Central nervous system-targeted genome editing through engineered extracellular vesicles

This project aims at developing novel modalities and delivery strategies for CRISPR-based gene therapies. The objectives are: 1) To identify novel pseudotypes to be used in combination with VesiCas to target cells in the central nervous system (CNS); 2) To develop a cell-based screening system to allow the streamlined identification of potential candidates with the desired tropism characteristics to target the CNS; 3) To improve the VesiCas production process by engineering the currently used producer cell line and by optimization of the downstream purification processes (through mass spectrometry, next-generation sequencing, microscopy analyses).

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A successful project will result in: 1) A VesiCas-based platform to allow the tissue/cell-specific delivery of different genome editing tools to neuronal cell populations in vitro and in vivo; 2) Demonstration of VesiCas cargo flexibility by incorporating different genome editors (base editors, prime editors, epigenome editors) as well as novel CRISPR nucleases; 3) Validation of the best identified pseudotypes and novel cargo proteins in a relevant CNS disease model in vitro including preliminary in vitro immunogenicity tests; 4) Optimization of the VesiCas producer cell line and purification protocols to increase the overall yields. 

 

Enrolment in Doctoral School: Università degli Studi di Trento

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Planned secondments:

• INSERM, France (Months 23-24): Introduction to nanoblades and comparison with VesiCas 

• KU Leuven, Belgium (Months 31-32): Vector production and purification from optimised cell lines