Entwicklung einer lichtinduzierten Proteinsynthese in optogenetisch aktivierbaren Säugerzellen für therapeutische Applikationen

authored by: Nina Louisa Wichert
supervised by: Cornelia Blume
Abstract: In optogenetics, a combination of optics and genetics, cells become light-sensitive through genetic manipulation, so cell functions can be specifically controlled by light, diseases can be analyzed and new approaches for cures can be developed. An optogenetic system consists usually of two transcription factors that assemble into a unit under light activation of a chromophore, which activates the promoter of the target sequence and starts protein synthesis. The aim of the present work was the development of a light-induced protein synthesis in optogenetically activatable human cells for therapeutic applications. For example patient-derived mesenchymal stem cells (hMSCs) could be placed on the CI (cochlear implant) during implantation and synthesize BDNF (brain-derived neurotropic factor) upon light induction. BDNF has a positive effect on spiral ganglion cells which in turn enhance auditory mediation of CIs. The good temporal and spatial resolution of optogenetic systems spares surrounding tissue and minimizes unwanted side effects. Mammalian cells were used as model cells. Establishment of the optogenetic PhyB (phytochrome B) system in CHO-K1 (Chinese hamster ovary) cells with EGFP (green fluorescent protein) marker resulted in 5-fold higher protein expression after optimization compared to leakage while a 26-fold increase in protein expression, was obtained for the CRY2 (cryptochrome 2)-system in HEK293 (human embryonic kidney) cells with luciferase marker. Analysis of gene expression by qPCR (quantitative polymerase chain reaction) confirmed these results. Both systems could be activated by LED (light emitting diode) induction as well as by laser induction and synthesized the protein BDNF optogenetically whereas only the CRY2-system produced a therapeutically relevant concentration. Various transfection methods were tested to transfer these systems into hMSCs here isolated from adipose tissue. Chemical transfection (using DreamFect™ Gold) provided the best results with a single transfection efficiency of about 35%, but co-transfection resulted in a transfection efficiency of only 6%. Thus it is not yet applicable for therapeutic applications in this form.
Organisation(s): Institute of Technical Chemistry
Type: Doctoral thesis
No. of pages: 228
Publication date: 2023
Publication status: Published
Sustainable Development Goals: SDG 3 - Good Health and Well-being
Electronic version(s): https://doi.org/10.15488/13719 (Access: Open)