Periodic Reporting for period 1 - BacStar (Design, Build and Test of novel bacteria-delivered proteins for cancer therapy)
Periodo di rendicontazione: 2023-01-01 al 2025-02-28
Sintesi del contesto e degli obiettivi generali del progetto
Context and Overall Objectives
BacStar sits at the intersection of cancer therapy, synthetic biology, and computational protein design. While bacterial-based cancer therapies show promise—especially engineered strains that colonise tumours and produce therapeutic proteins in situ—a key limitation remains: getting those proteins efficiently into cancer cells. Most current strategies rely on invasive strains or protein modifications like cell-penetrating peptides, both of which face safety, efficiency, and compatibility issues.
BacStar aims to solve this delivery bottleneck. The project’s goal is to develop a modular, safe, and robust platform to deliver de novo designed therapeutic proteins directly into the cytoplasm of cancer cells using engineered non-invasive bacteria. Originally, I proposed self-delivering proteins capable of crossing membranes autonomously. However, early in the project, a major advance by Kreitz et al. [9]—the demonstration of programmable protein delivery using bacterial Contractile Injection Systems (CIS)—led to a strategic shift. CIS technology offers a more direct and versatile solution for intracellular protein delivery, better aligning with BacStar’s objectives.
To achieve this, the project pursues four objectives:
• Design potent protein/peptide therapeutics candidates (e.g. mini-binders, macrocyclic peptides) using state-of-the-art computational tools (e.g. RFDiffusion, MPNN) and validate targeting cancer-relevant intracellular proteins (e.g. Trim21, MCL1, MDM2, GABARAP).
• Engineer bacterial strains with functional CIS machinery to deliver these payloads intracellularly.
• Create tools (e.g. imaging binders) that facilitate the monitoring of these bacterial deliveries.
Scale and significance of the project’s expected impacts:
Intracellular delivery is a major limitation for the development of biotherapeutics (e.g. proteins) known to have higher interaction accuracy, binding efficiency, and thus have the potential of acting more effectively than small molecules. Using live bacterial systems could be transformative. This project’s integrated synthetic biology, computational design, and bacterial engineering to bring forward a novel therapeutic platform with broad application in oncology and multiple other diseases, and in applications beyond health, for example in agriculture, forestry, biodiversity conservation and pest control.
Scientific Impact: BacStar proposes a new paradigm in live bacterial therapeutics by linking AI-based protein design with precision intracellular delivery. It will provide an open-source platform, validated CIS system, and new therapeutic candidates—enabling progress against targets previously considered undraggable.
Societal Impact: By enabling targeted delivery of potent protein drugs, BacStar supports the EU Cancer Mission and UN SDGs. The approach may offer more effective, less toxic therapies and could extend to vaccines or treatments for other diseases.
Economic Impact: The project is expected to generate IP on therapeutic designs and delivery tools, with commercial potential through biotech spin-outs or partnerships. This contributes to Europe’s leadership in synthetic biology and strengthens its bioeconomy.
BacStar sits at the intersection of cancer therapy, synthetic biology, and computational protein design. While bacterial-based cancer therapies show promise—especially engineered strains that colonise tumours and produce therapeutic proteins in situ—a key limitation remains: getting those proteins efficiently into cancer cells. Most current strategies rely on invasive strains or protein modifications like cell-penetrating peptides, both of which face safety, efficiency, and compatibility issues.
BacStar aims to solve this delivery bottleneck. The project’s goal is to develop a modular, safe, and robust platform to deliver de novo designed therapeutic proteins directly into the cytoplasm of cancer cells using engineered non-invasive bacteria. Originally, I proposed self-delivering proteins capable of crossing membranes autonomously. However, early in the project, a major advance by Kreitz et al. [9]—the demonstration of programmable protein delivery using bacterial Contractile Injection Systems (CIS)—led to a strategic shift. CIS technology offers a more direct and versatile solution for intracellular protein delivery, better aligning with BacStar’s objectives.
To achieve this, the project pursues four objectives:
• Design potent protein/peptide therapeutics candidates (e.g. mini-binders, macrocyclic peptides) using state-of-the-art computational tools (e.g. RFDiffusion, MPNN) and validate targeting cancer-relevant intracellular proteins (e.g. Trim21, MCL1, MDM2, GABARAP).
• Engineer bacterial strains with functional CIS machinery to deliver these payloads intracellularly.
• Create tools (e.g. imaging binders) that facilitate the monitoring of these bacterial deliveries.
Scale and significance of the project’s expected impacts:
Intracellular delivery is a major limitation for the development of biotherapeutics (e.g. proteins) known to have higher interaction accuracy, binding efficiency, and thus have the potential of acting more effectively than small molecules. Using live bacterial systems could be transformative. This project’s integrated synthetic biology, computational design, and bacterial engineering to bring forward a novel therapeutic platform with broad application in oncology and multiple other diseases, and in applications beyond health, for example in agriculture, forestry, biodiversity conservation and pest control.
