Efficient Methods for Ex Vivo Generation of Gene-edited Neutrophils

Neutrophils are the most numerous circulating leukocyte in humans and are key first responders to invading pathogens and effectors of tissue repair and homeostasis. The potent antimicrobial functions of mature neutrophils are tightly controlled due to their potential for tissue damage, and thus neutrophil dysregulation is a central feature in many inflammatory and infectious diseases. Mature human neutrophils are challenging to study due to their short lifespan in culture and propensity to activate to diverse stimuli making the genetic modification of mature human neutrophils impossible, an important technical limitation in the field of neutrophil biology. Here I demonstrate that primary human haematopoietic progenitors isolated from the circulation of healthy adults can be expanded and directed to differentiate into mature neutrophils using granulocyte colony stimulating factor, and that these cells recapitulate multiple functions of mature circulating neutrophils. Stable modification of progenitors by Cas9 ribonucleoproteins or lentiviral vectors followed by granulocytic differentiation yields uniform populations of gene edited neutrophils in an activation-free manner, allowing the interrogation of individual genes in the neutrophil context. I use lentiviral vectors to validate the pro-efferocytic function of neutrophil TIMD4 and show its trafficking to membranes during apoptosis, uncovering a novel mechanism by which apoptotic neutrophils actively regulate their clearance by phagocytes. I then show that Cas9 ribonucleoprotein delivery by nucleofection is capable of stable knockout of the highly expressed gene *ITGAM* and that loss of its product CD11b, a key component of the complement receptor CR3, does not impair neutrophil phagocytosis of serum opsonised bacterial bioparticles, contrary to current evidence. This method for the efficient, flexible, and scalable generation of gene-edited primary neutrophils represents a powerful tool capable of discovering truly novel facets of neutrophil biology, the understanding of which is critical in the rational design of therapeutic strategies in a diverse array of infectious and inflammatory diseases.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Ng, Anthony

Advisor dc:contributor.advisor

• Summers, Charlotte

• CRISPR
• Genome Editing
• Immunology
• Lentivirus
• Neutrophil
• TIMD4

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/eade690d-b5c8-4df3-99b4-e1283d731076/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.113137

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/375424