Human Practices

Stakeholder Discussions

From the very beginning of our project, we knew stakeholder engagement would be an integral part of Human Practices, but we had no idea how valuable it was until we scheduled our first interview. Our stakeholder discussions soon became the cornerstone of our Human Practices work, and the foundation on which we built our further research on Human Practices issues. By talking to real people, especially experts, and hearing their concerns, we realised that our project was so much more than a simple idea, but had implications and repercussions that could span decades of further research. Throughout this process, we learned about the many applications and therapeutic contexts in which our project could be good for the world, and gained insights that allowed us to make informed decisions about the future of our project. We became responsible researchers, who consider the impact of their work on society and how they can improve their designs in response to real-world concerns. We extend our deepest gratitude to the academics who kindly lent us their time and helped us investigate the extensive considerations and implications of our project. In fact, we have made some of our stakeholder discussions publicly available as a Therapeutics Interview Series, so that future iGEM teams (especially those in therapeutics) can derive as much value from them as we did. Learn more on our Contribution page!

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We analysed the insights gained after each interview using the Human Practices Cycle. The Human Practices Cycle can be thought of as the design-build-test-learn Engineering Cycle iterating using stakeholder insights instead of experimental results. We gathered key takeaways from each interview that we then used to inform future considerations of our project, spanning our experimental work during the iGEM timeframe, to manufacturing and clinical trial designs which may not be implemented until years later.

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Dr Sanjeev Kumar

Dr Sanjeev Kumar is a medical oncologist at Chris O’Brien Lifehouse, with an academic background in cancer drug development. In his work with breast cancer patients, he is on the frontlines of selecting and delivering drug treatments to patients, including those in clinical trials. Our interview with Dr Kumar was invaluable in detailing the key priorities in cancer treatment and how R bodies could potentially be used as a chemotherapeutic delivery system.

As patients are the end-users who will be directly affected by our project and the benefits it hopes to achieve, we knew it was important to consider their perspective, as well as that of clinicians who are responsible for recommending and delivering treatments to them. Dr Kumar identified the main priority for cancer patients as being quality of life, which drugs can help with by removing/shrinking the tumour or delaying its spread. He also advised that a drug should be easy to administer in a schedule that is acceptable to patients, which was not something we had previously considered as we were in the preliminary stages of experiments.

Using antibody-drug conjugates (which he identified as the closest analogue to our system in the chemotherapy world) as an example, Dr Kumar took us through some of the key issues in cancer drug design. Antibody-drug conjugates have an antibody, linker and cytotoxic payload (like our system when applied to chemotherapeutics), and the ratio and packaging of these components affects efficacy and toxicity. We were pleased to hear that our system’s ability to release the drug payload intracellularly, like antibody-drug conjugates do, could mitigate the cytotoxic side effects of traditional chemotherapy (e.g. hair loss). In addition, the targeting ability of the antibody would allow us to treat a broader spectrum of patients without having to test for the receptor that the antibody targets. However, while reducing classic chemotherapy toxicities, each construct can have its own toxicity that can be quite harmful, so it is important to optimise drug packaging to minimise those risks while still enhancing the efficacy of each component.

Dr Kumar warned us about off-target toxicity and side effects, using the HER2 receptor in breast cancer as an example. HER2 is expressed in other cells like alveoli, and antibody-drug conjugates can have a bystander effect on these non-target cells, resulting in interstitial fluid buildup in the lungs. One of his patients had actually died from this side effect, which was a harrowing reminder of how detrimental side effects can be.

Dr Kumar also emphasised the accessibility of drugs, not just to specialised oncologists in well-resourced academic centres but community oncologists which treat a wide range of cancers. Community oncologists will not be able to monitor patients as closely, and drugs should have an acceptable toxicity profile that can be managed by these oncologists as well as an academic tertiary oncologist.

As an oncologist at Lifehouse, which has a vibrant Phase 1 clinical trial unit, Dr Kumar noted several considerations for drugs like ours which will require many clinical trials before becoming a standard treatment option. We would need comprehensive pre-clinical investigations, including detailed safety data, toxicity profiles and pharmacokinetics/pharmacodynamics (PK/PD) analysis before moving on to clinical trials. Dr Kumar’s perspective was crucial to help us consider our project’s future as a cancer treatment, and identify issues that could be addressed earlier in our design rather than later.

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Dr Kristina Cook

Dr Kristina Cook is a researcher at the University of Sydney whose primary focus is oxygen-sensing pathways in cancer. She has a multi-disciplinary background in drug development, high-throughput screening and rational drug design. We were fortunate to have her share her extensive understanding of cancer drug design with us, and the issues she raised inspired us to reflect deeply on our project and how it could be implemented for maximum benefit in treating cancer.

Dr Cook echoed Dr Kumar’s points about quality of life being the main patient consideration for treatments. However, treatment priorities change depending on the cancer: for cancers like brain tumours with a life outlook of only 1-2 years, the priority is to find any treatment that works. For curable cancers, the treatment should be tolerable and the patient should have a high quality of life across the span of the treatment. Thus, patient priorities are very heterogeneous among circumstances and cancer types.

Dr Cook brought up the issue of how a drug can get into the body in the first place: oral delivery (via tablet) is non-invasive and convenient for patients, but R bodies might need to be delivered intravenously since most proteins get digested by the gut. IV delivery would require patients to go to hospital (and stay there if receiving daily treatments), and could be challenging for the health system to handle on a large scale.

Dr Cook agreed with the importance of the patient perspective, even though it’s easy to overlook their concerns as researchers working in the lab. She used the example of “scan-xiety”, which refers to the anxiety patients experience when waiting for scans or results as they are used for diagnosis and to monitor disease progression and treatment response. Extra scans may seem mundane and trivial to researchers, but the added psychological burden on patients must be considered. Another example Dr Cook discussed was that tightly fitted masks worn during radiotherapy ensure other parts of the brain are not irradiated, but can be very claustrophobic for patients. Dr Cook mentioned that many grant applications, especially for funding bodies for cancer research, now require patient advocates to give their input and sign off on proposals for clinical trials, and that these advocates (which can be patients themselves or carers, people who have had experience with the disease) often serve on ethics committees.

