Team:Heidelberg/Proof Of Concept


Proof of Concept





The journey of our novel drug

Background

Our proof of concept will be the combination of the wetlab and drylab parts to summarize the whole project. Every aspect of our research was important to create our final proof of concept. In order to explain this we will describe how our designed drug would travel through the whole body regarding its final application for the single-gene disease phenylketonuria (PKU). Every single step is described to make it easier to follow our overall treatment idea.
It is important to mention that it is not only based on our creative minds but also on previous research. The treatment of PKU and other metabolic diseases by influencing the microbiome is a great new topic in which a part of the basic research already has been done. In the study by Durrer et al. from 2017 a mouse model with PKU was treated with in vitro cloned bacteria carrying a phenylalanine ammonia lyase (PAL) which were brought into the intestine. The results show a promising outlook for further trials [1]. Moreover, a rat study tried to naturally transform rat gut bacteria by transporting DNA into their gastrointestinal tract. They tested the transport of the DNA into the intestine by feeding the rats with pellets containing transformant DNA which showed no functional results [2].
Much more research has been done to support our new approach but now we would like to present our drug application by describing it step by step to make it easier to follow.

Figure 1: Summary of our drug application. Step (1) describes the drug treatment via an orally taken capsule to transport the DNA into the intestine. In (2) the drug is released and should be taken up via natural transformation by competent bacteria. (3) describes the specific strains that could be tackled in diseased patients and in (4) the transfer of the plasmid is described. The next step (5) shows the expression of the gene of interest. (6) describes a diet that is based on a selective advantage through β-agarase degradation of agarose. Finally, step (7) represents the killswitch which should take care to stop every possible escaping event. Created with BioRender.com

1. How is the drug delivered?

The journey of the new drug starts in the form of a capsule for oral application releasing its content into the intestine. A varnish is required so that there is no premature dissolving in the stomach acid.
The capsule design and production big scale is discussed in detail in our proposed implementation chapter. The treatment should be as congruent as possible like the normal amino acid degradation in a healthy human. The phenylalanine degradation takes place as well in the intestine by the phenylalanine hydroxylase (PAH) which is produced in the liver [3] (see also AvPAL Assay).

2. How is the DNA taken up by the bacteria?

After the drug release the plasmid should be taken up by natural competent bacteria. In our experiments for cloning with natural transformation we proved that Acinetobacter baylyi ADP1 is naturally competent without any processing of the bacteria as well as of the plasmid itself. Additionally, the same protocol works as well in co-culture with more influences, stress and competition pressure. The colony forming unit (CFU) of the naturally transformed A. baylyi shows a lower efficiency in the co-culture than compared to monoculture experiments.
We performed preliminary co-culture experiments to control the growth behaviour and impact of the three laboratory strains on each other. Overall there was no severe impact from one of the strains on another except for Bacillus subtilis 168 which was growing faster and inhibited the growth of A. baylyi ADP1. An option to increase the efficiency of the DNA uptake could be for example using multicopy or linear plasmid structures. Important to mention is that the origin of replication must be suitable for the natural competent strain and bacterial strains which should express the genes of interest.

3. Which bacteria can take up the DNA?

In general bacteria which express type 4 pili (T4P) are more likely to take up DNA. We wanted to use this feature in order to single out bacterial strains which fulfill both of our conditions:
    They need to be competent, meaning that they can undergo natural transformation.
    They need to be more abundant in diseased patients than in healthy patients.
With this goal in mind, we trained a logistic regression model by giving it data of healthy and diseased patients. After training, we evaluated the relevance of each feature, for the outcome whether a test sample was diseased or healthy. The features used were the different bacterial strains or rather their abundance in our case, for the outcome whether a test sample was diseased or healthy. The only strain which fulfilled both criteria was Fusobacterium prausnitzii. Its abundance in healthy patients did not differ that much compared to the one in diseased patients but that could be due to the lack of data which could be found.

4. How is the DNA transferred between the bacteria?

To observe the spreading behaviour of the plasmid carrying the gene of interest we have to consider existing models of horizontal gene transfer in the microbiome. The best studied and known gene transfer is conjugation. It is known that conjugation takes place in the human gut microbiome [4]. It is difficult to observe and measure the frequency of distribution of the plasmid of interest in the intestine first with natural transformation and then by conjugation to finally keep a constant level of PAL. This is probably the most complicated task to engineer because as always “only the dose makes the poison” said by Paracelsus. Already in the United States of America PAL was used as a treatment, but as already discussed, an overdose can cause severe side effects (see Integrated Human Practices). This needs to be observed in animal trials by controlling the appearance and multiplication of the plasmid in the mouse stool. To sum up, we assume that there is at least a small horizontal gene transfer, especially if the selective advantage serves a benefit for the bacteria that have taken up the plasmid [4].

5. What is the function of the drug?

When the plasmid is taken up successfully, the phenylalanine ammonia lyase (PAL) BBa_K1983000 enzyme derived from Anabaena variabilis can be expressed. AvPAL is responsible for the phenylalanine degradation to trans-cinnamic acid (tCa). We performed the in vitro assay and measured tCa absorbance. With the measurements of tCA it can be assumed that AvPAL is functional. The product tCa can be metabolized by humans and excreted naturally [5]. We showed the AvPAL assay only in vitro because we were missing the L-phenylalanine permease (BBa_K1983013) gene which is required for an in vivo assay. On the part page BBa_K1983000 the iGEM Team Vilnius Lithuania from 2016 showed that an in vivo assay is possible if the L-phenylalanine can be transported into the bacteria.

