Zinc-finger-based artificial transcription factors and their applications

Advanced Drug Delivery Reviews 61 (2009) 513–526

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Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Zinc-finger-based artificial transcription factors and their applications☆ Takashi Sera ⁎ Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

a r t i c l e

i n f o

Article history: Received 28 November 2008 Accepted 10 March 2009 Available online 23 April 2009 Keywords: Artificial transcription factor Zinc-finger proteins DNA recognition Gene regulation Transcriptional effector domain Gene therapy Antiviral therapy

a b s t r a c t Artificial transcription factors (ATFs) are potentially a powerful molecular tool to modulate endogenous target gene expression in living cells and organisms. To date, many DNA-binding molecules have been developed as the DNA-binding domains for ATFs. Among them, ATFs comprising Cys2His2-type zinc-finger proteins (ZFPs) as the DNA-binding domain have been extensively explored. The zinc-finger-based ATFs specifically recognize targeting sites in chromosomes and effectively up- and downregulate expression of their target genes not only in vitro, but also in vivo. In this review, after briefly introducing Cys2His2-type ZFPs, I will review the studies of endogenous human gene regulation by zinc-finger-based ATFs and other applications as well. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . Interaction of Cys2His2 ZFP with DNA . . . . . . . . . . . . Construction of ATFs . . . . . . . . . . . . . . . . . . . . 3.1. Construction of ATFs by assembly of finger domains . . 3.1.1. Step 1. Determination of target DNA sequences 3.1.2. Step 2. Design of finger domains . . . . . . . 3.1.3. Step 3. Assembly of finger domains . . . . . 3.1.4. Step 4. Evaluation of ZFPs . . . . . . . . . . 3.1.5. Step 5. Fusion of other functional domains . . 3.1.6. Step 6. Evaluation of ATFs . . . . . . . . . . 3.2. Other methods to generate ZFPs . . . . . . . . . . . Application of ATFs. . . . . . . . . . . . . . . . . . . . . 4.1. Short history . . . . . . . . . . . . . . . . . . . . 4.2. Modulation of endogenous gene expression . . . . . . 4.2.1. ErbB . . . . . . . . . . . . . . . . . . . . 4.2.2. MDR1 multidrug resistance gene. . . . . . . 4.2.3. Erythropoietin . . . . . . . . . . . . . . . 4.2.4. Vascular endothelial growth factor A . . . . . 4.2.5. PPARγ . . . . . . . . . . . . . . . . . . . 4.2.6. IGF2 and H19 . . . . . . . . . . . . . . . . 4.2.7. Bax . . . . . . . . . . . . . . . . . . . . 4.2.8. Oct-4. . . . . . . . . . . . . . . . . . . . 4.2.9. Checkpoint kinase 2. . . . . . . . . . . . . 4.2.10. Cholecystokinin 2 receptor . . . . . . . . . 4.2.11. Parathyroid hormone receptor 1 . . . . . . . 4.2.12. γ-Globin . . . . . . . . . . . . . . . . . .

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☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Gene Regulation for Effective Gene Therapy”. ⁎ Tel.: +81 75 383 2769; fax: +81 75 383 2767. E-mail address: [email protected]. 0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2009.03.012

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4.2.13. Mammary serine protease inhibitor. . 4.2.14. Utrophin . . . . . . . . . . . . . . 4.2.15. Pigment epithelium-derived factor . . 4.3. Combination with chromatin remodeling drugs 4.4. ATF library . . . . . . . . . . . . . . . . . 4.5. Cell-permeable ATFs as potent protein drugs. . 4.6. Application to antiviral therapies . . . . . . . 4.6.1. RNA virus . . . . . . . . . . . . . . 4.6.2. DNA virus . . . . . . . . . . . . . . 5. Future challenges . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction A variety of biological processes including development, differentiation, and disease are regulated through gene expression. Gene expression is mainly modulated by endogenous transcription factors. This suggests that “artificial” transcription factors (ATFs) designed to bind to a promoter region of a gene of interest could modulate the target gene expression independently of organisms, thereby giving new insights into molecular biology and presenting novel gene therapy protocols. Transcription factors, in principle, comprise (1) a DNA-binding domain, (2) a transcriptional regulation domain or effector domain, and (3) a nuclear localization signal (NLS); a DNAbinding domain is required to bind to the promoter of a target gene, an effector domain to up- or downregulate the target gene, and an NLS to deliver an ATF into nuclei (because eukaryotic transcription occurs in nuclei) (Fig. 1). Therefore, if we can design and construct an artificial DNA-binding protein (or domain) that recognizes a target DNA specifically, we can create a desired ATF. Until now, many artificial DNA-binding proteins or small molecules such as pyrrole–imidazole polyamides have been reported. Among them, the Cys2His2-type zinc-finger protein (ZFP) is the most promising candidate for the DNA binder. The zinc-finger DNA-binding motif comprising multiple repeats of approximately 30 amino acids Xaa2-Cys-Xaa2−4-Cys-Xaa12-His-Xaa3,4-His-X2−6 was first discovered in the transcription factor TFIIIA of Xenopus laevis in 1985 [1]. To date, many of the motifs have been identified in many eukaryotes (e.g., N700 ZFPs for humans), and it is now one of the most common DNAbinding motifs. The unique features are (1) ZFPs bind to their DNA targets as a monomer, and (2) ZFPs contain several finger domains,

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generally from three to more than nine domains. The first feature is very important because almost DNA regions in genomes are nonpalindromic, which means that motifs that bind to DNA as homodimers, such as the helix-turn-helix motif, are not adequate for genomic DNA recognition. ZFPs have such no restriction. The second feature is also important for genome recognition. To target a specific genomic site, recognition of a long DNA sequence is necessary. For example, it is theoretically necessary to recognize N16-bp DNA for specific recognition of one DNA region in human cells because the genome size is 3 × 109 bp. Because ZFPs are known to contain many finger domains and recognize long DNA sequences, the consecutive linking of the motif seems to perturb the whole structure relatively little compared with other DNA-binding motifs, and thereby potentially recognize N16-bp targets with high affinities. In this review, I will first explain the design of the ATF and then review ATFs harboring ZFPs as a DNA-binding motif. 2. Interaction of Cys2His2 ZFP with DNA In this review, I will focus on ATFs comprising Cys2His2 zinc-finger domains as their DNA-binding moiety. For other zinc-finger domains, please see Klug's good review [2]. Classically, one unit of the Cys2His2 zinc-finger domain was thought to contact 3-bp of the DNA target. Pabo's group reported ´ the X-ray crystal structure of the DNA complex with Zif268 at 2.1 A˚ resolution [3]. In the complex, amino acids at positions − 1 and 6 of the α-helical region of the zinc-finger domain (position 1 is the starting amino acid in the α-helix) in the 1st and 3rd fingers contacted the 1st and 3rd bases, and amino acids at positions −1 and 3 in the

Fig. 1. Basic structure of ATF. An ATF is composed of three domains, a DNA-binding domain, an effector domain and a nuclear localization signal (NLS). The ATF enters a nucleus via the NLS, binds to a promoter region of a target gene, and then up- or downregulates the target gene expression in an effector-dependent manner.