In terms of cancer drug development, Dr Cook highlighted the challenges of tumour localisation and response. Vascular perfusion of drugs can be difficult as the surrounding vessels do not deliver blood to the tumour very well and tumours can outgrow their blood supply. Cancer cells are extremely dynamic and the tumour microenvironment is constantly growing and changing, making it difficult to monitor treatment response. Dr Cook informed us that transarterial chemo-embolisation (TACE), in which an interventional radiologist threads a tube through the arteries into the tumour so drugs can be injected directly through the tube, could be an option for R body delivery. Dr Cook encouraged us to investigate the pharmacology of R bodies as a drug delivery system, including its stability in the blood, how the body breaks it down, whether it stays for a long time or is removed quickly, and how it is metabolised and cleared.

Dr Cook emphasised cellular uptake as a consideration not only for our system, but for drug design in general, as even drugs that reach their target tissues will not be able to elicit an effect unless taken up by cells. It is easier for small molecules to get into cells than larger ones, but it is difficult to target specific protein-protein interactions (e.g. those driving tumour aggression in cells) with small molecules. Dr Cook revealed that cyclic peptides are better at inhibiting protein-protein interactions, but that cellular uptake of these peptides is a problem currently faced by researchers with little success. We were excited to hear that R bodies could be useful in facilitating peptide uptake! However, Dr Cook mentioned that cancer cells can upregulate transporters to pump drugs out, which would be a significant barrier to the effectiveness of R bodies and warrants further research.

Dr Cook also talked about the various safety considerations required for clinical development of drug therapies. Since immunogenicity (ability to provoke the body’s immune response) would likely be a factor with R bodies being foreign proteins, we would need many animal studies to characterise this before being confident to move onto human clinical trials. In addition, completely novel treatments need much more evidence in different animals before getting to humans. However, there are still differences between animals and humans that could mean some therapies work in animal models but not in humans, complicating trials for treatments like immunotherapies.

Finally, Dr Cook gave us a brief insight into the world of commercialising drug treatments. She brought up the issue of intellectual property, as companies want to be able to make money from your product and won’t invest if you lose intellectual property rights. This could involve having people sign non-disclosure agreements, and making sure IP is protected before presenting projects publicly (e.g. at conferences). While this was often a lot of paperwork, Dr Cook acknowledged that the hassle was necessary, as pharmaceutical companies can be an invaluable source of funding for further research and development, allowing researchers to bridge the gap between wanting to help people and getting the treatment to them. She also emphasised the importance of having robust and comprehensive data to demonstrate our confidence in our system’s effectiveness, so potential investors would see it as more of a guaranteed win rather than a gamble and be more likely to support us since they are more sure they can make back the money they invest.

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Dr Peter Wich

We spoke with Dr Peter Wich, a professor at the University of New South Wales, to discuss the viability of our approach on a molecular and cell level. He leads the UNSW Research Lab for Functional Biopolymers, and just like us, his team is focused on chemical modifications of proteins for drug delivery and pharmaceutical applications in particular.

Similarly to Dr Thordarson, Dr Wich emphasised that lack of clarity in the literature surrounding the process of endocytosis, even though it is crucial to biology generally. Additionally, most traditional drug delivery mechanisms, such as the COVID mRNA and lipid nanoparticle vaccines, and other anti-cancer nanoparticles, aim for their therapeutics to be 100-200 nm, as cell uptake of objects past 300nm in diameter is less likely. Our R bodies, even coiled up, are about one micron in length, which is a major concern for our project. However, their diameter is about 300 nm, which may be enough for endocytosis — our tests showed some evidence for R body internalisation, so it may be possible nonetheless.

Dr Wich warned that our antibody conjugation approach was unlikely to aid in internalisation, though it may pull R bodies close to the target cells, and thus promote somewhat more specific drug uptake anyway. Another approach to facilitating cell uptake may be cell-penetrating peptides, however, these may be too large to fit onto a R body without hindering its extension, as observed in the Reb206 proteins with fusion proteins on the ends of the monomers (Engineering cycles 1.3 and 1.4). Other properties of cancer cells in particular could help targeting, such as some cancers’ higher permeability and retention, though the clinical unreliability and heterogeneity of this effect may make reliance on this unfeasible (Sun et al, 2022), and the Warburg effect, where cancers maintain a slightly acidic environment — Dr Wich suggested that even if our R bodies are unable to enter cells, their spiking action in the tumour-produced acidic microenvironment could anchor them to a tumour (or other target cell), and facilitate release of the drug around multiple tumour cells, similarly to a drug which Dr Kumar mentioned, where efficacy seemed to be improved with slightly lower specificity due to attacking different subtypes of cells within a tumour which may not all have the target of the antibody. Thus, relying solely on cell uptake may be unreliable, so examining alternative mechanisms of action or focusing on improving cell uptake of R bodies will be important in future development. Considering ligands to conjugate apart from just antibodies, such as cell-permeating peptides, or other cleavable molecules promoting membrane permeation, could also be alternatives to investigate in the future.

Dr Wich, who has been involved in research assessing PEG (polyethylene glycol) in drug delivery and developing pH responsive PEG formulations, had some comments on our proposed use of PEG on the R bodies. PEGylation, which is the attachment of PEG to molecules, is a commonly-used method in the pharmaceutical industry to improve the pharmacokinetics of a drug. This kind of modification decreases a drug’s ‘visibility’ to the immune system by forming a hydration layer around the drug. This improves protein stability and increases bioavailability, reducing the likelihood of the immune system attaching to and reacting against it, the rate of renal clearance through increasing the effective size of particles, and reducing the accumulation rate of serum proteins, which can form protein coronas or clusters of proteins, reducing the efficacy of the drug. Thus, PEGylation may be wise to reduce the likely immunogenicity of our product. However, it may also reduce some cellular uptake, and tweaking formulations of PEG may be necessary for a compromise between drug uptake and a longer half-life and reduced immunogenicity. Additionally, some individuals may have immune reactions to PEG itself, though the evidence for this is slim. PEGylation could also affect R body extension, though this would be fairly straightforward to test. Finally, the PEGylation process requires highly purified R bodies to not create a mess with making a range of PEGylated proteins. Improving our purification process will be an important step in further developing our idea, as the purity of our samples can be variable (see the Expression and Purification cycles in Engineering). Exploring new alternatives to fill the role of PEGylation may also be worthwhile; however, given that it is an established pharmaceutical attachment, the lower barrier of entry to the market of using PEG may make it more feasible than an alternative.