Scientific Impact: BacStar proposes a new paradigm in live bacterial therapeutics by linking AI-based protein design with precision intracellular delivery. It will provide an open-source platform, validated CIS system, and new therapeutic candidates—enabling progress against targets previously considered undraggable.
Societal Impact: By enabling targeted delivery of potent protein drugs, BacStar supports the EU Cancer Mission and UN SDGs. The approach may offer more effective, less toxic therapies and could extend to vaccines or treatments for other diseases.
Economic Impact: The project is expected to generate IP on therapeutic designs and delivery tools, with commercial potential through biotech spin-outs or partnerships. This contributes to Europe’s leadership in synthetic biology and strengthens its bioeconomy.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
Work Performed and Main Achievements
Here I outline the scientific activities performed and their outputs and outcomes for the BacStar project in its first reporting period, after the outgoing phase, conducted at the Institute for Protein Design (IPD), University of Washington, was successfully completed. The core research and training objectives for this period have been met, and in some cases, the research strategy was adapted to leverage recent technological advancements, ensuring the project remains at the forefront of therapeutic protein delivery.
1. Progress Overview and Strategic Adaptation from Original Work Plan (Annex 1)
BacStar, as detailed in Annex 1, originally aimed to design multi-component proteins capable of self-transport from bacteria into tumour cells, leveraging integrated Cell Penetrating Peptides (CPPs) for cell entry and the bacterial Sec system for export. However, during the initial months of this fellowship, a major advance in the field—Kreitz et al [1] demonstration of programmable protein delivery using bacterial contractile injection systems (CIS)—led us to strategically evolve our delivery approach. This because, CIS presented a more direct and efficient alternative to the CPP-based strategies.
1. Decoupling the therapeutic payload from the delivery mechanism: allowing for independent optimization of the payload and simplifies production within the bacterial chassis.
2. Bio-availability of payload: CIS delivered cargo circumvent any challenges related to endosomal entrapment in CPP-based strategies, which would increase the bio-availability of active effectors in the cytoplasm.
3. Enhanced Engineerability & Feasibility: CIS offers a modular and more robust system for delivering a diverse range of protein effectors without the need to integrate complex CPP and secretion signal functionalities directly into each therapeutic candidate itself.
4. Broader Payload Compatibility: This approach allows for the design of highly potent peptides and mini-proteins optimised purely for therapeutic function, as their delivery is handled by a separate, dedicated system, circumventing challenges of cargo-dependent membrane-translocation design optimisation of CPP-based strategies.
While there is a deviation from the originally proposed protein delivery strategy, the new strategy fully aligns with, and increases the feasibility of achieving BacStar's overarching goal of overcoming drug-delivery challenges in bacteria-based cancer therapeutics by enabling the transport of active proteins into the cytoplasm of tumour cells.
As per Annex 1, the status of objectives and relevant Work Packages for this reporting period (end of outgoing phase) is as follows:
Research Objectives adapted for CIS strategy):
WP1(RO1): Design Build Test of Module 1 – Delivery method (originally genetically encoded CPPs )
Status – Fully Achieved (adapted).
CIS was tested for the delivery of fluorescent tags to HER+ cancer cells only, after specifying the binding to the receptor. For this RO, plasmids and materials from Kreitz et al [1], delivering fluorescent (GFP) tags expressed in E. coli, were cloned, expressed, purified and administered to cancer cells to verify delivery. The experiments confirmed findings by Kreitz, de-risking and establishing the core delivery platform for WP3, prompting us to focus on the design of potential therapeutics (WP2).
WP2(RO2 ): Design Build Test of Module 2: therapeutic proteins (originally self-transducible therapeutics)
Status – Fully Achieved (adapted).
As the design the direct application of CPPs for self-translocation of the entire therapeutic was not needed, we focused this work package in designing and validating potent, small, genetically encodable peptides and mini-proteins suitable as deliverable payloads was fully met through the design of binders, that inhibit or activate cascade actions in the cells. These protein effectors (therapeutic candidates) targeting cancer pathways were successfully designed and validated to bind the targets in vitro. These effectors will now serve as payloads for the CIS that will be test in WP3, in vivo.
WP3(RO3) (Design Build Test of Module 3: bacterial delivered proteins through CIS (originally: secreted proteins )
Status – on track (adapted)
To be executed in the return phase (WP3), now incorporating CIS delivery.
WP4. Project Management and Career development:
Status – Fully Achieved for outgoing phase.
Research Skills:
All planned training in programming and computational protein design at IPD was fully achieved and it was the primary focus of the training at IPD. During this time I learned: Programming in Python for bioinformatics applications, utilizing a suite of cutting-edge computational protein design programs developed and pioneered at the Bhardwaj and Baker Labs (IPD), including RFDiffusion, MPNN, FastRelax, AlphaFold, and Chai2, along with associated Python-based modules for structural bioinformatics and PDB file manipulation [7,9,10]. These tools are state-of-the-art, with many recently published or forthcoming, placing the acquired skills at the forefront. Using these tools I was able to generate mini-protein binders, macrocyclic peptide binders and enzymatic catalytic centres, that can be CIS-deliverable therapeutics.
