Entrepreneurship

Introduction

The probiotics market in China is expected to reach a size of USD 13,527.100 million by 2025, growing at a CAGR of 8.81 percent. In 2019, the market was valued at US8,149.466 million¹. Previous research findings have emphasized the significance of gut microbiome on human health, and this has opened up the marketing of probiotics. Taking advantage of the booming market and the maturation of bioengineering technology, our project aims to guard our guts through bacteria-bacteria interaction, a method that is an alternative to antibiotics. Some probiotic product utilizing bacteria-bacteria cell interaction has already gone through clinical trials and entered the market, such as Lactobacillus reuteri DSM17648 which treats H.pylori infection². Therefore, it is possible for our product to secure a place in the probiotic market.

The terminal of our designed product is a drug used by people. We understand that animal model testing as well as clinical trials are often involved in the process of drug development. What we have reached so far is only a prototype tested in vitro. After the completion of the in vitro testing, we will proceed to in vivo models. To improve our plan we interviewed Jisheng Lin from Sinovac Biotech company to collect feedback and suggestions about how to turn our products into commercial ones as well as caution on putting our products in a real-world situation. Here we provide considerations from various aspects of accessing our products into the market and our solutions so far.

Safety

"Safety is always the first concern," as suggested by Mr. Lin, "even if it doesn't work well, at least does no harm." Based on Mr. Lin's suggestions as well as our previous thoughts, several sources of safety concerns are listed as follows: First, the plasmid we used for selection contains a bacterial ampicillin resistance gene. though not expressed in yeast due to different promoter and codon structures, potential horizontal gene transfers still leave risks of spreading antibiotic-resistant genes to other prokaryotes in the intestine. Second, though our vectors cell lineage yeast strain EBY100 has been well studied, the genomic background of our engineered yeast is unknown. Our engineering process may alter the growth and metabolic characteristics. Efforts should be given to test if engineered yeast could harm human bodies. Besides, the human intestine is a complicated microenvironment comprised of microbiota yet not fully understood. It is also important to clarify how long our engineered yeast can survive in the human body, whether it gains competitive advantages over native gut microorganisms, and how it alters the structure of the gut microbiome. Third, similar to antibody therapies, allergy may be a question for some people. Therefore, an approach should be developed to pre-test patients' irritability. Fourth, as a commercial product, a standard of quality control during each step of engineering or drug production is also needed.

Effectiveness

Effectiveness demonstrates the values of our products. We have shown qualitatively the expression of the antibodies and the binding between the antigen and the antibodies. For a real-world application, we need to establish a standard to access and quantify these values. Moreover, the success in the simplified in vitro assay does not guarantee the effectiveness of the drug to work in a real scenario. Considering the real-world applications that our products function in human GI tracts, it requires the engineered yeast to be able to settle down, proliferate, and bind to Shigella spike proteins. Hence, the growth curve of the engineered yeast in human intestines should also be tested. More importantly, the performance of the drug should also be assessed. Potential questions include whether there is a dosage-dependent protection effect, how to assess these effects if they exist, and whether this product focuses on therapy or is precaution oriented.

Economics

The third aspect is related to the economics of our products. Apart from the reagents and consumable usage, stabilities, including plasmid stability and the stability of the phenotype expressions, are important factor influence the cost of production. A passaging lineage stably expressing the recombinant antibodies greatly reduces the cost of continuously re-engineering the yeast to harvest antibody expression. Efforts should be devoted to focusing on increasing the stability of antibody expression during passages.

Future

We should also seek further development of our products in the future, such as increasing the valency of our product so that it can target multiple pathogens at the same time.

Our Solution

As many goals are still far from being achieved, here we proposed our plan altogether with what we have reached. We used two different approaches to remove the influence of the ampicillin resistance gene. In the first approach, we designed a PCR fusion that jumps the whole ampicillin-resistant gene and the bacterial replication origin. The resting plasmid contains around 3200bp including the TRP1 gene which is a suitable size for PCR amplification and enables the selection of transformant yeast cells. The alternative approach used CRISPR-Cas9 to integrate the whole "promoter-Aga2-20ipaD" cassette into the yeast genome. The latter also increases phenotype stability as no plasmid is needed to maintain the phenotype. For expression level assessment, we have already established a system employing ELISA to quantify the expression. However, one drawback of this approach is that it does not differentiate surface-displayed antibodies from antibodies that are not correctly presented on the surface. An improved design uses biotins to label all surface proteins followed by avidin affinity binding to isolate these proteins from the protein repertoire. Isolated surface protein can be then analyzed via ELISA. To improve expression level, we have listed a set of developed constitutive yeast promoters as candidates. However, we also realized that promoter engineering processes may be required in the future. For the effectiveness test, we designed a cell invasion test in vitro. Engineered yeast cells together with Shigella will be applied to Vero cell cultures. Effectiveness will be measured by the proportion of cells being infected.

To remove the influence of cell lines on the result of the invasion test, we plan to substitute the Vero cell lines with epithelial cells in the future. After the completion of in vitro assay, further in vivo assay may be applied.

Conclusion

We realized that what we have built is only a prototype of a Shigella infection precaution drug. There is still a long way to go in optimizing the drug in vitro as well as testing the drug in vivo. However, via a series of design processes, we have developed plans to turn it into real-world applications.