In terms of testing R body action within a cell, Dr Wich recommended avoiding using our target drug, doxorubicin as a test due to safety concerns — it is a chemotherapeutic, and has mutagenic effects on non-cancer cells as well. Dealing with a toxin may also affect cell toxicity during experiments; setting up negative controls would be critical to ensure this does not occur. He suggested labelling the R bodies with fluorescent dyes would be easier, and would produce enough evidence to warrant further testing. He suggested that observing whether internalised fluorescence was punctate or diffuse with cellular imaging would be sufficient for demonstrating endosomal escape; however, Dr Thordarson suggested that an optical approach would be difficult due to the high sensitivity required from the microscopes. Independent experimentation may be able to fix this, however, we may need to investigate alternative approaches to resolve endosomal escape.

With respect to implementing our product, Dr Wich emphasised the importance of demonstrating high scalability and mass-producibility of our product early on, in addition to demonstrating some clinical potential. This is very important for securing industry support and funding for research and development. SImilarly to Dr Hunt’s statement on scientists needing to consider their target market in the very long-term, Dr Wich also noted that what may not be scalable currently may be so ten years in the future — for example, humanity developed the capacity to mass-produce COVID-19 vaccines in a very short timespan, when previously it had never been considered possible. Simplicity is a great aid here — in our case, relying on E. coli to produce our proteins, and using stable proteins like R bodies, which can resist a variety of conditions, are great boons to commercial viability, where manufacture and transport of drugs is a massive concern not considered so much in a laboratory. Characterising the stability of R bodies under different conditions, such as being freeze-dried, would be useful in demonstrating the further versatility of R bodies in transport, reducing cost of transport. Once pharmaceutical companies are involved in our idea, we can use their great testing facilities and resources to further our product. Improving the efficiency of drug release may also lighten the financial load of drug procurement relative to the inefficiency of raw drug intake, though the ratio of R body to drug must be considered to maximise this advantage.

Dr Wich suggested targeting immune cells due to their ability to phagocytose larger objects like our R bodies. However, modulating these cells may be more difficult than merely killing them, as would be the case in cancer cells. In any case, a simple enough system such as ours may be useful in various contexts — Dr Wich gave the example of mRNA vaccines being developed with cancer in mind, and then being transferred to antiviral vaccine technology in the COVID-19 pandemic. Genetic therapies may also be more complicated to encapsulate, protect, and release with our R bodies than small-molecule drugs; focusing on the latter, which is cheaper and better understood, may yield more fruit as an initial focus of development. Our pH responsive system would also be a bonus in the small and growing market of nanoparticle drug delivery, as it allows our system to adapt to different situations to optimise pharmacokinetics in various situations — such as endosomal escape through pH responsiveness.

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Dr Lifeng Kang

Dr Lifeng Kang is a University of Sydney School of Pharmacy Senior Lecturer who does research on microscale technologies for drug delivery, and has also licensed several of his patents to companies and start-ups. Our discussion with him was helpful in picking apart details of our project that we had previously overlooked, as well as expanding our knowledge of how therapeutics are commercialised.

Dr Kang warned that endocytosis can be a rare process in cells, and although the main advantage of R bodies may be their ability to burst endosomes inside cells, targeting and uptake would still be concerns. We had been concentrating so much of our research on the intracellular activity of R bodies that we had neglected to characterise how they might be taken up by the cell in the first place. Dr Kang’s reminder spurred us to add endocytosis experiments to our plan.

Like previous interviewees, Dr Kang highlighted safety as a big concern for our project. He told us that since proteins are biological molecules, they can be quite immunogenic as drugs, so R bodies could cause biological issues and be poisonous to humans. Using the example of insulin overdose, he cautioned us that putting any protein into a human can even cause death. At the stage we were at with our preliminary experiments, Dr Kang suggested we do some in vitro studies regarding mechanical properties and stability at different temperatures and pH ranges, as well as cellular studies to see the effect of R bodies on cells (e.g. whether it causes them to lyse or release inflammatory chemicals), and we incorporated these into our future plans. Any future animal studies would first focus on whether the animal survives before doing further research.

One thing Dr Kang strongly emphasised was the importance of extensive characterisation. We should investigate as much as we can about R bodies, including their size, structure, the characteristics of the bacteria they come from, how they are produced, underlying molecular mechanisms, and how it interacts with cells and other molecules before using them as a carrier in our drug delivery system. Dr Kang encouraged us to “become Wikipedia” for R bodies and read all the existing literature to have a comprehensive understanding of the properties of the protein.

Finally, Dr Kang shared his experiences with patenting and commercialisation. He told us that such matters are usually handled in conjunction with the University’s legal department, and the key issue for patenting is whether a project is good lead for further development. If we were looking to commercialise, we would need extensive data to support the use of R bodies as a drug delivery system, and specifics on how we are using the R bodies (e.g. for breast cancer treatment) as we wouldn’t be able to patent things like their structure which was already published. Dr Kang advised that once you receive a patent, you can start a company to license out the patent, but it can take many years before you get somewhere with commercialisation. Startups collaborate with sponsors/donors who usually look for a share of the company in exchange for funding. Funding is essential at every stage as you need money from the very beginning to pay for the consumables, manpower and time required to move a project forward until you can make money yourself by selling your product. Dr Kang expressed doubts about the commercial future of our project, as proteins are quite expensive and less stable. They can aggregate and form precipitates with other molecules, so they need to be freeze-dried. Other polymers would be much cheaper so we need to demonstrate a substantial benefit for R bodies in drug delivery.