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2nd finger contacted the 1st and 2nd bases. However, no other amino acid–base contact was observed at that resolution. Next, observation ´ of the structure of the Zif-268-DNA complex at 1.6 A˚ resolution suggested constant interaction of amino acids at position 2 with the 4th base [4]; however, some base–amino acid interactions were still not apparent even at 1.6 A resolution. After Pabo's reports, Berg's group reported important structural information on DNA recognition by ZFPs. They revealed the crystal ´ structure of the DNA complex of their designed ZFP at 2.2 A˚ resolution [5]. The ZFP comprising a consensus finger framework [6] was selected from a family of three-finger proteins that had been prepared and characterized with regard to their DNA-binding specificities [7–10]. The X-ray crystal structural analysis of the DNA complex revealed the following important features: (1) each zinc-finger domain recognizes an overlapping 4-bp DNA sequence, where the last base pair of each 4-bp target is the first base pair of the next 4-bp target (Fig. 2A); (2) in all three fingers of the protein, amino acids at specific positions contact DNA bases at specific positions in a regular fashion. Namely, amino acids at positions −1, 2, 3, and 6 contact the 3rd, 4th, 2nd, and 1st bases of the overlapping 4-bp DNA targets, respectively (only the 4th base in the antisense strand) (Fig. 2B). Whether a one-finger domain recognizes a 3- or 4-bp DNA remains unclear. Although Berg's group found regular DNA contact of amino acids at position 2, they reported that a particular amino acid at that position is not important for DNA recognition [10], and finger domains developed for ZFP construction (so-called “modules” or “building blocks”) are described as 3-bp binders (for examples, see [11,12]) (please also see Section 3.1). In contrast, a series of studies done by Klug and Choo's group clearly indicated the contribution of the amino acid at position 2 to DNA binding and 4-bp recognition by a one-finger domain [13–15]. We also confirmed that contribution of the amino acid at position 2 to binding affinities and specifities is not minor. Therefore, we included amino acids for recognition of the 4th base pair in our nondegenerate recognition code table [16] (see Section 3.1). Currently, many papers and reviews still report that one zincfinger domain recognizes 3 or 4 bp. The binding mode may depend on the finger framework used for ZFP design.

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3. Construction of ATFs Design of the ZFP as the DNA-binding domain is the most important and difficult part in the construction of an ATF. In 1992, Berg's group reported the first example of ZFPs with altered binding specificities [17]. They mutagenized the amino acid at position 3 of the 2nd finger of Sp1 based on a database of zinc-finger sequences available at the time, and presented a first partial “recognition code” of GNG and GNT triplets. Shortly after Berg's first report, Young's group also reported engineered ZFPs [18]. They mutagenized recognition amino acids of the 1st finger of the yeast transcription factor ADR1. They also examined the possibility of gene activation using ADR1 mutants and showed that one of the mutants actually activated its mutant target gene in transient reporter assays. Their result clearly indicated that engineered ZFPs with new binding specificities could be used to modulate expression of genes of interest for the first time. In 1994, Pabo's group reported the selection of ZFPs with new DNA-binding specificities by phage display [19]. Soon after that report, Well's, Klug's, and Barbas's groups reported other zinc-finger proteins selected by phage display [20−22], making generation of ZFP-based ATFs practical. 3.1. Construction of ATFs by assembly of finger domains Currently, many good reviews on the design and construction of engineered ZFPs are available (for examples, please see [23–29]). Therefore, I will outline the simplest method for construction of ATFs via modular assembly in this section. Generally, the construction is divided into 6 steps as follows (Fig. 3). Step 1. Step 2. Step 3. Step 4. Step 5. Step 6.

Determination of target DNA sequences Design of finger domains Assembly of finger domains Evaluation of ZFPs Fusion of other functional domains Evaluation of ATFs

3.1.1. Step 1. Determination of target DNA sequences This step is probably the most important or critical one to obtain good ATFs. After choosing an endogenous target gene for modulation, we have to determine the genomic region to be targeted. As Wolffe's group showed, the accessibility of genomic DNA in chromatin to ATFs seems to be very important [30]. Before their study, transient reporter assays were often used to select good ATFs. In the reporter assays, a reporter gene such as luciferase was placed under the control of the promoter of the target gene. However, as shown in the study done by Wolffe's group, certain ATFs that showed good performance in transient reporter assays did not modulate the endogenous gene expression effectively, indicating that the chromatin structures of promoters in reporter plasmids are different from those in actual chromosomes. Therefore, they first identified DNase I hypersensitive sites and then constructed ATFs targeting these sites. The same group also reported that their ATFs could activate the expression of silenced genes, in which the target sites were not hypersensitive to DNase I digestion [31,32], suggesting that certain ATFs may be able to sneak in and bind to their target sites crowded in chromosomes. Therefore, the most practical way to obtain effective ATFs is to screen a panel of ATFs that individually target different sequences in the promoter region of the gene of interest in a high-throughput manner, as they reported (for example, please see [33]). Fig. 2. Interaction of 3-finger ZFP with double-stranded DNA. (A) Interaction observed in the DNA complex of Berg's designed ZFP. The 4-bp DNA targets are overlapped. (B) Interaction of a zinc-finger domain with 4-bp DNA target observed in the complex. In the complex, one-to-one interactions between all recognition amino acids and DNA bases at specific positions were observed for the first time.

3.1.2. Step 2. Design of finger domains After determining the sequence and length [i.e., 3N or (3N + 1)bp, where N is the number of finger domains used] of a target site for an ATF, the site is divided into N pieces of 3-bp or overlapping 4-bp DNA

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Fig. 3. Construction of ATF. The scheme illustrates how to generate an ATF. Please see the text for a detailed explanation.

segments, where the last base of each 4-bp target is the first base of the next 4-bp target. Then, an individual finger domain recognizing each 3- or 4-bp segment is selected from building modules, such as Barbas's modules for 5′-GNN-3′, 5′-ANN-3′, 5′-CNN-3′, and a partial 5′-TNN-3′ [11,34,35], Kim's modules [36], or Young's modules for 5′-GNN-3′ and a partial 5′-TNN-3′ [12]. Barbas's modules can be also designed by using the Zinc Finger Tools website [37]. An alternative method to construct each finger domain is to choose four recognition amino acids of positions −1, 2, 3, and 6 of the α-helix region of the zinc-finger domain from recognition code tables. Klug's and Pabo's groups individually constructed degenerate recognition code tables based on finger domains selected by their phage display, which enable generation of selected finger domains [38,39]. We constructed a nondegenerate recognition code table based on known and potential DNA-base–amino acid interactions [16]. Because our table is nondegenerate, it is easy to make all finger domains for specific 4-bp targets, enabling generation of artificial ZFPs (AZPs) in a highthroughput manner. Our AZPs can recognize DNA sequences containing more than three guanines (at any positions) in the first 9 bp of 10bp targets with high affinities [16,40–43]. We call it “3G rule.” Now two-finger building blocks made by Sangamo are commercially available via Sigma-Aldrich [44].

