The key values of our project, namely our social and scientific morals, were continually addressed throughout our engineering cycles. We focused on designing an inclusive and functionally restorative therapy for the debilitating condition that is SCI. We aimed at designing, building and furthering scientific research within Synthetic Biology and Regenerative Medicine.
We approached key stakeholders, including clinicians, academics and industry personnel to confirm whether we were developing a project, which was responsible and good for the world . By incorporating their guidance, we were able to assess how each component of our design would interact in a system and identify other applications of our research, with an aim to contribute to the scientific community. For example, using ChABC in the treatment of neurodegenerative diseases. We discuss alternative uses to our therapy on our Proposed Implementation Page.
Our Human Practices work shaped our project at multiple different stages:
We were motivated to develop a project, which was responsible and good for the world and tackled this by investigating the potential interactions between our proposed therapy and the immune system. We not only identified potential risks to our patient pool but also assessed our therapies impact on the environment, as discussed in our Safety and Security Page. We have selected the use of our synthetic bioadhesive, PVFP-5 as it is non-cytotoxic, selected for our poly-caprolactone scaffold as it is biodegradable and incorporated the use of our mutated ChABC to promote axonal regeneration and ensure it remains functional at physiological temperatures.
We responded to the feedback we received from our Silver medal Human Practices by incorporating our stakeholder guidance into our engineering cycles. Throughout our research, we obtained consent and confirmation from all participating parties before conducting an official interview. In addition, we did not directly contact patient groups, this ensured we limited any risk of patients misunderstanding the nature of our research. We did not want to disseminate false hopes before testing our therapeutic in Clinical Trials.
We also informed our technical decisions through our human practices, namely refining our pore geometries and scaffold mechanics, troubleshooting our experimental protocols and developing our method for thermostabilising ChABC. Similarly, we developed our safety and ethical decisions by altering our therapies target region. In Phase II of our project, we have decided to treat patients presenting with C6-T11, complete spinal cord injuries. This was to ensure we did not disrupt respiratory function or risk losing the minimal function patients with higher cervical lesions have. Through our many discussions, we were motivated by our stakeholders to uphold the Hippocratic Oath and tailored our decisions to ensure the wellbeing of SCI patients.
Furthermore, in developing our project design we had to prioritise decisions to sustain the safety, feasibility and validity of our project. For example, in deciding on the method of delivery of our enzyme, ChABC, we had to decide between microinjections or hydrogels. While there is extensive literature on the benefits of using hydrogels, after in-depth discussions with academics, we discovered that microinjections would ultimately work better within the context of our therapy, especially when combined with our scaffold. We decided to prioritize the cohesiveness of our therapy, rather than developing a ‘perfect’ delivery method in isolation.
Through the effective use of the information gathered from our Human Practices, we were able to “close the loop” and create a project that is functional and safe .
In September, we collaborated with the University of Manchester iGEM team in a human practices inspired initiative towards deepening our understanding concerning the differing views between clinicians and researchers on the topic of novel therapies. Following research into concepts such as Learning Healthcare Systems, we distributed questionnaires among clinicians and researchers alongside having conducted a live interview with Dr Adrian Heald. We asked him questions about novel therapies for which he graciously offered us his valuable insight. Together, we aim to utilise information from both perspectives to bridge the gap between the two poles of therapeutic advancement. This is essential for breaking down barriers to help guide the synthetic biology community into a brighter future. Moreover, we would eventually advance this initiative by promoting it to more clinicians and researchers alongside potentially publishing our findings in an article to benefit the synthetic biology community.
These human practice contacts helped us understand specific parts of the problem we were tackling better and guided our research to overcome these problems.
Antonia Pontiki is a PhD student and graduate teaching assistant at King’s College London in the department of Biomedical Engineering with an academic research interest in chest wall reconstruction for cancer patients, having devised a 3D printing method to produce a patient specific chest wall reconstruction for patients undergoing chest wall resection for malignancy that is cost-effective.
Due to her experience with 3D printing and collaborating with hospitals and trusts to implement her product in the medical world we decided to get in contact with her with the hope of becoming more familiar with printing procedures, testing and better utilising the resources available from King’s College London and partner hospitals.
Antonia recommended two main types of tests for our scaffold. Mechanical testing including Young’s modulus, shear stress tests and potentially bending tests using equipment from the Biomedical Engineering and the Engineering department. The other type of tests are biocompatibility ones including cytotoxicity. She advised us to get in contact with the lead of the tissue engineering department at KCL, Dr Lucy Di Silvio, who could potentially help. She provided a contact for a PolyJet 3D printer available at St Thomas Hospital which would be quite efficient in printing our scaffold. She also provided contacts for clinical trial experts and guided us through the process of implementing devices in hospitals.
In order for the hospital to agree to implement a therapy we would need to get in contact with a member of the trust at said hospital- potentially a surgeon- that would be interested in the clinical application of our product and get approval from the board. This approval would only be acceptable within said trust and in order to have ethical approval for our device we would have to apply for one with IRAS (Integrated Research Application System). She estimated that an approval would take no less than 6 months for a new implantable device. We would also need to be in contact with clinicians within said hospital that would sterilise the product.
She also informed us that to her knowledge the only gamma radiation facility within a hospital in the UK is established in Scotland so the manufacturing and sterilisation process for our medical device would not be able to be conducted within one hospital.
Finally, we discussed a problem with 3D printing our scaffold related to a high amount of stringing in the end product that needs to be manually removed. She suggested changing the settings in the printer to reduce the amount of stringing therefore helping us make the printing process more efficient.
