Future Directions

Where can we take our idea next?

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Next Steps: Engineering Bigger Picture: Human Practices Beyond: Entrepreneurship

Next Steps: Engineering

During the course of our project we managed to develop nine new basic parts and eight new composite parts. We faced many challenges throughout the year, but our Engineering cycles allowed us to learn something from every experiment, whether it succeeded or failed. While we were proud of what we had achieved within the iGEM timeframe, one thing was clear: we had lots more work to do, and in fact our idea could take us through years of further research.

During the first iteration of our project, we found that Reb206 constructs were non-functional, possibly due to interference by the mNeonGreen fusion with the mechanical extension of the R bodies. This fusion may have disrupted the normal folding or assembly of the R bodies or may have simply hindered their ability to extend and contract under the required pH conditions. In contrast, some of the Reb1 R bodies could extend at low pH, whilst others did not. This could be a result of differences in protein assembly, potentially arising from temperature-dependent misassembly leading to functional variability between otherwise identical constructs. In the future, exploring different expression conditions may help determine whether temperature affects R body protein folding and assembly. Lowering expression temperature, or adjusting other growth conditions could help reduce misassembly and enhance functional protein yield. Understanding why the Reb206 R body functionality is affected may provide insight into the mechanics behind R body extension and contraction. Additionally, refining the purification process may help preserve the functional integrity of R bodies. Much work in optimising R body purification and maintaining protein functionality remains. Through optimisation, we may be able to achieve more consistent functionality in the constructs, providing further understanding of the mechanical properties of R bodies and increasing their viability in therapeutic delivery systems.

The following future directions would help us refine conjugation strategies, enhance scalability, and enable robust characterisation of R bodies for therapeutic applications:

  • Development of biochemical quantification methods: Establishing methods to quantify R body yields and conjugation efficiency is critical for future therapeutic applications and characterization.
  • Optimization of reagent concentrations: Reducing and optimising the concentration of reagents (e.g., GGG-mNeonGreen and eSrtA) to improve reaction efficiency and scalability, especially for potential entrepreneurial efforts.
  • Expansion of non-canonical amino acid (ncAA) use: Testing different ncAAs to broaden the range of R body conjugation strategies.
  • 2-PCA conjugation compatibility: Ensuring that 2-PCA conjugation is compatible with other R body constructs, particularly N-terminal modifications like TAG-RebA and TAG-RebB. This could enable two orthogonal conjugation strategies at a single site.
  • Chemical analysis of intermediate products: Changing the order of reaction steps to enable analysis of intermediates using techniques like NMR or MS, allowing for better characterization of the conjugation process.

The final future direction will prioritise characterising R bodies' ability to escape the endosome and deliver cargo, a crucial test that we were unable to complete within the iGEM time frame. Moving forward, in vitro testing using a cathepsin B-cleavable linker, such as RebA-LPETGGG-mNeonGreen, will help demonstrate endosomal escape by testing for diffuse fluorescence in the cytoplasm, providing proof of concept for R body-mediated cargo liberation.

Bigger Picture: Human Practices

From our discussions with experts in Human Practices, we realised that the experiments we had done this year were barely the tip of the iceberg. We identified many future investigations that would improve our research and build extensive pre-clinical data for clinical and commercial development. Integrating our insights from Human Practices, we compiled a list of future actionables to expand the scope of our project:

  • Characterise R bodies’ mechanical properties and stability, underlying molecular mechanisms, how they interact with cells and other molecules, and how this changes when different drugs and ligands are conjugated. We had already started to characterise some of these in our experiments (see Results) but much more work needs to be done.
  • In future work, one key area of focus will be the immunogenicity of R bodies, as their repeating epitopes pose a significant risk of triggering immune responses. To address this, a comprehensive study using mouse models will be crucial for evaluating potential immune reactions to R bodies. By monitoring immune markers and antibody production, we can assess whether R bodies elicit a strong immunogenic response, which could limit their therapeutic use.
  • To mitigate immunogenicity, we intend to explore PEGylation—a strategy that involves attaching polyethylene glycol (PEG) molecules to the R bodies. PEGylation has been shown to reduce immunogenicity in other therapies by masking antigenic sites, and we will also experiment with similar molecules that may provide additional protective effects.
  • Investigate the endocytic pathways by which R bodies are taken up into cells, how we can probe these, and the pathways of endosomes containing R bodies once they are taken up into the cell (whether they are recycled or exocytosed).
  • Quantify endosomal escape of R bodies using assays, such as those by Associate Professor Angus Johnston at Monash University.
  • While we used the anti-cancer drug doxorubicin for our proof-of-concept within the iGEM timeframe, we have yet to investigate the many other applications of our system, including other anti-cancer drugs, peptide delivery, RNA delivery, vaccines and gene therapy.
  • Experimenting with different ratios of targeting molecules, payloads and linkers, making sure the packaging of our construct is optimal before taking it into a patient context.
  • Investigate metabolism and clearance: how stable is our R body system in the bloodstream – will it stay for a long time or be removed quickly? How and where will R bodies be filtered and degraded in the body?
  • Optimise imaging and monitoring of R body constructs, so patient treatment response (including efficacy and distribution) can be characterised in the future.
  • Investigate if R body constructs be freeze-dried for transport and storage, and still used effectively for their purpose. This affects their scalability, manufacturing and distribution.
  • As our R body system is a novel therapeutic, we will need to check in with regulators to ensure all guidelines are being followed, and actively liaise with them as we expand our project. See our Entrepreneurship page for more about regulatory compliance.
  • Interdisciplinary collaboration: work with biologists, immunologists, engineers, chemists and pharmaceutical representatives and integrating their expert perspectives on issues such as immunogenicity, targeting, chemical formulations and marketing/commercialisation.
  • Work with patient advocates: while we reached out to a number of patient organisations, we were not able to organise correspondence with them within the iGEM timeframe. However, we intend to pursue ongoing future collaborations with patient advocates as their perspective on our potential treatment is integral.

Beyond: Entrepreneurship

We intend to further develop our research as a start-up, anticipating clinical development and commercialisation with the ultimate goal of implementing our R body system as a new standard of therapeutic care (see our Entrepreneurship page for details). However, these stages bring with them their own set of considerations that must be addressed in future.

For one, we will need to conduct preclinical experiments to thoroughly evaluate the pharmacokinetics and bioavailability of R bodies. Experiments will involve:

  • Developing a physiologically-based pharmacokinetic (PBPK) model to predict how R bodies behave in vivo, considering factors like absorption, distribution, metabolism and excretion.
  • Testing for the concentration of the drug or biologic cargo in plasma, enabling us to assess systemic exposure over time.
  • Focus on bioavailability, ensuring that a sufficient amount of the therapeutic cargo reaches the target tissues.

These preclinical experiments will not only provide insight into the safety and effectiveness of R bodies but also support regulatory compliance, which is another concern. Adherence to Good Manufacturing Practices (GMP) will be a top priority as we scale up production. Developing a risk management plan that includes obtaining a GMP licence and establishing an adverse reaction reporting system to monitor any potential side effects is critical. This system will be essential for ensuring patient safety and addressing ethical concerns associated with therapeutic applications of R bodies.