Team:Montpellier

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IMPLEMENTATION

      The problem

Cancer is one of humanity's biggest scourges. Even with early diagnosis and treatments, some forms remain deadly.

Physicians most often fight the disease with four types of cancer treatments: surgery, radiotherapy, chemotherapy, and immunotherapy. Others exist of course: tumor perturbation with sonic waves, hormonal treatments… But the majority consists of the four described above. Unfortunately, cancer is versatile and resilient and thus difficult to cure:

Surgery is inefficient on metastasis, microscopic tumors, or tumors adjacent to vital organs.
Chemotherapy heavily burdens the patient’s body and mind; moreover, cancer cells can actually adapt to it and become resistant.
Radiotherapy relies heavily on tumor imaging, can damage surrounding tissue, and cannot kill large tumors.
Immunotherapy has side effects, is very expensive, and is impaired in immune suppressor tumors.

Knowing these limitations, our project aims at providing physicians with a different approach that will complement the others while being convenient and safe.

Briefly, the concept of Phagent is to use innocuous phages to reprogram our microbiome into cancer fighters. For this, we will package genes coding for anti-cancer proteins into phages targeting intratumoral bacteria. The infected bacteria will produce active molecules right at the cancer location.

      The power of our project

Numerous scientific articles demonstrate that we can transfer genes to bacteria with phages. Genes coding for therapeutic proteins is no different from other genes therefore we could transmit them to the bacteria in the tumors.

Phages are very specific: they always target single bacterial species and often a species sub-group. This is a great advantage: it limits the risk of off-targeting and prevents any chance of human cell infection.

But this also means that we have to know the most abundant bacteria in each cancer type in order to select the phage most fitted. This was one of our main challenges but thanks to several scientific articles we understand more and more about tumor microbiome and we can select phages accordingly.

**Table 1: Group of phage for a specific bacterium.**
Tumor site	Bacteria genus	Proposed phage
Prostate	Propionibacterium	Phage 174
Colon	Rectal and distal: Alistipes, Akkermansia, Halomonas and Shewanella. Proximal: Faecalibacterium, Blautia and Clostridium.	/
Lung	Veillonella and Megasphaera	Phage N2, N11 and N20
Ovary	Pediococcus	cIP
Pancreas	Pseudomonas putida	PF16 and phiPMW [7]

Two new challenges await us: finding a suitable bacterial target with a high concentration in the tumor microbiome and finding a phage that we could engineer. Indeed phages can be difficult to engineer for our project for several reasons: limitation in gene capacity, wrong injection of its genome in the bacteria, killing too fast its target, or lack of characterization.

One way to bypass this issue is to infect E. coli which is prevalent in the gut microbiome and can migrate to other organs such as the pancreas; hence we could reprogram the gut microbiome and let it migrate to the tumor site. Consequently, colon and prostate cancer could be suitable targets for Phagent.

Phagent is a versatile concept that can in principle be adapted to cure any cancer, provided that the tumor microbiome and a compatible phage are characterized. The project implementation may slightly vary depending on the targeted tumor and the chosen phage. Here we will focus on a scenario to target pancreas cancer using Pseudomonas Putida strain and pf16 phage (due to its phylogenetic and genomic closeness to T4, a well studied Escherichia Coli phage).

      Safety assessment

Safety is primordial for our project. We decided to tackle each potential issue one at a time:

1. Phage administration innocuity

Since data regarding phage therapy have been largely lost or is only available in Russian, the doubt about phage innocuity persists. In our case, it is also a GMO that interplays with the immune response (possible inflammation) and may burden the kidney with protein filtration (phage being essentially made of proteins). So we will need to conduct pharmacological tests.

**Clinical trial schematic by James Lind institute in 2018 (https://www.jliedu.com/blog/clinical-trial-phases/)**

Preclinical tests (preferably on the same cancer type as the therapeutic molecule tested) need to be run with two animal models in order to obtain the therapeutic and adverse effects dosage used for clinical tests.

For therapeutic dosage evaluation, we need animals with microbiome and cancer response close to our’s, and for possible adverse effects, we need mammals with the immune system and protein filtration system compatibility. Therefore rats, mice, and dogs are suitable models.