Following our conversation with Professor James Fawcett we met with Dr Alejandro Carnicer-Lombarte, a Postdoctoral Research Fellow at the University of Cambridge with an interest in neural-computer interfaces to restore function after injury to the CNS. We met with Dr Carnicer-Lombarte to discuss the implication of introducing a foreign material to an injury site in the central nervous system where the body responds to implantation with inflammation and fibrosis – a foreign body reaction (FBR). Through this conversation we were hoping to engineer our scaffold to reduce the amount of FBR at the lesion site and therefore reduce risks for patients and increase the efficiency of our therapy.
Dr Carnicer-Lombarte advised us to implant a less stiff material - or redesign the scaffold material to match the stiffness of the spinal cord to prevent mechanical mismatch as this can increase the amount of damage to the tissue and lead to FBR; Furthermore he confirmed that the Young’s modulus of our scaffold was acceptable for our proposed implementation.
As our scaffold is biocompatible and biodegradable Dr Carnicer-Lombarte has informed us that the levels of FBR would be reduced. It is especially important to reduce FBR at the ends of the lesion. That could cause scarring and would prevent axons entering the scaffold. He recommended filling the lesion cavity to the best of our abilities with our scaffold and decreasing degradation rate to allow for tissue formation. As he was unfamiliar with our PVFP-5 protein he was unable to advise on the implications of using it within our scaffold, however he did ultimately suggest that coating our scaffold with such a protein could be of interest, since if it helps in stabilising the implant at the lesion site, it would reduce the amount of FBR.
Professor Elizabeth Bradbury and Dr Elizabeth Muir have been conducting research on ChABC for many years and are currently working on developing an effective treatment for SCI by delivering ChABC through gene therapy. In our meeting we discussed the potential implementation of ChABC within our project and strategies to overcome some of the difficulties we may encounter with its implementation.
Regarding our scaffold design, Professor Bradbury suggested that we implement ChABC within or at the edge of our scaffold to optimize neurite outgrowth, whereby the neuroprotective nature of ChABC will hopefully reduce the post-lesion cavity size and stimulate reparatory macrophages (M2) as opposed to inflammatory macrophages (M1). In this regard, Professor Bradbury cited results from her own research (Burnside et al., 2018) indicating that administration of ChABC in rat models lead to successful recovery of the corticospinal tract (CST), an essential change for the significant improvement of motor function. Professor Bradbury suggested that such an approach could potentially achieve the same effect in human SCI patients, if combined with other forms of activity-based training, such as physical rehabilitation and electrical stimulation to strengthen the nerve pathways. As a result, we decided to deliver ChABC to the lesion site alongside our designed scaffold to create a permissive environment for axonal regeneration. Implementing a combinatorial approach with ChABC, we decided to devise rehabilitation protocols to maximize the functional improvements of SCI patients, thus leading to improved sensory, motor and autonomic outcomes.
One point of consideration within our project at the time of this meeting was to encourage axonal regeneration and elongation. Professor Bradbury suggested the application of neurotrophin 3 at the caudal end of the scaffold, with a concentration gradient away from the scaffold to ensure the axons leave the permissive scaffold. As a result, we decided to compare different growth factors that have been studied in synergy with ChABC, contrasting our scaffold and ChABC approach with them.
Professor Bradbury also confirmed our scaffold design would likely encourage significant sensory and motor ascending and descending axonal synapses/connections with host axons, potentially restoring some sensory, motor and autonomic functions.
Additionally, we discussed whether or not to remove the glial scar prior to implantation of our scaffold. Understanding that the glial scar has an important role in the early stages of SCI progression, Professor Bradbury suggested that we change the composition of the scar instead of removing it entirely. Taking this into account, we decided to communicate with neurosurgeons to ask for insights into the surgical protocols of spinal cord surgeries, specifically about the removal of the glial scar.
Dr Jack Lee is a lecturer for the KCL Biomedical Engineering Department, whose research is primarily focused on computational modelling and patient-specific simulations for clinical translation. In order for us to validate how feasible it is for the scaffold to enhance axonal regeneration in the spinal cord, we discussed how we could complete our proposed method of modelling axonal regrowth and regeneration, with particular reference to an existing mathematical model previously reported in related literature (Zhu et al, 2018). In this regard, he advised that we would not be able to follow the first step in this model due to the complexity of our scaffold microarchitecture. However, instead of suggesting that we change our scaffold microarchitecture, he minimised the impact of our concerns by suggesting alternate methods with which we could overcome this, such as altering the steps of the model to allow the scaffold to be mathematically quantified in a different way to what was outlined in the paper.
We also discussed our research into computational fluid dynamics (CFD) which led to the recommendation of various software packages we could utilise to perform such calculations ourselves, such as ANSYS. Additionally, Dr Lee provided us with advice relating to how to initially set up CFD modelling in terms of the assumptions we must make and common practices. He also suggested modelling smaller sections of our scaffold instead of the whole object, as well as investigating the type of flow CSF the fluid we would be modelling would follow.
Dr Jerry Silver is a Professor in the Department of Neuroscience at Case Western University. He supported the development of our project by advising our team to expand the scope of our therapy to include the thoracic region of the spinal cord. During our meeting he highlighted the importance of making informed safety decisions when developing a therapy for spinal cord injuries; we initially targeted the cervical region but were made aware that we could risk the safety of our patients. He discussed the importance of targeting lower cervical regions, as the higher C1-C4 regions are responsible for respiratory function, which may be compromised by resecting the glial scar. Dr Silver identified that the use of ChABC would be optimal at the rostral and caudal ends of the lesion, helping to ensure axonal regrowth into our scaffold. He also suggested the use of fibroblast growth factor (FGF) to encourage other supporting cells to migrate into the scaffold, which we evaluated in our research found on our SCI Biochemistry page.
