| dc.contributor.advisor | Zelarayán, Laura C. PD Dr. | |
| dc.contributor.author | Schoger, Eric | |
| dc.date.accessioned | 2022-11-17T15:39:38Z | |
| dc.date.available | 2022-11-18T00:50:08Z | |
| dc.date.issued | 2022-11-17 | |
| dc.identifier.uri | http://resolver.sub.uni-goettingen.de/purl?ediss-11858/14347 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-9564 | |
| dc.language.iso | deu | de |
| dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | |
| dc.subject.ddc | 610 | |
| dc.title | In vivo application of CRISPR/Cas9 mediated gene re-expression in the failing mouse and human myocardium | de |
| dc.type | cumulativeThesis | de |
| dc.contributor.referee | Schwappach, Blanche Prof. Dr. | |
| dc.date.examination | 2021-11-19 | de |
| dc.description.abstracteng | Transcriptional adaptation in cardiomyocytes precedes cellular deterioration and functional decline upon myocardial stress and damage resulting in heart failure progression. The re-activation of WNT/β-catenin (CTNNB1) signaling, an evolutionary conserved developmental cascade, was identified as a driver of disease progression in the hypertrophic myocardium. In the homeostatic adult heart, WNT/CTNNB1 signaling is inhibited by the transcription factor Krüppel-like factor 15 (KLF15). Consequently, transcriptional loss of KLF15, which is occurring upon disease progression, leads to the activation of WNT/CTNNB1 signaling concomitant with cardiomyocyte hypertrophy and pathological remodeling. Aiming to identify cardiac-specific regulators of the ubiquitously involved WNT/CTNNB1 pathway, a nuclear, cardiomyocyte-specific, multimeric protein complex consisting of CTNNB1, TCF7L2, KLF15 and BZW2 was elucidated and characterized. Upon improved understanding of the molecular determinants of WNT/CTNNB1 modulation in heart tissue, I aimed to investigate mechanisms for re-establishment of gene regulatory networks by restoration of transcription-controlling homeostatic protein-complexes. Although therapeutically desired, the implementation of this concept is technically challenging. To tackle this problem, CRISPR/Cas9 based gene activity modulation with catalytically inactive Cas9 (dCas9) targeted to gene regulatory regions by guide RNAs (gRNA) in combination with transcriptional activators (VPR) or repressors (KRAB) were repurposed as a platform for synthetic gene activation (CRISPRa) and inactivation (CRISPRi). In this work, a mouse model for endogenous, dCas9VPR-mediated gene activation was established, which efficiently and safely expressed dCas9VPR exclusively in cardiomyocytes. As a proof-of-concept for control over endogenous gene activity in vivo, I demonstrated induced expression of Myocyte enhancer factor 2 D (Mef2d) to recapitulate a hypertrophic cardiomyopathy phenotype within eight weeks after gRNA delivery. Furthermore, titratable, endogenous activation of epigenetically silenced Klf15 was achieved in neonatal mice. Furthermore, I aimed to re-activate Klf15 expression in murine hearts upon pressure overload using the established model to achieve transcriptional re-establishment of Klf15 levels in stressed cardiomyocytes. This resulted in blunted cardiac hypertrophy and reduced organ deterioration. To study the relevance of this approach in human cells, CRISPR/dCas9 systems were introduced into human induced pluripotent stem cell (hiPSC) derived cardiomyocytes. I generated hiPSC expressing dCas9VPR (CRISPRa) or dCas9KRAB (CRISPRi) for full control over endogenous gene activity by targeted transgene integration at the AAVS1 locus using CRISPR/Cas9 gene editing and homology directed repair. Pluripotency and differentiation potential of these hiPSC lines was not affected by transgene integration or constitutive dCas9VPR or dCas9KRAB expression. These cells were differentiated into spontaneously beating hiPSC-derived cardiomyocytes (hiPSC-cardiomyocytes). Expression of KLF15 was inducible or repressible, respectively, as well as titratable by applying single or multiple gRNA lentivirally administered or by using cells with distinct dCas9 expression levels. With these cells, engineered human myocardium (EHM) was generated to test transcriptional enhancement of KLF15 in a 3D model of surrogate human heart tissue. I modelled myocardial stress in EHM by forcing tissues to isometric contractions and confirmed a molecular stress response including a transcriptional loss of KLF15 reminiscent of the in vivo disease condition which was mimicked by TGFB1 stimulation in hiPSC-cardiomyocytes in vitro. Moreover, the validation of multiple gRNAs targeted to a total of 10 mouse and 5 human genes allowed to re-define the selection criteria for suitable gRNA design. This was related to the gRNA target site relative to the transcriptional start site (TSS) for dCas9VPR gene activation purposes. Altogether, I contributed to the investigation of potential regulatory target gene expression modulation including KLF15. I showed that CRISPR/dCas9 based transcriptional activation is a suitable tool for titratable, endogenous gene activation in both human and mouse cardiomyocytes. The transcriptional loss was rescued by synthetic gene activation, restoring KLF15 mRNA levels comparable to non-stressed conditions in vivo and in vitro. This was accompanied by reduced maladaptive cardiomyocyte remodeling and a reduced cellular response to myocardial stress, which validated the approach. Overall, this work integrated knowledge of fundamental biology to generate tools, that allowed precise and efficient gene activity modulation to prevent heart failure progression and provided a new platform for the identification and validation of potential therapeutic targets. | de |
| dc.contributor.coReferee | Behr, Rüdiger Prof. Dr. | |
| dc.contributor.thirdReferee | Meyer, Thomas Prof. Dr. | |
| dc.contributor.thirdReferee | Lutz, Susanne Prof. Dr. | |
| dc.contributor.thirdReferee | Streckfuß-Bömeke, Katrin Prof. Dr. | |
| dc.subject.eng | CRISPR/Cas9 | de |
| dc.subject.eng | Gene activity control | de |
| dc.subject.eng | Cardiac remodeling | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-ediss-14347-4 | |
| dc.affiliation.institute | Medizinische Fakultät | |
| dc.subject.gokfull | Pharmakologie / Toxikologie / Pharmakotherapie - Allgemein- und Gesamtdarstellungen (PPN61987550X) | de |
| dc.subject.gokfull | Gentherapie (PPN619875194) | de |
| dc.subject.gokfull | Methoden und Techniken in der Medizin (PPN619875143) | de |
| dc.description.embargoed | 2022-11-18 | de |
| dc.identifier.ppn | 1822865476 | |
| dc.identifier.orcid | 0000-0002-5485-9828 | de |
| dc.notes.confirmationsent | Confirmation sent 2022-11-17T15:45:01 | de |