Mushroom poisonings caused by Amanita phalloides (the "death cap") remain one of the deadliest intoxications
worldwide. The primary toxin, α-amanitin, irreversibly inhibits RNA polymerase II, blocking protein biosynthesis and
leading to acute liver failure. With no approved antidote available, treatment often requires liver transplantation,
and mortality remains high even with intensive care.
Our project DeathCapTrap aims to develop a modular therapeutic platform that combines nanobody engineering with
advanced drug delivery systems. By creating a targeted antidote for α-amanitin, we hope to pioneer an approach that
can later be adapted to other toxins, addressing an urgent unmet medical need.
- Why? To efficiently generate and purify nanobodies as the foundation of our
therapeutic system.
- How? We aim to construct plasmids containing DNA sequences for GFP (green
fluorescent protein) and an anti-GFP nanobody, express these in E. coli, and develop protocols for
expression, purification, and functional testing.
- Outcome: This system serves as a testbed for producing nanobodies in a
reproducible and scalable way.
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Why? α-amanitin's structure and toxicity require a highly specific binder that
does not interfere with normal cell function.
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How? Using structure-based, computer-aided modeling, we aim to develop novel
nanobodies that we anticipate will bind α-amanitin with high affinity.
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Outcome: A shortlist of candidate nanobody sequences for recombinant expression
and testing.
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Why? To confirm that our production pipeline works for therapeutic nanobodies, not just test systems.
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How? Our initial goal is to establish a GFP-anti-GFP workflow, which we can then use for our newly
developed anti-α-amanitin nanobody.
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Outcome: Proof that our nanobody can be generated using our own modular production system.
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Why? To demonstrate that our nanobody can neutralize α-amanitin.
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How? After successfully designing the alpha-amanitin nanobody using in silico methods, and following its
expression and purification, we plan to investigate its functionality through in vitro experiments. This
will include simulations and validation of the binding interactions.
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Outcome: Foundational evidence that our nanobody can be applied in two therapeutic strategies:
- Extracellular neutralization of α-amanitin in the bloodstream.
- Intracellular protection via delivery with lipid nanoparticles into
hepatocytes.
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Why? Nanobody delivery requires a safe and efficient transport vehicle. LNPs must be optimized for size,
homogeneity, lipid composition, and stability.
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How? We aim to produce LNPs, load them with our nanobodies, and adjust their lipid composition to ensure
efficient targeting of liver cells. Modifications with PEGylation or other ligands will enhance
specificity and circulation properties.
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Outcome: A flexible nanoparticle system for targeted nanobody delivery.
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Medical relevance: Poisoning by Amanita phalloides is one of the most severe and widespread toxicological
problems, with no specific antidote available.
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Synthetic biology potential: Nanobody engineering offers a powerful yet underexplored avenue for developing
precision antidotes.
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Inspiration: Previous studies showed that molecules like Polymyxin B can mitigate amanitin toxicity in mice,
inspiring us to expand this approach using modern synthetic biology tools.
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Transferability: Our modular system could be adapted to neutralize other toxins, broadening its impact beyond
mushroom poisoning.
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Future perspectives: Our LNP nanoparticles can also be loaded with supporting drugs such as polymyxin B, enabling
synergistic therapy and enhancing the efficacy of the nanobodies. Furthermore, nanobodies targeting other toxins
can be developed, produced, and validated using our workflow.
With DeathCapTrap, our goal is not only to develop a potential life-saving antidote against α-amanitin, but also to
establish a broadly applicable platform for the rapid design of nanobody-based therapies, delivered to humans
through lipid nanoparticles.