I also trained in advanced protein analysis techniques, such as HPLC, MS/ LC-MS, CD spectroscopy, spectroscopy, imaging, high-throughput methods for expression and testing of designed proteins, techniques to study protein interactions e.g. high-throughput screening with yeast display, and obtaining binding kinetics, with surface plasmon resonance.
Transferable Skills:
During my outgoing phase, I obtained hands-on experience in mentoring, as I mentored 3 PhD students, I also was able to develop a large network of co-workers and collaborators, with whom I will share publications, I attended and presented my work at the RosettaCon meeting – the largest meeting of scientist in computational protein design. Further, through the independence granted by the MSCA, I also acquired skills in science communications, science advice, science leadership, advocacy, and policy (more below)
Project Management:
The carrier management plan, the data management plan and project management plans were presented after the grant commencement. All measures are taken to follow and fulfil the plans accordingly.
WP5. Exploitation, Dissemination and Communication of results:
Dissemination:
Status – Partially Achieved & On Track.
Significant outputs (publications, conference presentations) have been generated and are being generated. Full achievement will continue throughout the fellowship. At the moment, the work has led to:
-Six peer-reviewed manuscripts: One in press in Nature Chemical Biology [2], two are currently under consideration at Nature Communications [3] and another leading journal. Three are in advanced manuscript preparation [4, 5, 6]. One project involving enzymatic design [7, 8] is undergoing final experimental validation ahead of manuscript preparation.
-Research findings were disseminated through poster presentations at two international conferences: Summer RosettaCon 2024 [9] and the 2025 ARPA-E Energy Innovation Summit [8].
Communications:
Since the Nobel Prize award of my co-supervisor (Prof. David Baker), I have engaged in popular media communication to communicate protein design and its reach (e.g. https://www.siliconrepublic.com/innovation/computational-protein-research-honduras-nobel-ucc-cork(si apre in una nuova finestra)) have led the production of a science + art project that culminated in an exhibition at the GYA General Assembly in Rwanda (see: https://www.youtube.com/watch?v=5w3bvmBLOlg(si apre in una nuova finestra)) I also designed and delivered computational biology workshop for undergraduates in Rwanda (see pg. 16 of the report): https://globalyoungacademy.net/wp-content/uploads/2023/11/AGM-and-Conference-Report-2023.pdf(si apre in una nuova finestra) in addition I have contributed to the elaboration/edition of the United Nations Secretary General Scientific Advisory Board brief in Synthetic Biology: https://www.un.org/scientific-advisory-board/en/synthetic-biology(si apre in una nuova finestra) I also presented the potential of the fields and implications at the Science and Technology Society Forum, Kyoto, Japan, Oct 7th, 2024 (See pg. 133: https://www.stsforum.org/file/2024/11/STS2024_Summary.pdf(si apre in una nuova finestra)). At IPD, I have also engaged in the undergraduate mentoring (Jupyter) summer programme, where early stage undergraduates get to experience the work done at IPD and we teach them and provide them with hands on experience.
Exploitation:
This is in track to be followed at the incoming stage, at UCC in year 3 and 4 and will be presented in the final report.
Conclusion and Next Steps
As evidenced by the outputs, the outgoing phase of this fellowship has been highly productive. The research and training objectives for this period, as outlined in Annex 1 and adapted for the CIS strategy, have been successfully achieved, and Work Packages 1 and 2 are complete. A strong portfolio of potential therapeutic payloads has been designed and validated, and the CIS delivery platform has been established in our hands. This body of work provides an excellent foundation for the next phase of the project (WP3), which will focus on engineering bacteria for CIS-mediated in vivo delivery of these therapeutics and developing imaging markers (such as the FLPP3 binders [4]) to track bacteria and/or CIS activity in tumours.
Beyond the scope of this research project:
The fellowship has enabled a level of independence that has been key in strengthening my engagement in science leadership and policy, enabling me to take on active roles across diverse organisations, deepening my involvement in science advice, science and society, and research policy at both national and global levels. In this context, I have had the opportunity to serve in the following capacities:
• 2024/5 Co-Chair of the Global Young Academy (GYA), hosted by the Leopoldina Academy of Sciences in Halle, Germany. The GYA is a global network of 200 early-career researchers from over 100 countries, selected for their scientific excellence and societal engagement. In this role, I have contributed to international dialogues on the role of science in addressing global challenges and supported efforts to promote equity in research environments.
• 2023 Steering Board Member of the Coalition for Advancing Research Assessment (CoARA), where I contributed to shaping policies aimed at reforming research evaluation systems to better recognise diverse contributions across disciplines and career stages.