3.1.3. Step 3. Assembly of finger domains DNA fragments encoding selected zinc-finger domains are assembled by PCR to generate DNA fragments encoding ZFPs. In many cases, the canonical peptide linker of TGEKP works well to link finger domains. However, certain linkers are known to enhance the binding affinities of ZFPs. For the detailed information on engineered linkers, please see [23,25,26,29] for examples. 3.1.4. Step 4. Evaluation of ZFPs After cloning DNA encoding a ZFP into an E. coli expression plasmid such as pET (Novagen), the ZFP is expressed and purified. DNAbinding properties of purified ZFPs are examined in several ways. The most popular method to determine the apparent dissociation constant (Kd) is an electromobility-shift assay (EMSA) or gel shift assay. The assay also enables examination of the specificity by using mutant DNA probes. Apparent Kds are also determined by using surface plasmon resonance or a BIAcore system [45]. This assay gives us the kinetic information on the DNA binding at the same time. The DNA-binding specificities can also be examined by enzymelinked immunosorbent assay (ELISA) [38]. A 96-well format enables evaluation of relative affinities against multi-mutant probes. The use of systematic evolution of ligands by exponential enrichment (SELEX)

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or cyclic amplification and selection of targets (CAST) [46] presents all possible binding sequences of a ZFP by the combination of EMSA with a permutated DNA probe library and PCR. The use of DNA microarrays to determine the DNA-binding specificities was also reported [47]. A bacterial one-hybrid system, in which ZFP is fused to the α-subunit of RNA polymerase, was also reported [48]. 3.1.5. Step 5. Fusion of other functional domains After evaluation of the DNA-binding properties, the best or better ZFPs are fused to a nuclear localization signal (NLS) and an effector domain to generate ATFs. The short peptide PKKKRKV from the simian virus 40 large T antigen [49] has been used most extensively as the NLS, and it works well. Effector domains used for ATFs are listed in Table 1. Among them, the most frequently used activation domain is the herpes simplex virus VP-16 activation domain [50]. The most popular repressor domain is a Kruppel-associated box (KRAB) domain of KOX1 [51]. To monitor ATF expression, several epitope tags such as FLAG, c-myc, and HA are incorporated at N or C termini (or both) of ATFs. Optionally, a switch domain is connected to ATF to regulate the timing of gene modulation by addition of a small molecule to the medium. To date, fusion of ligand-binding domain(s) of hormone receptors such as an estrogen receptor [52,53], thyroid hormone receptor α [54], or a progesterone receptor [55] to ATF was reported. Barbas's group also generated single-chain ligand-binding domains derived from an estrogen-receptor homodimer and an ecdysone-receptor/retinoid X receptor heterodimer, and observed ligand-dependent gene regulation in mammalian cells [52,56]. Pollock's group reported a gene switch using a small-molecule heterodimerizer [57,58]. In their system, a ZFP is fused to FK506-binding protein (FKBP), and an effector domain is fused to the rapamycin-binding domain of FKBP rapamycin-associated protein. The heterodimerizer assembles the ZFP and the effector domain noncovalently, and the resulting ATF can modulate target gene expression in a heterodimerizer-dependent manner [58]. Further, a cell-penetrating peptide (reviewed in [59]) or protein transduction domain is conjugated to ATF, rendering ATF molecules able to permeate cells [40]. 3.1.6. Step 6. Evaluation of ATFs Finally, the resulting ATF is cloned into a mammalian expression plasmid or viral vector. The efficiency of the ATF is investigated by transient reporter assays or more ideally by analyzing expression

Table 1 Effector domains used for construction of ATFs.

Activation domain

Name

Comments

Ref

VP16

C-terminal transcriptional activation (approximately 75 amino residues) of herpes simplex virus VP-16 trans-activator, very effective and most popular Tetrameric repeat of DALDDFDLDML, more active than VP16 C-terminus of the human NK-κB transcription factor p65 subunit (residues 288–548) Chimeric activation domain comprising the p65 subunit (residues 281–551) and human heat shock factor 1 (residues 406–529) Eleven tandem copies of EDTDL derived from the C-terminal transcriptional activation domain of β-catenin Krüppel-associated box domain of KOX1 (residues 1–75), most popular and powerful Residues 1–36 of the Mad mSIN3 interaction domain Residues 473–530 of the est2 repressor factor Ligand–binding domain of the thyroid hormone receptor v-erbA Derived from the Arabidopsis thaliana transcription factor SUPERMAN

[50]

VP64 p65 S3H

(FDTDL)11

Repressor domain

KRAB SID ERD vErbA SRDX

[68] [112] [57]

[40]

[51] [113] [114] [54] [40]

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levels of endogenous target genes. The mRNA levels of target genes are analyzed by Northern blotting or quantitated by real-time PCR. The protein levels of target genes are analyzed by flow cytometry, Western blotting, or ELISA. The transfection or transduction efficiencies of ATFexpression plasmids or viral vectors and expression levels of ATFs should be normalized when several ATFs are compared. DNA microarray analysis is a very powerful method to evaluate specific modulation of target genes by ATFs. Further, binding of ATFs to their target sites in cells is examined by in vivo DNase I footprinting or chromatin immunoprecipitation (ChIP). Now ChIP assays are used more frequently. 3.2. Other methods to generate ZFPs Selection of ZFPs from a library is a powerful alternative approach to generation of ZFPs. In addition to phage display selection (many reviews available) and ribosome display [60], other selection systems have been applied or newly developed to generate ZFPs. A yeast onehybrid system has been used to select ZFPs [61]. A bacterial twohybrid system was developed by Pabo's group [62]. In their system, two expression plasmids were used for fusion of ZFP–Gal11P and fusion of Gal4 with the α-subunit of RNA polymerase. The specific interaction between Gal11P and Gal4 recruits RNA polymerase to activate a selection marker gene in vivo. By using this system, Young's group selected ZFPs successfully [63]. Chandrasegaran's group reported a bacterial one-hybrid selection system, where the α-subunit of RNA polymerase was directly fused to a ZFP [64]. Furthermore, Choo's group exploited cell-free selection of ZFPs by using emulsionbased in vitro compartmentalization [65]. I will introduce studies on direct selection of ATFs in Section 4.4. 4. Application of ATFs 4.1. Short history As described above, the first study of gene regulation by altered transcription factors in living cells (yeast) was reported in 1992 by Young's group [18]. They mutagenized the Cys2His2 zinc-finger domains of the yeast transcription factor ADR1 to alter the binding specificities and demonstrated that one of the ADR1 mutants recognized their target DNA and transiently activated a reporter gene harboring its target DNA in yeast. In 1994, gene regulation using a ZFP designed to recognize a 9-bp genomic sequence in mammalian cells was reported by Klug's group [66]. In their study, a 3-finger ZFP alone (but not an ATF!) was used for gene regulation. The ZFP was designed to bind to a unique 9-bp region of a BCR-ABL fusion oncogene, and indeed it repressed p190BCR-ABL expression in murine cells by blocking RNA polymerase movement. This study demonstrated for the first time that ZFP could modulate endogenous gene expression in living cells. Concurrently, they also demonstrated for the first time that an ATF, in which the engineered ZFP was fused to a VP16 transcriptional activation domain, could activate CAT-reporter gene expression transiently in living cells. After these reports, many groups reported engineered 3-finger ATFs and evaluated their functions in transient reporter assays. However, ATFs are required to specifically recognize N16-bp genomic sequences in human cells due to the genome size (i.e., 3 × 109 bp) in order to modulate their target gene expression. In 1997, Barbas's group reported the first 6-finger ATF applicable to endogenous gene regulation in human cells [67]. They generated a 6-finger ZFP by assembling modular blocks recognizing 5′-GNN-3′ triplets and demonstrated that the 6-finger ATF modulated expression of a reporter gene under the control of an artificial promoter harboring six tandem copies of its 18-bp binding site. In 1998, the same group succeeded in modulation of a reporter gene under the control of a native erbB-2/HER-2 promoter by using a 6-finger ATF [68].

