Exosomes as Potential Transport Vehicles of Tetrahydrobiopterin, 6-Pyrovyoltetrahydrobiopterin-Synthase and Tripeptidyl-Peptidase I

by Kristina Lang
Doctoral thesis
Date of Examination:2018-11-30
Date of issue:2018-12-14
Advisor:Prof. Dr. Dr. Robert Steinfeld
Referee:Prof. Dr. Blanche Schwappach
Referee:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Ralf Dressel
Referee:Prof. Dr. Hubertus Jarry
Referee:Dr. Nuno Raimundo
Persistent Address: http://dx.doi.org/10.53846/goediss-7199

 

 

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Abstract

English

Extracellular vesicles (EV) for the therapeutic application are of growing scientific interest. But even though the number of clinical trials on EVs almost doubled from 29 to 52 within 2017, the number of trials with therapeutically applied EVs was only two. In this project, the performance of folate receptor alpha (FRα) presenting EVs as drug vehicles in neuropediatric disorders was investigated. FRα was proven to be the key player for the transport of folates to the brain. On that account, FRα-presenting EVs are predestined to be used as vehicles for therapeutics which are naturally not processed across the blood-cerebrospinal fluid-barrier. The application of EVs as carriers is hampered by the complex nature of this biological material and a still strongly evolving field of methodology. In this project, central questions of the production of EV therapeutics were addressed: preparation, cargo loading and biological screening. For a reliable purification of large amounts of highly pure EVs, a unique fast performance liquid chromatography purification method for his-tagged vesicles was established. This method is made up by size exclusion chromatography followed by immobilized metal affinity chromatography. The preparation was comprehensively characterized and verified as exosome-like vesicles. In the second phase, the loading of therapeutic cargo was performed. While artificial loading of therapeutic cargo based on the introduction of pores into the vesicles and addition of the drug protein had low efficiency, better results could be achieved by endogenous loading by means of a membrane targeted drug protein. In choroid plexus cells from rat an increased uptake of these modified EVs in comparison to native vesicles could be proven. This uptake tendency could not be manifested for the brain in in vivo experiments using adult mice. Finally, the neuronal drug delivery capacity of these promising vehicles was screened in a mouse model of a biopterin disorder. In these experiments, an enzyme replacement therapy delivered by EVs could be realized for the first time. The mice had a significant improvement in their health status and performed better in a behavioral assay in comparison to not exosome-treated animals. By this successful treatment, evidence for an EV based drug delivery to the central nervous system of an enzyme replacement therapy was given.
Keywords: Exosomes; Extracellular vesicles; Drug delivery; Neuronal ceroid Lipofuscinosis; Biopterin disorder; Folate receptor
 
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