Image-guided genome editing of tuberous sclerosis complex using CRISPR-CAS9 system

• Applicant: Dr. Abdallah Salemdawod
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Radiology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 501126763

Image-guided genome editing of tuberous sclerosis complex using CRISPR-CAS9 system Scientific problem: Tuberous sclerosis complex (TSC) is caused by defective TSC1 or TSC2 genes, which lead to over-activation of the mammalian target of rapamycin (mTOR) protein complex. Clinically, it manifests as a complex syndrome including drug-resistant epilepsy, epileptogenic tumors, neurodevelopmental delay, and extracranial abnormalities. Highly epileptogenic benign brain tumors represent the greatest therapeutic challenge . Current treatment options targeting the TSC are limited to cytostatic drug application leading to rapid tumor regrowth after cessation of treatment, underscoring the need to identify new approaches for long-lasting clinical effect. Rationale: The monogenetic basis of TSC inspires to capture the potential of CRISPR/Cas9 gene-editing technology to replace for the defective gene. It is well-aligned with many ongoing attempts to exploit genome editing technologies to cure genetic diseases. Here, we face an additional challenge of effective therapy delivery to the brain. The advanced image guidance is expected to help providing a precise delivery of therapeutic agents and therefore bring hope to expand genome editing to brain diseases. The well-established TSC mouse model developed and modified by Feliciano et al. serves as an ideal test field. By eliminating TSC1/TSC2 in selected neuronal populations in mice, hyperactivity of mTOR1 leads to the exhibition of cortical tuber-like lesions containing cytomegalic and multinucleated neurons with abnormal dendritic trees resembling giant cells (Feliciano et al. 2011). Experimental design: The project is divided into five following specific aims (SAs). SA1: Designing of ribonucleoprotein (RNP) complex capable of replacing defective TSC gene and testing it in the cell-free in vitro plasmid model. SA2: Replacement of defective gene in vitro in cells obtained from an animal model of TSC using RNP. SA3: Radiolabeling of RNP for image-guided neurointerventions SA4: Local delivery of RNP to the brain as an in vivo proof-of-concept SA5: Comparison of the intra-arterial route and focused ultrasound as strategies for global RNP delivery to the brain. Significance:This project will examine whether the introduction of a functional TSC1/TSC2-gene has a therapeutic effect in the TSC animal model. The outcome will be considered a breakthrough in TSC therapy as it is expected to definitively eliminate the cause of disasese by replacing a mutated gene. Furthermore, it might also have further important implications for engineering efficient gene therapeutics in other mono- or oligo-genetic neurological conditions.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Dr. Miroslaw Janowski