Microbial Synthetic in vivo Cell Therapy Systems

Project Information

MiStiC

Grant agreement ID: 101078461

DOI

10.3030/101078461

EC signature date 4 April 2023

Start date 1 September 2023

End date 31 August 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 499 938,75

EU contribution

€ 1 499 938,75

Coordinated by

VIB VZW
Belgium

Periodic Reporting for period 1 - MiStiC (Microbial Synthetic in vivo Cell Therapy Systems)

Reporting period: 2023-09-01 to 2026-02-28

Summary of the context and overall objectives of the project

Microorganisms have been the source of many of humanity’s most powerful medicines, including antibiotics, anticancer agents and immunosuppressants. However, producing these complex natural molecules through conventional chemical synthesis is often challenging, costly and unsustainable. At the same time, the rise of chronic intestinal diseases, such as inflammatory bowel disease and colorectal cancer, has created an urgent need for more effective and precisely targeted therapies.

Recent advances in microbiome research have revealed that our gut microbiota plays a crucial role in maintaining health and modulating disease. This has inspired a new concept in human disease treatment: using engineered beneficial bacteria as “living medicines” that can sense disease signals and release therapeutic molecules directly at the site of need. However, current engineered bacteria used for such cell therapies, like Escherichia coli or Lactococcus lactis, have important limitations. They do not stably colonize the human gut and can only deliver simple protein drugs, not complex natural products with strong therapeutic potential.

MiStiC (Microbial Synthetic in vivo Cell Therapy Systems) aims to overcome these limitations by pioneering the use of Clostridia, naturally abundant and stable human gut symbionts, as a next generation therapeutic chassis. By integrating cutting-edge synthetic biology, genome engineering, and natural product biosynthesis, MiStiC will develop Clostridium strains that can (1) detect disease-associated biomarkers, such as those linked to colorectal cancer, (2) respond by producing natural product-based anticancer agents, and (3) self-eliminate when treatment is complete or the bacteria leave the host.
The expected impact of MiStiC is twofold. Scientifically, it will establish a synthetic biology toolkit for commensal Clostridia, enabling advanced microbiome engineering and expanding the frontiers of in vivo biotechnology. Societally, it will open new avenues for safe, precise and long-term microbial therapies against major chronic diseases, reducing side effects and improving patients’ quality of life. By transforming a natural gut resident into a programmable therapeutic platform, MiStiC sets the stage for a new era of personalized, sustainable and microbiome-based medicine in Europe and beyond.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Over the last two years, we established experimental and computational foundations for engineering the beneficial gut bacteria as safe, programmable platforms for therapeutic applications. Our work focuses on Clostridium leptum and closely related gut commensals, key members of the human microbiome that naturally produce beneficial metabolites but have long remained difficult to modify genetically. While Clostridium leptum is our primary target, initial challenges with genetic transformation prompted us to conduct a comparative pangenomic analysis across the Clostridia class to identify other promising Clostridial lineages for engineering, thereby increasing our chances of success. Based on these insights, we selected six additional strains and optimized their cultivation and transformation protocols. For several species, we achieved stable DNA delivery and gene expression for the first time, representing a major step forward in Clostridium genetic engineering. In parallel, we are developing a versatile genetic toolbox for these bacteria, comprising shuttle and expression plasmids, promoter-ribosome binding sites, sensitive reporter systems and optimized CRISPR–Cas genome editing methods. These tools are now being used to delete native biosynthetic gene clusters to enhance safety and metabolic efficiency. A second focus is the construction of a modular nanobody-based biosensing system, enabling Clostridium to detect extracellular cancer-related biomarkers and trigger the production of an anti-cancer agent. Preliminary experiments using a positive control variant confirmed functionality, and ongoing work is now optimizing signal strength and regulatory control. Progress in WP3 has focused on enabling the efficient biosynthesis of complex natural products. We developed experimental and computational tools to monitor and optimize precursor supply and constructed genome-scale metabolic models that accurately predict growth and metabolic flux distributions. These efforts will guide pathway refactoring. We have also developed an open-access computational tool: CAGEcleaner, a software package that streamlines the discovery of natural product biosynthetic pathways. Finally, we initiated work on biosafety systems that ensure environmental containment, including inducible CRISPR-based kill switches and sporulation control modules. Together, these advances bring us closer to realizing the vision of safe, programmable therapeutic probiotics.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our results so far represent a major step forward in the field of microbiome engineering by making previously inaccessible gut bacteria genetically tractable, programmable and potentially therapeutic.
A first breakthrough is the successful transformation of several gut commensal Clostridia species that had long been considered genetically intractable. By systematically optimizing DNA delivery methods and constructing a tailored E. coli donor strain, we achieved stable DNA transfer and gene expression in several Clostridium species. To our knowledge, some of these species had never been transformed before. This achievement not only expands the experimental possibilities for studying beneficial gut microbes, but also lays the foundation for their use as engineered therapeutic delivery vehicles.

A second significant advance is the creation of a modular nanobody-based biosensing platform in Clostridium leptum. This system allows bacteria to detect external molecules and translate this signal into a controlled genetic response. While such biosensing circuits exist in aerobic model organisms, demonstrating initial functionality in an obligate anaerobe marks a genuine step beyond the state of the art. It opens exciting perspectives for developing various engineered probiotics that can sense disease-associated signals in the gut and respond by producing a therapeutic compound precisely where it is needed.

Finally, our CAGEcleaner software represents a computational advance for natural product discovery. It addresses a key limitation of existing genome-mining tools, which produce large, redundant datasets that are difficult to interpret. CAGEcleaner introduces an innovative algorithm that removes redundancy while preserving biological diversity, dramatically improving the clarity and speed of analysis. The tool is openly available and already being used by other laboratories, supporting broader knowledge transfer and collaboration in the field of microbial genomics.

Together, these advances establish new experimental, synthetic, and computational foundations for engineering beneficial gut bacteria. Looking ahead, we aim to refine these tools for clinical and industrial applications, supported by continued research, validation under physiological conditions, and collaboration with partners in biotechnology and health innovation.