Renervate Therapeutics, Phase II of our project, was inspired by the work completed by the King’s College London (KCL) International Genetically Engineered Machine (iGEM) 2020 team, Renervate. In 2020 (Phase I of the project), the team completed a theoretical design focusing on the use of a novel biomaterial in the treatment of spinal cord injuries (SCI). In this research, they identified the benefits of implanting a 3D printed scaffold coated in a novel mussel-foot protein (MFP). The Phase I team hypothesised that this approach would not only provide a valuable contribution to the field of SCI treatments, but also aid the progression of therapeutic applications of synthetic biology, thus providing inspiration for our Phase II team.
Following Phase I of our project, our team leaders sought to expand upon our initial design-build-test cycles through improving and validating our initial therapy. In particular, we aimed to look into the suitability of synthesizing and utilizing our mussel foot protein, creating our polycaprolactone (PCL) scaffold, and investigating SCI pathology in order to further investigate avenues of improving our treatment.
The original inspiration for our project stemmed from the 2019 iGEM Jamboree, where the Team Leaders of Phase I were inspired by the research completed by Leiden and Great Bay SCIE. Here, Leiden worked towards using a suckerin protein-based hydrogel to treat burn wounds, whereas Great Bay SCIE worked towards categorising MFPs as potential bioadhesives within therapeutics.
Together, we collated research completed by the aforementioned iGEM teams, as well as by present-day start-ups, like Spiderwort, who focus on using novel approaches such as plant-based scaffolds to promote axonal regrowth. This information helped us formulate a comprehensive project design that aimed to help solve the global health challenge of SCIs.
Although we faced many challenges throughout the pandemic, they only served as further motivation for the completion of Phase II, as we constantly adapted to every barrier we faced throughout the summer. The resilience, determination, and flexibility of our team never faltered, and with the aid of endless lateral flow tests, we have further developed our project, which we are proud to present.
SCI is characterised by damage to the spinal cord that disrupts axonal function as a result of mechanical impact or injury, typically resulting in a lesion (Bennett et al., 2021). Up to 90% of SCIs are a result of traumatic injury, such as traffic accidents, with the remaining cases being a result of non-traumatic origins, such as spina bifida and osteoarthritis (Spinal Injury Association, 2019; National Spinal Cord Injury Statistical Center, 2013).
Following the initial SCI trauma there are several phases of progression, the acute, subacute, and chronic phases. During the acute phase of injury, a cavity forms within the spinal cord, which is then encapsulated by a glial scar from the subacute phase onwards. This is followed by the chronic phase of SCI, which involves further axonal dieback and the maturation of the glial scar, where sustained changes in the spinal cord microenvironment increases growth inhibition. This glial scar acts as a physical barrier to axonal regeneration within the lesion, suppressing recovery and serves as a main target for our therapeutic treatment; see our SCI Biochemistry page for further description (Song et al., 2019).
Given the large size of the spinal cord, damage within different regions typically results in different consequences. These can be broadly grouped under deficits in motor, sensory and autonomic function; see Figure 1 for a more detailed overview.
Figure 1: A sagittal view of the spinal cord indicating structure and respective control over body parts, together with the innervating nerves.
SCI can also affect an individual's mental wellbeing, wherein a large existing body of literature indicates dramatic effects, such as a 1.33 times greater incidence of new-onset depression and anxiety (Lim et al., 2017). SCI is also associated with other complications such as post-traumatic stress disorder (Nas et al., 2015), all of which collectively contribute to a reported reduction in quality of life (Craig and Middleton, 2009).
Additionally, factors such as an inability to work likely contribute to negative mental health outcomes due to loss of income (Khazaeipour et al., 2014; Zürcher et al., 2019; Lim et al., 2017). Studies have suggested that only 35% of SCI patients return to active employment (Tomassen et al., 2000; Young and Murphy, 2002; Ottomanelli and Lind, 2009). This poses a pervasive problem in regions where free healthcare is not provided, such as the USA, where an inability to pay medical bills is the leading cause of bankruptcy (Himmelstein et al., 2009).
Collectively, there is a clear need to develop regenerative treatments for SCI, as opposed to those that focus on symptom management.
