1. Molecular Therapy - Nucleic Acids
Activation of Fetal γ-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains 
Mir A Hossain, Yong Shen, Isaac Knudson, Shaleen Thakur, Jared R Stees, Yi Qiu, Betty S Pace, Kenneth R Peterson, Jörg Bungert 
Molecular Therapy - Nucleic Acids 
Volume 5, (January 2016)
DOI: /mtna
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Generation and analysis of ZF-DBDs targeting the -567 GATA site in the Gγ-globin gene promoter. (a) Target sequence for the 6 ZF (blue) and 8 ZF (blue and orange) -567 Gγ ZF-DBDs and outline of the general research strategy. As illustrated the ZF-DBDs are expected to compete for binding with a GATA1-repressor complex. The research strategy begins with the assessment of binding specificity and affinity in vitro, with the examination of binding specificity in the context of cells, with the optimization of protein delivery protocols, and with the functional assessment of the proteins with respect to activating γ-globin gene expression in primary erythroid cells. (b) Expression constructs for the 6 or 8 ZF -567Gγ ZF-DBDs. The His-tagged 6 and 8 ZF-DBDs were used in in vitro DNA-binding and protein delivery experiments. Constructs expressing NLS and 3× FLAG-tagged proteins were used in transfection studies. His-tag, 3× FLAG-tag, and NLS containing constructs were used in protein delivery studies. (c) Expression and purification of the recombinant 6 ZF -567Gγ ZF-DBD as shown by SDS-PAGE followed by coomassie blue staining. NLS, nuclear localization sequence; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; ZF-DBDs, zinc-finger DNA-binding domains.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Assessment of the DNA-binding affinity of the 6 and 8 ZF -567Gγ ZF-DBDs. The DNA probe encompassing the -567 GATA site from the Gγ-globin promoter was incubated with increasing concentrations of the 6 ZF (a) or 8 ZF (b) -567Gγ ZF-DBD, as indicated. The bound DNA-fractions were calculated and plotted against the concentrations of ZF-DBDs to derive the binding curves shown on the right. The protein concentration at which half-maximal binding occurred reflects the dissociation constant Kd (±SD, n = 3). ZF-DBDs, zinc-finger DNA-binding domains; kd, dissociation constant.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Assessment of the DNA-binding specificity of the 6 and 8 ZF -567Gγ ZF-DBDs. (a) Determination of DNA-binding specificity in vitro. The 6 or 8 ZF -567Gγ ZF-DBDs (100 nmol/l) were incubated with the WT DNA probe encompassing the -567 GATA site of the Gγ-globin gene promoter. Binding was challenged with unlabeled WT or mutant oligonucleotides (Mut1–3 and Mut1–8) at the indicated molar excess. Target sequences for the 6 ZF (blue) and 8 ZF (blue and orange) are highlighted. Mutated sequences are shown in red color. (b) Determination of the DNA-binding specificity of the 6 ZF -567Gγ ZF-DBD in K562 cells using ChIP. Right panel, western blotting of cytoplasmic (C) or nuclear (N) proteins from K562 cells stably transfected with the 3× FLAG-tagged 6 ZF -567Gγ ZF-DBD using antibodies specific for FLAG-tag, CTCF, or GAPDH. Left panel, ChIP assay using monoclonal anti-FLAG or control IgG antibodies. The isolated DNA was subjected to qPCR using primers specific for LCR hypersensitive site (HS) 2, the promoters of ▵-globin, Gγ-globin, Aγ-globin, β-globin, GAPDH, MyoD1, HCK, and WNT1, as well as for six potential off-target sites as indicated. Error bars represent mean ± SD. The experiment was performed twice with PCRs performed in triplicate. ChIP, chromatin immunoprecipitation; CTCF, CCCTC-Binding Factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ZF-DBDs, zinc-finger DNA-binding domains; qPCR, quantitative polymerase chain reaction.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Assessment of protein delivery and stability of the 6 ZF -567Gγ ZF-DBD in K562 cells. (a) A general outline of the protein delivery method. (b) The 6 ZF -567Gγ ZF-DBD containing NLS, His- and 3× FLAG-tag was delivered to K562 cells at increasing concentrations in the absence of serum. Cytoplasmic (C) or nuclear (N) proteins were extracted from the transduced or untransduced (Buffer) cells and were analyzed using western blotting. (c) Protein delivery in the presence or absence of the NLS. The 6 ZF-DBD with or without NLS and 3× FLAG-tag were delivered to K562 cells and were analyzed as in panel b. (d) Protein stability of the NLS containing -567Gγ ZF-DBD delivered to K562 cells. The -567Gγ ZF-DBD was delivered to K562 cells at a