• 2024, 2025 External Expert for Working Group 6 on “Credit and Accountability”, under the Strategic Council for Research Excellence, Integrity, and Trust of the US National Academies. My role supports the development of frameworks that promote fair attribution and responsibility in collaborative research.
• GYA Representative to the United Nations Secretary-General’s Scientific Advisory Board, where I have participated in high-level discussions to ensure that youth and early-career researcher perspectives are included in science-policy interfaces at the global level.
References:
1. Kreitz, J., Friedrich, M.J. Guru, A. et al. Programmable protein delivery with a bacterial contractile injection system. Nature 616, 357–364 (2023). https://doi.org/10.1038/s41586-023-05870-7(si apre in una nuova finestra)
2. Rettie, S. A., Juergens, D., Adebomi, V., Flores Bueso, Y., Zhao, Q., Leveille, A. N., Liu, A., Bera, A. K., Wilms, J. A., Greisen, P., Jr, Sankaran, B., Langan, R. A., Kang, A., Lee, H., Brunette, T. J., Chow, K. M., Borst, A. J., Goreshnik, I., Moyer, A., ... Baker, D. (2024). Accurate de novo design of high-affinity protein binding macrocycles using deep learning. Accepted for publication in Nature Chemical Biology, 631(8020), 456–464. Pre-print published online 2024 May 22. https://www.google.com/url?sa=E&source=gmail&q=https://doi.org/10.1038/s41586-024-07463-5(si apre in una nuova finestra)
3. Rettie, S., Campbell, K., Bera, A., Kang, A., Kozlov, S., De La Cruz, J., Ahlrichs, M., Adebomi, V., Zhou, G., DiMaio, F., Ovchinnikov, S., Flores Bueso, Y., Cheng, S., Gerben, S., Murray, A., Lamb, M., & Bhardwaj, Accepted for publication in Nature Communications.
4. Gokce, G., Rettie, S., Flores Bueso, Y., et al. Design of disulphide stappled macrocyclic peptides using deep learning. Manuscript under preparation.
5. Flores Bueso, Y. Gokce, G., Rettie, S., Jeung, J. et.al. A platform for expressing, and testing encoded disulphide stappled peptide by E. coli. Manuscript under preparation.
6. Gokce, G., Huang, B., Kreuk, L. S. M., Liu, A., Adebomi, V., Flores Bueso, Y., Bera, A., Kang, A., Gerben, S., Rettie, S., Vafeados, D., Roullier, N., Goreshnik, I., Li, X., Baker, D., Mougous, J. D., Woodward, J. J., & Bhardwaj, G. (2025). De novo design of high-affinity miniprotein binders targeting Francisella tularensis virulence factor. Manuscript under preparation.
7. Ahern, W., Yim, J., Tischer, D., Salike, S., Woodbury, S. M., Kim, D., Kalvet, I., Kipnis, Y., Coventry, B., Altae-Tran, H. R., Bauer, M., Barzilay, R., Jaakkola, T. S., Krishna, R., & Baker, D. (2025, April 9). Atom level enzyme active site scaffolding using RFdiffusion2. [Preprint]. bioRxiv. https://doi.org/10.1101/2025.04.09.648075v1(si apre in una nuova finestra).
8. Ennist, N., Swartz, A., Lund, P., Flores Bueso, Y., Sun, E., Sehgal, E., & Baker, D. (2025, March 17-19). De novo designed proteins for artificial photosynthesis [Poster presentation]. 2025 ARPA-E Energy Innovation Summit, National Harbor, MD.
9. Flores Bueso, Y., Rettie, S., Kreuk, L., Thomason, M., Woodward, J., & Bhardwaj, G. (2024, August 7). Targeted Protein Degradation with Trim 21 [Poster presentation]. Summer RosettaCon 2024, Suncadia, WA.
10. Watson, J.L. Juergens, D., Bennett, N.R. et al. De novo design of protein structure and function with RFdiffusion. Nature 620, 1089–1100 (2023). https://doi.org/10.1038/s41586-023-06415-8(si apre in una nuova finestra)
11. J. Dauparas et al. ,Robust deep learning–based protein sequence design using ProteinMPNN.Science378,49-56(2022).DOI:10.1126/science.add2187
Here I outline the scientific activities performed and their outputs and outcomes for the BacStar project in its first reporting period, after the outgoing phase, conducted at the Institute for Protein Design (IPD), University of Washington, was successfully completed. The core research and training objectives for this period have been met, and in some cases, the research strategy was adapted to leverage recent technological advancements, ensuring the project remains at the forefront of therapeutic protein delivery.
1. Progress Overview and Strategic Adaptation from Original Work Plan (Annex 1)
BacStar, as detailed in Annex 1, originally aimed to design multi-component proteins capable of self-transport from bacteria into tumour cells, leveraging integrated Cell Penetrating Peptides (CPPs) for cell entry and the bacterial Sec system for export. However, during the initial months of this fellowship, a major advance in the field—Kreitz et al [1] demonstration of programmable protein delivery using bacterial contractile injection systems (CIS)—led us to strategically evolve our delivery approach. This because, CIS presented a more direct and efficient alternative to the CPP-based strategies.