Given the pervasive influence of SCI and the broad range of associated deficits, there is a great impact on wider society. In this regard, existing literature has highlighted the difficulties faced by caregivers of those suffering from SCI, with a review by Lynch and Cahalan (2017) indicating significant rises in depression and anxiety, as well as reports of isolation and loss of identity; collectively these factors contributed to a general reduction in life satisfaction.
In addition to the psychosocial effects on individuals in caregiver positions, there are also significant economic impacts on society, with a mean cost per SCI case in the U.K. of £1.12 million , rising to £1.87 million for more severe ASIA Impairment Scale (AIS) grade A-C injuries (McDaid et al., 2019), putting a large strain on healthcare systems to provide the necessary resources.
In this regard, the rehabilitation process for SCI is lengthy and often unclearly defined (Burns et al., 2017), with a high rate of mental complications, such as a post-traumatic stress disorder, which has an estimated incidence of 17% in the first 5 years post-injury (Nas et al., 2015), further complicating treatment and increasing costs. Additionally, SCI is associated with a high risk of re-hospitalisation, with some studies reporting estimates as high as 36-45% within the first year post-injury (Merritt et al., 2019), exacerbating the issue. Our team has developed a guide covering the rehabilitation process, which can be found on our Education Page.
Again, such factors contribute to the need to develop regenerative treatments that can reduce the strain SCI places on wider society, improving not only the quality of life of those in caregiver positions, but also through reducing healthcare costs via shortened rehabilitation times.
Naturally, SCI is an ongoing global issue, with an estimated incidence rate ranging between 13.0 to 163.4 per million, although this varies greatly depending on region (Löfvenmark et al., 2015; Pickett et al., 2006; Kang et al., 2018; Singh et al., 2014). Similarly, there is a great range in prevalence estimates of SCI, particularly as this statistic is less well reported (Wyndaele and Wyndaele, 2006). Here, estimates within European regions are typically in the range of 250-280 per million, with an estimated 906 per million in the USA (Singh et al., 2014).
Regarding the demographic variables for those suffering from SCI, there are stark gender differences in incidence rates, wherein the sex distribution of SCIs in men compared to women is 3.8:1. The mean age of patients currently suffering from SCI is approximately 33 years old (Wyndaele and Wyndaele, 2006), with the World Health Organisation (WHO) indicating that the most affected age ranges are from adolescence to young adulthood, as well as those over the age of 60 (World Health Organisation, 2013).
In Phase II of our project we aimed to further develop three elements of our initial design:
In order to efficiently address the aforementioned Project Goals and reach our finalised design, we split our team into three subgroups: SCI Biochemistry, Bioprinting, and MFP . Each subgroup addressed different project aims before collaborating together to form a finalised therapy.
Our SCI subgroup investigated methods of making the SCI environment more permissive of axonal regrowth, as well as clearly identifying the type of SCI we will treat. The Bioprinting subgroup focused on creating a printable scaffold design, whilst the MFP subgroup focused on utilising our adhesive to ensure our scaffold remained in place. Additionally, each subgroup had the goal of validating their Phase I proposals, as well as any new additions to the therapy, such as the redesigning of our scaffold microarchitecture and addition of ChABC injections, which will be discussed later.
Each subgroup planned to achieve their goals by conducting literature reviews, communicating with both experts in the field and our stakeholders, as well as performing any necessary validations via proof of concept investigations.
This year, we aim to initiate the first steps of moving our therapeutic from the dry lab into real-world application. Building upon the research completed in Phase I, we identified the following aims:
Continuing with last year’s Phase I project, we aimed to validate various aspects of our project in the lab, including our PCL scaffold, our MFP, and ChABC, a novel enzyme that shows promise in promoting axonal regrowth following SCI. Further detailing on our research can be found on our Project Design.
This involved determining the specific pathology our scaffold is optimized to treat, as well as investigating the entrepreneurial potential of our therapy within the biotechnology market. We looked towards developing a full business plan and pitch deck to engage with investors. Further detailing on our entrepreneurial research is available on our Entrepreneurship Page.
