Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice

Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the β-globin gene (HBB). Here we show that prime editing can correct the SCD allele (HBBS) to wild type (HBBA) at frequencies of 15%–41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBBA levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors. An average of 42% of human erythroblasts and reticulocytes isolated 17 weeks after transplantation of prime-edited HSPCs from four SCD patient donors expressed HBBA, exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBBA-derived adult haemoglobin at 28%–43% of normal levels and resisted hypoxia-induced sickling. Minimal off-target editing was detected at over 100 sites nominated experimentally via unbiased genome-wide analysis. Our findings support the feasibility of a one-time prime editing SCD treatment that corrects HBBS to HBBA, does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks.

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Policy information about availability of computer code Data collection Illumina Miseq software (version 2.6.2.1) was used on the Illumina Miseq sequencer to collect the high-throughput DNA sequencing data.

Data analysis
Sequences were analyzed by single-end reads and analysing amplicons for the desired sequence and indels using CRISPResso2 software (version 2.2.11a, https://github.com/pinellolab/CRISPResso2). The editing frequency for each target site was calculated as the ratio between the number of aligned reads with the desired edit and without indels to the total number of aligned reads. The statistical significance of the sickling reduction between 2xPE3max-edited and untreated cells was calculated with one-sided multiplepaired t-tests correcting for multiple comparisons using the Holm-Šídák correction method with Prism 9 (version 9.4.1). CIRCLE-seq data analyses were performed using open-source CIRCLE-seq analysis software (version 1.1) and the default recommended parameters (https://github.com/tsailabSJ/circleseq). The editing frequency at each off-target site was calculated via a custom script (link provided in Code Availability). To calculate the statistical significance of off-target editing for 2xPE3max, we applied one-sided multiple-paired t-tests correcting for multiple comparisons using the Holm-Šídák correction method with Prism 9 (version 9.4.1). Droplets generated for ddPCR quantification of the 7.4-kb deletion between HBB and HBD were analysed using QuantaSoft (version 1.4). To calculate the statistical significance of the abundance of the deletion between 2xPE3max-edited and untreated cells, we applied a one-sided ttest using Prism 9 (version 9.4.1).
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March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy All data supporting the results of this study are available within the paper and its Supplementary Information. High-throughput sequencing data is available from the NCBI Sequence Read Archive database (PRJNA915048). Source data for the figures are provided with this paper. Key plasmids are available from Addgene (depositor: David R. Liu), or from the corresponding authors on request.

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Blinding was not used, and mice were treated only a single time each. Mice were housed, fed and handled identically.
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