1. Decoupling the therapeutic payload from the delivery mechanism: allowing for independent optimization of the payload and simplifies production within the bacterial chassis.
2. Bio-availability of payload: CIS delivered cargo circumvent any challenges related to endosomal entrapment in CPP-based strategies, which would increase the bio-availability of active effectors in the cytoplasm.
3. Enhanced Engineerability & Feasibility: CIS offers a modular and more robust system for delivering a diverse range of protein effectors without the need to integrate complex CPP and secretion signal functionalities directly into each therapeutic candidate itself.
4. Broader Payload Compatibility: This approach allows for the design of highly potent peptides and mini-proteins optimised purely for therapeutic function, as their delivery is handled by a separate, dedicated system, circumventing challenges of cargo-dependent membrane-translocation design optimisation of CPP-based strategies.
While there is a deviation from the originally proposed protein delivery strategy, the new strategy fully aligns with, and increases the feasibility of achieving BacStar's overarching goal of overcoming drug-delivery challenges in bacteria-based cancer therapeutics by enabling the transport of active proteins into the cytoplasm of tumour cells.
As per Annex 1, the status of objectives and relevant Work Packages for this reporting period (end of outgoing phase) is as follows:
Research Objectives adapted for CIS strategy):
WP1(RO1): Design Build Test of Module 1 – Delivery method (originally genetically encoded CPPs )
Status – Fully Achieved (adapted).
CIS was tested for the delivery of fluorescent tags to HER+ cancer cells only, after specifying the binding to the receptor. For this RO, plasmids and materials from Kreitz et al [1], delivering fluorescent (GFP) tags expressed in E. coli, were cloned, expressed, purified and administered to cancer cells to verify delivery. The experiments confirmed findings by Kreitz, de-risking and establishing the core delivery platform for WP3, prompting us to focus on the design of potential therapeutics (WP2).
WP2(RO2 ): Design Build Test of Module 2: therapeutic proteins (originally self-transducible therapeutics)
Status – Fully Achieved (adapted).
As the design the direct application of CPPs for self-translocation of the entire therapeutic was not needed, we focused this work package in designing and validating potent, small, genetically encodable peptides and mini-proteins suitable as deliverable payloads was fully met through the design of binders, that inhibit or activate cascade actions in the cells. These protein effectors (therapeutic candidates) targeting cancer pathways were successfully designed and validated to bind the targets in vitro. These effectors will now serve as payloads for the CIS that will be test in WP3, in vivo.
WP3(RO3) (Design Build Test of Module 3: bacterial delivered proteins through CIS (originally: secreted proteins )
Status – on track (adapted)
To be executed in the return phase (WP3), now incorporating CIS delivery.
WP4. Project Management and Career development:
Status – Fully Achieved for outgoing phase.
Research Skills:
All planned training in programming and computational protein design at IPD was fully achieved and it was the primary focus of the training at IPD. During this time I learned: Programming in Python for bioinformatics applications, utilizing a suite of cutting-edge computational protein design programs developed and pioneered at the Bhardwaj and Baker Labs (IPD), including RFDiffusion, MPNN, FastRelax, AlphaFold, and Chai2, along with associated Python-based modules for structural bioinformatics and PDB file manipulation [7,9,10]. These tools are state-of-the-art, with many recently published or forthcoming, placing the acquired skills at the forefront. Using these tools I was able to generate mini-protein binders, macrocyclic peptide binders and enzymatic catalytic centres, that can be CIS-deliverable therapeutics.
I also trained in advanced protein analysis techniques, such as HPLC, MS/ LC-MS, CD spectroscopy, spectroscopy, imaging, high-throughput methods for expression and testing of designed proteins, techniques to study protein interactions e.g. high-throughput screening with yeast display, and obtaining binding kinetics, with surface plasmon resonance.
Transferable Skills:
During my outgoing phase, I obtained hands-on experience in mentoring, as I mentored 3 PhD students, I also was able to develop a large network of co-workers and collaborators, with whom I will share publications, I attended and presented my work at the RosettaCon meeting – the largest meeting of scientist in computational protein design. Further, through the independence granted by the MSCA, I also acquired skills in science communications, science advice, science leadership, advocacy, and policy (more below)
Project Management:
The carrier management plan, the data management plan and project management plans were presented after the grant commencement. All measures are taken to follow and fulfil the plans accordingly.
WP5. Exploitation, Dissemination and Communication of results:
Dissemination:
Status – Partially Achieved & On Track.
Significant outputs (publications, conference presentations) have been generated and are being generated. Full achievement will continue throughout the fellowship. At the moment, the work has led to:
-Six peer-reviewed manuscripts: One in press in Nature Chemical Biology [2], two are currently under consideration at Nature Communications [3] and another leading journal. Three are in advanced manuscript preparation [4, 5, 6]. One project involving enzymatic design [7, 8] is undergoing final experimental validation ahead of manuscript preparation.
