Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells

Abstract

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34⁺ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the β-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.

Figure 1. Editing the SCD SNP in K562 cells

A) Schematic depicting the experimental approach to editing in K562 cells. A panel of 10 sgRNAs that cut within 100 bp of the SCD SNP was selected. A WT-to-SCD edit was programmed by an ssDNA template (T1) bearing silent PAM mutations for the sgRNAs (except G7), which also introduces a SfcI restriction site. B) T7 endonuclease assay showing indel formation in pools of cells edited by candidate RNPs. C) HDR editing of candidate sgRNAs detected by SfcI digestion. G5, G10 and a truncated variant, trG10, efficiently yield HDR products in K562 cells. D) Gene modification of select sgRNAs and templates at the SCD SNP, assessed by NGS. See Fig. S1 for definitions of donors T1 and T2. (average of 3 biological replicates, error bars indicate standard deviation). E) Analysis of off-target cutting by the G10 RNP at sites predicted by the online CRISPR-Design tool (reference), in K562 cells, determined by NGS.

Figure 2. Editing of wild-type human CD34+ HSPCs by the Cas9 RNP

A) Analysis of editing in un-expanded HSPCs (left) and erythroid-expanded HSPCs (right), using trG10 RNP and conditions as indicated. Templates, which are asymmetric about the G10 cut site, were designed as described in the text. All samples are 3 biological replicates, error bars are ± standard deviation. B) Modification (HDR+Indel) at off-target sites in HSPCs edited with the trG10 RNP and template T88-107, compared to untreated cells. Samples for HBB, OT1, FSCN3, and MNT are from 3 biological replicates, with error bars ± standard deviation. Targets were selected using the online CRISPR-design tool (reference). C) Indel formation at on-and off-target sites by Cas9 mutants with increased specificity (HF1 and espCas9 1.1), compared to WT Cas9, all complexed to the G10 sgRNA and no ssODN. All samples are n=3 biological replicates, with error bars ± standard deviation.

Figure 3. Correction of the SCD mutation in SCD HSPCs

A) Editing the SCD mutation in un-expanded CD34+ HSPCs from the whole blood of SCD patients, assessed by NGS. B) HPLC trace depicting hemoglobin production in SCD HSPCs edited with 200 pmol of the trG10 RNP and the T111-27S donor, compared to untreated HSPCs, after differentiation into erythroblasts. Significant increases in HbA, HbF, and HbA2 are apparent. C) Stacked bars showing HPLC results, with HSPCs edited as indicated after differentiation into erythroblasts. D) Globin gene levels in SCD HSPCs edited as in Fig. 3C, determined by RNA-seq.

Figure 4. Engraftment of edited HSPCs into NSG mice

A) Engraftment of human CD45+ cells in NSG mice injected with edited HSPCs, compared to 2 un-injected mice. B) Analysis by NGS of editing at the SCD SNP in cells prior to engraftment. Error bars indicate mean ± standard deviation of three separate experiments with cells from two healthy donors. C) HDR-mediated editing (left) and indel formation (right) at the SCD SNP in human cells engrafted in mouse blood, spleen, and bone marrow (BM), at 5 and 16 weeks post-injection. Error bars indicate mean ± standard deviation over either 6 mice (spleen) or 7 mice (BM and blood).