Dr Silver confirmed our multiple time staggered injection timeline of ChABC administration, suggesting the use of imaging to visualise when to administer the second set of injections. The first set would allow neurites to grow into the implemented scaffold. The second set would enable their exit as well as allow the formation of new synapses and functional recovery.
Mr Gordan Grahovac is a consultant neurosurgeon and complex spine surgeon based in London. He provided us with further insight regarding the procedures involved in inserting a scaffold within the spinal cord. Mr Grahovac advised that we initially design our therapy for thoracic injuries, specifically the C7 and T1 regions, as these are more surgically accessible. He also highlighted the importance of targeting patients with complete ASIA A SCIs in the thoracic region, subsequently reducing the risk of worsening symptoms. Mr Grahovac also elucidated the difference between the stages of SCI, with particular reference to needing to resect the glial scar at an early time point before it becomes too difficult to remove.
We then discussed the importance of science communication when presenting our novel therapy to patients and ensuring they understand the associated risks. Mr Grahovac also supported the use of our MFP adhesive by highlighting that if we did not have an adhesive present our scaffold could move within the lesion due to CSF, thus compromising the efficacy of our therapy.
We approached Hilary Sutcliffe, Director of Society Inside, to inform us of our ethical decisions relative to our project design. She informed our science communication and project delivery, by highlighting the importance of using plain language, focussing on the problem we are trying to solve and presenting our project intent to our audience. Through our conversation with Hilary, we re-directed our focus to prioritise the problem of SCI rather than our technology. She encouraged us to clearly identify whether our solution is fit for purpose. With all of this in mind we were able to tailor our science communication, to develop trust with our audience and focus on delivering a safe and responsible project.
Professor Steve Thompson spearheads the King’s College London (KCL) Extended Medical Degree Programme and is Widening Participation lead for King’s Health Partners, making him the perfect candidate for discussion of our Biologix competition, particularly in terms of inclusivity. We hoped that this conversation would help us to bridge the gap between our proposed design and the desired outcome.
One point of discussion was of the types of skills we could transfer to participants throughout the competition that may be applicable in higher education. Such skills included confidence building, communication, presentation and networking. Additionally, Professor Thompson also suggested we incorporate values from the KCL Outreach for Medicine programme, such as ‘changing mindsets and inspiring students’. In this regard, the relatively small age gap between our team and likely competition participants allowed us to share our experiences in synthetic biology, helping to motivate students and develop a greater understanding of the field.
Another important recommendation was that we consider the accessibility of equipment to students participating in our remote competition. Here, he encouraged us to contact schools to ensure our participants were able to access the relevant equipment and had a quiet location within which they could focus on the competition.
We also discussed possible collaborations with KCL initiatives, including K+, in order for students to further their skills and support their university application. Such collaborations may result in the addition of more creative elements to the project, as was advised by Steve, thus generating a more enjoyable and engaging experience.
Finally, Steve proposed we measure the immediate impact of our initiative through surveys and a long-term follow-up, allowing us to measure any impact over time. Due to the short time frame within which iGEM is run, it may be difficult for us to assess these long term impacts. As such, we hope that future KCL iGEM teams will be able to evaluate the impact of our project in future years.
We approached NeuroStimSpinal to understand and learn from their scientific and entrepreneurial journey of developing a solution to treat spinal cord injuries using a combinatorial approach. We recognised many parallels between both our projects; they plan to use a graphene based scaffold, adipose decellularized tissue scaffold and electrical stimulation to encourage recovery.
We spoke to Paula Marques, the coordinator of NeuroStimspinal. She informed us on the ethical approvals required for a therapeutic product which require a certain level of technical readiness level to enter clinical trials. She led us to consider the ethical implications of creating spinal cord injuries in animal models, we would need to consider different methods to cause contusions in the spinal cord, for instance, hemisection and or transection where the fromer results in incomplete injury and the retainment of some limb movement, which is more easily approved by the ethical committee. We also need to consider treatment of the animal throughout the in-vivo studies. There is a need to carefully design and refine studies to reduce animal suffering. She also highlighted the limitations of Brexit in attaining different EU funding grants. As a result, as a UK-based team, we decided to expand our fundraising plan and apply for non-EU grants. Paula emphasized the importance of having conversations with the stakeholders, for instance neurosurgeons to incorporate clinical advice on our therapeutic product. Neurosurgeons tend to compare our scaffold approach to hydrogel treatments. As hydrogels are less invasive and injectable, this approach is favoured by many neurosurgeons for SCI at an early stage. However, we identified that one of the strengths of our scaffold lies in the intricacy of the microarchitecture mimicking the spinal cord tissue properties and providing structural support to growing axons.
We identified the unique selling point of their project being the novel graphene based nature of their scaffold which is responsive to external electrical signalling. Together with their designed typography of the scaffold, they aim to promote neurite outgrowth and functional recovery. With the same aim and similar scaffold approach, we decided to include our unique selling point in our business plan to out compete other emerging therapeutic approaches like this. We identified our unique selling point as the coating of MFP on the surface of our PCL scaffold, utilizing the underwater adhesiveness and non-immunogenic nature of MFP to adhere the scaffold in the lesion site and prevent inflammatory foreign body responses.