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Professor Palli Thordarson

Professor Palli Thordarson and his group at UNSW design new bio-mimetic drug delivery systems, as well as establishing protocols and assays to produce and evaluate their effectiveness. Luckily for us, he had recently presented a second-year pharmacology lecture on lipid nanoparticles, which facilitated delivery of the mRNA COVID-19 vaccines by enabling their passage through the endosome. This was highly relevant to our project’s objective of achieving endosomal escape.

Our interview with Professor Thordarson largely highlighted present uncertainties in the academic field about current mechanisms of endosomal escape. Most small molecule drugs pass through the endosome, but little is known about the details: the latter stages of endocytosis, the mechanisms of endosomal escape of small molecules, and especially the factors explaining our current delivery systems’ effect on endosomal escape. For example, the lipid nanoparticles used in the vaccines against COVID-19 improve endosomal escape for mRNA particles, but only to 10% efficiency, for reasons unknown. Professor Thordarson posited that this may be due to residual lipid nanoparticle elements remaining in the cell, with the aggregates inhibiting mRNA translation. He also noted that the mechanism by which mRNA leaves the lipid nanoparticles and endosome intact is unknown. This uncertainty around mechanisms of natural and engineered endosomal escape make it difficult to rationally design new drug delivery systems. He praised our project as having a rational design, with a clear and intuitive mechanism of action to enable endosomal escape.

The uncertainty around endosomal escape makes it important to perform reliable assays quantifying this phenomenon. Simple fluorescence assays would require real-time cell monitoring with high resolution microscopes, however, this would also require high sensitivity (the fluorescence in the cytosol could be as low as 1-10% of the fluorescence in the endosome), and measuring every endosome would be difficult due to the large number of endosomes in a cell. Professor Thordarson identified Associate Professor Angus Johnston, from Monash University, and his group as having described a split luciferase assay to quantify endosomal escape. We knew we would have to experimentally determine and measure endosomal escape to confirm that our system had achieved what it set out to, but we had been having trouble coming up with ideas on how to do that, so we were inspired to look into Associate Professor Johnston’s research. The assay looked promising and we incorporated it into our future experimental plan, but were not able to perform it within the iGEM timeframe.

Professor Thordarson also noted various difficulties in developing targeting systems for drug delivery systems. The exact factors determining nanoparticle selectivity for different targets is unknown, with lipid nanoparticles for example having specificity for different tissues based on minor and unpredictable changes in formulation. Targeting ligands improve selectivity for different tissues, with whole antibodies being the obvious ‘sledgehammer’ approach to targeting. Other ligands such as antibody fragments, small peptides, polysaccharides, and aptamers are other approaches currently being researched, though these have not yet reached clinical trial stages. Even exploiting the inexplicable selectivity of lipid nanoparticles to target tissue types without ligands may be successful. Additionally, different cell types may be differentially susceptible to targeting by nanoparticles — the liver, which is involved in cleaning the bloodstream and thus soaks up molecules in the blood, is easy to target, whereas muscle cells, being thin and rigid, may be harder to target. Again, due to the many unknowns here, high-throughput screening (and possibly AI) may be the most useful tool to find the most successful targeting strategies for delivery systems like ours, and this is something we will definitely consider when selecting model targeting approaches for our project.

Professor Thordarson also noted that conjugating PEG to the outside of our R bodies is a sensible choice, as PEG likely reduces the chance of immune reactions (though the immune system may respond to the PEG molecules themselves), improves solubility, and increases the circulation time of the drug by reducing the likelihood of being filtered and destroyed by the liver.

Exploring targeting effectiveness of different externally conjugated ligands would be important to ensuring tissue specificity (and thus drug delivery specificity) — this improves safety by reducing off-target toxicity, which would otherwise limit safe dosage levels. A striking example we found highlighting the importance of specificity in drug design is in comparing the modified cancer drug aldoxorubicin to its unmodified progenitor doxorubicin — with a moderate increase in specificity from attaching to albumin proteins in the bloodstream, aldoxorubicin has a safe cumulative dosage level over 12 times higher than doxorubicin with reduced cardiac toxicity, and even reduced alopecia (Gong et al., 2018). Additionally, with our delivery system potentially drastically increasing the efficiency of endosomal escape, any payload’s effective potency would be significantly increased, magnifying the importance of designing safe targeting systems to minimise off-target effects. However, considering that even our initial aim of improving endosomal escape is already fraught with uncertainty, we decided that testing targeting systems was outside the scope of our project. Nevertheless, future research on using R bodies for drug delivery should carefully consider R bodies’ compatibility with available external ligands for improving drug pharmacokinetics and specificity.

One issue that Professor Thordarson raised was the size of RNA payloads — mRNA molecules can be ten times larger than the proteins they might encode. This was not something we (and many others, Professor Thordarson assured us) had ever considered before! Our R bodies are large proteins, with an inner diameter more than large enough to handle RNA strands (113nm inner diameter when extended, and 318 when coiled; Cai, 2023). However, RNA length may be an issue: R bodies have a length of 231nm when coiled and 6um when extended, but considering that most human transcripts are at least 2000bp long (Lopes et al., 2021), which is about 680nm, our choices for mRNA transcripts to attach to R bodies for therapies is limited to those that will be able to fold to fit into that space, and still have enough mRNA attached to yield some therapeutic benefit. This consideration allowed us to determine that while mRNA delivery may be difficult, RNAi therapies would definitely be suitable for our system, with anti-sense oligonucleotides, siRNA, and shRNA all using around 20bp lengths of RNA, about a twentieth the length of mRNA transcripts.