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Then, in 2000, Barbas's group reported that their 6-finger ATFs specifically up- and downregulated the endogenous erbB-2 gene in living cells [69]. At almost the same time, Juliano's group reported repression of the endogenous MDR1 multidrug resistance gene by a 5-finger ATF [70]. After these studies, various ATFs were generated to modulate endogenous gene expression both in vitro and in vivo. 4.2. Modulation of endogenous gene expression I will list the studies of endogenous gene regulation by using ATFs to date. I will introduce them in a chronological order. 4.2.1. ErbB As described in Section 4.4.1, in 1998, Barbas's group reported the first 6-finger ATF that is targetable to a specific human genomic site [68]. They used the ErbB-2 gene as a model target for evaluation of their ATF. Members of the ErbB receptor family play important roles in the development of human malignancies. In particular, ErbB-2 is overexpressed as a result of gene amplification and/or transcriptional deregulation in a high percentage of human adenocarcinomas arising at numerous sites, including breast, ovary, lung, stomach, and salivary gland. ErbB-2 is an important therapeutic target for breast cancer. The antibody for the receptor is now used clinically for therapy. Barbas's group designed a 6-finger ZFP [apparent dissociation constants (Kds) of 0.5−0.75 nM] for recognition of the an 18-bp sequence on the ErbB-2 (or HER-2) 5′-UTR and fused it to a VP16 or VP64 activation domain, or to an ERD, KRAB, or SID repressor domain [68]. They found that the ATF(VP64) activated a reporter gene under the control of the native ErbB-2 promoter/5′-UTR region by 27-fold, and ATF(KRAB) repressed the reporter gene to the background level. Then in 2000, they demonstrated that the same ATFs modulated the endogenous ErbB-2 gene expression in living cells [69]. The ATFs transduced by a retroviral vector specifically up- or downregulated endogenous ErbB-2 gene expression in the human carcinoma cell line A431, as shown by flow cytometric analysis. The ATF did not change the expression profile of the other family members, ErbB-1 and ErbB-3. Another ATF (apparent Kd of 0.35 nM) designed for regulation of ErbB-3 also discriminated ErbB-3 from ErbB-1 and ErbB-2. The ATF modulated ErbB-3 gene expression specifically without affecting expression of ErbB-1 and ErbB-2. The 18-bp target sequence of the ATF designed for ErbB-2 differed from that of the ATF designed for ErbB-3 by only three nucleotides. The ATF for ErbB-2 bound to its target with about 15-fold higher affinity than to the ErbB-3 target, and the ATF for ErbB-3 bound to its target with about 30-fold higher affinity than to the ErbB-2 target. Therefore, their results indicate that ATFs having such binding selectivity can discriminate their targets from very similar sequences in mammalian nuclei. By targeting the ErbB-gene family, Barbas' group regulated two family members with a single ATF and thereby obtained new insight into the functions of ErbB gene products by combination with ATFs targeting a specific family member [71]. Furthermore, they evaluated their single-chain hormone receptor as the gene switch for ATFs by using the gene family [56]. 4.2.2. MDR1 multidrug resistance gene In 2000, Juliano's group reported modulation of the MDR1 gene to control multidrug resistance in cancer cells [70]. The human MDR1 gene encodes the P-glycoprotein, a 170-kDa membrane ATPase that can transport many types of drugs from cells. Increased levels of P-glycoprotein expression in tumor cells results in the phenomenon of multidrug resistance, a significant problem in cancer chemotherapy. They targeted a region overlapping of the DNA-binding sites of three transcription factors, EGR1, SP1, and WT1, that regulate the MDR1 expression. They constructed a 5-finger ZFP and fused it to a VP16 activation domain or two tandem copies of a KRAB repressor domain.

The ATF activator increased expression of a reporter gene under the control of two tandem copies of the 15-bp target by 463-fold, and the ATF repressor reduced the expression to b5% of a control in KB-8-5 cells. The same ATF repressor reduced expression of a reporter gene under the control of a portion (− 88/+105) of the MDR1 native promoter to 25% of a control in KB-8-5 cells. They analyzed repression of the endogenous MDR1 expression by the ATF repressor using flow cytometry, which revealed that the ATF repressor effectively blocked tissue plasminogen activator-mediated induction of P-glycoprotein, but did not change the expression profile of the α5β1 integrin used as a control. 4.2.3. Erythropoietin In 2000, Wolffe's group reported activation of the human erythropoietin gene [31]. They generated ten 3-finger ZFPs (apparent Kds of 0.23–23 nM) and fused each of them to a VP16 activation domain. All ATFs activated a reporter gene under the control of the erythropoietin promoter, but not endogenous erythropoietin; only ATFs targeting a distinct genomic region could activate the endogenous gene. They also showed that binding affinities did not determine the activation rate. Next, they investigated why ATFs (designated ATF-862) targeting the region around −850 (relative to the transcriptional start site) activated the erythropoietin gene. They constructed a stable ATF expression system that was induced by addition of doxycycline (Dox) to the medium. In the absence of Dox, the −850 region in HEK293 cells was not hypersensitive to DNase I, corresponding to previous studies reporting that the promoter is normally silent in HEK293 cells. However, the addition of Dox (i.e., expression of ATF-862) induced hypersensitive sites around the −850 region. ChIP assays revealed that the addition of Dox enriched DNA fragments around the −850 region bound by ATF-862 by 3070-fold. These results suggest that their ATFs could remodel chromatin in a targeted manner. 4.2.4. Vascular endothelial growth factor A In 2001, Wolffe's group reported activation of vascular endothelial growth factor A (VEGF-A) [30]. VEGF-A is an endothelial cell-specific mitogen that is a key inducer of new blood vessel growth, both during embryogenesis and in later processes such as wound healing. VEGF-A levels are dramatically increased by hypoxia, triggering angiogenesis and microvascular permeability. Therefore, both activation and repression of VEGF-A are attractive therapeutic approaches. Namely, treatments that reduce VEGF-A levels may prevent angiogenesis associated with tumor growth, rheumatoid arthritis, and diabetic retinopathy. In contrast, enhancement of VEGF-A levels may stimulate neovascularization to treat ischemia, arteriosclerosis obliterans, and wound healing. They first identified the “open” chromatin regions on the promoter/5′-UTR of VEGF-A by DNase I mapping. Then, they designed ten 3-finger ZFPs targeting the open chromatin regions and characterized their DNA-binding affinities (apparent Kds of 0.005 to 2.8 nM). They fused these ZFPs to a VP16 activation domain and confirmed that the resulting ATFs activated the endogenous VEGF-A expression by 2- to 15-fold. In this study, they also compared the activities of VP16 and p65 activation domains. In some ATFs, the p65 fusion showed higher activation than the corresponding VP16 fusion (e.g., 3- to 4-fold activation). Furthermore, they demonstrated synergetic activation of the VEGF-A gene by using one ZFP-VP16 fusion and another ZFP-p65 fusion simultaneously. Wolffe's group's study also raised another important issue. Comparison of the endogenous activation profile with the corresponding reporter activation profile clearly indicated that the accessibility of ATFs to a promoter (and/or 5′-UTR) on a closed circular plasmid differs from that to a native promoter (and/or 5′-UTR) on chromosomes, depending on the location. Their study demonstrated the importance of chromatin structure in the design of ATFs/ZFPs. In 2002, Sangamo's group reported the first evaluation of the in vivo efficacy of an ATF for endogenous gene regulation [72]. They first

