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Dr. Julia Ladewig

DFG - Deutsche Forschungsgemeinschaft : Deciphering alcohol addiction-associated gene regulation changes on a single cell level. 01/2020-12/2022.

Changes in brain structure and function that results from chronic exposure to alcohol suggest that neuroadaptive alterations in gene regulation substantially contribute to addictive behavior. Transcriptional and epigenetic profiling of brain tissue from animal models and post-mortem human samples has identified multiple candidate genes to be dysregulated upon chronic alcohol exposure. However, a detailed assignment of those changes to specific cell types has not been performed due to technical limitations and lack of appropriate tissue. In this planned consortium, we will apply paralleled single cell sequencing to decipher transcriptional and epigenetic changes underlying alcohol addiction. We will perform single nuclei RNAsequencing (snRNA-seq) and snATAC-seq epigenetic profiling using postmortem tissue from a well-defined, high-quality brain bank of deceased alcohol addicts. We have previously established a novel snRNA-seq platform that allows isolating and sequencing of individual nuclei from snap-frozen brain samples obtained from patients with other neuropsychiatric diseases. In parallel we will use standardized human iPSC-derived forebrain organoids from controls and alcohol addicts to monitor alcohol-induced changes in gene regulation and gene expression in an isogenic (non-exposed vs. exposed; acute, chronic intermitting, acute withdrawal) forward approach. We expect that the proposed project will deliver the largest available database on alcohol addiction-associated gene regulation changes on a single cell level and help define critical contributors in the pathogenesis of alcohol addiction eventually eading to new therapeutic paradigms.

Ladewig J. DFG - Deutsche Forschungsgemeinschaft LA 2933/3-1 : MECPer-3D: "Personalisierte MECP2-Gentherapie unter Verwendung der CRISPR / Cas9-Technologie gekoppelt mit AAVvermittelter Abgabe in 3D-Zellkultur- und KI-Mäusen. 01/2024-02/2021.

Rett syndrome is one of the most common causes of intellectual disability in girls, resulting in severe cognitive and physical disabilities. The classic form is caused by mutations in the transcriptional regulator MECP2. Effective therapies are not currently available and gene editing based on CRISPR/Cas9 combined with Homology Directed Repair appears an appealing option for the development of new therapeutic approaches. We already engineered a gene editing toolkit and demonstrated its ability to efficiently correct one of the most common MECP2 mutation, c.473C>T - (p.(T158M)), in patient cells. Based on these results, in this project we will further validate constructs for this mutation and develop and validate toolkits for three other MECP2 mutation hotspots, namely c.502C>T (p(R168X)), c.763C>T (p.(R255X)), c.916C>T (p.(R306C)). To characterize the potential of our approach in a relevant context and define its efficiency in a human 3D model, we will employ brain organoids differentiated from patient-derived induced pluripotent stem cells. Thanks to the ability of specific AAV serotypes to cross the Blood Brain Barrier (BBB) following intravenous injection, we will test our system in KI mice to validate efficacy and safety in vivo. Moreover, since available AAV serotypes have an imperfect brain tropism, with significant distribution to other organs, new serotypes will be developed and validated for their ability to cross the BBB in the mouse and their efficacy and specificity in human cells. These experiments will allow us to demonstrate the full potential of gene editing as a therapeutic option for Rett and for other neurodevelopmental disorders currently lacking an effective treatment.

Ladewig J. DFG - Deutsche Forschungsgemeinschaft LA 2933/2-1: Cerebral organoids to decipher molecular mechanisms perturbed in EML1 induced ribbonlike subcortical heterotopia . 08/2020-07/2020.

Ladewig J. BMBF - Bundesministerium für Bildung und Forschung 01EW1611: Neuron Verbund STEM-MCD: Stammzellen und Mechanismen die zu humanen kortikalen. 04/2019-05/2020.

Die Entwicklung des menschlichen Gehirns sowie seine Architektur sind durch eine immense Vergrößerung der Oberfläche bedingt durch eine komplexe Faltung in Gyri und Sulci charakterisiert. Kortikale Fehlbildungen können sehr schwerwiegend sein und zu geistiger Behinderung sowie Epilepsie führen. In Mausmodelle können bestimmte Aspekte der fehlerhaften Entwicklung nachvollzogen werden, jedoch fehlen im Maus Gehirn Zelltypen die für die menschliche Gehirnentwicklung entscheidend sind. Diese Zellen spielen eine wichtige Rolle während der Proliferation, der Vervielfältigung und der Organisation von Nervenzellen. Um diese Prozesse während der Entwicklung des menschlichen Gehirns und die Variabilität von entsprechenden kortikalen Fehlbildungen zu studieren, können Patienten abgeleitete reprogrammierte Zellen genutzt werden (iPSZ), welche gewisse Aspekte der menschliche Gehirnentwicklung in der Zellkulturschale wiederspiegeln können. Unter Verwendung von modernsten Mikroskopie-Technologien sollen Entwicklungsstörungen des menschlichen Gehirns visualisiert werden. Molekulare Mechanismen der Fehlbildungen sollen mit Hilfe von globaler Genexpression sowie neusten Technologien zur Hochdurchsatz-Sequenzierung von RNA aus einzelnen Zellen geklärt werden. Die Identifizierung von molekularen und zellulären Mechanismen wird uns gestatten auf gestörte subzelluläre Prozesse zu fokussieren und nach korrigierenden Strategien zu suchen.


Zentralinstitut für Seelische Gesundheit (ZI) - https://www.zi-mannheim.de