Considering the vast unknowns surrounding crucial aspects of drug delivery, Professor Thordarson noted that collaboration with many different people is key — including biologists, who understand the translation and expression of RNA; immunologists, who understand immunogenic responses to treatments, a major challenge for all nanoparticle-based therapies; engineers and chemists (like himself), who put the ideas into practical formulations and designs; clinical scientists, who are essential for identifying clinical needs and directing clinical trials; and finally pharmaceutical companies, who ground the ideals in practical reality, and provide direct funding and research assistance to support the development of drugs. However, he also stated that even if pharmaceutical companies are able to perform cutting-edge research with far greater resources than their academic counterparts, the economic reality making this research possible also limits the spread of their findings. Investors, seeking some certainty in the incredibly risky market of pharmaceuticals (roughly 9 out of 10 biotech companies fail), seek patents which cause the academic literature to lag significantly behind industry in terms of knowledge. For example, Moderna created a lipid nanoparticle formulation which stabilised their mRNA vaccine such that it could be stored at -20C, rather than Pfizer’s -80C — however, they only published this a year after it was invented, limiting the capability for researchers to understand and learn from this invention. Additionally, researchers are unable to accurately compare their own formulations with the gold-standard industry products seeing real-world use, as the details of their proprietary products are intentionally hidden. Professor Thordarson wistfully imagined an open-access drug delivery platform, upon which researchers might openly collaborate to create more effective medications — however, he also recognised the economic realities of the pharmaceutical industry preventing such a development from being viable, and concluded that academic and industry collaboration is essential and to be appreciated, despite the economic caveats hindering faster development of more effective treatments.

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Dr Nicholas Hunt

We reached out to Dr Hunt, a senior lecturer at the University of Sydney and member of The Centre for Drug Discovery Innovation, both for his knowledge of the basic science surrounding drug pharmacokinetics and delivery, and for his experience in the pharmaceutical industry, as he is developing a new treatment for diabetes. We talked to him to better understand the biological considerations for drug delivery, as well as the current state of gene therapies in the pharmaceutical regulatory and industry fields, as we were interested in the implications of using our R bodies for gene therapies.

Dr Hunt noted that there are many roadblocks to efficient drug delivery, including the various mechanisms of drug clearance by the liver and immune system, the requirement for specificity to reduce off-target effects, and also various considerations for developing endosomal escape platforms. Complement proteins around the body identify foreign proteins, particularly bacterial membrane proteins, and surround them, cleaving them with complement enzymes as well. Immune cells may also enter the scene, phagocytosing the surrounded proteins — this is ideal as a mechanism of clearance, as it clears the protein without a significant immunogenic reaction. However, if antigens are then retrieved and integrated into the adaptive immune network, these proteins will be destroyed quicker. Our R body proteins, being large bacterial proteins, will likely be susceptible to the adaptive immune system recognising bacterial antigens, causing immunogenic reactions which endanger patients, and reducing the efficiency of future treatments. Adeno-associated viruses (AAVs), the current gold standard viral vectors for gene therapies, largely avoid these negative immune responses through their natural non-pathogenic occurrence in humans. However, as Dr Wich and Prof Thordarson also noted, our PEGylation conjugation approach could negate these adverse factors to some extent by increasing drug circulation time, and temporarily avoiding immune surveillance.

Sensitive biological materials such as nucleic acids are also easily degraded in lysosomes, and the ability of our R body protein to facilitate endosomal escape of its payload would improve the efficiency of drug delivery. One further approach we could explore is developing selective endosomal escape systems, where endosomal escape would be tuned to specific cell types. This adds another layer of specificity for our drug delivery platforms, reducing the likelihood of off-target effects. Reducing off-target effects is particularly important for gene therapies to minimise off-target mutations — which are technically introducing cancers into cells.

One key advantage of our R body platform for gene therapies would be its cost-effectiveness, in comparison with AAVs. Administering 1mg of AAV could cost millions, making clinical trials a massively expensive endeavour. In comparison, our R body systems would for the most part rely on well-established technology — PEGylation is a commonly-used strategy, and can be modelled in pre-clinical testing. Antibodies and targeting molecules are also available which have already gone through years of screening. Producing small-molecule drugs is also inexpensive, though producing gene therapies, especially those involving CRISPR-Cas9 systems, remains costly. If our R body systems can effectively improve mRNA’s potency through its endosomal escape action and effective specificity from antibodies or other targeting molecules, while maintaining acceptable immunogenicity through PEGylation, they could introduce a competitive low-cost platform for gene therapy to the pharmaceutical industry.

The lower cost for producing R body-based therapeutics will be incredibly important in advancing through clinical trial stages — in clinical trials for cancer therapeutics, Dr Hunt estimates the phase 1 trials for human safety and dosage as costing $12M, phase 2 trials for effectiveness as costing $20-40M, and phase 3 trials for understanding broader efficacy as costing anywhere between $200M and $1B. Each of these clinical trials phases is incredibly risky for drug developers, with half of all drugs failing phase 1 trials, almost 75% failing phase 2 trials, and 70% of the remainder failing phase 3 trials. Utilising already-established technologies will makes drug development cheaper, as production systems for each component are already set up. However, R bodies are unknown to regulators and industry, and development of therapeutics will require continuous engagement with these parties as well as additional experimentation, increasing required capital to bring these drugs to market.

Before testing in animal and clinical trials, much of the future development and research of R bodies would be in high-throughput screening, to assess characteristics of R body-drug constructs, and optimise them for targeting different cell types or receptors. There would also be a lot of pre-clinical modelling to do, to establish the pharmacokinetic profile of the drug and its resultant safety — this would be critical for convincing regulators and industry collaborators of the future viability of this product, and accumulating further regulatory and testing support.

For producing pharmaceutical-grade R bodies suitable for clinical testing, manufacturing protocols and facilities must be used, certified as running in accordance with the Good Manufacturing Practice (GMP) standards set by the Australian regulator, the Therapeutic Goods Administration (TGA). This may be difficult to find in Australia, particularly as producing novel therapeutics is more risky for manufacturers than existing products. After high-throughput cell screening — which may take years — a drug candidate is found, and testing can advance to testing in animal models, or organoids as an intermediate between cell lines and animals. This process might take even more time, particularly because there is the risk that candidate drugs that work in cell lines fail in later stages, bringing testing back to the designing and high-throughput screening phase. Animal models will help establish safe dosage windows, and provide some pharmacokinetic and pharmacodynamic information, informing us of the drug’s circulation and clearance properties, and the extent to which it achieves the desired therapeutic effect respectively. One experimental design to establish safety is to administer the drug to an animal, and observe the drug’s pharmacokinetic and pharmacodynamic profile for 28-96 days, watching for markers of toxicity or reactivity such as haematological complications. Ideally, the therapeutic action of the therapeutic should be evident at 100-1000 times below the safe dosage limit, to minimise the possibility of overdose.