We aimed to achieve this by developing our synthetic biology competition, Biologix, to target local and international high schools. This competition was aimed at introducing students to the vast potential of synthetic biology; to learn more about the launch of our competition, please see our Education Page. Additionally, we aimed to further our human practices by integrating experts within our research practice. We were also motivated to promote an open dialogue with individuals within the healthcare and ethics fields, thus developing a more rounded understanding of the nature of our project.
The SCI subgroup had the overarching goal of identifying and validating an appropriate method of improving the SCI environment to be more permissive of axonal regrowth, as well as identifying the precise type of SCI to which our therapeutic treatment could be applied.
Addressing the first goal, we decided to implement chondroitinase ABC (ChABC) in our therapy. ChABC is a bacterial enzyme that degrades chondroitin sulphate proteoglycans (CSPGs), which are axonal-growth inhibitory molecules that limit axonal regeneration and elongation, thus hindering functional recovery. However, ChABC is thermally unstable and loses its enzymatic activity at >37°C, the physiological temperature (Lee et al., 2010). As such, with the help from experts in the field we aimed to computationally mutate, express and purify a more thermostable variant of ChABC, thus increasing the efficacy of our treatment. You can learn more about the process of thermostabilzing ChABC on our Model Page. Furthermore, we aimed to test the enzymatic activity of our mutated ChABC in the lab to validate its thermostability and efficacy.
Addressing our second primary goal, we considered the practical, ethical and safety aspects of ChABC implementation in our therapy. To develop a more holistic and inclusive therapy, we aimed to e expand our target patient pool from cervical which often results in tetraplegia in Phase I, to incorporating C6-C7 cervical lesions and the entire thoracic lesions which causes paraplegia. We decided to exclude higher cervical lesions for safety consideration when surgically implanting our scaffold, as surgical procedures at the uppermost regions of the cervical spinal cord (C1-C5) carry too great a risk due to its crucial role in controlling respiratory function (Berlowitz et al., 2016). These safety decisions allowed us to provide a safer treatment and treat a larger number of patients (Lujan and DiCarlo, 2020).
Furthermore, we aimed to deliver ChABC in the least invasive and effective manner, by conducting research into different delivery methods of ChABC into target injury sites. To learn more about our research regarding the administration of ChABC, visit our SCI Biochemistry Page. Finally, we were motivated to fully elucidate the mechanisms behind axonal growth and the biochemical cascades of which ChABC functions.
Importantly, although not an initial aim, we also devised surgical and post-surgical rehabilitation protocols as a result of our communication with stakeholders, such as neurosurgeons and physiotherapists; see our Rehabilitation Guide on our Education page for further detailing.
Following the in silico simulations of our scaffold design in Phase I, the primary goal of the bioprinting subgroup this year was to fabricate and validate a printable polycaprolactone (PCL) scaffold with customisable dimensions specific to the patients’ clinical profile.
To fabricate the scaffold our team performed extensive literature reviews to re-evaluate the micro- and macro-architecture to ensure that chosen design is printable. We were determined to create a design that would be easily tailored to each patient while maintaining the key therapeutic properties. Our team also looked into several fabrication methods mainly focusing on different types of 3D printers to assure accessibility of our therapy. For further details please visit the Scaffold Engineering page.
To further validate our scaffold design, as an alternative to complex and expensive experimental testing, we aimed to validate our scaffold through Computational Fluid Dynamics (CFD) simulations (Ali and Sen, 2018). Through these simulations, we planned to investigate the permeability of our scaffold and predict the wall shear stress exerted on it by the CSF. Both of these properties provided an insight into how well the architecture and topology of the scaffold can facilitate axonal regeneration (Singh et al., 2018). For further explanation of our CFD modelling please see our Modelling page.
The subgroup has one overarching aim: to produce a usable PVFP-5-based bioadhesive and to experimentally validate the research from Phase I. One of the first goals of the subgroup was to conduct a review of all available PVFP-5 sequences and create a new structural model using the updated sequence (UniProtKB: U5Y3S6). Using the structural model, we further aimed to generate a binding model between the PVFP-5 protein and polycaprolactone (PCL) to predict the adhesive behaviour between our PCL scaffold and PVFP-5.
