A genome-wide CRISPRi screen to unravel the role of liposomal phospholipid composition in phagocytic cell uptake and subsequent tolerogenic immune responses
Abstract
The phospholipid composition of antigen-carrying liposomal vaccines has profound effects on the resulting antigen-specific immune response. With this knowledge, nanoparticulate vaccines can be designed to either promote a pro-inflammatory response (i.e. for anti-tumor or anti-pathogenic effects), or anti-inflammatory response (i.e. against chronic inflammation or autoimmunity). While it is generally achievable to induce pro-inflammatory immune responses using adjuvant-free antigen-carrying nanoparticles, it is more challenging to induce tolerance.1)2)
The majority of tolerance-inducing vaccines consist of antigens associated with anionic phospholipid-based nanoparticles, such as liposomes, while pro-inflammatory vaccines consist of cationic phospholipids. Antigen-presenting cells efficiently take up these nanoparticles, which alters the phenotype of the antigen-presenting cell towards either a tolerogenic or immune-activating phenotype. However, fundamental insights into the mechanism of uptake of these nanoparticles are lacking. This is a significant obstacle to the development of nanoparticulate vaccines. This proposal aims to identify relevant molecular pathways for nanoparticle adhesion and uptake by a CRISPR/Cas interference (CRISPRi) screen, screening several different liposomal compositions. The CRISPRi screen will give unbiased information about the positive and negative regulators of liposomal uptake, which have so far been out of reach using other methods. Hits from this unbiased screen will be validated and novel liposomal formulations will be designed to improve tolerance induction by liposomal vaccines.
In this proof-of-concept study, we will validate the hits for liposomal uptake, and link the loss of receptor-mediated uptake of liposomes to a loss of tolerance induction. We will select genes from the CRISPRi screen for validation, silence these in THP-1 cells, and measure the changes in uptake of fluorescently labelled DSPC:DSPG:CHOL liposomes using flow cytometry. Further, we will select top hits with the strongest phenotypes and validate the immune effects by ELISA, qPCR and flow cytometry.
Benefit for the community
Our CRISPRi screening approach will identify genes that are important for the uptake of tolerance-inducing liposomes by phagocytic cells. The fundamental information we gather in this proposal will be novel and highly interesting to the phospholipid community and researchers in the field of nanoparticulate vaccines. Our findings will be a significant step forward to bringing tolerance-inducing nanoparticulate vaccines to patients.
Visit the supervisors lab
Contact to Naomi Benne and Rutger D. Luteijn.