Once the pre-clinical experimentation shows the potential for safe usage and efficacy in animal models, the human clinical trials may begin. These will start with phase 1 testing, involving a dose escalation design on a few healthy patients (perhaps 10-20) to dial in a safe dosage window, and then a slightly larger study on the target patient population to assess whether the drug is likely to demonstrate some efficacy in future studies. Phase 2 also assesses effectiveness, and expands the patient population to 100-300 patients from the target patient population — this phase can take a very long time depending on the disease and the expected speed of treatment. Finally, Stage 3 testing again expands the testing population, now to 300-400 patients, and examines effectiveness of the drug in a more wide-scale and natural setting. The pre-clinical trials and clinical trials are lengthy processes, that makes collaborating with industry members and investors extremely necessary for supporting drug development. Dr Hunt emphasised that, unlike industry members, who mainly consider the value of their pharmaceutical product in the shorter term, scientists must consider the full range of development, from idea, to publication, to industry development, and pre-clinical and clinical testing, and the great risks in between, and factor in the long timeframe until their drug may be in reach of market viability — looking a decade ahead is the scientist's role.

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Professor John Rasko

We had the immense privilege of talking to Professor John Rasko AO, who is chair of the Gene Technology Technical Advisory Committee at the Office of the Gene Technology Regulator (which is responsible for regulating genetically modified organisms in Australia), and previously chaired the Advisory Committee on Biologicals at the Therapeutic Goods Administration (the Australian government agency which regulates biological therapeutics). He is a leading researcher in gene and stem cell therapy, and recently headed the establishment of Royal Prince Alfred Hospital’s chimeric antigen receptor (CAR) T-cell therapy program. In our short chat with him, he engagingly and sensitively described various complex issues that we had never even thought about before, and we felt absolutely honoured at having the opportunity to discuss them with him.

Professor Rasko affirmed the positive contribution our project could make in the field of gene therapy. Safety is still a major concern when using the current standard of engineered adeno-associated viruses (AAV) for gene therapy, as viruses are highly evolved to disrupt cellular systems and there is always a risk of off-target complications. If applied to gene therapy, our project could avoid the inherent risks associated with viruses. Immune responses can be lethal, and he cited the example of Jesse Gelsinger, the first person publicly identified as having died in a clinical trial following adenovirus-mediated gene therapy. AAVs and other viral vectors also have the potential to integrate genes into the host’s genome and may increase their risk of cancer, though this has yet to be demonstrated in human patients following AAV gene therapy. R bodies are unlikely to enter the nucleus at all, so integration of genes from R body payloads is nigh impossible. In addition, our idea of using a protein carrier would allow our system to be manufactured free of contaminants or unknowns, while achieving purity - something more difficult to attain with viruses. We resolved to be proactive in identifying the worst possible risks and complications of our system, including but not limited to immunogenicity.

One thing we had never considered was the optics of getting patient consent. Professor Rasko stated that public perception of viruses makes this difficult for AAV as clinicians must be honest about what is going into patients’ bodies, yet it is complicated to say to a patient that they will be injected with a virus – the overall public perception of viruses (e.g. HIV) leads to an immediate reaction of caution. Our system could mitigate this significantly as it is much more straightforward to tell patients about injection with proteins, which are associated with beneficial effects (e.g. physical fitness).

We learned that the cost of AAV is massively prohibitive (up to $3.5 million USD per injection!) and it is only viable because it has the potential to cure/prevent disease. The world is still figuring out the best way to approach the cost of these technologies, as we have never before had such tools to cure or prevent diseases, yet plenty of projects have failed to progress due to lack of funding or patenting. Failure to consider the economic aspects of our treatment could render it useless. Professor Rasko emphasised the importance of considering health economics, and warned that many people currently consider gene therapies to be “a scientific and medical success, but an economic failure”. This inspired us to look into projected costs for our system and how to keep it financially accessible, as well as the specific benefits we were hoping to achieve with it (prevention/cure or treatment) since this could help balance out the cost burden.

We also discussed the challenges of communicating with and convincing the public about gene technologies. Weaponising genes is a recurring theme in mainstream portrayals of genetics (mutant superheroes and Frankenstein often come to mind), and this narrative was perpetuated in COVID-19 conspiracy theories. The public mind regards gene technologies negatively even when they have undeniably positive effects – for example, the idea that genetically modified organisms (GMOs) are problematic. GM crops are essential to sustain the massive amounts of food required to feed the planet and have never been shown to cause health problems, yet ‘organic’ food is always perceived as beneficial and people often choose GM-free options.

Professor Rasko related the difficulties he would experience trying to convince people based on scientific facts and arguments, as fear and uncertainty is a much more compelling driver. Persuading the public would require a sophisticated understanding of population dynamics and society. Science is difficult to educate with, but ethics and morality are intuitively understandable to everyone. He urged us to be responsible scientists and acknowledge potential ethical and moral issues and that our work has consequences. We should make it clear that we’re not trained in any of these issues and can’t know all the answers upfront, but we need to understand that there are many perspectives, and be open to having discussions and collaborating with others. Even acknowledging these issues would put us ahead of other scientific groups which are too caught up in the thrill of discovery and fail to acknowledge them.

Finally, we were curious about Professor Rasko’s experience introducing CAR T-cell therapy to RPA Hospital, as the final goal for our project would be to establish it as a new therapy in hospitals. He told us that he had been educating hospital management to comfortably talk about gene therapy for the past 20 years, which was a challenge because bureaucrats are extremely risk-averse. This demonstrated to us the role of time and familiarity as something that would be on our side when educating the public about science, as a possible buffer to fear and emotion. The endeavour also involved training staff (e.g. nurses who were understandably scared of accidents involving the therapy), and discussing with regulators to make sure rules were followed. Only once the infrastructure was in place, was CAR T-cell therapy able to become another pillar of anti-cancer technology at RPA. However, Professor Rasko acknowledged the ongoing process of education and addressing new concerns that will inevitably arise as more people want the treatment and more money is put into it.

