Lipid Nanoparticles Allow Efficient and Harmless Ex Vivo Gene Editing of Human Hematopoietic Cells [NHEJ]
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1029179 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26413200.fastq.gz | Fastqsanger.gz | |||
| SRR26413300.fastq.gz | Fastqsanger.gz | |||
| SRR26413201.fastq.gz | Fastqsanger.gz | |||
| SRR26413301.fastq.gz | Fastqsanger.gz | |||
| SRR26413202.fastq.gz | Fastqsanger.gz |
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Project description:Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo by electroporation and, when aiming to homology-driven correction, of a DNA template often provided by viral vectors together with a nuclease editor. Whereas HSPCs activate robust p53-dependent DNA damage response (DDR) upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multi-omics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism and inducing inflammatory response. Nuclease RNA 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 clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared to electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for treatment of human diseases.
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Project description:Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo by electroporation and, when aiming to homology-driven correction, of a DNA template often provided by viral vectors together with a nuclease editor. Whereas HSPCs activate robust p53-dependent DNA damage response (DDR) upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multi-omics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism and inducing inflammatory response. Nuclease RNA 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 clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared to electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for treatment of human diseases.
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Project description:Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo by electroporation and, when aiming to homology-driven correction, of a DNA template often provided by viral vectors together with a nuclease editor. Whereas HSPCs activate robust p53-dependent DNA damage response (DDR) upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multi-omics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism and inducing inflammatory response. Nuclease RNA 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 clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared to electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for treatment of human diseases.
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Project description: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.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2023-06-30 | GSE216250 | GEO
Project description:Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo via electroporation, and when aiming for homology-driven correction of a DNA template, often provided by viral vectors together with a nuclease editor. Although HSPCs activate a robust p53-dependent DNA damage response upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multiomics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism, and inducing an inflammatory response. Nuclease RNA delivery using lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding a higher number of edited cells compared with using 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 clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared with electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for the treatment of human diseases.
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Project description:DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.
2022-09-11 | E-MTAB-12037 | biostudies-arrayexpress