-Research findings were disseminated through poster presentations at two international conferences: Summer RosettaCon 2024 [9] and the 2025 ARPA-E Energy Innovation Summit [8].
Communications:
Since the Nobel Prize award of my co-supervisor (Prof. David Baker), I have engaged in popular media communication to communicate protein design and its reach (e.g. https://www.siliconrepublic.com/innovation/computational-protein-research-honduras-nobel-ucc-cork(si apre in una nuova finestra)) have led the production of a science + art project that culminated in an exhibition at the GYA General Assembly in Rwanda (see: https://www.youtube.com/watch?v=5w3bvmBLOlg(si apre in una nuova finestra)) I also designed and delivered computational biology workshop for undergraduates in Rwanda (see pg. 16 of the report): https://globalyoungacademy.net/wp-content/uploads/2023/11/AGM-and-Conference-Report-2023.pdf(si apre in una nuova finestra) in addition I have contributed to the elaboration/edition of the United Nations Secretary General Scientific Advisory Board brief in Synthetic Biology: https://www.un.org/scientific-advisory-board/en/synthetic-biology(si apre in una nuova finestra) I also presented the potential of the fields and implications at the Science and Technology Society Forum, Kyoto, Japan, Oct 7th, 2024 (See pg. 133: https://www.stsforum.org/file/2024/11/STS2024_Summary.pdf(si apre in una nuova finestra)). At IPD, I have also engaged in the undergraduate mentoring (Jupyter) summer programme, where early stage undergraduates get to experience the work done at IPD and we teach them and provide them with hands on experience.
Exploitation:
This is in track to be followed at the incoming stage, at UCC in year 3 and 4 and will be presented in the final report.
Conclusion and Next Steps
As evidenced by the outputs, the outgoing phase of this fellowship has been highly productive. The research and training objectives for this period, as outlined in Annex 1 and adapted for the CIS strategy, have been successfully achieved, and Work Packages 1 and 2 are complete. A strong portfolio of potential therapeutic payloads has been designed and validated, and the CIS delivery platform has been established in our hands. This body of work provides an excellent foundation for the next phase of the project (WP3), which will focus on engineering bacteria for CIS-mediated in vivo delivery of these therapeutics and developing imaging markers (such as the FLPP3 binders [4]) to track bacteria and/or CIS activity in tumours.
Beyond the scope of this research project:
The fellowship has enabled a level of independence that has been key in strengthening my engagement in science leadership and policy, enabling me to take on active roles across diverse organisations, deepening my involvement in science advice, science and society, and research policy at both national and global levels. In this context, I have had the opportunity to serve in the following capacities:
• 2024/5 Co-Chair of the Global Young Academy (GYA), hosted by the Leopoldina Academy of Sciences in Halle, Germany. The GYA is a global network of 200 early-career researchers from over 100 countries, selected for their scientific excellence and societal engagement. In this role, I have contributed to international dialogues on the role of science in addressing global challenges and supported efforts to promote equity in research environments.
• 2023 Steering Board Member of the Coalition for Advancing Research Assessment (CoARA), where I contributed to shaping policies aimed at reforming research evaluation systems to better recognise diverse contributions across disciplines and career stages.
• 2024, 2025 External Expert for Working Group 6 on “Credit and Accountability”, under the Strategic Council for Research Excellence, Integrity, and Trust of the US National Academies. My role supports the development of frameworks that promote fair attribution and responsibility in collaborative research.
• GYA Representative to the United Nations Secretary-General’s Scientific Advisory Board, where I have participated in high-level discussions to ensure that youth and early-career researcher perspectives are included in science-policy interfaces at the global level.
References:
1. Kreitz, J., Friedrich, M.J. Guru, A. et al. Programmable protein delivery with a bacterial contractile injection system. Nature 616, 357–364 (2023). https://doi.org/10.1038/s41586-023-05870-7(si apre in una nuova finestra)
2. Rettie, S. A., Juergens, D., Adebomi, V., Flores Bueso, Y., Zhao, Q., Leveille, A. N., Liu, A., Bera, A. K., Wilms, J. A., Greisen, P., Jr, Sankaran, B., Langan, R. A., Kang, A., Lee, H., Brunette, T. J., Chow, K. M., Borst, A. J., Goreshnik, I., Moyer, A., ... Baker, D. (2024). Accurate de novo design of high-affinity protein binding macrocycles using deep learning. Accepted for publication in Nature Chemical Biology, 631(8020), 456–464. Pre-print published online 2024 May 22. https://www.google.com/url?sa=E&source=gmail&q=https://doi.org/10.1038/s41586-024-07463-5(si apre in una nuova finestra)
3. Rettie, S., Campbell, K., Bera, A., Kang, A., Kozlov, S., De La Cruz, J., Ahlrichs, M., Adebomi, V., Zhou, G., DiMaio, F., Ovchinnikov, S., Flores Bueso, Y., Cheng, S., Gerben, S., Murray, A., Lamb, M., & Bhardwaj, Accepted for publication in Nature Communications.