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Associate Professor Khoon Lim

Associate Professor Khoon Lim works on the delivery of bioactive molecules, and his research has generated a number of intellectual property and patent applications. We were very grateful for his expertise in therapeutics and commercialisation, and he gave us a lot to think about for our project.

Associate Professor Lim noted that conjugating targeting molecules/drugs to R bodies, as we plan to do, could affect the structure of the R body protein, including extension and hydrophobicity. Though we were already planning to test extension with conjugated R bodies, we realised we should also explore other properties of the conjugated proteins, such as hydrophobicity.

He advised that in the chemotherapeutic space, we need to remember that tumours are solid 3-dimensional masses, which researchers sometimes overlook. Associate Professor Lim urged us to consider the tumour microenvironment, as cancer tissue can harden/stiffen and deposit more collagen and fibrous proteins to form a preventative barrier that could block drugs from getting in. He also brought up the issue of metabolism, as proteins like ours may be subject to enzymatic degradation.

In terms of his experience with commercialisation, Associate Professor Lim told us that drugs need to undergo comprehensive pre-clinical and clinical development before being commercialised and sold on the shelf, and that this process can take more than a decade. Regulation can be a barrier as it is advancing rapidly with the advent of new biotechnologies, and we would also need to consider manufacturing as projects need to restart if they don’t comply with good practice. Continuous development would require constant investment, with pharmaceutical companies being a major financial contributor if you can get them interested in your research. Associate Professor Lim emphasised that companies first and foremost look for how quickly they can achieve return on their investments.

Finally, Associate Professor Lim reiterated the importance of collaboration. It is beneficial to work very closely with clinicians, as they can very quickly observe issues from their perspective. He related a meeting with an orthopaedic surgeon about a proposed new surgical intervention, who helped him realise that prolonged surgery times would not be favourable unless quality of life would be much better than current treatments. Collaborating with companies can also be useful, as they have experienced teams that can help with co-development, co-design and marketing. In particular, “big pharma” companies have a very clear agenda of what they want: they know exactly which regulations they might face and how to get their products into patients.

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Professor Georges Grau

We had the pleasure of speaking to Professor Georges Grau, who has studied extracellular vesicles in immunopathology for over 20 years. Having recently had a friend diagnosed with cancer, Professor Grau appraised our goal of cancer treatment as a noble aim, and found our research on endosomal escape extremely exciting, with high possibility of interest from large pharmaceutical companies.

He encouraged us to consider what happens after endocytosis of R bodies, because although some compartments do form the late endosome, others can be targeted to exosomes for exocytosis, or to recycling endosomes. Before, we had not considered possibilities other than the late endosome, but the passage of endosomes could be investigated in future experiments by labelling and imaging, as suggested by Professor Grau. He affirmed that the stability of R bodies was a big benefit of our project, as the proteolytic systems in endosomal compartments can be quite strong.

Professor Grau told us about his experience using PEG to increase stability in the bloodstream, as we intended to do with R bodies. He stated that he was unsure if PEGylation would modulate immunogenicity, especially since we have both an innate immune system (involving monocytes and macrophages) and adaptive immune system (involving T and B lymphocytes). Professor Grau suggested that we might be able to reduce the antigenicity of R bodies by making them tolerogenic, i.e. incresaing the likelihood that the immune system develops a tolerance to them.

Finally, Professor Grau validated many of the issues we had come across before. While it would be simple to assess the efficacy of R bodies in vitro, in vivo experiments and animal studies would still be the best way to assess safety before moving to humans, but he warned that this could be difficult due to animal ethics issues. He also related his experience working with big pharmaceutical companies, noting that it can take months or years to organise contracts and deal with legal issues such as intellectual property, but encouraged us to generate as much experimental data as possible before approaching potential investors.

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Further research and integration

We noticed that some key takeaways from our interviews were mentioned by multiple experts, signalling to us that they were highly important to consider in our Human Practices work and the rest of our project. This inspired us to do further research on the following issues:

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Immunogenicity

Anti-drug antibodies (ADA) pose a significant challenge in the biotherapeutics industry and clinical medicine as they can reduce the efficacy of a biotherapeutic or cause adverse immune responses. Protein aggregation and repeating peptide epitopes can increase the immunogenicity of biotherapeutics, and can be a key factor that results in immunogenicity in the clinic (Lundahl et al 2021).

R bodies are composed of a repeating structure of millions of monomers (Cai 2023). Repeating epitopes can increase recognition by antigen-presenting cells, stimulating a classical humoral response via T cell-mediated B cell activation. Similarly, antigens possessing repeating epitopes can cause B cells receptors to cross-link, directly triggering activating signals which can lead to antibody production. PEGylation, or PEG-alternatives may be a potential strategy to reduce immune recognition of individual R body proteins (Shi et al. 2022).

Additionally, as they are synthesised in bacteria, R bodies do not contain PTMs like glycosylations, which can help stabilise protein-protein interactions to reduce aggregation (Duran-Romana 2024). Non-native protein aggregation is frequently observed at many stages of bioprocessing, including protein expression, purification, and storage. This process is generally irreversible, subsequently, minimising aggregation during the manufacturing process requires tightly-controlled conditions (Weiss et al., 2009). Excipients like surfactants, amino acids and pH buffers may reduce protein aggregation by competing with the therapeutic protein for adsorption to the air-liquid interface (Ratanji 2014).

While R bodies pose a significant immunogenic risk, appropriate processing methods and the use of immune-modulating molecules can improve clinical outcomes by mitigating ADA-mediated drug inefficacy.

