Implementation

End Users and Beneficiaries of our Product

The end users of our product would be of course the patients that suffer from chronic wounds. However, commercialising and selling would be targeted towards clinicians, nurses and physicians, because they can best assess, if a patient would be in need of our therapeutic.

Our product has to be used in a combinatorial therapy. This is because it does not in itself contribute to wound healing, rather it only slows its deterioration by inhibiting biofilm from building up. This is not a disadvantage in our product, but rather an advantage, because it is an addition that strengthens other products and therapeutics. A biofilm is known to be tolerant to antibiotics as well as the host immune system (Høiby et al., 2010). The tolerance is due to many factors involving the metabolism of the bacteria as well as the polysaccharide matrix making it more difficult for therapeutic molecules and antibodies to pass through (Stewart et al., 2019).

QBlock's mechanism of action

There are several different approaches to biofilm treatment, because individual therapies are often also not very successful (Schilcher and Horswill, 2020). A common step early in the wound care process is debriding. Debriding a wound is defined as removing (usually via scraping) the necrotic tissue, foreign material, senescent cells, and bacteria from the open wound site (Manna and Morrison, 2019). It aims to remove enough of these inhibitory factors so that the wound can progress beyond the inflammatory stage towards healing. Removing biofilm is one of the more difficult things to do, because it is firmly adherent to surrounding tissue, and is tolerant to and poorly penetrated by antibiotics (Roy et al., 2018). Furthermore, debriding may also cause pushing the biofilm deeper into the tissue and worsening the state of the wound (Sen et al., 2021). Our solution is intended to be a combinatorial treatment with other mechanical (debriding) and pharmacological (antimicrobial) therapeutics.

Since QBlock directly affects the early biofilm accumulation process, our hypothesis is that it can be applied early after debriding to limit new biofilm formation and allow other therapeutic agents to contribute towards the actual healing of the wound. To improve the success of this initial debriding process and to increase the chances of subsequent antimicrobial therapy, the flow of treatment would be as follows:

1. Debride the wound as much as possible
2. Add QBlock onto the decreased-thickness, less firmly-adherent biofilm, to stop its re-accumulation. This can be combined with our Dresden partner team's phage therapy, so that any remaining biofilm is digested by the applied phages.
3. Target the now thinner biofilm, which should not have re-accumulated as much as it would have without DARPin application, with
   I. Antimicrobials, which should now have better penetration
   II. Our Dresden partner's growth factors, to promote disinfection simultaneously with tissue regrowth
4. Repeat steps if necessary

Improving QBlock

In order to implement QBlock in the real world, we still have to take some steps. We have now identified potential candidate DARPins as the basis for QBlock. As stated on the modelling page, we originally had a DARPin library size of 1012. However, due to funding constraints, we needed to downscale it to a library of 42 DARPins. With these we still received sufficient results to state which specific DARPins would be ideal to target AIP.

Therefore, the first steps that we would need to take is to confirm that this is indeed the best performing DARPin out of our original 1012 library. This can be done by increasing the size of the library again, repeating the experiment and checking whether a DARPin with a higher affinity towards the target AIP exists.

We were able to show that our bioreporter is inducible with AIP and maybe could test it against the DARPIn. However, the inducible promoter used for GFP seemed to be leaky, therefore, improvement of the expression system is still needed. We can also show the effectiveness of the DARPin in other ways than testing it with a bioreporter. In order to check that the DARPin is inhibiting biofilm formation, we should continue with our planned in vitro model and check QBlock's effect on S. epidermidis. This was not feasible in this project due to time constraints, but should be performed in future to measure biofilm formation.

Additionally, we would eventually plan to design other products similar to QBlock targeting other quorum sensing pathways of other biofilm-forming pathogens on chronic wounds.

QBlock's Optimisation

To be able to eventually utilise QBlock in a wound care setting, we have to finalise a suitable delivery mechanism first.

According to the modelling results that were achieved in the collaboration with TU Dresden, DARPins would have a half-life of 10 hours to slow down AIP1 signalling when 30 µM of DARPins would be administered via a hydrogel to the wound. Therefore, we plan to expand our collaboration with the Dresden iGEM on the hydrogel delivery aspect. This means continuing to test the diffusion constant, aggregation tendency, half-life and stability of our DARPins if introduced to a hydrogel dressing. We would keep adjusting the hydrogel's thickness and composition until a suitable speed of diffusion and other suitable characteristics of delivery are reached. The advantage of continuing our partnership with Dresden is that we can also combine our treatments, because we would benefit each other.

Testing QBlock Preclinically

Once we have optimised our DARPin delivery mechanism, we would need to validate these tests on tissue models, where we would apply the DARPin-infused hydrogel to animals with skin surface wounds (murine and porcine models) as well as to injured organoids infected by a S. epidermidis biofilm and possibly even artificial skin - an innovative collagen scaffold that induces regeneration (Yoon et al, 2016; Garfein et al., 2009). This will also be an opportunity to check whether QBlock binds to other non-target molecules and if we need to backtrack and tweak the affinity of our top DARPin. If the model testing is successful and shows that our hydrogel adequately releases DARPins into tissue sites while maintaining favourable stability and degradation properties, we would have a first prototype. At this stage, we would have a viable, safety-compliant product in the pre-clinical phase.