Two types of the administration seem very promising for our project, each with pros and cons. The intratumoral injection has the advantage of needing a lower dosage than intravenous but intravenous can reach the whole body and possible metastases. Intravenous could prevent cancer recurrence this way but it also increases risks (inflammation, gene transfer, microbiota imbalances…)

And finally, we’ll run clinical tests on cancer patients in close collaboration with clinicians and authorized labs. For these tests, however, we’ll need to find money either by public funds or by an industrial partnership.

2. Bacteria's gene editing

In the scenario where we target E.coli in the colon cancer microbiome, we chose to use M13 phage. After infection, the M13 genome does not integrate into the genome of the bacteria; there is therefore no edition of the bacterial genome per se. However, the alteration of its metabolism could result in different and possibly detrimental phenotypes.

This concern can be addressed by in vitro testing of our product on healthy cells and by the pharmacological tests described above.

However, it is necessary to repeat this approach for other combinations of protein, phage and bacteria. For example, if we switch to pf16 for pancreas cancer therapy we’ll have to redo the tests, even if the therapeutic proteins are the same.

3. Protein expression innocuity

According to Murphy’s law even if phage injection is proven benign we still have to use containment techniques to avoid possible long-term effects and maximize safety.

Therefore we want to use two inducible promoters specific to the tumor microenvironment (one for acidic environment the other for hypoxia) in order to be certain that the expression is restricted to the tumor. We are also considering implementing a kill switch to lower the risk.

4. Phage-driven spread of other bacterial genes

One concern about phage is its ability to vehicle genes between bacteria, some of which can be detrimental for the host.

The most efficient tool we have to limit this is actually what impairs the efficiency of our project: the burden of the phage genome and its metabolic cost will give a competitive disadvantage to bacteria receiving genes. With inducible promoters, we impair the ability of bacteria to express our design outside of the tumors. However advantageous genes could be encapsulated by mistake and transmitted by the phages to other bacterias. In this case, antibiotic resistance genes could be transmitted to other populations of bacteria which is troublesome as cancer patients are often treated in clinics where antibiotic is used for fighting nocive bacteria.

To avoid this we should implement a suicide switch killing bacteria infected by our phage if they receive a specific compound. This switch will be activated after the therapy, preventing gene transmission afterward.

If we recapitulate what we will need to do in order to address these issues:

Phage engineering and safety part introduction
In vitro testing with healthy cells
In vivo tests with two mammals models
Clinical trials with patients

      Implementation in the real-life

Our application will be reserved for hospitals and clinics only.
The price of the product will depend on R&D cost, funds, production charges, and the type of partnership we find for clinical tests.
Depending on the cancer stage, a suspension of engineered phages will be administered by intravenous (for metastatic stages) or intratumoral injection. Viral titration needs to be precisely measured for each batch of treatment and dosage has to be precisely determined during clinical trials.
Treatment duration and frequency will be determined during clinical trials, as well as the response timing. Patients will be followed for an appropriate duration.
Once the tumor is cleared, a killswitch can be activated in order to clear the remaining phages and infected bacteria. This will be an additional precaution to prevent any long term side-effect.

      Other challenges: social implementation

Social implementation is also a critical point of our project because as a GMO it already has social obstacles.

One is the need for GMO acceptance by the Government which depends on its laws.

According to the European GMO legislation, we need to prove our project therapeutic effect with clinical tests as well as its innocuity. In addition, we need to implement strategies to contain its possible spread in the environment: this can be achieved by new protocols in clinics for cleaning operating devices and treating waste.

Besides ethics and laws, there is also a huge work to do on social acceptance, which also varies depending on states and culture. This is one reason why we wanted to evaluate the acceptance of our project, what part of it is difficult to accept, and how we could improve our project or communicate better to overcome this. We designed a poll to measure acceptance levels for phage-based anti-cancer treatments and correlate acceptance scores with social descriptors.

We know now that in order to promote our project we need to improve the knowledge of the population regarding the use of GMOs and phages. Therefore we aim at creating educational content for everyone: we started with comics raising awareness of cancer, phages, and microbiome and designed a video game.