These human practice contacts helped us decide and implement some of our ideas. They also gave us a greater insight into industry practices and troubleshoot our proposed approaches.
Katharine Morgan who works as a Widening Participation Officer at KCL delivering outreach opportunities to students interested in pursuing a degree in medicine and dentistry, provided considerable insight into various aspects of the Biologix Competition that had not yet been finalised.
Firstly, she provided us with a list of schools, mainly South East London based, that should be the primary targets for our competition. Moreover, she recommended specific schools to contact, with an emphasis on those which had the lowest university participation rate following secondary education.
We also asked for her advice on potential methodologies that we could adopt to build a direct rapport with teachers, however, Katharine explained to us that these relationships are processes that require years of work. For this reason, Katharine agreed to act as a bridge between our team and the school teachers, helping to aid the credibility of our project and competition. She also advised that we target students directly, as well as career advisors, the head of sciences and sixth forms. Additionally, Kate also offered to include Biologix within a newsletter that is sent periodically to schools. This was an extremely beneficial opportunity and provided us with an ideal method of raising awareness and gaining participants. This method of reaching a wide range of individuals was particularly important as Kate suggested we would need to target a much larger number of students than initially expected given likely high dropout rates. Additionally, she also discouraged our collaboration with the K+ project, as the target audience of the two programs is different.
Secondly, we inquired about the feasibility of our current design for the competition. In this regard, she recommended launching Biologix from the last week of July to the first week of August to increase participation from students. Kate also suggested that we make our competition compatible with all commonly used electronic devices, such as phones, tablets and computers. In this regard, she advised that we aim to record all the lectures we want to deliver ahead of time to help avoid technical issues.
We were also encouraged to make our competition as enjoyable as possible, with a minimal workload. Kate explained that the students we most want to engage with may get discouraged by the larger workloads and that we should aim to set clear tasks with defined time limits.
Thirdly, Kate helped us consolidate our ideas regarding possible prizes for the competition winners, such as medals, certificates, t-shirts and mugs. Alongside these, she suggested planning a “thank you” event at the end of the competition. Kate also suggested that she would be willing to help us run a follow-up workshop to assist students with their personal statement and UCAS applications, helping students not only develop their understanding of synthetic biology, but also prepare them for higher education.
Finally, in consideration of the aforementioned time constraints, we were advised to work alongside the KCLSU and the KCL Biotechnology and Biology Society to help ensure the longevity of our competition and facilitate the application of long-term follow-up questionnaires.
During this meeting, we had the chance to speak to Sarah Tattam, the Faculty Student Experience Manager (Engagement and Support), and Anisha Boghaita, who holds a Senior Student Experience Officer position. Both are heavily involved with the Faculty of Life Sciences and Medicine. Building upon our previous encounter with Professor Steve Thompson, we were able to delineate some of the more delicate intricacies regarding Biologix, with a specific focus on the legalities of the competition which hadn’t yet been pinpointed in our current design.
Firstly - as Biologix is being run under a KCL society - Anisha brought up the availability of our KCL Student Union’s (KCLSU) Widening Participation Coordinator, who could potentially help us target the appropriate schools, subsequently allowing us to draw on KCL’s strong networks with the local community. Through this stream, we would also be able to address the marketing component of Biologix, as well as gain fully informed consent to run the competition, as required by the General Data Protection Regulations (GDPR). Moreover, we were also encouraged to inquire about the possibility of adding an introductory session to Biologix in KCL’s K+ initiative; this would be addressed during our next meeting with Kate Morgan.
To further ensure the well-being of the student participants, both Sarah and Anisha recommended having a ‘housekeeping’ segment at the beginning of each teaching session, detailing the avenues they could reach out to for support or for any questions/concerns. Additionally, we were also advised to include precise details within our survey relating to time commitments and resource requirements to help ensure student accessibility. Additionally, Sarah and Anisha both suggested the use of questionnaires such as this was an appropriate method of gathering quantitative data to assess the impact of our competition.
Sarah also elaborated on specific skills within the Life Sciences field, with particular reference to the KASE (Knowledge, Attributes, Skills, and Experience) framework from the King’s Careers and Employability department. Here, they emphasise the importance of incentivisation and student engagement, indicating that clearly outlining our target audience would help us best identify transferable skills that would be valuable when applying for higher education.
Finally, they discussed the addition of prizes for the winning schools. Here, we were informed that we must take into consideration potential conflicts of interest. Overall, the meeting helped us develop existing features of the competition, as well as highlight previously unconsidered facets of this project, helping us to better plan its execution and bridge the gap between the initial proposed design and the desired outcome.
Through the Praxis Institute, a Canadian-based not-for-profit organisation focussing on SCI research, we were put into contact with Andrew Forshner. Andrew is the Manager of Commercialisation & Partnership at the Praxis and was able to inform our team on potential routes to upscale our early-stage project. He introduced us to their Ideation Clinic and informed our entrepreneurial research by highlighting the importance of obtaining funding early. Andrew also encouraged us to consider the strategic alignment of our project by being receptive to feedback, are patient focused and are driven by our consumers. He also highlighted the importance of participating in events with stakeholders involved in SCI and the importance of providing proper evidence for curative therapy designs, as most clinical trials in mice do not directly demonstrate the technologies interaction with humans.