The MFP subgroup aimed to expand beyond the dry-lab work of phase I, by designing, expressing and purifying our PVFP-5 plasmid. We aimed to test the suitability of the PVFP-5 plasmid when expressed in various E.coli cell lines including BL21 (DE3), Rosetta-Gami B (DE3) and SHuffle (DE3) to determine which cell line was most optimal for the expression of our protein in its soluble form.
Expanding on our protein expression, we were motivated to design a co-expression system with PVFP-5 and the tyrosinase enzyme from Bacillus megaterium to optimise the use of PVFP-5 as a bioadhesive through increased post-translational modifications of tyrosine to L-DOPA.
To further validate the use of PVFP-5 in conjunction with our PCL scaffold, we aimed at investigating factors relating to the feasibility of its application as a therapy, with particular reference to cost-effectiveness. In this regard, analysis of the PVFP-5 to PCL binding ratio was performed, as well as investigation of methods to increase PVFP-5 yield - please look at our Model Page for further information.
In Phase II of our project, Renervate Therapeutics, we applied Synthetic Biology through the use of recombinant gene technology to synthesize new biological parts, such as our recombinant MFP, PVFP-5. Our MFP uses l-3,4-dihydroxyphenylalanine (L-DOPA) to adhere to surfaces, even underwater. This unique property, as well as low immunogenicity and cytotoxicity (Santonocito et al., 2019), making it a strong candidate for use as a novel bioadhesive. Further supporting the use of MFPs in therapeutics, existing academic bodies have already shown its potential in fields such as wound closure and breast cancer, where gold nanoparticles were bound to epidermal growth factor antibodies using an MFP inspired adhesive (Mehdizadeh et al., 2012; Black et al., 2013).
Within our project we have successfully utilised PVFP-5 in conjunction with our PCL scaffold, thus providing further evidence for the potential applications for MFPs as adhesive agents, with particular relevance within therapeutic applications. We hope that our research and project can help pave the way for novel applications of MFPs in a wide range of clinical trials, thus furthering the potential applications of synthetic biology in the therapeutic field.
Naturally, one major effect of COVID-19 on our project was developing a healthy team dynamic from the beginning of the season, whilst working remotely. Our team leaders scheduled multiple initial Zoom meetings dedicated solely to socialising and getting to know one another. This approach helped to develop a healthy work environment and ensure each member felt more comfortable communicating.
Figure 2: A zoom call with Renervate Therapeutics!
Beyond the social aspects of our project, COVID-19 had a pervasive impact on our ability to access the lab during Phase II. This posed a significant challenge, as Phase I was solely focused on in silico research due to similar restrictions, the results from which we hoped to validate in Phase II. Unfortunately, given the persistence of the pandemic, many academic groups again experienced limited access to labs, with senior researchers being prioritised. Last year, we worked closely with Dr Pastore and expected to be in her lab this summer, however, this did not come to fruition. Indeed, gaining any lab access at all was uncertainty during the early months of our project.
Following our final exam period in May, we were informed by our supervisors that we may gain laboratory access in mid-June. As such, we began quickly preparing our experimental procedures (See Contributions ) and consistently remained up to date with all COVID-19 regulations, ensuring that these were followed by all team members; see our Safety and Security page for more details. Unfortunately, however, the laboratory space which we had initially planned to work in delayed our access multiple times due to a lack of space and a tight regulation over the number of individuals who could be there at any given time. By the end of June, we were informed that this laboratory would be completely unavailable to us, this was of particular detriment as this laboratory has significant experience working with PVFP-5, the MFP of choice within our therapy.
We instead used our PI, Dr Anatoliy Markiv’s laboratory, which at the time was also limited to only a couple of our team members, due to King’s College London’s safety regulations. This limited the number of experiments we could perform; therefore the team chose to prioritise the development of simple protein expressions to validate our system.
In addition to the aforementioned issues, we experienced repeated unexpected periods of self-isolation, which lead to frequent switching from remote to in-person work, thus disrupting the progression of our project in the laboratory. Fortunately, this experience was familiar to the remaining team members from Phase I, who were able to provide support and advice for the newer members.