with no other safety hazards.

Therefore, we could ensure that when our product is applied to the public, every person, including those with high risk factors, should be able to use it without any concern.

The challenges we would need to consider

1. The first challenge is to further evaluate the sensitivity and the accuracy of our project. This is what people value the most when it comes to diagnostic tests, and we also strive to improve these two values. Through mathematical modeling, we concluded that MAP3K7-LUC is more sensitive than ZNF91-LUC in both slicing reporters (MAP3K7-LUC or ZNF91-LUC). However, due to the conditional restrictions, we couldn't do clinical trials.

2. The second challenge is the design of the specific scenario of our project application in the diagnosis of MDS disease. While we were starting the survey about the project, we interviewed two experts in the field of MDS diagnosis, Dr. Wei Xue and Director Sun Li. They pointed out that the mutation of SF3B1 is not the root and only factor that leads to MDS, because there are other factors, such as molecular methylation and affected-intercellular information pathways. So, if you only use plasmid sensors to detect RNA dysregulation to confirm the diagnosis of MDS, more thought and improvement is needed. Moreover, the two experts also mentioned that there are now many ways to help diagnose a disease, especially a blood disease. Therefore, in the future, we need to make different attempts in clinical application, together with other methods to assist in the diagnosis of MDS, such as bone marrow biopsy, PCR, flow cytometry analysis, etc.. And, it is worth noting that mutations in the SF3B1 gene are also closely related to the prognosis of MDS. Our project will also provide some assessment of the prognosis of MDS patients.

3. The third challenge is to increase the availability and visibility of our product. When we conducted a survey on our project, though the majority are supportive of our project, there was still a group of skeptical people. Therefore, to increase the credibility of our project is another challenge. Compared to other common diseases like leukemia, people do not know much about MDS disease. So, the illness is in serious often because the treatment of MDS is delayed.

To solve the issue, we could make some posters and host some educational workshops to let people know more about our project. We firstly popularized the concept of MDS disease and the consequences of delaying the disease, and then emphasized the advantages of our project, we hope that more people would be flexible with this new approach.

References

[1] Sato, Shintaro, et al. "Essential function for the kinase TAK1 in innate and adaptive immune responses." Nature immunology 6.11 (2005): 1087-1095.
[2]Saminathan, Thangasamy, et al. "Differential gene expression and alternative splicing between diploid and tetraploid watermelon." Journal of experimental botany 66.5 (2015): 1369-1385.
[3]Ponnazhagan, Selvarangan, et al. "Adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived CD34+ hematopoietic progenitor cells: donor variation and correlation of transgene expression with cellular diffeentiation." Journal of virology 71.11 (1997): 8262-8267.
[4]Tenenbaum, Liliane, Enni Lehtonen, and Paul E. Monahan. "Evaluation of risks related to the use of adeno-associated virus-based vectors." Current gene therapy 3.6 (2003): 545-565.