Figure 3
ZFN-mediated integration of α-globin expression constructs in AAVS1 site. (A) Integration strategy. Four different α-globin constructs were tested. Therapeutic cassettes were inserted between exons 1 and 2 of the PPP1R12C by cotransfection of 2 ZFN-containing plasmids and of a targeting construct containing short homologies to part of intron 1 flanking the therapeutic transgenes. Successful targeting leads to expression of the puromycin gene under the control of the PPP1R12C promoter because of the presence of the splice acceptor (SA). (B) Location of the primer sets for demonstration of ZFN-mediated integration of α-globin constructs after selection of puromycin-resistant colonies. P1 hybridizes to a region just 5′ of the left homology arm. P2 hybridizes to the puromycin-resistance genes. P3 hybridizes to the 3′ part of the α-globin gene; P4 hybridizes to a region just 3′ of the right homology arm. P5 hybridizes to the 5′ region of the LCR. P1/P2, P3/P4, and P5/P6 only yield a PCR product if the transgenes are specifically inserted at AAVS1. P1/P4 detects the unmodified PPP1R12C gene. (C) PCR results demonstrating insertions of α-globin constructs at AAVS1. The PPP1R21C (P1/P4) PCR amplification can be used to identify heterozygous or homozygous insertions. α1-Hom indicates homozygous transgene in same orientation as PPP1R12C, driven by α-globin promoter; β1-Het, heterozygous transgene in same orientation as PPP1R12C, driven by β-globin promoter; β1-Hom, homozygous transgene in same orientation as PPP1R12C, driven by β-globin promoter; and β2-Het and β2-Hom, same as above but transgene is in opposite orientation. (D) Expression of PPP1R12C after insertion of the therapeutic transgenes. Heterozygous clones express PPP1R12C at approximately 50% the level of the unmodified locus. Homozygous clones do not express any detectable levels of PPP1R12C (n = 3). These results confirm the results of the analysis in panel C. (E) Histograms illustrating the number of copies of the corrective α-globin construct inserted in the genome. Q-PCR analyses were performed to compare the number of copies of α and β-globin present in the genome after. Y-axis = 2 × 2(Ctα-globin − Ctβ-globin). Together with the Southern blot analysis, these results demonstrate that these clones did not harbor any off-target integrations.

ZFN-mediated integration of α-globin expression constructs in AAVS1 site. (A) Integration strategy. Four different α-globin constructs were tested. Therapeutic cassettes were inserted between exons 1 and 2 of the PPP1R12C by cotransfection of 2 ZFN-containing plasmids and of a targeting construct containing short homologies to part of intron 1 flanking the therapeutic transgenes. Successful targeting leads to expression of the puromycin gene under the control of the PPP1R12C promoter because of the presence of the splice acceptor (SA). (B) Location of the primer sets for demonstration of ZFN-mediated integration of α-globin constructs after selection of puromycin-resistant colonies. P1 hybridizes to a region just 5′ of the left homology arm. P2 hybridizes to the puromycin-resistance genes. P3 hybridizes to the 3′ part of the α-globin gene; P4 hybridizes to a region just 3′ of the right homology arm. P5 hybridizes to the 5′ region of the LCR. P1/P2, P3/P4, and P5/P6 only yield a PCR product if the transgenes are specifically inserted at AAVS1. P1/P4 detects the unmodified PPP1R12C gene. (C) PCR results demonstrating insertions of α-globin constructs at AAVS1. The PPP1R21C (P1/P4) PCR amplification can be used to identify heterozygous or homozygous insertions. α1-Hom indicates homozygous transgene in same orientation as PPP1R12C, driven by α-globin promoter; β1-Het, heterozygous transgene in same orientation as PPP1R12C, driven by β-globin promoter; β1-Hom, homozygous transgene in same orientation as PPP1R12C, driven by β-globin promoter; and β2-Het and β2-Hom, same as above but transgene is in opposite orientation. (D) Expression of PPP1R12C after insertion of the therapeutic transgenes. Heterozygous clones express PPP1R12C at approximately 50% the level of the unmodified locus. Homozygous clones do not express any detectable levels of PPP1R12C (n = 3). These results confirm the results of the analysis in panel C. (E) Histograms illustrating the number of copies of the corrective α-globin construct inserted in the genome. Q-PCR analyses were performed to compare the number of copies of α and β-globin present in the genome after. Y-axis = 2 × 2(Ctα-globin − Ctβ-globin). Together with the Southern blot analysis, these results demonstrate that these clones did not harbor any off-target integrations.

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