This also suggests the possibility of combining biofilm formation inhibition through DARPins with Dresden's delivery method. We imagine, medical workers could have a set of three hydrogel patches: the first one would contain DARPins and stop biofilm formation, so that the antimicrobial treatment will become efficient, the second one would deliver bacteriophages to eliminate bacterial infection, and the last patch would bring growth factors to the wound stopping inflammation and increasing general wound healing.

We would need to move onto additional safety checks to verify the pharmacological viability of our product. This would involve ensuring that the DARPin does not have any other targets in the human body.

Testing QBlock Clinically

We then need to perform clinical trials with the DARPin-infused hydrogel to be fully convinced of its benefits and low risk profile.

We would start by contacting hospitals in the Helsinki area as well as more broadly in Finland to conduct clinical trials on real patients suffering from chronic wounds.

Inclusion criteria

The patients would be selected into different age and sex groups, with the conditions that all of them suffer from a diagnosed diabetic-aetiology chronic wound. The patients don't need to have followed the same treatment history, but they do need to have had some biofilm previously formed on their wounds, which should be predominantly made out of S. epidermidis.

Operation of the trial

This biofilm would be debrided (scraped off) from all patients on day 0 of the trial, and their progress with re-formation of biofilm would be tracked over the course of two weeks. This would be a double blind trial, in which a non-DARPin-containing hydrogel (placebo) is applied to the wounds of certain patients and the DARPin-containing hydrogel applied to the wounds of others. If the clinical trial results on day 14 are favourable and confirm that using DARPin infused hydrogels decreases the biofilm accumulation rate, as well as confirm the product's low risk profile, we would begin the process of sending our solution to the market.

Reaching out to Business Contacts

Within our network, this includes Jouni Lounasmaa, former leader of the Finnish Startup Foundation, who has previously advised us on the management side of our project (see: Human Practices) and who has already offered to connect us to other regenerative biotechnology startups. Another avenue will be reaching out to our contacts at Slush, a global start-up conference held annually in Helsinki, to receive guidance about building our marketing and sales plan. Finally, we would seek further advice from the clinicians we previously spoke to at the HUS Wound and Burn Centre, particularly researchers like Dr. Esko Kankuri who are in strong contact with the pharmaceutical and medical devices industry. This would further help us to understand how to best position our product when pitching it to both target clients and end-users (hospitals and physicians/nurses) and manufacturing partners.

Final Preparations for Commercialisation

Though our main goal is to make treatment accessible and inexpensive to all, we acknowledge that we would have better chances to have investors if we have a patent for our product - see Entrepreneurship. Depending on the market, drugs can have different durations for patents. In Europe, the maximum patent duration is 20 years. We will be applying for the patent to ensure our competitiveness in the market when we first enter it, with the knowledge that it would expire anyway. So, by the time the medication is more well-known, it would be an open access formula for other manufacturers to refine and develop further.

The production of DARPins can be easily achieved and is cost-efficient. Expression of the DARPin can be easily done in E. coli as we have shown. Then extraction needs to be scaled-up and improved, but in general it is low in cost, because very little materials are required. The addition of the DARPin to a hydrogel can be easily achieved due to our close partnership with TU Dresden and we would therefore have a low cost product. Of course, if we expand our partnership with TU Dresden, we will have to take the business aspect into account and consider future shares of the profit

In order to achieve a scaled-up production of our DARPin we would need to either utilise our investors' funds to increase manufacturing ourselves, or to approach pharmaceutical companies with existing manufacturing facilities to outsource this task to them.

We would imagine that QBlock would be rather prescribed by the doctor than be available over the counter. This is due to the fact that QBlock is a combinatorial treatment. In order to make combinatorial treatments work, there should be some testing and experience that the products complement each other. We would of course provide this information to the doctors, pharmacists and nurses, but would then expect that they can make the right decisions of combining our treatment with the correct medication that we would recommend. This would prevent misuse of QBlock, which is very important to not induce tolerance or resistance in the bacteria both for QBlock and the combined therapy e.g. antibiotics.

We see QBlock as a very advantageous treatment, because it enhances other therapeutics by weakening biofilm formation in the chronic wound. Therefore, we foresee considerable buy-in from clinicians, investors and pharmaceutical collaborators alike.

Challenges of QBlock

There are many different products in the wound care market targeting biofilms, targeting chronic wound healing. Therefore, QBlock is added to a very niche market, which might already have some over-saturation of products, therefore we need to ensure our marketing of the product to challenge other products (see: Entrepreneurship)

Additionally, QBlock has currently some biosafety concerns regarding the functionality of the product, for example causing unforeseen side-effects and contributing to - rather than preventing - biofilm formation. We need to consider these and test them well in the laboratory prior to proceeding with clinical trials, but this we considered in our implementation and biosafety.

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