Figure 1.

Studies with human CD34⁺cells. (A) Schematic structure of globin locus with localization of the sgRNA in the promoters of the γ-globin genes, the CRISPR/Cas9 cleavage site (arrowhead), the 13-bp (−114 to −102) HPFH deletion (box), and the BCL11A-binding motif (“TGACCA” bold sequence). Note that only the distal TGACCA motif (red) within the indicated sequence is critical for γ-globin silencing.³² The CRISPR/Cas9 cleavage site is located 2 bp upstream of the BCL11A binding motif. ε, Gγ, Aγ, δ, and β are globin genes. (B) HDAd-HBG-CRISPR vector structure. The sgRNA gene is transcribed by PolIII from the U6 promoter, and the spCas9 gene is under the control of the EF1α promoter. Cas9 expression is controlled by miR-183-5p and miR-218-5p, which suppress Cas9 expression in HDAd producer cells but do not negatively affect Cas9 expression in CD34⁺ cells.³⁵ The corresponding microRNA target sites (miR-T) were embedded into a 3′untranslated region (3′UTR). (C) Experimental design. CD34⁺ cells were transduced with HDAd-HBG-CRISPR at a multiplicity of infection of 2000 vp’s per cell. Two days later, cells were infected with HDAd–anti-CRISPR³⁶ at the same multiplicity of infection to terminate CRISPR/Cas9 activity and, thus, minimize cytotoxicity to HSPCs. (The 2 anti-CRISPR peptides [AcrII4 and AcrII2] expressed from this vector are capable of binding to the CRISPR/Cas9 complex, thus blocking its activity.⁵⁷) Sixteen hours after the last infection, CD34⁺ cells were transplanted into irradiated NSG mice. Untransduced cells were incubated for 2 days + 16 hours in the same medium as transduced cells. At week 10 after transplantation, human CD45⁺ cells were isolated from the bone marrow by magnetic-activated cell sorting and subjected to T7E1 assay and erythroid in vitro differentiation for globin analysis by flow cytometry and HPLC. (D) Target site cleavage frequency (measured by T7E1 assay) in human CD45⁺ cells isolated from bone marrow at week 10 after transplantation. Each lane is an individual mouse. (E) γ-Globin flow cytometry of cells after erythroid in vitro differentiation. (F) Ratio of γ-globin to adult α- or β-globin measured by HPLC after erythroid differentiation. n = 3. HS, DNase I hypersensitivity sites; untr, mice transplanted with untransduced CD34⁺ cells.