concentration of 100 nmol/l. After changing the medium, cells were incubated without the ZF-DBD for the indicated time points. Western blotting was performed as described in panel b. (e) Protein stability of the -567Gγ ZF-DBD lacking an NLS. Experiment was performed as described in panel d. NLS, nuclear localization sequence; ZF-DBDs, zinc-finger DNA-binding domains; CTCF, CCCTC-Binding Factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Assessment of protein delivery and stability as well as γ-globin gene expression in β-YAC BM CID cells. (a) Protein delivery of the -567Gγ ZF-DBD containing His and 3× FLAG-tags as well as an NLS in β-YAC BM CID cells in the presence or absence of serum. Cells were incubated with or without the ZF-DBD (100 nmol/l) as indicated and were subjected to western blotting analysis as described in Figure 4b. (b) Protein stability of the -567Gγ ZF-DBD in β-YAC BM CID cells. The -567Gγ ZF-DBD containing NLS as well as His- and 3× FLAG-tags was delivered to β-YAC BM CID cells in the presence of serum at a concentration of 150 nmol/l. After initial delivery cells were either incubated in the absence (withdrawal) or presence (prolonged incubation) of the ZF-DBD (150 nmol/l). Total cell lysates were then subjected to western blotting using antibodies specific for FLAG-tag or GAPDH. (c) Gene expression analysis in β-YAC BM CID cells transduced with the 6 ZF -567Gγ ZF-DBD lacking NLS and 3× FLAG-tag (2 μmol/l) or with ZF-DBD storage buffer (Buffer). RNA was extracted from the cells and subjected to cDNA synthesis. The cDNA was analyzed by qPCR using primers specific for the human Gγ-globin, total γ-globin (human γ-globin), and β-globin genes as well as for the mouse α-globin, β-major globin, βh1-globin, E/y-globin, and dematin genes. (d) Gene expression analysis in β-YAC BM CID cells transduced with the 8 ZF -567Gγ ZF-DBD lacking NLS and 3× FLAG-tag (2 μmol/l) or with ZF-DBD storage buffer (Buffer). The experiment was performed as in panel c. Error bars in panels c and d represents mean ± SD of four (panel c, n = 4) or three (panel d, n = 3) independent experiments with PCRs performed in triplicate. * indicates P-value of < BM, bone marrow; CID, chemical induced dimerization; CTCF, CCCTC-Binding Factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NLS, nuclear localization sequence; qPCR, quantitative polymerase chain reaction; ZF-DBDs, zinc-finger DNA-binding domains; β-YAC BM CID, β-globin yeast artificial chromosome.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Increased γ-globin expression in PBMC derived erythroid cells exposed to the 6 or 8 ZF -567Gγ ZF-DBD. (a) Protein delivery of the 6 or 8 ZF -567Gγ ZF-DBD lacking NLS and 3× FLAG-tag in PBMC derived erythroid cells. Cells were incubated for 1 hour with or without the ZF-DBD (500 nmol/l) and were analyzed as in Figure 4b. (b) Gene expression analysis of PBMC derived erythroid cells after 3 days treatment with sodium butyrate (50 μmol/l), or 6 or 8 ZF -567Gγ ZF-DBD lacking NLS and 3× FLAG-tag (from 500 nmol/l to 3 μmol/l). RNA was extracted and analyzed as in Figure 5c, using primers specific for human total γ-globin, β-globin, α-globin, and USF1 genes. Error bars represent mean ± SD of four independent experiments (n = 4) with PCRs performed in triplicate. # and * indicate P-values of < * refers to significant differences between data obtained from treated cells compared with those of the untreated cells. # refers to significant differences between data obtained from cells exposed to the 8 ZF-DBD and those obtained from cells exposed to the 6 ZF-DBD, as indicated. (c) Fraction of γ-globin expression among β-type globin gene expression in PBMC derived erythroid cells after various treatments. Percentage of γ-globin (γ-globin%) was determined from the data shown in panel b using γ-globin% = (γ-globin/(γ-globin + β-globin)) × 100. (d) Western blot analysis of γ-globin, β-globin and GAPDH in K562 cells and in PBMCs exposed to buffer (untreated), exposed to the 8ZF -567Gγ ZF-DBD (0.5 or 1 μmol/l), or exposed to sodium butyrate (50 μmol/l). The cells were first induced to differentiate and at day 7 they were exposed to sodium butyrate or to the ZF-DBD for 3 days. Every 24 hours the medium was changed including fresh sodium butyrate or ZF-DBDs. CTCF, CCCTC-Binding Factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NLS, nuclear localization sequence; PBMCs, peripheral blood mononuclear cells; ZF-DBDs, zinc-finger DNA-binding domains.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions