Lipid nanoparticles improves ex vivo gene editing of human hematopoietic cells

ABSTRACT: Ex-vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases by non-homologous end joining (NHEJ) gene disruption or homology-driven repair (HDR) gene correction. Gene editing encompasses delivery of nucleases by electroporation and, when aiming to HDR, of a DNA template often provided by viral vectors. Whereas HSPCs activate robust p53-dependent DNA damage response (DDR) upon editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multi-omics analyses and found that electroporation is the culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism and inducing inflammatory response. Nuclease delivery by lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding higher number of edited cells compared to electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher reconstitution by long-term repopulating HSPCs compared to electroporation, reaching similar editing efficiencies. Overall, LNPs may allow efficient and stealthier ex-vivo gene editing in hematopoietic cells for treatment of human diseases.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: alessandro cuomo  

LAB HEAD: Luigi Naldini

PROVIDER: PXD037529 | Pride | 2024-01-26

REPOSITORIES: Pride