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Development of an in vitro method for the evaluation of neurotoxic substances based on neurons differentiated from induced pluripotent stem cells

Methods:

Differentiation protocols for the differentiation of human induced pluripotent stem cells (iPSCs) or neural progenitor cells to motor neurons and the analysis of target genes
Gene editing via CRISPR/Cas9 for stable integration of a reporter gene construct for gaussia luciferase

Project description:
Botulinum neurotoxin (BoNT) is one of the most potent known bacterial toxins, which prevents the release of neurotransmitters at the neuromuscular end plate and causes flaccid paralysis of the skeletal muscles. Besides the botulism caused by BoNT, a feared food poisoning, BoNT is used in the pharmaceutical industry. BoNT is used for the treatment of various diseases and especially in aesthetic surgery for smoothing mimic wrinkles. For pharmaceutical application BoNT is obtained from bacterial cultures.  Due to their potentially extremely pronounced toxicity, the individual batches must be examined for their individual potency. With the previous gold standard, the mouse LD50 test, several hundred thousand animals have to be used worldwide every year. Immunological alternative methods developed to date are serotype-specific. However, eight different serotypes of BoNT are known so far.  A new approach to circumvent this limitation is to express luciferase specifically in neuro-secretory vesicles of neuronal cell lines.  Depolarisation of the cell leads to the release of luciferase, which can be quantified by luminescence measurement.  The application of BoNT in pharmacologically relevant concentrations can prevent the exocytosis of the vesicles and thus to the release of luciferase. This process is not serotype-dependent, which facilitates the identification of BoNT and is an advantage over other existing alternative methods. The cell lines used so far are neuronal tumour cell lines. This research project aims to further develop the existing test system by using induced pluripotent stem cells (iPSCs), which will then be transfected with the corresponding reporter gene, in order to develop an alternative, human-relevant test system for testing the neurotoxic potential of BoNT and its serotypes.

 

Funding:

MoNLight-BoNT, German Ministry of Education and Research

 

Project Lead:
Prof. Bettina Seeger, PhD (bettina.seeger@tiho-hannover.de; 0511-856-7602)
Institute for Food Toxicology, University of Veterinary Medicine Hannover, Foundation, Germany

Maren Schenke, PhD (bettina.seeger@tiho-hannover.de; 0511-856-7603)
Institute for Food Toxicology, University of Veterinary Medicine Hannover, Foundation, Germany

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