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identified open chromatin regions in the mouse VEGF-A locus by DNase I mapping, as they did in their previous study for human VEGF-A [30]. Based on that information, they designed two 3-finger ZFPs (Kd of b10 pM) and one 6-finger ZFP (Kd of 31 pM) and fused each of them to a VP16 activation domain. The 6-finger ATF and one 3-finger ATF increased VEGF-A mRNA in the mouse-derived C1271 cell line by 2- and 2.5-fold, respectively. The 6-finger ATF introduced into the quadriceps muscle of CD-1 mice via an adenovirus vector increased VEGF-A mRNA by 2.7-fold, but no or little enhancement of control genes (i.e., Lgh1, Pgk1, and Glut1) induced by hypoxic response was observed. Sangamo's group then investigated effects of the 3-finger ATFs on vascularization in a mouse ear angiogenesis model. As shown in their photographs of ATF-treated ears, injection of the ATF-expressing adenoviral vector into the external ear of CD-1 mice induced neovascularization. Immunohistochemical counting of vessels revealed the 2.5-fold enhancement of vessel formation by the ATF. The angiogenesis stimulated by the ATF did not produce a hyperpermeable neovasculature, as determined by Evans blue dye extravasation. On the other hand, the neovasculature induced by adenovirus transduction of VEGF-A164, which is the major splice variant, showed spontaneous hemorrhage and Evans blue extravasation (i.e., generation of leaky blood vessels). As previously reported [30], the ATF for mouse VEGF-A also produced naturally occurring splice variants in their normal stoichiometry. Several studies have suggested that the proper isoform balance is crucial for VEGF-A function. This result presents one of advantages of the ATF approach for gene therapy: modulation of gene transcription from natural promoters. They also demonstrated acceleration of wound healing by the 6-finger ATF in a mouse model. In 2003, Sangamo's group examined whether their ATFs would be sufficient to reduce the high levels of VEGF expression associated with vascularizing tumors to a therapeutically relevant degree [54]. They fused three effective 3-finger ZFPs to a ligand-binding domain of chicken thyroid hormone receptor α1 or its viral relative, v-ErbA as the repressor domain. Transient expression of these inducible ATFs resulted in ∼50% repression of the endogenous VEGF-A expression in HEK293 cells. To eliminate the contribution of untransfected cells in the transient transfection assays, they constructed stable U87MG cell lines in which the T-Rex system (Invitrogen) provided inducible expression of the ATFs. The human glioblastoma U87MG cell line is highly tumorigenic; the expression of VEGF-A protein is ∼20-fold higher in U87MG cells than in HEK293 cells. In the Dox-inducible cell line, they found Dox-dependent repression of the endogenous VEGF-A expression. For example, in the presence of 2 ng/ml Dox, the repressed level of VEGF-A mediated by their ATF was reduced ∼20-fold compared with a non-Dox-treated control. The reduced VEGF-A protein level (∼300 pg/ml) was comparable to that (∼350 pg/ml) of the nontumorigenic human glioblastoma cell lines, U251MG and T98G. In addition, Sangamo group's evaluated the in vivo efficacy of their ATF in mice [73], rats [74], and rabbits [75]. The ATF is in a phase II trial now [44]. In 2004, we reported the first cell-permeable ATFs, which we called designed regulatory proteins (DRPs) [40]. We designed a 6finger AZP targeting a 19-bp sequence of the 5′-UTR of human VEGF-A, which is also conserved in mouse VEGF-A, by using our nondegenerate recognition code table [16]. We fused the AZP to an activation domain of VP16 or 11 tandem copies of the short peptide FDTDL, which was derived from the C-terminal transcription activation domain of β-catenin (FDTDL11), or to a repressor domain of KRAB, SID, or an SRDX domain derived from the plant transcription factor SUPERMAN [76]. Transfection of the ATF activators and repressors resulted in N10-fold activation and N80% repression of the endogenous VEGF-A, respectively, compared with a control. We also found that the ATF(FDTDL11) was more effective than the ATF(VP16), and that the ATF(SRDX) reduced the VEGF-A expression under hypoxia-mimicking

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conditions induced by cobalt treatment, not under normoxia. Next, we fused a cell-penetrating peptide (CPP) of the basic residues 47–57 from the HIV-1 Tat protein [77], an 11-amino-acid synthetic peptide, YARAAARQARA (PTD4) [78], or a 9-mer of arginine (R9) [79] to both the ATF activator and repressor proteins for the first time. We first confirmed by immunofluorescent staining that the CPP−ATF or DRP exogenously added to the medium entered the nuclei. Then, we found that multiple administrations of the R9−ATF(VP16) protein increased the VEGF-A level by N20-fold, and that the PTD4−ATF(KRAB) protein reduced the VEGF-A level to 20% of that of a control. Our DRPs provide a convenient way to modulate the direction, time, location, magnitude, and duration of the desired change in gene expression. DRPs will serve as novel protein drugs for various therapeutic targets. In 2008, Yun's group reported in vivo repression of VEGF-A [80] by using the ATF repressor generated by Kim's group [81]. They constructed a replication-competent oncolytic adenoviral vector [82] expressing the ATF. They assessed the antitumor efficacy of their ATFexpressing adenoviral vector in the U87 human glioma xenograft model. Treatment with their ATF-expressing adenoviral vector enhanced survival rates of male athymic nu/nu mice. By day 40 after treatment, 100% of the mice were still alive, whereas no control mouse was alive. 4.2.5. PPARγ In 2002, Sangamo's group reported repression of the nuclear hormone receptor PPARγ, which is essential for cellular differentiation and lipid accumulation during adipogenesis [83]. They first identified open chromatin regions by DNase I mapping. Then, they generated two 6-finger ZFPs (apparent Kds of 20 and 44 pM) targeting one of the open regions and fused each of them to a RAB repressor domain. They transduced mouse 3T3-L1 preadipocyte cells with retroviral vectors harboring these ATF genes and observed that one ATF significantly reduced PPARγ expression. Consistent with the observation, the ATF totally blocked cellular lipid accumulation. 4.2.6. IGF2 and H19 In 2003, Sangamo's group reported up- and downregulation of the IGF2 and H19 genes, which is related to neoplasms and Beckwith– Wiedemann syndrome [32]. They designed five 3-finger ZFPs (apparent Kds of b100 pM) and fused each to VP16 activation domains. These ATFs transiently activated the IGF2 mRNA levels by 1.8- to 50-fold. The ZFP (Kd of ∼30 pM) of the most active ATF was fused to a p65 activation domain, and the resulting ATF increased the H19 mRNA levels by 120-fold. They also confirmed that their ATFs repressed the IGF2 and H19 genes. They fused the ZFP to a v-ErbA repressor domain and established a stable Dox-inducible cell line expressing the ATF repressor. In their cell line, expression of IGF2 and H19 was reduced in a Dox-dependent manner; addition of 2 ng/ml Dox reduced expression levels of IGF2 and H19 to 20% and 11% of those of a control, respectively. They confirmed by ChIP assays that the ATF bound to the IGF2 and H19 promoters. Finally, they demonstrated that ATF(VP16) reactivated the transcriptionally silent IGF2 and H19 alleles in HEK293 and murine cell hybrids. 4.2.7. Bax In 2003, Juliano's group reported up-regulation of the proapoptotic bax gene that drives cancer cells into programmed cell death [84]. The bax gene is regulated by the tumor suppressor p53 and activates the downstream caspase cascade, eventually resulting in apoptosis. Because the p53 gene product is lost or inactivated by mutation in over 50% of cancers, p53-regulated pro-apoptotic genes, including bax, fail to be induced. They designed a 5-finger ZFP and fused it to a VP16 activation domain to construct an ATF for activation of the bax gene. The transfection of the ATF-expression plasmid into p53-deficient Saos-2 cells resulted in 9.2-fold activation of the luciferase reporter gene under the control of an artificial promoter

