Achievements
We designed different plasmid constructs for an anti-GFP nanobody and GFP, carrying a secretion signal for Designing a Theoretical In Vitro Pipeline to Test Our Nanobody-Based Antidote
Although we could not implement all experiments within the iGEM timeframe, we achieved a major milestone: the theoretical design of a complete in vitro experimental pipeline to investigate the feasibility of our therapeutic nanobody strategy against the deadly mushroom toxin α-amanitin.
Framework
This conceptual framework, developed in close exchange with the Liver Lab at the University Hospital Hamburg-Eppendorf (UKE), allowed us to map out how our antidote could be tested step by step from the bloodstream to the nucleus of liver cells. By addressing both extracellular and intracellular compartments, we created a rational, multi-layered workflow that future researchers can directly adapt and implement.
Why this is important
- Scientific novelty: Our concept integrates freely circulating nanobodies with intracellularly delivered nanobodies via lipid nanoparticles (LNPs). This two-compartment neutralization strategy is unique.
- Feasibility assessment: By breaking the project into testable steps, we ensured that critical questions are addressed systematically before moving toward animal or clinical studies.
- Transferability: Our pipeline is modular, allowing replacement of the toxin–nanobody pair with any other therapeutic protein and target system.
Our key design achievements
- Defined experimental readouts for each step, from ELISA binding assays in blood to live-cell imaging of nuclear nanobody–toxin interactions.
- Outlined state-of-the-art analytical methods, including ELISA, confocal microscopy, ATP-based viability assays, and FOCI co-localization analysis.
- Integrated a mock system (GFP/anti-GFP nanobody) as a safe validation tool before handling α-amanitin.
- Considered biosafety and ethics by proposing cell-based and organoid assays as alternatives to animal testing.
This theoretical pipeline forms the scientific backbone of our project. It demonstrates that our approach is not only imaginative but also experimentally actionable. With additional time and resources, each step can be realized, bringing us closer to a novel antidote for α-amanitin poisoning.
MENTAL LAB
The deadly mushroom toxin α-amanitin enters the bloodstream after ingestion and accumulates in the liver, where it concentrates in the nucleus of hepatocytes and inhibits RNA polymerase II. Our therapeutic concept aims to neutralize the toxin in two compartments: (1) in the bloodstream, using freely circulating nanobodies, and (2) inside hepatocytes, particularly in the nucleus, using nanobodies delivered by lipid nanoparticles (LNPs).
Two Battlefields Against Amanitin: Bloodstream and Nucleus
To test the feasibility of this strategy, we developed an experimental in vitro pipeline. However, we didn't have enough time within the IGEM framework to implement it in practice. Here we describe a strategy we conceived theoretically, with the support of the University Hospital Hamburg-Eppendorf (UKE) Liver Lab. In doing so, we addressed the following key questions:
- Does the nanobody bind the toxin in the blood?
- Do LNPs successfully dock to liver cells and get taken up?
- Is the nanobody released from the LNPs after uptake?
- Is the nanobody imported into the nucleus via its nuclear localization sequence (NLS)?
- Does the nanobody bind the toxin in the nucleus?
- What is the survival rate of hepatocytes with and without therapeutic treatment?
To assess the interaction between nanobodies and α-amanitin, we propose an ELISA-based assay. Nanobodies will be immobilized on a solid support surface, and samples containing defined concentrations of α-amanitin will be applied. We will also include controls without nanobodies in parallel. Following incubation, the amount of unbound toxin will be quantified using a commercially available amanitin-specific ELISA kit. By comparing the initial toxin input with the residual unbound fraction, we can determine the binding efficiency of the nanobodies.
Additionally, we plan to validate our experimental pipeline using a mock–toxin, in our case GFP and an anti-GFP nanobody, which we previously employed in the wet lab as a test system for protein production.
Commercial ELISA kits serve as a benchmark for sensitivity.
ELISA Assay with our designed anti-alpha-amanitin nanobody
The Cell Model
For cellular assays, we selected the HepG2 human hepatocellular carcinoma cell line. These immortalized hepatocytes are widely used for experiments regarding drug uptake, imaging, apoptosis, and cytotoxicity assays. Although not fully representative of healthy liver tissue, they are robust, well characterized, and safer to handle than primary hepatocytes. Standard cultivation requires media such as EMEM, DMSO, FBS, antibiotics, and growth factors, with ethical concerns associated with FBS. Cells would be expanded 1–2 weeks prior to experiments to ensure optimal viability.
Effects on Cell Viability
Cell survival will be quantified using a luminescence-based ATP assay (e.g., CellTiter-Glo), which offers greater sensitivity than the commonly used Trypan Blue exclusion assay. Cells will be exposed to varying concentrations of α-amanitin with and without therapeutic treatment, and viability will be measured at multiple time points.This approach allows us to determine both the toxicity thresholds of poisons and the therapeutic efficacy of antidotes.
To investigate cellular uptake (for more information see LNP design and description), LNPs will be loaded with a fluorescent reporter (e.g., GFP) and incubated with HepG2 cells. Uptake and intracellular localization will be monitored using confocal fluorescence microscopy. Co-localization with endosomal markers can reveal whether LNPs remain trapped in endosomes, which may hinder therapeutic release. For higher-resolution analysis, electron microscopy could be employed, though this method is technically demanding.
Release of Nanobody Inside Cells
Nanobodies labeled with a suitable fluorophore will be packaged into LNPs and delivered to cells. Confocal microscopy will distinguish between punctate fluorescence (nanobody confined to vesicles) and diffuse cytoplasmic/nuclear fluorescence (nanobody successfully released).
Nuclear Import of Nanobody
To evaluate nuclear import mediated by the nuclear localization sequence (NLS), fluorescently-labeled nanobodies will be visualized alongside DAPI-stained nuclei using confocal fluorescence microscopy. Co-localization of nanobody fluorescence with the nuclear signal would demonstrate successful import.
Nanobody–Toxin Interaction in Nucleus
A multi-co-localization (FOCI) analysis will assess whether nanobodies bind α-amanitin in the nucleus. Cells will be treated with toxin (which is also fluorescently labeled or GFP as a surrogate) and fluorescently labeled nanobodies. Overlap of fluorescence signals from nucleus (DAPI), toxin, and nanobody will indicate nuclear binding events.
Live-Cell Imaging
As an alternative or complementary strategy, time-lapse live-cell imaging with LNPs, nanobodies, and the toxin labeled with different fluorophores can track the entire delivery cascade (docking, uptake, release, localization) in real time.
Beyond 2D Cell Culture: Advanced Models
While initial proof-of-concept studies rely on HepG2 monolayers, more complex models could provide higher translational value:
- 3D Liver Organoids: derived from human cells, they replicate tissue architecture and cell–cell interactions,, avoiding animal use.
- Animal Models (e.g., mice): could reveal systemic pharmacokinetics and organ crosstalk, but raise ethical concerns as they require injection of the toxin.
