Schematic illustration of selection strategy. For the selection of γ-globin promoter activators, cells were transfected with VSV-G envelope and a 5-ZF-VP64-library expressed by a retroviral transfer vector. Cells were cotransfected with the pPur-γ-gp plasmid encoding the MLV structural genes gag and pol under the control of γ-globin promoter and enhancer elements. Cells that expressed an activator of the γ-globin promoter showed increased expression of MLV gag/pol genes which lead to increased transduction efficiency of target cells. To start the next selection round, target cells were transfected directly with pPur-γ-gp and VSV-G envelope for the production of new viral particles. Five rounds of selection were carried out.

Selected TF_ZF libraries show increased GFP⁺ transduced cells after each round of TF_ZF selection. Retroviral particles were produced in 293T cells co-transfected with pPur-γ-gp and selected TF_ZF-libraries (Lib 1^st – Lib 5^th), used for transduction of fresh 293T target cells and compared to cells transduced with the non-DNA-binding control-ZF (Ala-VP64), cells transduced with unselected pMX-5ZF-library (Lib) and the positive control gg1-VP64. Three days after transduction, cells were analyzed by FACS to assess GFP expression. Percentage of GFP⁺ cells is depicted in yellow (right scale). Selected TF_ZF libraries show increased transcriptional activation on γ-globin promoter after each selection round. HeLa cells were transfected with control-ZFs (Ala-VP64 and gg1-VP64) and TF_ZF libraries (Lib 1^st – Lib 5^th) together with a luciferase reporter plasmid expressing firefly luciferase under the control of γ-globin and Renilla luciferase under the control of the β-globin promoter. Specific activation of γ-globin promoter was calculated by normalizing to Renilla luciferase values. Normalized luciferase activity of the selected TF_ZF libraries was compared to that of unselected pMX-5ZF-library (Lib = 1) and the positive control gg1-VP64. The normalized luciferase activities are shown in green (± s.d.) (right scale). For Lib-VP64 vs Lib-1st-VP64, p= 0.00015; for Lib-VP64 vs Lib-5th-VP64, p=0.00073.

Selected TF_ZFs shows repression of γ-globin promoter expression with when fused to the repressor domain KRAB. HeLa cells were transfected with TF_ZFs fused to KRAB (#γA-KRAB, #γA-opt-KRAB, #γL-KRAB, #γK-KRAB and #γM-KRAB gg1-KRAB) and a luciferase reporter plasmid expressing firefly luciferase under the control of γ-globin and Renilla luciferase under the control of the β-globin promoter. Specific activation of the γ-globin promoter was calculated by normalizing to Renilla luciferase values. Error bars represent standard deviation from the mean. Normalized luciferase activity of the selected TF_ZFs was compared to that of cells transfected with the non-DNA binding negative control Ala-KRAB and the positive control gg1-KRAB. For Ala-KRAB vs #γA-KRAB, p= 0.00018; For Ala-KRAB vs #γA-opt-KRAB, p= 9.6E-05.

Activation of human γ-globin in β-YAC BMCs containing selected zinc-fingers measured by RT-PCR. β-YAC BMCs were transfected with TF_ZFs (Ala-VP64, #γA-VP64, #γA-opt-VP64, #γL-VP64, #γK-VP64 and #γM-VP64 gg1-VP64) expressed in pcDNA-hygro. TF_ZF-expressing cells were selected for hygromycin resistance. RNA was isolated and γ-globin expression was measured by RT-PCR normalized to GAPDH expression. The data shown are the averages of duplicates or triplicates from at least two independent experiments for each sample. Error bars represent standard deviation from the mean. For Ala-VP64 vs γA-opt-VP64, p= 0.0012.