Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells [RNA-seq]

ABSTRACT: Gene editing (GE) using homology-directed repair (HDR) in hematopoietic stem and progenitor cells (HSPCs) offers promise for long-range gene correction of inherited genetic disorders. However, adverse cellular responses induced by CRISPR-Cas9/AAV6 engineering impair the long-term repopulating potential of HDR-edited HSPCs, limiting clinical translation. Our study uncovers a senescence-like response in genetically-engineered HSPCs triggered by p53 and IL-1/NF-κB activation, which restricts graft size and clonal diversity in long-term transplantation assays. We show that transient p53 inhibition or blocking inflammatory pathways can mitigate senescence-associated responses, enhancing the repopulating capacity of edited HSPCs. Importantly, we identify Anakinra, an IL-1 signaling antagonist, as a safer and effective strategy to enhance polyclonal output in HDR-edited cells while minimizing genotoxicity risks associated with the gene-editing procedure. These findings present strategies to overcome key hurdles in HDR-based HSPC therapies, providing a framework for enhancing the efficacy and safety of these approaches in future clinical applications.

ORGANISM(S): Homo sapiens

PROVIDER: GSE244249 | GEO | 2025/03/18

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

Similar Datasets

Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells [NHEJ]

Project description:Gene editing (GE) using homology-directed repair (HDR) in hematopoietic stem and progenitor cells (HSPCs) offers promise for long-range gene correction of inherited genetic disorders. However, adverse cellular responses induced by CRISPR-Cas9/AAV6 engineering impair the long-term repopulating potential of HDR-edited HSPCs, limiting clinical translation. Our study uncovers a senescence-like response in genetically-engineered HSPCs triggered by p53 and IL-1/NF-κB activation, which restricts graft size and clonal diversity in long-term transplantation assays. We show that transient p53 inhibition or blocking inflammatory pathways can mitigate senescence-associated responses, enhancing the repopulating capacity of edited HSPCs. Importantly, we identify Anakinra, an IL-1 signaling antagonist, as a safer and effective strategy to enhance polyclonal output in HDR-edited cells while minimizing genotoxicity risks associated with the gene-editing procedure. These findings present strategies to overcome key hurdles in HDR-based HSPC therapies, providing a framework for enhancing the efficacy and safety of these approaches in future clinical applications.

2025-03-18 | GSE287805 | GEO

Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells [RNA-seq]

2025-03-18 | GSE244247 | GEO

Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells [BAR-seq]

2025-03-18 | GSE287803 | GEO

Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells [RNA-seq]

2025-03-18 | GSE244248 | GEO

Lipid nanoparticles improves ex vivo gene editing of human hematopoietic cells

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.

2024-01-26 | PXD037529 | Pride

Transient Inhibition of 53BP1 Increases the Frequency of Targeted Integration in Human Hematopoietic Stem and Progenitor Cells

Project description:Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach to increasing the frequency of HDR in human HSPCs by the delivery of an inhibitor of 53BP1 (named "i53") as a recombinant peptide. We show that the use of i53 peptide effectively increases the frequency of HDR-mediated genome editing at a variety of therapeutically relevant loci in HSPCs as well as other primary human cell types. We show that incorporating the use of i53 recombinant protein allows high frequencies of HDR while lowering the amounts of AAV6 needed by 8-fold. HDR edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in NSG mice, suggesting that i53 recombinant protein might be safely integrated into the standard CRISPR/AAV6-mediated genome editing protocol to gain greater numbers of edited cells for transplantation of clinically meaningful cell populations.

2023-12-31 | GSE246766 | GEO

Choice of Template Delivery Mitigates the Genotoxic Risk and Adverse Impact of Editing in Human Hematopoietic Stem Cells (RAAVIoli sequencing)

Project description:Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long- term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

2022-10-06 | GSE197388 | GEO

Choice of Template Delivery Mitigates the Genotoxic Risk and Adverse Impact of Editing in Human Hematopoietic Stem Cells (Targeted deep sequencing)

2022-10-06 | GSE197386 | GEO

Clonal Tracking Uncovers Barriers and Validate New Strategies to Enhance Gene Editing in Human Hematopoietic Stem Cells [BAR-seq]

Project description:By applying a barcoding strategy to clonal tracking of edited cells (BAR-seq), we show that p53 activation triggered by HDR editing significantly shrink the HSPC clonal repertoire in hematochimeric mice, despite engrafted edited clones preserved multilineage and self-renewing capacity. Transient inhibition of p53 restored polyclonal graft composition. We then overcame HSC constraints to HDR by forcing cell cycle progression and upregulating components of the HDR machinery through transient expression of Adenovirus 5 E4orf6/7 protein, which operates the major cell cycle controller E2F. Combined E4orf6/7 expression and p53 inhibition resulted in high and stable HDR editing efficiencies in the human graft, without perturbing repopulation and self-renewing properties of edited HSCs.

2020-04-22 | GSE144340 | GEO

Clonal Tracking Uncovers Barriers and Validate New Strategies to Enhance Gene Editing in Human Hematopoietic Stem Cells [RNA-seq]

2020-04-22 | GSE143995 | GEO