4. Gokce, G., Rettie, S., Flores Bueso, Y., et al. Design of disulphide stappled macrocyclic peptides using deep learning. Manuscript under preparation.
5. Flores Bueso, Y. Gokce, G., Rettie, S., Jeung, J. et.al. A platform for expressing, and testing encoded disulphide stappled peptide by E. coli. Manuscript under preparation.
6. Gokce, G., Huang, B., Kreuk, L. S. M., Liu, A., Adebomi, V., Flores Bueso, Y., Bera, A., Kang, A., Gerben, S., Rettie, S., Vafeados, D., Roullier, N., Goreshnik, I., Li, X., Baker, D., Mougous, J. D., Woodward, J. J., & Bhardwaj, G. (2025). De novo design of high-affinity miniprotein binders targeting Francisella tularensis virulence factor. Manuscript under preparation.
7. Ahern, W., Yim, J., Tischer, D., Salike, S., Woodbury, S. M., Kim, D., Kalvet, I., Kipnis, Y., Coventry, B., Altae-Tran, H. R., Bauer, M., Barzilay, R., Jaakkola, T. S., Krishna, R., & Baker, D. (2025, April 9). Atom level enzyme active site scaffolding using RFdiffusion2. [Preprint]. bioRxiv. https://doi.org/10.1101/2025.04.09.648075v1(si apre in una nuova finestra).
8. Ennist, N., Swartz, A., Lund, P., Flores Bueso, Y., Sun, E., Sehgal, E., & Baker, D. (2025, March 17-19). De novo designed proteins for artificial photosynthesis [Poster presentation]. 2025 ARPA-E Energy Innovation Summit, National Harbor, MD.
9. Flores Bueso, Y., Rettie, S., Kreuk, L., Thomason, M., Woodward, J., & Bhardwaj, G. (2024, August 7). Targeted Protein Degradation with Trim 21 [Poster presentation]. Summer RosettaCon 2024, Suncadia, WA.
10. Watson, J.L. Juergens, D., Bennett, N.R. et al. De novo design of protein structure and function with RFdiffusion. Nature 620, 1089–1100 (2023). https://doi.org/10.1038/s41586-023-06415-8(si apre in una nuova finestra)
11. J. Dauparas et al. ,Robust deep learning–based protein sequence design using ProteinMPNN.Science378,49-56(2022).DOI:10.1126/science.add2187
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
Concrete research outputs of Outgoing phase (deliverables planned to date):
1. Contributed to the design and characterised potent macrocyclic peptide binders targeting critical intracellular cancer-associated proteins MCL1, GABARAP, and MDM2 [2, 3]. This work aligns with the original proposal's goal of targeting specific cancer.
2. Contributed to the design and testing for peptides that are genetically encodable and can be cyclized in vivo by bacteria (e.g. in the periplasm). This is highly relevant for cost-effective production within the bacterial delivery vehicle [4, 5].
3. Contribute to the testing of designs against the bacterial lipoprotein FLPP3 [6], which can potentially serve as tools for imaging the bacterial delivery vehicle within the tumour microenvironment.
4. Contributed to the de novo design of protein scaffolds to host enzymatic catalytic reaction centres using the advanced RFdiffusion2 methodology [7]. This work, currently ongoing with a poster presentation [8], demonstrates the breadth of design capabilities acquired and their potential for creating enzyme-based therapeutics or reporter systems.
5. Mini-protein binders against Trim21 displaying picomolar binding activity. These are intended to activate targeted protein degradation cascades, a novel therapeutic modality [9].
Potential Impacts:
This work establishes a validated pipeline combining advanced computational design with CIS-mediated delivery, opening avenues to target intracellular pathways previously inaccessible to protein therapeutics delivered via bacteria. It will potentially provide novel workflows and diverse molecular scaffolds (mini-proteins, macrocycles) of broad applications, disseminated via publications and platforms like RosettaCommons.
This project also creates links between leading EU-US scientific labs in cutting-edge biotechnology, introducing the field of computational protein design (a field that is revolutionising biotechnology, awarded 2024 Nobel Prize) to Ireland, a country with a large bio-pharmaceutical sector.
In the long term, this platform could lead to new classes of highly targeted cancer therapies with potentially reduced side effects. The approach holds potential for cost-effective production and delivery, potentially applicable to other diseases, including immune, neurodegenerative and infectious disease vaccines (using motifs for antigen delivery). Once the full pipeline has been validated, the developed technology and IP have clear potential for commercialisation through start-ups or licensing, contributing to the biotech economy.
Key Needs for Further Uptake and Success:
Further Research:
In vivo Validation (WP3): testing of the designed payloads delivered via CIS-engineered bacteria in relevant preclinical cancer models as planned for WP3, is key. This includes assessing efficacy, biodistribution, pharmacokinetics, and safety, down the line, it would be important to consider host immune response systemically, and within the tumour microenvironment in order to predict efficacy and potential limitations.