PEGylation

Polyethylene glycol (PEG) has a long history in the biopharmaceutical industry, commonly used to increase drug half-life in the blood, and reduce immunogenicity. PEG is highly hydrophilic, and as such, soluble in a wide variety of solvents, making it useful in many therapeutic formulations (Shi 2022). The versatility of PEG structures allows for optimisation of drug delivery systems to ensure maximum efficacy. Studies have shown that PEGylation can reduce the uptake of certain drugs by dendritic cells, thereby decreasing antigen presentation to T cells (de Bourayne, 2022). Additionally, PEGylation has been proven to enable nanoparticles to induce antigen-specific immune tolerance (Li, 2021) which results in improved patient outcomes.

However, PEGylation has also been associated with immunogenicity (Wang 2023, Taiki 2024), low-level inflammation (Asoudeh, 2024), and hypersensitivity to PEGylated nanomedicines (Chen, 2023). A recent study suggest that a PEG-like polymer may avoid inducing an immune response while exhibiting enhanced pharmacokinetics (Ozen 2022). Thus, there remains an apparent need for developing novel conjugate structures that can further reduce the immunogenicity of drugs while maintaining therapeutic properties.

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Ethical issues

Gene technologies, especially gene therapy, pose many ethical dilemmas.

• Is it ethical to alter nature at all? Many religious groups are opposed to genetic modification on principle. For example, embryonic stem cell research is banned in Italy, a highly Catholic country.
• Gene therapy can replace disease-causing mutations and prevent them from being passed onto offspring. But how can we even approach the issue of consent for people who haven’t even been born yet?
• Where does the line between gene editing and eugenics end and begin? Are we subtly sending the message that certain phenotypes are preferable to others? Are we undermining the valuable sense of community that people with disabilities can experience?
• Are we reinforcing inequalities and economic disparities that already exist? How can we possibly say we are striving towards equality when we live in a world where some people are able to screen for embryos free of diseases and others cannot, simply because they can’t afford it?

There are no easy answers to these questions, and we seek to learn more about them from people who are affected by them in different ways, including those whose views we may not necessarily agree with. In light of these complex issues, and inspired by Professor Rasko’s encouragement to acknowledge their importance to our research, we present the following Ethics Statement by our team:

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Pre-clinical experiments

We developed some pharmacokinetic experiments to validate our system, using the example of R bodies conjugated to doxorubicin, a cancer drug. These experiments would provide the source of our preclinical data, to submit as a feasibility study for clinical trials. The presentation of pre-clinical data is a requirement for TGA-conducted human clinical trials.

Experiment 1: Develop a physiologically-based pharmacokinetic (PBPK) model

Develop a physiologically-based pharmacokinetic (PBPK) model to predict:

• R body doxorubicin release rate
• Diffusion and accumulation profile of doxorubicin in tumor tissue

In doing so, the bioavailability, elimination half-life, clearance, and distribution of both the R body chassis and the released doxorubicin can be measured.

Experiment 2: Testing drug/biologic content in plasma

Test for doxorubicin and R-body elimination and half-life rates by:

• Delivering a single dose of product to monkeys based on weight.
• Taking blood samples from monkeys at 10-minute intervals during the first hour, then every 30 minutes for the next 3 hours.
• Centrifuging samples to obtain plasma.
• Analysing drug concentrations in plasma.

Experiment 3: Bioavailability test

Compare oral vs intravenous administration of our product with the following steps:

• Fasting the test subjects (monkeys) for 24 hours.
• Delivering the R body-drug conjugate orally (single dose).
• Collecting blood samples every 30 minutes for 3 hours, then centrifuging to obtain plasma.
• Analyzing plasma profile for drug concentrations.
• Completing a washout period of the R body/drug from the animal’s system (24 hours).
• Delivering the R body-drug conjugate intravenously and repeating analysis.
• Comparing drug bioavailability for oral vs intravenous delivery.

(This information can also be found on our Entrepreneurship page, as it forms part of our future plans for validation of our product.)

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Regulatory compliance

We also need to make sure we are complying with all relevant regulations, including good manufacturing practice (GMP). We aim to incorporate necessary on-site and documentation Therapeutic Goods Administration (TGA) requirements into our workflow and quality management.

Under the TGA, our system would be a Higher Risk Product, which generally encompasses sterile and non sterile medicines, and cellular therapies. We need to apply for a GMP license, which includes registering a laboratory production facility and each team member for training.

Under the GMP, our team, being the initial main manufacturing source for our product, will:

• Have a quality management system in place to control all manufacturing activities.
• Provide information on how the laboratory equipment is maintained and controlled (e.g., phase microscope usage).
• Control manufacturing activities and experiments through the use of detailed procedures, in the form of an electronic lab notebook.
• Record manufacturing events using record-keeping practices (electronic lab notebook).

Other action items we will initiate during post market surveillance of our system, as required by the TGA, include:

Developing a risk management plan: To identify and characterize known or potential safety concerns if our product and contingency plans to minimize risk. Assesses the risk-to-benefit ratio of our product for consumers with middle to late stages of cancer.

Developing an adverse reaction (AR) reporting system: To report harmful and unintended responses (no later than 15 days from occurrence) during clinical trials and when in market.

Ensuring pharmacovigilance audits: A pharmacovigilance system will help detect and investigate issues in consumer responses to our product in a timely manner. We will nominate a team member to act as a Qualified Person responsible for Pharmacovigilance Audit (QPPVA) undertakings. They will be responsible for communicating pharmacovigilance findings between us and the TGA. Pharmacovigilance training will be provided for all members in the team, educating them on ways to identify adverse events and how to mitigate them.

(This information can also be found on our Entrepreneurship page, as it forms part of our future plans for expansion of our product and quality management.)

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References

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Cai, G. (2023). An Interdisciplinary Investigation of Conformational Changes in Quasi-Crystalline Protein Array R-bodies in Response to pH. University of Washington.

Chen, W.A., Chang, D.Y., Chen, B.M., Lin, Y.C., Barenholz, Y., and Roffler, S.R. 2023. Antibodies against poly(ethylene glycol) activate innate immune cells and induce hypersensitivity reactions to PEGylated nanomedicines. ACS Nano. 17(6). https://doi.org/10.1021/acsnano.2c12193

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