We contacted the Intellectual Property and Licensing Team at King's College London to gain an insight on how we could protect the intellectual property we develop as undergraduate students at the university and what factors we would have to consider during this process. We spoke to Dr Ceri Mathews, the IP & Licensing Manager, who was able to give us a clearer view of what the patenting process would be like for our team specifically. As our IP would be created with our student-led team, as opposed to an employee of the University like an academic, our novel ideas do not directly become KCL-owned IP. This means there would be an opportunity to negotiate and gain full ownership of our IP for the purpose of patenting.
Additionally, he provided us with technical guidance regarding the protection and effective use of our intellectual property. As our project is multidisciplinary, with many components, he advised we would have to apply for multiple patents and ensure they are all filed on the same day to ensure one patent does not expose another. Though this would be a greater financial expense, this fact helped us develop our licensing strategy as outlined in our business plan. He highlighted several situations in which we would have to be careful not to disclose information that would prevent patentability of our intellectual property, prior to having filed an application. For example, if we outlined, even briefly, future improvements to our project that could be considered to be novel in our Wiki or any scientific paper we publish, it would be counted as a public disclosure and therefore we would no longer be able to patent the invention that comes from this idea. Overall, Dr Mathews’s technical advice regarding intellectual property was instrumental to our understanding of the patenting process and guided the strategies we created to protect and utilise our IP.
To gain an understanding of the transition from research to a start-up company we scheduled a meeting with NurExone Biologic, a company developing an intranasal treatment for complete SCI using exosomes loaded with PTEN siRNA containing mesenchymal stem cells. Here we discussed the transition from research group to company and the procedures for clinical trials, patents, manufacturing and distribution. In addition to this, we were also able to gain an insight into market competitors and potential partnerships.
NurExone Biologic were able to advise us on the steps required to enter clinical trials as they are currently proceeding in their preclinical trials and aim to start clinical trials in the near future. More specifically, they were able to provide an insight into the technical and ethical development of our protocols. We were told to look at the possibility of outsourcing scaffold implantation protocols as this would provide us with the strong validation of results required to gain market approval from regulatory bodies. In our case, Renervate Therapeutics will require approval from the FDA before our scaffold can enter the market to ensure both safety and effectiveness of treatment.
We also enquired about potential future partnerships for the development of our company and effectiveness of treatment. NurExone Biologic aims to deliver PTEN siRNA containing mesenchymal stem cells to SCI patients using exosomes to regenerate damaged axons, to which they hold a Patent Cooperation Treaty (PCT) application for this unique delivery system. This is delivered intranasally, and so, is non-invasive, this holds the potential to become an easily accessible treatment for hospitals. As such, we enquired about potentially partnering with them to utilise their delivery system for our thermally stabilised ChABC. Though the delivery system will require modification to transport ChABC, a partnership in the future is feasible. Their support has meant we could develop our business plan with confidence, knowing there are routes for potential partnership has allowed us to consider our future goals and our gradual expansion as a company in greater depth. As a result we have been able to generate a fully comprehensive business plan outlining the steps we plan to take to transition from research to start up and eventually, a well-established company.
During the summer, we spoke with Dr Byeong Seon Yang, a postdoctoral researcher at the University of Basel, looking for specific guidance on the fusion of our MFP adhesive with a spidroin protein from Nephila clavipes. As Dr Yang has a patent on the co-expression system for the synthesis of MFP alongside tyrosinase, this meeting also provided us with an opportunity to refine our protocols and decide on whether an in vivo or in vitro approach would be most suitable for our project.
Together, we evaluated the technical benefits of spidroin fusion with MFPs, discussing the main differences between utilising a spidroin-based polymerisation method over traditional DOPA-quinone linkages via oxidative measures. We identified that the main benefit of using spidroin proteins is their ability to self-assemble into fibres, avoiding the complex redox manipulation typically required to balance cohesion and adhesion in the natural process alongside an increased flexibility.
We then questioned whether a spidroin fusion could interfere with the structure of PVFP-5, as well as its ability to surface bind. Dr Yang advised that due to the unstructured nature of MFPs, the addition of another large protein would not significantly affect adhesiveness, as was previously supported by his own research findings. However, Dr Yang’s research also identified that the degradation of the adhesive properties was more significant in the fusion protein, although he believed this to be a result of the linker used.
We were then provided with references to methods of expressing and purifying MAPs, facilitating the development of our wet-lab protocols. Specifically, as MAPs are sticky upon purification and during chromatography, we were provided with approaches that minimise these issues.
Finally, we were made aware of the scale of production required for both experimental and large-scale implementation within our project. Dr Yang advised that we could only obtain roughly 50-100mg of MFP per litre of culture, whereas medical applications require units within gram scales. We therefore identified this as a potential target for the entrepreneurial element of our project, wherein we could evaluate the cost-effectiveness of our adhesive and the amount of adhesive required per area of scaffold.
These human practice contacts helped brainstorm ideas for the scientific aspects of our project and guided our design cycle.
In efforts to increase the thermostability of our protein, Chondroitinase ABC, we reached out to Dr Marian Hettiaratchi, one of the primary authors of the paper ‘Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability.’ (Hettiaratchi et al., 2020) we discussed the thermostability of ChABC and subsequent laboratory protocols relating to mutating and expressing this enzyme.
One of the primary issues with the application of ChABC is its thermal instability at temperatures greater than 37 degrees celsius. One method of overcoming this issue is through computational mutation in order to identify more stable variants. In this regard, Dr Hettiaratchi indicated that due to the large size of ChABC, making multiple mutations would be more effective than single-point based mutations.
