Postdoc on EV-mediated CRISPR-Cas9 delivery (2024)

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CRISPR-Cas9 can be employed to create dsDNA breaks, generate precise mutations, or incorporate specific sequences, regulate RNA transcription. One major hurdle in the development of Cas9-based therapies is the intracellular delivery of Cas9-sgRNA complexes. To address this challenge, your project will focus on the development of extracellular vesicle-mediated strategies for CRISPR-Cas9 delivery, and apply these strategies to integrate DNA sequences into target cells.

Your job
Over the last decade, CRISPR/Cas9-based techniques have been developed for various genomic engineering strategies in eukaryotic cells. Using single guide RNAs (sgRNA) containing specific targeting sequences, Cas9 can target precise genomic locations. Thus, CRISPR/Cas9-based techniques are of substantial interest for the development of novel therapies to treat genetic diseases. Moreover, as they can be targeted to precise genomic targets with high specificity, they have considerable therapeutic potential for currently “un-druggable” pathologies. Unfortunately, one of the major hurdles in the development of CRISPR/Cas9-based therapies is the intracellular delivery of Cas9-sgRNA complexes. Cas9-sgRNA complexes are unable to cross the cell membrane due to their large size, as well as their negative charge. Moreover, Cas9-sgRNA complexes need to be shielded from opsonization and degradation in circulation, do to their immunogenicity. Your project will focus on the development of extracellular vesicle (EV)-mediated strategies for CRISPR/Cas9 delivery to address these challenges.

Under the combined supervision of Dr Olivier de Jong (UU) and Dr Pieter Vader (UMCU) you will develop and optimise extracellular vesicle-mediated loading and delivery strategies of CRISPR-Cas9 ribonucleoprotein (RNP) complexes and DNA templates and study the uptake pathways and kinetics in target cells. As a functional readout, the engineered EVs will be studied for their efficiency in genomic engineering based on hom*ology-independent targeted insertion (HITI) and hom*ology-directed repair (HDR) pathways. You will design and test fusion proteins through conventional cloning techniques in DNA plasmids. These fusion proteins will consist of EV-enriched proteins, Cas9, and previously confirmed cleavable linker sequences. These plasmids will then be transfected into EV-producing cells to test their efficacy in Cas9 loading and delivery. Initial DNA templates will encode fluorescent (eGFP) reporter genes and will be tested on various cell lines. If successful, DNA sequences of CAR-T cell receptors will be loaded into EVs and delivered to various relevant cell types, including Jurkat cells and PBMC-derived T-cells. To gain a better understanding of the cellular processes that play a role in EV-mediated cargo delivery, cellular uptake, intracellular trafficking, and delivery will be studied using confocal microscopy. Experimental work will be performed at the department of Pharmaceutics at Utrecht University, as well as at the CDL Research department at the University Medical Center.

This project is funded by the Eurostars funding programme, and this postdoctoral research position will be part of a collaboration between various academic partners (Utrecht University, dept. of Pharmaceutics; University Medical Center Utrecht, CDL Research dept.) and industrial partners (ExoVectory and EVerZom). The goal of this consortium is to combine the expertise and technologies of the consortium members to develop novel strategies for EV-mediated delivery and genomic integration of DNA sequences using CRISPR-Cas9. These expertises consist of EV-mediated Cas9 delivery (UU), EV uptake and trafficking (UMCU), DNA loading and delivery (ExoVectory), and up- and downstream processing for scalable EV production (EVerZom), respectively.