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harboring two copies of 40 bp (−489 to − 449) of the bax promoter. They also confirmed that the ATF caused apoptosis. The ATF reduced viability of Saos-2 cells to approximately 40% in cell survival assays. Because the addition of the caspase inhibitor B-D-FMK to this system increased the viability to 80%, they concluded that the ATF actually induced apoptosis. Interestingly, the same ATF did not affect the viability of p53-replete U-2 OS cells. 4.2.8. Oct-4 Sangamo's group reported regulation of the Oct-4 gene to control stem cell fate in 2003 [85]. Oct-4, which is expressed in ES and germ cells, is important for the maintenance of pluripotency and selfrenewal. In mouse ES cells, activation or repression of Oct-4 resulted in cell differentiation. They constructed 10 different 6-finger ZFPs by assembling three 2-finger domains and fused them to a VP16 activation domain or KRAB repressor domain to up- or downregulate the Oct-4 gene. They screened these ATFs by ELISA and Taqman assays to select the best one. The most effective ATF bound to a 19-bp sequence, which was located between bp −25 and − 7 relative to the transcription start site in the promoter. Transfection of the ATFexpression plasmid under the control of a human elongation factor 1a promoter resulted in 1.6-fold activation or 0.3-fold repression of Oct-4 mRNA. Then, they examined expression levels of other genes affected by Oct-4 expression. It has been shown that OTX1 is increased and HAND1 is repressed by Oct-4 expression. As expected, the ATF activator for Oct-4 increased OTX1 activation 1.8-fold, and the ATF repressor for Oct-4 increased HAND1 activation 56-fold. Finally, they examined possibility of differentiation of mouse ES cells by their ATFs and observed morphological changes due to ATFs in the presence of murine leukemia inhibitory factor. Although they concluded that their ATFs were able to induce ES cell differentiation by modulating Oct-4 levels, they did not characterize the “differentiated” cells further. 4.2.9. Checkpoint kinase 2 Repression of the checkpoint kinase 2 (CHK2) gene was reported by Sangamo's group in 2003 [86]. CHK2 acts as a key integrator of DNA-damage signals regulating cell-cycle progression, DNA repair, and cell death by phosphorylating a variety of substrates, including the p53 tumor suppressor protein. They designed a 6-finger ZFP that recognized an 18-bp target in the DNase-hypersensitive region on the CHK2 promoter (Kd of ∼ 70 pM) and fused it to a KRAB repressor domain. The resulting ATF showed 50% repression of the CHK2 mRNA in transient transfection assays. Then, to eliminate the contribution of untransfected cells in the transient transfection assays of repression, they constructed stable cell lines expressing the ATF in a Doxindependent manner. Twelve of 16 of clones showed Dox-dependent repression of CHK2 mRNA levels (i.e., N10-fold repression). They confirmed that the ATF functionally abolished CHK2 activity by checking BXA and MDM2 mRNA, which were induced by the addition of a DNA-damaging agent camptothecin activating a p53-depedent DNA-damage pathway. Importantly, they showed that the ATF repressed only their target gene CHK2 by using the Affymetrix U133A array, which provides information on 22,225 probe sets or ∼16,000 genes. No other gene on the array was identified as changed up or down in two different stable cell lines. Importantly, this result demonstrates that ATF can be designed to modulate only its target gene. 4.2.10. Cholecystokinin 2 receptor In 2004, Sangamo's group reported generation of a cell line for drug discovery to overcome patent issues concerning the use of cDNA [87]. They designed a three-finger ATF targeting a DNase I hypersensitive site in the promoter region of the cholecystokinin 2 receptor (CCK2R), a widely investigated and patented G-protein-coupled receptor, and fused it to a VP16 activation domain. After they confirmed ∼ 200-fold activation of the endogenous CCK2R gene by transfection of the ATF plasmid, they established a stable Dox-

inducible cell line expressing the ATF. They confirmed the Doxdependent activation of CCK2R in the cell line. Using the cell line, they performed the high-throughput screening of a small-molecule library. 4.2.11. Parathyroid hormone receptor 1 Sangamo's group reported generation of an isogenic human cell line for drug discovery in 2005 [88]. They chose the human parathyroid hormone receptor 1 (PTHR1) gene because the gene plays important roles in regulating serum calcium homeostasis and bone metabolism. They designed a three-finger ZFP (apparent Kd of 20 pM) and fused it to a VP16 or p65 activation domain. Transfection of the expression plamsmids transiently enhanced the endogenous PTHR1 expression in five different cell lines; the ATF(VP16) showed greater activity than the ATF(p65) in four cell lines. Then they established a stable Doxinducible cell line expressing the ATF, and confirmed the Doxdependent activation of PTHR1 in the cell line. Dox (2 ng/ml) enhanced the PTHR1 mRNA level by ∼ 25-fold. 4.2.12. γ-Globin In 2005, Barbas's group reported upregulated γ-globin gene to test their hypothesis that targeting genomic regions proximal to the binding sites of natural transcription factors is effective for gene regulation by ATFs [89]. Because several genetic diseases are associated with defective β-globin expression, the most common being β-thalassemias and sickle cell disease, these diseases can be mitigated by the fetal γ-globin, which forms tetrameric fetal hemoglobin (HbF). They designed three 6-finger ZFPs that recognize the region proximal to the position −117 of γ-globin promoter (apparent Kds of 0.7 to 5.4 nM) and fused them to a VP64 activation domain. The region around the position −117 is proximal to the binding sites (position of −140) of the γ-globin transcriptional activators FKLF and FKLF-2. In transient reporter assays, the ATF with the highest affinity increased a reporter gene under the control of the γ-globin promoter by 47-fold, but not another reporter gene under the control of the β-globin promoter. The ATF also upregulated the endogenous γ-globin gene. It transiently increased HbF production in the erythroleukemia K562 cell line by 8-fold. Transduction of the ATF-expression retroviral vector resulted in 14-fold enhancement of the HbF level. Furthermore, a ChIP assay revealed that the ATF bound to the γ-globin promoter in living K562 cells. Such enrichment of immunoprecipitated chromatin was not detected in a ChIP assay for an unspecific promoter of the ICAM-1 gene used as a negative control. This study indicates that targeting a genomic region around known cis elements that are accessible by native transcription factors is a good choice when information on the chromatin structures of target genes is not available. 4.2.13. Mammary serine protease inhibitor Two years later, Blancafort's group reported reactivation of the silenced mammary serine protease inhibitor (maspin) gene in tumor cells for cancer therapy [90]. Maspin is one of the tumor suppressor genes, and a high level of expression is associated not only with reduction of tumor growth, but also decreased angiogenesis, cell motility and invasion, and metastatic dissemination. This gene is silenced by transcriptional and aberrant promoter methylation in aggressive epithelial tumors. They generated three 6-finger ZFPs that bound to different 18-bp targets on the maspin promoter (apparent Kds of 0.9 to 3.1 nM) and fused each of them to a VP64 activation domain. All three ATFs, which were transduced using a retroviral vector, activated maspin expression in three breast cancer cell lines. In particular, the most effective ATF (designated ATF-126) increased endogenous maspin expression by N60-fold in the MDA-MB-231 cell line, which comprises a methylated and silenced maspin promoter. Consistent with the activation properties, these ATFs reduced viability of an MDA-MB-231 cell line: ATF-126 reduced the viability to 30% of that of a control by