Upon sharing experiences and challenges Dr Hettiaratchi’s team encountered, she indicated that the large size of ChABC also poses a difficulty in finding a suitable plasmid to carry its genomic sequence, thus limiting the number of different sequences that they were able to investigate.
In the beginning of our project, Dr Hettiaratchi reminded us of what we could feasibly achieve within the time-frame of the iGEM competition, resulting in our decision to computationally mutate ChABC using 8 of the mutations highlighted in her paper, aiming to improve the thermal stability of ChABC in physiological conditions, increasing its efficacy in our therapy. Throughout the laboratory process of expressing and purifying our mutated ChABC, Dr Hettiaratchi consistently provided constructive advice and potential improvements to our project and protocol. One specific point of note was the recommendation that we adapt the ChABC enzymatic activity protocols applied within their studies by expressing ChABC with a His tag and a FLAG tag for Nickel affinity, followed by size exclusion chromatography in order to obtain a pure sample. Finally, Dr Hettiaratchi helped refine the direction of our project by suggesting we incorporate thermostabilized ChABC into a hydrogel for a sustained, localized release at the SCI lesion.
Professor James Fawcett is a senior author on many papers concerning CSPGs and ChABC in SCI and was thus a very useful contact in consolidating our understanding of its application, as well as our therapy as a whole. One problem we encountered was that our MFP bioadhesive binds to the glycosaminoglycan side chains of CSPGs in the ECM, which is what ChABC cleaves to create a more permissive environment. Professor Fawcett addressed this issue, indicating that the ‘stub’ structure left behind after GAG degradation by ChABC has the same structural and chemical make up as an intact chain, thus still providing a binding site for MFP. He also explained that at body pH, ChABC is specific to chondroitin sulfate as opposed to other proteoglycans which is beneficial as heparan sulfate proteoglycans are growth permitting.
Professor Fawcett additionally brought up the issue of the foreign body reaction (FBR) in response to the hard scaffold surface, something we had not yet considered. This led to us conducting additional research and a meeting with Dr Alejandro Carnicer-Lombarte, a researcher focused on the interface between implants and neuronal tissue, to discuss the FBR further.
Dr Paul Brown is an honorary lecturer at King’s College London. We decided to contact him after finding a low yield of protein following purification, which meant we did not have a sufficient amount of PVFP-5 for the coating of our PCL scaffold. After asking his help in understanding potential issues in our current expression methodology, Dr Brown offered advice into improvements that could be implemented to design a better expression system.
After an evaluation of our protein expression system, Dr Brown first suggested that we utilise an entirely different expression vector to optimise the production of our protein within our expression system. However, as a result of limited access to our lab and time constraints, this was not entirely feasible as we already had our pSB1A3-PVFP-5 expression vectors prepared. Dr Brown then further suggested that we explore the use of different E. coli strains that are tailored to optimise the expression of our protein. Furthermore, Dr Brown stated that a lower temperature during expression of our protein might improve its solubility and increase yield. Learning from his advice, we found the SHuffle (DE3) E.coli l strain, which has been optimised to improve formation of disulphide bonds in a target protein. After implementation of his advice in our experimental design, we were able to improve the expression and yield of our protein.
Dr Jia An advised us on the design of our scaffold, initially warning us as to the possible influence of scaffold porosity decreasing the mechanical properties of the scaffold which could have led to further implications in patients. He advised us on comparing the mechanical properties of the scaffold with the spinal cord to make sure they match, which would benefit the patients and minimise the risk of injury. He also suggested that the selective laser sintering (SLS) printing method may work better than filament printers, as the SLS method does not require any printing support and has a better resolution, thus allowing us to create complex anatomical shapes with smaller pores, however, due to the lack of SLS printers available to us, we decided against this. Dr An also helped us consider different pore geometries, suggesting that we change from using non-convex pores as these are more difficult to print than convex, which led us to change the shape of our pores (to a cross-hatch design) in order for them to be easier to print. Finally, Dr An provided guidance on the hydrogel-scaffold approach, suggesting we make use of robotics to make the process as precise as possible.
Dr Lorenzo Vechini is a lecturer in Reconstructive Sciences at King’s College London. He has authored papers on bioactive ceramic scaffolds and high content imaging to phenotype human primary and iPSC-derived cells. His research at King’s focuses on understanding the development of tissue-specific blood and lymphatic vessels as well as focusing on using human induced Pluripotent Stem Cells (hiPSC) as a model system.
From our meeting, Dr Veschini helped us identify a solution to the scaffold issue identified by Professor Coward and Mr Gonella leading to a new design; a cross-hatch scaffold that would be printable with our equipment.
Professor Coward is a Professor in Maxillofacial & Craniofacial Rehabilitation at KCL. Professor Coward has shared his resources, expertise, and services in 3D printing with us. We were able to go into his laboratory in Guy’s Hospital to observe 3D printers and develop our understanding of how they operate.
Professor Coward also provided us with technical feedback relating to our Phase I scaffold design, advising us that the proposed microarchitecture would likely be too complex to slice and print, which led to further discussions about changing the design to make it more simple. To address this issue, one of Trevor’s colleagues, Dr Lorenzo Veschini, helped us apply the cross-hatch method (Meng et al., 2020) over our scaffold macroarchitecture to create linear pores, ready for printing. He also elucidated the fact that a more simple design would benefit our project, as most printers would not have the specifications to print a complex design. Therefore, we changed the design of our microarchitecture to make the scaffold more viable for hospitals to print which increases the inclusivity and accessibility of our project.
