Tübingen 2018

Development of a shuttle mechanism using detoxified botulium toxin

In 2017 Vaquez-Cintron et al published a series of point mutations in botulinus toxin C that reduced the toxicity by a factor of approximately 5 x 10⁶, while still being able to transport the light chain into the target cell cytosol.

This construct therefore should enable molecule trafficking to a single cell type with botulinus toxins' intrinsic, excellent specificity and activity. Targeting neurons only, this method would offer various advantages over antibody-drug conjugates, which have been a great inspiration for this project.

1. Botulinus toxins are already poised to internalize, which can be a major limitation for antibodies.
2. Contrary to antibodies, botulinus toxins do not need glycosylation, allowing for quick, easy and cheap expression in bacterial or insect cells.
3. As shown by Bade et al 2004, botulinus toxins can in theory carry even moderately large Proteins into the target cell cytosol. This could allow for transient internalization of marker and/or rescue proteins for short term treatments.
4. It is easily imaginable that an antibody-drug conjugate that targets neurons could interact with lymphocytes, which would cause major problems to the organism.

Our project tries to explore the range of capabilities for internalization of the nontoxic botulinus toxin C, by fusing various proteins and small molecular compounds to it.

We aim to be able not only to target specific cells, but also their organelles, such as the nucleus or mitochondria with our final delivery system. By trying out cleavable linkers, we will attempt to release our fused molecules from the botulinus light chain, once it has invaded the target cell cytoplasm, thereby increasing the activity of especially the small molecular compounds. Finally we wish to make our delivery system as modular as possible. The complexities of all parts of the process increase, as the protein size increases. Therefore we aim to introduce sites for sortase-mediated protein fusion and genetic code expansion into this botulinus toxin.

Such an approach would simplify usage of our construct to a large degree, as expression conditions would not need to be optimized for each variation. Any peptide containing a C-terminal recognition motif LPXTG could be fused in vitro using the sortase competent toxin, whereas other molecules could be fused to our genetic code expanded toxin, applying the large variety of bio-orthogonal chemistry, such as Azide-alkyne, inverse electron demand diels-alder, aldehyde-hydroxamine/hydrazone, staudinger ligation or similar methods.