Ontology highlight
ABSTRACT:
PROVIDER: PRJNA838341 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23186900_1.fastq.gz | Fastqsanger.gz | |||
| SRR23186900_2.fastq.gz | Fastqsanger.gz | |||
| SRR23186901_1.fastq.gz | Fastqsanger.gz | |||
| SRR23186901_2.fastq.gz | Fastqsanger.gz | |||
| SRR23186902_1.fastq.gz | Fastqsanger.gz |
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Project description:Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle Cell Disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of ZFNs and CRISPR/Cas9 for editing and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODN), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLV) and adenovirus 5/35 serotype (Ad5/35) to transvert the base-pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base-pair transversions were significantly reduced when analyzing cells in vivo in immune-deficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODN. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high efficiency gene correction in HSPCs
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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: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.
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