6. Is there a benefit for the cloned bacteria?

Regarding maintaining a stable concentration of the enzyme, the plasmid needs to stay in the gut microbiome long term. It is a calculation of costs and benefits which indicates that without any selection advantage the plasmid will be sorted out [6], [7]. To get ahead of this process a diet friendly selection advantage must be found. The hypothesis was to include a β-agarase on the plasmid. The final product of the agarose degradation should be a usable sugar to metabolize for energy gain of the bacteria. Therefore it would be necessary to increase the amount of agarose in the diet. The results of the selective advantage experiments were not clearly pro or contra an selective advantage for the bacteria carrying a β-agarase. For further results more research is required.
Nevertheless, we took this task seriously as we talked to Dr. Kölker who told us that pharmacy is not always focused on good taste and consistency for children's medicine. We created delicious recipes containing agarose to create child friendly treatments (see Special Excellence). In addition, the glucose concentration must be lowered to increase the dependence of the bacteria from the β-agarase to receive sugars.

7. Which final safety step is required?

One day the bacteria with the plasmid will be digested and excreted by the patient. It is important to consider a possible killswitch to stop the spread of the new plasmid immediately after leaving the microbiome of the patient. One suitable killswitch described by the Fudan iGEM Team from 2020 is about the toxin/antitoxin system with MazE/MazF. It has the capability to produce antitoxin as long as the bacteria are alive under body temperature of 37 °C. When the temperature drops under 30 °C the antitoxin is no longer produced and the bacterium dies. The same killswitch exists with the RelE/RelB system but RelE was defined as an unsuitable toxin because eukaryotic cells can be endangered which could cause severe side effects (Team Fudan 2020).
The problem with this specific killswitch is the international application. We discussed this problem extensively in the MicrobioCOSMOS meeting. In the European latitudes it is unlikely to have temperatures for a longer period of time above 30 °C. In other latitudes for example around the equator it is likely to have over 30 °C air temperature for a longer time span. Therefore, the discussed killswitch could only be used regionally and for warmer climates and traveling patients another killswitch needs to be considered.

Figure 2: Cold triggered MazF/MazE killswitch. Part BBa_K3606028 is described. When the bacterium is under body temperature conditions the antitoxin is produced (1). When the temperature drops the antitoxin is no longer produced and the bacteria are killed by the toxin (2). Created with BioRender.com

Conclusion and outlook

For proof of concept, we have achieved some basic research aspects, but there are still many open questions. We showed that the natural transformation is working with competent bacteria and suitable plasmids. The AvPAL enzyme is functional as well as the β-agarase. Further research must be done for the natural diet based selective advantage and the closer definition of natural competent bacteria in each individual patient.
In future trials the focus will be on natural transformation protocols in vivo and keeping a constant AvPAL expression to prevent side effects as much as possible. At last, a suitable and harmless killswitch only against prokaryotes in the microbiome must be designed and tested. In the best case, the killswitch is turned off inside the patient's body and active outside of it.

References

[1] Durrer, K. E., Allen, M. S., & Hunt von Herbing, I. (2017). Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU. PloS one, 12(5), e0176286. https://doi.org/10.1371/journal.pone.0176286

[2] Nordgård, L., Brusetti, L., Raddadi, N., Traavik, T., Averhoff, B., & Nielsen, K. M. (2012). An investigation of horizontal transfer of feed introduced DNA to the aerobic microbiota of the gastrointestinal tract of rats. BMC research notes, 5, 170. https://doi.org/10.1186/1756-0500-5-170

[3] Vockley, J., Andersson, H. C., Antshel, K. M., Braverman, N. E., Burton, B. K., Frazier, D. M., Mitchell, J., Smith, W. E., Thompson, B. H., Berry, S. A., & American College of Medical Genetics and Genomics Therapeutics Committee (2014). Phenylalanine hydroxylase deficiency: diagnosis and management guideline. Genetics in medicine : official journal of the American College of Medical Genetics, 16(2), 188–200. https://doi.org/10.1038/gim.2013.157

[4] Huddleston J. R. (2014). Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes. Infection and drug resistance, 7, 167–176. https://doi.org/10.2147/IDR.S48820

[5] Garrait, G., Jarrige, J. F., Blanquet, S., Beyssac, E., Cardot, J. M., & Alric, M. (2006). Gastrointestinal absorption and urinary excretion of trans-cinnamic and p-coumaric acids in rats. Journal of agricultural and food chemistry, 54(8), 2944–2950. https://doi.org/10.1021/jf053169a

[6] Hülter, N., Sørum, V., Borch-Pedersen, K., Liljegren, M. M., Utnes, A. L., Primicerio, R., Harms, K., & Johnsen, P. J. (2017). Costs and benefits of natural transformation in Acinetobacter baylyi. BMC microbiology, 17(1), 34. https://doi.org/10.1186/s12866-017-0953-2

[7] Wein, T., Hülter, N. F., Mizrahi, I., & Dagan, T. (2019). Emergence of plasmid stability under non-selective conditions maintains antibiotic resistance. Nature communications, 10(1), 2595. https://doi.org/10.1038/s41467-019-10600-7