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inducing apoptosis. Furthermore, ATF-126 reduced cell invasion of MDA-MB-231 to 34% of that of a control in Matrigel invasion assays. Finally, Blancafort's group investigated the in vivo efficiency of ATF-126. They injected MDA-MB-231 variant cells transduced with an ATF-126 retroviral vector into the flank of immunodeficient mice and monitored tumor growth by caliper measurements. All mice injected with control cells (transduced with an empty retroviral vector) developed primary tumors, whereas none of the mice injected with ATF-126-expressing cells generated tumors, demonstrating that ATF-126 suppressed xenograft growth of the MDA-MB-231 cell line in nude mice. It is notable that a different ATF (designated ATF-97) showed greater activation than the most effective ATF (ATF-126) for endogenous activation in transient reporter assays in their studies, implying that we have to decipher the results of reporter assays carefully. It is likely that chromatin structures of promoters in (reporter) plasmids do not reflect in vivo chromatin structures. 4.2.14. Utrophin In 2007, Passananti and Corbi's group reported activation of the dystrophin-related utrophin gene to complement the lack of dystrophin function in Duchenne muscular dystrophy [91]. Several series of evidence support the hypothesis that utrophin expression precedes dystrophin expression in development. Indeed, ectopic expression of utrophin prevents muscular dystrophy in both dystrophin-deficient mdx mice and utrophin–dystrophin-deficient mice. They generated a 4-finger ZFP that bound to a 12-bp target on the human utrophin promoter A and fused it to a VP16 activation domain. The resulting ATF increased activation of a reporter gene under the control of the utrophin promoter by approximately 7-fold. The ATF also increased the endogenous utrophin expression by 1.7-fold. Their ChIP assay suggested that the ATF bound to the utrophin promoter in vivo. There was no control experiment using an unrelated promoter. Subsequently, Passananti and Corbi's group examined the efficacy of their 3-finger (not 4-finger) ATF in vivo [92]. They generated transgenic mice expressing the 3-finger ATF under the control of the myosin light chain promoter/enhancer. Molecular analysis of the two transgenic lines revealed skeletal muscle-specific expression of the ATF in one line and additional expression in heart tissues in another line. They confirmed the binding of the ATF to the utrophin promoter in the skeletal muscle tissues, but not to the dystrophin promoter. A quantitative analysis of utrophin mRNA revealed 3- to 4-fold activation of the mRNA in the ATF-transgenic lines. Consistent with this result, they observed an increase of utrophin protein and the consequent relocalization of it along the sarcolemma in the transgenic mice. I did not find description of toxicity or side-effects due to the ATF expression in mice. The 3-finger ATF could bind to the 9-bp sequence on many different loci in mice and thereby seems to affect more unrelated genes than 6-finger ATFs. Although they performed a microarray analysis, the analysis did not give sufficient information on the specific utrophin activation by their 3-finger ATF in mice. 4.2.15. Pigment epithelium-derived factor In 2007, Sangamo's group reported activation of the pigment epithelium-derived factor (PEDF) gene both in vitro and in vivo to suppress induced choroidal neovascularization (CNV) [33]. PEDF is a natural antiangiogenic protein that is produced by retinal pigment epithelial (RPE) cells and other cells in the eye. PEDF induces apoptosis of dividing endothelial cells in new blood vessels, without affecting quiescent endothelial cells, and it has been postulated to be part of the endogenous system for the control of new vessel growth within the eye. They constructed a panel of 50 6-finger ZFPs binding to 18-bp sequences within the PEDF promoter region and fused them to p65 activation domains. They screened them in HEK293 cells in transient transfection assays, and found that the most effective ATF activated the PEDF gene by 14-fold. They also confirmed the efficacy in

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ARPE-19 cells, a line derived from human RPE cells, the major source of endogenous PEDF in the eye. Although the basal level of PEDF mRNA in ARPE-19 cells was approximately tenfold higher than that in HEK293 cells, the ATF doubled the PEDF mRNA in ARPE-19 cells. Genome-wide microarray analysis revealed that only one other gene (SERPINA1) was upregulated by the ATF in U87MG cells when compared with a control, indicating the exceptionally high specificity of the ATF. Next, Sangamo's group screened a panel of mouse PEDF-specific ATFs, and constructed an adeno-associated virus type 2 (AAV-2) vector expressing the most effective mouse ATF. They found a ∼ 1.4- or ∼2-fold increase of PEDF mRNA in the posterior eyecup or retina 6 weeks after subretinal or intravitreous injection, respectively. Corresponding to these results, the ATF reduced CNV to ∼ 50% or ∼60% of that of a control by subretinal or intravitreous injection, respectively. Importantly, the ATF delivered via an AAV-2 vector had maintained such anti-angiogenic activity 3 months after intravitreous injection. 4.3. Combination with chromatin remodeling drugs Recently, an interesting study was reported [93]. Blancafort's group combined both epigenetic and genetic (sequence-specific ATFs) strategies to specifically reactivate the expression of an epigenetically silenced mammary serine protease inhibitor (maspin) gene in tumor cells. Maspin is an important tumor suppressor gene whose expression is associated not only with tumor growth inhibition but also with decreased angiogenesis and metastasis. They opened silenced chromatin regions to give ATFs access to their target sites more easily by using remodeling drugs simultaneously rather than searching for open chromatin regions on promoter regions of target genes to obtain the most efficient ATF. To reactivate the mapsin gene in MDA-MB-231 breast cancer cells, they generated three 6-finger ATFs possessing a VP64 transcriptional activation domain [68] that were designed to recognize different 18-bp sequences on the mapsin promoter. They examined the effects of the triple treatment with ATFs, 5-aza-2′deoxycytidine (5-aza-dC), and suberoylanilide hydroxamic acid (SAHA) at different doses on mapsin gene expression. They found that one ATF together with 5-aza-dC (1.0 mg/ml), and SAHA (0.5 mg/ ml) reactivated the gene expression by N500 fold. Furthermore, the combination of the ATF with 5-aza-dC (3.8 mg/ml), and SAHA (1.3 mg/ml) inhibited breast tumor cell proliferation by 95%. Chromatin remodeling drugs such as 5-aza-dC and SAHA have been reported to reactivate tumor suppressor genes that are aberrantly methylated in aggressive tumor cells. They are approved for therapeutic treatment or clinical trials. However, because these drugs are not specific for tumor cells/tissues in humans, the use of these drugs in cancer patients may be limited. Therefore, the synergized use with ATFs is expected to increase the targeting efficiency and specificity of current anticancer drugs, and thereby decrease the dose and cytotoxicity of anticancer drugs. 4.4. ATF library Barbas' group reported construction of the first ATF libraries and their application to genome-wide gene regulation in 2003 [94]. To demonstrate their concept, they targeted 10 different cell-surface marker genes such as vascular endothelial cadherin and integrin, which facilitate cell sorting. They assembled finger domains for recognition of triplets, 5'-GNN-3' [11], 5'-ANN-3' [34], and a partial 5'TNN-3' [34], to generate a 3-finger ZFP library theoretically comprising 9177 variants. Using the 3-finger ZFP library, they also generated a 6-finger library theoretically comprising 8.4 × 107 variants. Then, they fused each library to a VP64 activation domain and cloned it into a retroviral vector possessing a GFP gene. After transduction into cells, GFP-positive cells overexpressing a target marker gene were selected

