| dc.contributor.advisor | Cyganek, Lukas Dr. | |
| dc.contributor.author | Zimmermann, Maximilian | |
| dc.date.accessioned | 2019-12-02T07:19:39Z | |
| dc.date.available | 2019-12-09T23:50:03Z | |
| dc.date.issued | 2019-12-02 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0005-12BB-4 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7693 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7693 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 610 | de |
| dc.title | Genetic modification in CPVT patient specific induced pluripotent stem cells with CRISPR/Cas9 | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Guan-Schmidt, Kaomei Prof. Dr. | |
| dc.date.examination | 2019-12-02 | |
| dc.description.abstracteng | CPVT is a genetic cardiac disorder leading to sudden cardiac death in young patients without structural abnormalities of the heart. Abnormal Ca2+ handling in CMs causes severe arrhythmia during physical or emotional stress through missense mutations in the RyR2 or CASQ2 gene. Over 150 mutations in the RyR2 gene causing CPVT1 have been identified so far. The exact underlying pathomechanism causing a typical disease pattern induced by point mutations is not yet fully explored. The objective of this research is to contribute to the establishment of in vitro disease models for CPVT with hiPSCs as a source for CMs.
CPVT disease models of this project were based on hiPSCs from CPVT patients b1 and c2, who respectively had a missense mutation A2254V or E4076K at different loci. In this study pluripotency was proven in hiPSCs from CPVT patient c2. The differentiation potential of hiPSCs into all three germ layers ectoderm, mesoderm and endoderm was verified as well. The hiPSCs from CPVT patient b1 were characterized in previous studies.
For further investigation of the point mutation-based phenotype in CPVT patients CRISPR/Cas9 technology was applied for genome editing in hiPSCs from the CPVT patient b1. The study failed to fulfill the aim to correct the single point mutation in hiPSCs from patient b1 to generate an isogenic corrected control cell line. Nevertheless, homogeneous CRISPR/Cas9-edited hiPSCs with a knockout of the RYR2 protein were generated, characterized for pluripotency-associated marker and differentiated into CMs. The CRISPR/Cas9-edited CMs with homozygous deletions in RYR2 showed normal RYR2 mRNA expression, but no expression of the RYR2 protein. The CRISPR/Cas9 edited CMs showed a typical expression pattern for the remaining cardiac marker on mRNA and protein level. Surprisingly, RyR2_X4_KO-CMs were able to contract without the expression of RYR2 protein compared to the control CPVT-CMs and showed no CPVT typical Ca2+ leak.
In conclusion, the data of this study show that patient-specific hiPSCs are useful tools to create CPVT disease models. Even though an isogenic corrected control cell line could not be generated in this study, CRISPR/Cas9 systems are a promising tool for genetic manipulation in hiPSCs.
RyR2_X4_KO-CMs offer the possibility to analyze the compensatory adaption mechanism for a sufficient Ca2+cycling without functioning RYR2 protein. As a result, future drug targets can be developed. | de |
| dc.contributor.coReferee | Hülsmann, Michael Prof. Dr. | |
| dc.contributor.thirdReferee | Dressel, Ralf Prof. Dr. | |
| dc.subject.eng | iPSC | de |
| dc.subject.eng | CPVT | de |
| dc.subject.eng | CRISPR/Cas9 | de |
| dc.subject.eng | stem cell | de |
| dc.subject.eng | cardiology | de |
| dc.subject.eng | Ryanodine receptor 2 | de |
| dc.subject.eng | pluripotency | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-12BB-4-1 | |
| dc.affiliation.institute | Medizinische Fakultät | de |
| dc.subject.gokfull | Medizin (PPN619874732) | de |
| dc.description.embargoed | 2019-12-09 | |
| dc.identifier.ppn | 1684041244 | |