During our meeting with Dr Bryn Martin (Vice President of Research, Precision Delivery, and CSF Sciences at Alcyone Therapeutics) we discussed various elements of the CFD modelling of our scaffold. Specifically, Dr Martin has authored a number of papers relating to CFD modelling within the spinal cord environment, meaning that he could advise us on key considerations such as boundary conditions, assumptions and parameters.
Firstly, he suggested that we attempt to simplify our model as much as possible through the application of various assumptions about our system, thus reducing the required computational power. Such simplifications are also necessary given the extreme complexity of the spinal cord environment we were trying to simulate; indeed, he suggested that it is likely impossible to produce an entirely accurate model at present. As such, we decided to apply several of the assumptions outlined by Dr Martin, i.e., laminar and incompressible flow, a constant viscosity, and that the scaffold is a rigid porous media that cannot deform.
Additionally, in accordance with Dr Martin’s guidance, we assumed that the CSF has the same physical properties as water, as was stated within the literature he provided us with (Gupta et al., 2010; Haga et al., 2017; Kurtcuoglu et al., 2019; Martin et al., 2012; Pizzichelli et al., 2017; Sass et al., 2020). He also suggested we should implement a pressure inlet using a low value (~1mmHg) because the pressure is almost undetectable, whilst avoiding investigation of inertial effects, as these are mitigated within CSF. Dr Martin also highlighted the importance of validating our model before its final application by assessing mesh independence—i.e the result of the simulations (values for permeability and wall shear stress, in our case) should be the same regardless of the mesh setup. See our modelling page here for more information on our CFD model.
In ideal conditions we would be able to test our scaffold experimentally, however due to Covid-19 restrictions this was not possible. In this regard, Dr Martin’s verification that the application of CFD modelling was an appropriate method of assessing the suitability of our scaffold was reassuring and provided us with confidence moving forwards.
These human practice contacts validated the decisions we made throughout our project thereby ensuring our project is responsible and good for the world.
Dr Andrew Beavil is a senior lecturer in Molecular Biophysics at King’s College London, with a focus on the application of molecular biophysical and protein engineering techniques to study protein structure and function. Dr Beavil provided our team with assistance and advice as we attempted to create a new, updated structural model for our chosen MFP, PVFP-5. Our team had little success using a full sequence homology modelling approach due to the fact that we required 9 distinct disulphide bonds. Dr Beavil advised us to shift our approach to individually model the 3 EGF-like domains that our protein is composed of, helping to ensure more accurate modelling of our structure, as well as increasing our chances of forming the required disulphide bonds.
Dr Beavil also highlighted the importance of using an accurate and methodical approach when producing structural models due to the value they hold in future characterisation and functional studies. Lastly, he supported our final approach in creating our structural model, which consisted of homology modelling our 3 protein domains individually, assembling them into a single structure, then running them through a molecular dynamics simulation to clear any atomic irregularities. In addition to providing advice with our structural modelling approach, Dr Beavil used the then newly released protein structure prediction program, AlphaFold2 to validate our structural predictions. AlphaFold2 has been touted to have solved the ‘protein folding problem’, therefore we were confident with our own structural modelling approach when the resulting model from AlphaFold2 consisted of all 9 disulphide bonds we predicted.
Mr Gonnella is a student who has completed his studies in MRes Tissue Engineering at King’s College London. His primary research focuses on the fabrication and the characterization of 3D bioprinted hydrogels/hydrogel-PCL for both cartilage and subchondral bone repair. We corresponded with Giovanni across the majority of our project and he aided us in the printing of our PCL scaffold. With his technical expertise, he suggested changing our methods in order to overcome the issue of PCL melting at a much lower temperature during printing which caused the scaffold to deform and not print properly. This led to us creating a successful prototype of our scaffold so that we could validate the viability of our design compared to the alternative designs that were discussed. The printed scaffold could then undergo mechanical testing to gain values for variables like Young’s Modulus to ensure that our scaffold’s mechanical properties match the mechanical properties of the spinal cord and would not further damage it post-implantation. By creating a successful prototype, it also advanced our project, as we could then perform mechanical tests on the scaffold in order to provide a suitable proof of concept.
Professor Herbert Waite is a principal investigator in the Marine Biotechnology Lab at UC Santa Barbara, a research group that has extensively studied the surface interactions and binding of mussel adhesive proteins. During the meeting, we discussed the binding between our chosen MFP, PVFP-5, and our PCL scaffold, as well as possible improvements to our protein expression protocols.
Regarding our MFP/PCL binding model, Professor Waite agreed that the main binding mode between the two materials was likely to be hydrogen bonding between the ester groups of PCL and the hydroxyl groups of L-DOPA. As predicted, he favoured this mode by either bidentate ligand-fashion or a 1:2 hydrogen bonding ratio of PCL to L-DOPA. To test this prediction, Professor Waite suggested the use of Fourier Infrared Spectroscopy (FTIR), a scientific characterisation technique that could be used in future work to test the validity of the model . He also highlighted the importance of cysteine in adhesion due to its involvement in cysteine-dependent covalent chemistry. However, as there have not been any previous
reports of the binding modes of PVFP-5 to PCL, interactions such as thioester formation are still subject to speculation. To detect the presence of free thiols within our protein (and therefore the number of covalent linkages that can form with PCL), Professor Waite suggested the use of either vinyl pyridine or an Ellman’s reagent test.
