Organ transplantation is a serious domestic social issue that is widely concerned by the public due to the low complementation rate. Organ donation must be evaluated by a hospital, witnessed by a third party, and signed by family members. Although the number of organ donors is increasing, a considerable amount of organ donations cannot meet the required clinical conditions, making organ transplantation difficult to be implemented.
Number of organ donations from 2015 to 2020 in China[1]
Another reason is that the cost of organ transplants is too high for patients with the average income level to afford. In 2016, it is reported that the cost of kidney transplantation can reach 300,000 yuan and heart transplantation costs 600,000 yuan, while the average disposal income in terms of per capita income at that time was merely 23, 821 yuan[2][3]. Immunosuppressive drugs alone, which can prevent the activity of the immune system, can cost 30,000 to 50,00 yuan per year[4].
Therefore, our group proposes to optimize the metabolic regulatory network of Rapamycin, the main ingredient of anti-rejection drugs, and improve the fermentation level of rapamycin to improve drug manufacturing efficiency. This helps to achieve our short-term goal of significantly reducing the cost of Rapamune (also known as Sirolimus, an immunosuppressant widely used in kidney transplants) and increasing its productivity. In the future, we hope that our product can promote the fair distribution of public medical and health resources for organ transplantation and make it more affordable for patients in provinces with more impoverished populations.
To achieve the goals we mentioned above, we designed the experiment as follows. To construct the engineered strain, we amplified the upstream and downstream homologous arm of gene M271_14685/M271_14690, cloned it into pKC1139 plasmid, and then transfer it into ET12567/pUZ8002 competent cell. Screen the correct strain and co-culture with Streptomyces rapamycinicus, choose the double cross-over strain and test the fermentation yield of rapamycin by HPLC.
△M271_14685/m271_14690 and NRRL 5491 have produced rapamycin during 11 days.
It can be seen from the above figure that the rapamycin produced by our knockout △M271_14685/m271_14690 is much higher than that produced by NRRL 5491. And the amount of rapamycin produced on the ninth day reached 120mg/L. This suggests that the subject is feasible to improve metabolic pathways by knocking out the two-component system, which can be applied for clinical application in the near future.
So our product is an anti-rejection drug for patients who have undergone organ transplantation. Rapamycin exerts an immunosuppressive effect by blocking signaling through different cytokine receptors, blocking the progression of T lymphocytes and other cells from the G1 to S phase. There had never been an injectable version of rapamycin before. The injectable version of rapamycin is developed with many advantages. For example, it shows a much faster onset of action and greater bioavailability than rapamycin taken orally because it is annotated to deliver almost 100% of the drug directly into the bloodstream, whereas orally it tends to be broken. Moreover, injectables are sealed in special containers to keep the liquid or powder isolated from the outside air and are manufactured in a sterile or aseptic manner, making them more resistant to storage than other liquid preparations.
The target users of our products are mainly patients who suffer from diseases requiring organ transplantation and their families. As shown in the picture below, organ transplantation organizations are mainly located in relatively developed provinces, such as Guangdong, Beijing, and Shanghai. Thus, on the basis of geographical segmentation, it can be assumed that patients who mainly come from these regions could be our clients. And since the most prominent competitive advantage of our product is the low price we set, from the perspective of psychographic segmentation, patients who are sensitive to price are also our potential customers.
Reginal distribution of organ transplantation medical organization in 2019 in China[5]
The whole process of our team's experiments and human practice activities fully comply with iGem safety guidelines, both in terms of biosecurity and the Covid-19 situation.
In the vast majority of experiments, we used some safe and common laboratory strains, such as competent cells Escherichia coli DH5α, competent cells Escherichia coli ET12567/pUZ8002, wild-type Streptomyces rapamycinicus NRRL 5491 and mutant strain ΔM271_14685/M271_14690. These are commonly used model organisms that have been shown to be very safe, causing little or no harm to humans.
We have also fully considered the further safety issues that may arise in the future commercialization of the product. Although with the help of biotechnology, the price we set can be lower than the competitors because of the increase of production rate with lower costs. However, it should be noted that there are still several potential challenges waiting to be addressed by us.
As mentioned in the sections above, the main selling point of our product is the low price. Other than that, as an injectable product, it is more favorable than oral products for it is more effective and convenient. At present, no injectable Rapamune or other types of immunosuppressive drugs has been developed, suggesting a huge gap in the market.
As a start-up enterprise, our marketing plan is mainly to improve brand awareness. We plan to carry out free or inexpensive consultations for potential patients of organ transplantation or those who are interested in this aspect. During the consultation, we can objectively promote our products. Outdoor advertising near organ transplantation medical organizations, such as ads on the nearest bus stop or MTR station, can also be implemented.
Regarding product development, we will consult more experts to optimize the conditions for higher production of Rapamune through our engineered Streptomyces rapamycinicus and let it can be applied to industrial scale production in the near future.