Commercialisation & IPR Support:
Filing and management of intellectual property for novel protein designs, scaffolds, and delivery strategies would be required at the end of WP3 if the pipeline shows optimistic results. This will also be supported with a business plan (as planned in WP5 ) and securing seed funding (e.g. via UCC Sprint Accelerator or similar) to explore spin-out potential, licensing opportunities, or industry partnerships by engaging with biotech/pharma partners (like through the planned placement ).
Access to Finance:
Importantly, the introduction of the field of Computational Protein Design in Ireland will require securing follow-on funding (e.g. ERC Starting Grant, Wellcome Early-Career Award, and/or venture capital) for establishing the platforms for high-throughput design and validations.
Regulatory Framework:
It is necessary that we proactively engage with regulatory agencies (e.g. EMA) since early stages to work together towards the clinical requirements for the development of a novel class of Live Biotherapeutic Products incorporating engineered delivery systems and de novo protein payloads. Contributing to establishing appropriate safety and efficacy standards.
Internationalisation:
Maintaining and expanding the international collaborations established during this fellowship (IPD-UCC) will be key for accessing diverse expertise, resources, and know-how.
1. Contributed to the design and characterised potent macrocyclic peptide binders targeting critical intracellular cancer-associated proteins MCL1, GABARAP, and MDM2 [2, 3]. This work aligns with the original proposal's goal of targeting specific cancer.
2. Contributed to the design and testing for peptides that are genetically encodable and can be cyclized in vivo by bacteria (e.g. in the periplasm). This is highly relevant for cost-effective production within the bacterial delivery vehicle [4, 5].
3. Contribute to the testing of designs against the bacterial lipoprotein FLPP3 [6], which can potentially serve as tools for imaging the bacterial delivery vehicle within the tumour microenvironment.
4. Contributed to the de novo design of protein scaffolds to host enzymatic catalytic reaction centres using the advanced RFdiffusion2 methodology [7]. This work, currently ongoing with a poster presentation [8], demonstrates the breadth of design capabilities acquired and their potential for creating enzyme-based therapeutics or reporter systems.
5. Mini-protein binders against Trim21 displaying picomolar binding activity. These are intended to activate targeted protein degradation cascades, a novel therapeutic modality [9].
Potential Impacts:
This work establishes a validated pipeline combining advanced computational design with CIS-mediated delivery, opening avenues to target intracellular pathways previously inaccessible to protein therapeutics delivered via bacteria. It will potentially provide novel workflows and diverse molecular scaffolds (mini-proteins, macrocycles) of broad applications, disseminated via publications and platforms like RosettaCommons.
This project also creates links between leading EU-US scientific labs in cutting-edge biotechnology, introducing the field of computational protein design (a field that is revolutionising biotechnology, awarded 2024 Nobel Prize) to Ireland, a country with a large bio-pharmaceutical sector.
In the long term, this platform could lead to new classes of highly targeted cancer therapies with potentially reduced side effects. The approach holds potential for cost-effective production and delivery, potentially applicable to other diseases, including immune, neurodegenerative and infectious disease vaccines (using motifs for antigen delivery). Once the full pipeline has been validated, the developed technology and IP have clear potential for commercialisation through start-ups or licensing, contributing to the biotech economy.
Key Needs for Further Uptake and Success:
Further Research:
In vivo Validation (WP3): testing of the designed payloads delivered via CIS-engineered bacteria in relevant preclinical cancer models as planned for WP3, is key. This includes assessing efficacy, biodistribution, pharmacokinetics, and safety, down the line, it would be important to consider host immune response systemically, and within the tumour microenvironment in order to predict efficacy and potential limitations.
Commercialisation & IPR Support:
Filing and management of intellectual property for novel protein designs, scaffolds, and delivery strategies would be required at the end of WP3 if the pipeline shows optimistic results. This will also be supported with a business plan (as planned in WP5 ) and securing seed funding (e.g. via UCC Sprint Accelerator or similar) to explore spin-out potential, licensing opportunities, or industry partnerships by engaging with biotech/pharma partners (like through the planned placement ).
Access to Finance:
Importantly, the introduction of the field of Computational Protein Design in Ireland will require securing follow-on funding (e.g. ERC Starting Grant, Wellcome Early-Career Award, and/or venture capital) for establishing the platforms for high-throughput design and validations.
Regulatory Framework:
It is necessary that we proactively engage with regulatory agencies (e.g. EMA) since early stages to work together towards the clinical requirements for the development of a novel class of Live Biotherapeutic Products incorporating engineered delivery systems and de novo protein payloads. Contributing to establishing appropriate safety and efficacy standards.
Internationalisation:
Maintaining and expanding the international collaborations established during this fellowship (IPD-UCC) will be key for accessing diverse expertise, resources, and know-how.