Cleavage and correction at the β-globin locus in CD34⁺ cells using an IDLV donor. (A) A portion of exon I of β-globin showing the ZFN target site (underlined) atop the start codon (bold) and the location of the sickle mutation (bold, italic). (B) Representative gel showing targeted cleavage at the β-globin locus in CB CD34⁺ cells. Cells were analyzed 3 days after electroporation with in vitro transcribed mRNA encoding the ZFNs. Mock represents untreated CD34⁺ cells. Arrows indicate cut bands following PCR amplification and digestion with Surveyor Nuclease. (C) Schematic of site-specific gene correction at the sickle mutation. Details of the donor construct and resulting genomic DNA on cleavage by ZFNs and repair by HDR. Location of sickle mutation and HhaI RFLP (asterisk) is indicated. Translated regions of exons to scale, introns, and 5′ untranslated region not to scale. (D) Representative RFLP gel for targeted gene modification of β-globin. CB CD34⁺ cells were electroporated with in vitro transcribed ZFN mRNA and transduced with donor IDLV. Cells were harvested 4 days after treatment, PCR amplified from outside the donor region, digested with HhaI enzyme, and resolved on an agarose gel. Arrow shows cleaved product, indicating incorporation of the RFLP into the genome at the target site. (E) Gene modification percentages in CD34⁺ cells. CB CD34⁺ cells were electroporated with in vitro transcribed ZFN mRNA (10 μg/mL) and transduced with donor IDLV (2 × 10⁷ TU/mL). Cells were harvested 3 days after treatment and PCR amplified from outside the donor region, and qPCR was completed with primers designed to specifically detect the incorporation of the silent base change generating the HhaI RFLP and normalized to primers binding in exon II of the β-globin locus in the amplicon (n = 4 for all conditions). Error bars, mean ± standard deviation.