Professor Waite also gave an important insight into our protein synthesis and purification protocols, specifically emphasising the importance of maintaining a low pH (below 5) during purification to prevent early polymerisation of PVFP-5. One point of debate at the time of this meeting was the use of acetic acid versus the use of hydrochloric acid for this purpose. Professor Waite informed us that whilst acetic acid would usually be the preferred choice, when producing MFPs specifically for physiological uses, a dilute acid such as HCl would be ideal due to its weak buffering capacity under neutral pH. He also suggested the use of urea in the lysis buffer over guanidinium-HCl due to the likelihood of guanidinium interference with many purification/characterization steps, e.g. Ni-NTA chromatography and SDS PAGE. From this guidance, we made the scientific decision to devise a laboratory purification protocol that could be used for future purposes.
After discussing the polymerisation of our MFP, Professor Waite suggested an invitro method for the conversion of tyrosine to L-DOPA, which involves the addition of commercially available mushroom tyrosinase to a solution of our protein after purification, in the presence of a borate buffer. Borate would act as a temporary protecting group for the catechol in L-DOPA, preventing it from oxidising into DOPA-quinone and polymerising. This method is beneficial as the binding of borate to L-DOPA is pH reversible, meaning only a low pH (~3) would be needed to remove the borate. This advice prompted us to look into the use of boronate as a protecting group during protein expression, which we had also considered last year. Following the polymerisation, Professor Waite recommended the use of Arnow’s reagent to detect the presence of L-DOPA quantitatively.
Dr Sarah Barry is an active Chemistry researcher and lecturer at King’s College London. Her research interests include Biochemistry, as well as having particular expertise in Organic Chemistry, therefore we decided to reach out to her to further validate our PVFP-5/PCL binding model. According to the video we created (link), hydrogen bonding represents the main linkage between the catechol groups of our MFP, and the ester units of the polycaprolactone (PCL) scaffold we designed. Dr Barry confirmed this prediction.
Additionally, she suggested developing our model further by including the hydrophobic interactions between the two compounds. She explained the high likelihood of hydrophobicity due to the straight aliphatic chain nature of PCL, with non-polar residues in PVFP-5. To help validate this, Dr Barry suggested the use of Pymol to show the polar surface area of PVFP-5 in order to identify hydrophobic regions of our protein that are able to bind to the alkyl chain of PCL. From this, we made the technical decision to update our PVFP-5/PCL model to include hydrophobic interactions.
She also suggested looking into other residues within our PVFP-5 protein that may be involved in binding. After mentioning cysteine-dependent covalent chemistry (as spoken about with Professor Herbert Waite), Dr Barry informed us that whilst this can happen, it is not likely to occur within the body as a very reactive electrophile would be needed to attack the ester group of PCL. Instead, she recommended looking into metal binding coordination, such as the binding of Zn or Mg between the catechols of L-DOPA, leaving free coordination sites for other materials such as PCL to bind. To confirm our predictions, Dr Barry suggested conducting site-directed mutagenesis studies in the future.
Professor Sundararajan Madihally is a Faculty Fellow and Professor in Chemical Engineering at Oklahoma State University. He has authored papers relating to modelling porous scaffolds through the use of CFD simulations and was thus able to develop our understanding and ensure that we were moving in the right direction with our model.
Throughout our meeting he emphasised the importance of simplifying the initial CFD simulation as much as possible, thus reducing the lengthy and computationally demanding process of simulating and troubleshooting complex models. In response to our concerns regarding oversimplification he advised that once we have a running model, we could add variables to this framework and develop one which is progressively more accurate.
Additionally, Professor Madihally helped us identify which assumptions we should make to simplify our model, for example, assuming that the fluid has a Newtonian viscosity and that gravity has a negligible influence.
Finally, he gave us technical guidance on setting up a CFD simulation, with specific reference to the calculation of the number of nodes the mesh of our scaffold should have, as well as choosing the mesh shell shape, collectively providing an excellent starting point for our model.
Following our Phase I project, we further developed our entrepreneurial prospects through communication with key individuals like Adrian Signell, a Student Investment Partner at the Creator Fund. The Creator Fund is an early stage venture capital fund investing in groups who are building business out of university innovation. We saw this as an opportunity to investigate our potential in upscaling our iGEM project into a commercialised product.
We initially presented and brainstormed our business plan to Adrian in August. This meeting focussed on the importance of developing open-dialogue with our investors; he advised that we should focus on the problem we are targeting, alternative funding routes and clearly presenting our content on our pitch deck. Additionally, he identified our unique selling point and encouraged us to place more of an emphasis on our adhesive PVFP-5 protein, chondroitinase ABC enzyme and polycaprolactone scaffold. In response to this we developed our two tiered business plan, which included licensing our product to other groups and searching for potential partnerships to support the growth of our company.
Following this preliminary discussion, we worked towards incorporating all his feedback into our business plan and pitch deck. We recognised that we needed to identify and segment our target market prior to engaging in any other conversations. Once we had finalised our business plan including our financial projections, market and competitor analysis, we reached out to Adrian to officially pitch our project. During this pitch, Adrian assessed the delivery of our project, the quality of our presentation and thought content of our proposal. He emphasised the importance of including secondary data, as we have not finalised our proof of concept. As previously aforementioned, he advised us to clearly communicate our problem through our pitch deck, incorporating relevant statistics to engage stakeholders. We understood this was one of the main limitations of our presentation and have re-assessed both our business plan and pitch deck in response to his feedback. They now follow a clear structure and roadmap of implementing our project under clear milestones.