Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models

Abstract

Sickle cell disease (SCD) is caused by a 20A > T mutation in the β-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34⁺ cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact β-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34⁺ cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus β-to-βs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34⁺ cells 10-12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34⁺ HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials.

Keywords: CRISPR/Cas9; electroporation; gene correction; genome editing; hematopoietic stem cell; large animal model; non-human primate; sickle cell disease; transplantation; β-globin gene.

Graphical abstract

Figure 1

Efficient gene correction of the sickle cell disease (SCD) mutation in CD34⁺ cells in vitro (A) Schematic design of guide RNA targeting the SCD mutation in β-globin gene and donor single-stranded DNA (ssDNA) encoding normal β-globin gene. (B) A βs-to-β-globin gene correction model in SCD CD34⁺ cells by electroporation-mediated delivery of guide RNA, Cas9 endonuclease, and donor ssDNA, followed by in vitro erythroid differentiation. (C) CD34⁺ cell viability 1 and 2 days after electroporation, evaluated by trypan blue stain. ∗p < 0.05, ∗∗p < 0.01, evaluated by Dunnett’s test, compared with no electroporation (n = 1–6). Standard error of the mean (SEM) shown as error bars. (D) βs-to-β-globin gene correction (homology-directed repair [HDR]) and indels (small insertions and deletions), evaluated by targeted deep sequencing in cultured edited cells 2–5 days post-electroporation. ∗∗p < 0.01, evaluated by Dunnett’s test, compared with no electroporation (n = 1–6). SEM shown as error bars. (E) Biallelic correction (HDR/HDR) and monoallelic correction (HDR/indel and HDR/sickle) in colony-forming units (CFUs), evaluated by quantitative polymerase chain reaction (qPCR) single-nucleotide polymorphism (SNP) genotyping (n = 1). (F–H) Normal β-globin protein production in SCD CD34⁺ cell-derived erythroid cells following gene correction, evaluated by hemoglobin electrophoresis (F), and reversed-phase high-performance liquid chromatography (RP-HPLC) (G and H). ∗∗p < 0.01, evaluated by Dunnett’s test, compared with no electroporation (n = 1–6). SEM shown as error bars.

Figure 2

Similar-level β-to-βs-globin gene conversion among subpopulations of CD34⁺ cells (A) A β-to-βs-globin gene conversion model to evaluate HDR and indels among sorted subpopulations of edited CD34⁺ cells 2 days post-electroporation. (B) HDR and indels among subpopulations (CD34⁺CD133⁺CD90⁺, CD34⁺CD133⁺CD90⁻, CD34⁺CD133⁻, and CD34⁻) of edited CD34⁺ cells. Not significant, evaluated by Dunnett’s test, compared with bulk cells (n = 3). SEM shown as error bars. (C) HDR and indels among cell cycles (G0/G1, S, and G2/M) in edited CD34⁺ cells. Not significant, evaluated by Dunnett’s test, compared with bulk cells (n = 3). SEM shown as error bars.

Figure 3

Engraftment of gene-corrected SCD CD34⁺ cells in xenograft mice (A) Xenograft mouse transplantation for SCD CD34⁺ cells from 3 donors following βs-to-β-globin gene correction. (B) Gene correction (HDR) and indels in edited SCD CD34⁺ cells in vitro, evaluated by targeted deep sequencing (n = 1). (C) Normal β-globin protein production in erythroid cells in vitro differentiated from edited SCD CD34⁺ cells, evaluated by RP-HPLC (n = 1). (D) Engraftment of edited SCD CD34⁺ cells 4–24 weeks post-xenograft transplantation, evaluated by human CD45 expression in flow cytometry. ∗p < 0.05, ∗∗p < 0.01, evaluated by Dunnett’s test, compared with no electroporation (n = 2–5). SEM shown as error bars. (E) Gene correction (HDR) and indels in peripheral blood cells 4–24 weeks post-xenograft transplantation, evaluated by targeted deep sequencing. ∗p < 0.05, ∗∗p < 0.01, evaluated by Dunnett’s test, compared with no electroporation (n = 1–5). SEM shown as error bars.

Figure 4

Engraftment of gene-edited CD34⁺ cells with β-to-βs-globin conversion in rhesus macaques (A) Rhesus transplantation of autologous CD34⁺ cells following β-to-βs-globin gene conversion in 2 animals (13U005 and 12U011). (B) Gene conversion (HDR) and indels in edited rhesus CD34⁺ cells in in vitro culture, evaluated by targeted deep sequencing. (C) βs-globin protein production in erythroid cells in vitro differentiated from edited rhesus CD34⁺ cells, evaluated by RP-HPLC. (D) Gene correction (HDR) and indels in peripheral blood cells post-transplant, evaluated by targeted deep sequencing. (E and F) βs-globin protein production in red blood cells post-transplant, evaluated by hemoglobin electrophoresis.