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by flow cytometry, DNA encoding the zinc-finger domain was recovered by PCR, and it was re-cloned in the retroviral vector for subsequent selection. After three or four rounds of selection, both ATFactivator libraries had increased expression of five marker genes, as shown by FACS analysis. Detailed molecular analysis of selected ATF clones revealed that the ATFs recognized and activated their target marker genes. They also demonstrated that the ZFP library that was fused to a KRAB repression domain reduced CDH5 gene expression in FACS analysis. Importantly, they showed that a 3-finger ATF library can be used for screening. A 3-finger ATF with a very weak affinity (apparent Kd of 1 μM!) was also selected. Although 3-finger ATFs have lower affinities than 6-finger ATFs, the libraries with fewer fingers have advantages: (1) higher representation of individual library members, (2) greater possibility of binding more sites in regulatory regions, and (3) a mode of regulation analogous to the action of natural transcription factors. In 2004, Barbas's group reported in vivo selection of ATFs for modulation of the intercellular cell adhesion molecule 1 (ICAM-1) as a model system from combinatorial libraries [95]. The binding of ICAM1 to β2 integrins promotes adhesion and signaling for transendothelial migration of leukocytes and for T-cell co-activation during inflammatory and immune responses. They used their 6-finger ATF activator library [94]. After three rounds of selection with flow cytometry, a total of 35 individual ATFs were isolated. Among them, four ATFs reproducibly upregulated endogenous the ICAM-1 gene by 2- to 4fold. They also fused each of these selected ZFPs (apparent Kds of 1 to 8.5 nM) to a KRAB or SID repressor domain. These ATF repressors worked well. The most effective ATF repressor reduced ICAM-1 expression to 15% of that of a control. In this study, SID worked better than KRAB as the repressor domain. The most effective ATFs increased the expression by 63-fold and reduced it to 17% in primary cells. They also demonstrated that the improved ATF derived from the selected one (apparent Kd of 0.16 nM) increased the ICAM-1 expression further by 0.7- to 2.7-fold as compared to the selected ATF. Seol's group reported application of an ATF library to identify genes inducing Taxol resistance (TR) in 2004 [96]. Taxol, a brand name of paclitaxel, is one of the most prescribed chemotherapeutic agents. Although it is widely used to treat breast, non-small cell lung, and ovarian cancers, the development of drug resistance is a major problem in paclitaxel chemotherapy. Drugs that inhibit the expression of these genes may be useful for blocking the development of TR. They used 25 zinc-finger domains out of the possible 64 (= 4 × 4 × 4) domains to generate a 4-finger ZFP library [97], and then fuse it to a p65 activation domain. After transient transfection of the plasmid expressing the ATF library into HeLa cells, they treated the resulting cells with paclitaxel (100–200 nM). ATF plasmids isolated from paclitaxel-resistant cells were used for subsequent selection. After eight rounds of selection, they sequenced N100 selected plasmids, and obtained six identical pairs of ATF plasmids. Among them, five plasmids promoted TR. Two ATF clones promoting the highest TR increased the survival rate of HeLa cells in the presence of paclitaxel from ∼ 20% (a control) to ∼ 70%. In cDNA microarray analysis, they found that 37 genes were co-activated N2-fold by transfection with both of the most effective ATF clones. Although the genes identified should be investigated further, the gene pool includes genes known to be related to TR (such as cytochrome P450 3A5 and aurora-C kinase), indicating the effectiveness of their approach. In 2005, Barbas's group reported application of an ATF library to identify genes involved in tumor progression [98]. They transduced HeLa cells with the retroviral vector of their 6-finger ATF activator library [94]. After screening them for paclitaxel resistance, paclitaxelresistant cells were morphologically examined for the appearance of more complex morphological transformations, resembling epithelial– mesenchymal transition. Among several ATF clones promoting paclitaxel resistance, one ATF induced elongated, fibroblast-like morphologies. This ATF enhanced both cell migration in laminin-coated

transwell assays and cell invasion by 20-fold in Matrigel invasion assays. Furthermore, they confirmed that the ATF clone enhanced lung metastasis in nude mice. Finally, they identified eight endogenous genes affected by transfection of the ATF clone by using a microarray with ∼18,500 genes. Among them, E48 (500- to 1000-fold upregulation), AGT, and IL-13Rα1 (8- to 10-fold upregulation) were highly regulated by the ATF clone. Interestingly, the transiently transfected ATF promoted cell invasion more efficiently than cotranfection of these three isolated genes, indicating that a single ATF can regulate multiple target genes by binding to similar or identical DNA sequences located in several regulatory regions. Barbas's group also reported, in 2008, the use of an ATF library to isolate efficient regulator genes of TR in tumor cells [99]. They used HeLa cells for the screening, as Seol's group did [96]. However, they transduced the cells by using a retroviral vector harboring their 3- of 6-finger ATF activator library [94]. In this study, the best ATF for enhancement of ER was a 3-finger ATF (the ZFP's Kd of 3.3 nM) rather than a 6-finger ATF (the ZFP's Kd of 1.2 nM). Although they performed detailed molecular analysis of the best 3-finger ATF clone (e.g., enhancement of TR in different cancer cell lines), analysis of 31 genes regulated by the ATF was not performed in detail. It is unclear whether genes identified by Barbas's group contribute more effectively to enhancement of TR than genes identified by Seol's group [96]. 4.5. Cell-permeable ATFs as potent protein drugs Until 2003, ATFs had been introduced into cells or animals only by delivery of the expression vectors. Another potential method for the delivery is the direct introduction of ATF proteins into target cells or tissues. To achieve this, we reported conjugation of cell-penetrating peptides (CPPs) to ATF proteins in 2004 [40]. We demonstrated the concept by modulation of the endogenous VEGF-A gene, as described in Section 4.3.4. Since the first report of the HIV-1 Tat protein as a CPP in 1988 [100], various CPPs including the basic residues 47–57 from the HIV-1 Tat protein [77], an 11-amino-acid synthetic peptide, YARAAARQARA (PTD4) [78], and a 9-mer of arginine (R9) [79] have been reported, and the novel CPPs continue to be identified or developed (reviewed in [101]). Although the mechanism of CPP-mediated transduction across the cell membrane is not fully understood, various molecules (from small drugs to large proteins) that are conjugated to CPPs are successfully transported into mammalian cells. Certain studies have suggested that internalization of CPPs may be an artifact of fixation: CPPs attached to cell surfaces may diffuse into cells that are made permeable by fixation [102,103]. We confirmed the effectiveness of the CPP−ATFs in living cells experimentally. After our report, in 2008, Kim's group investigated efficacy of the CPP−ATFs in mice [104]. Although they used their ATF conjugated with TAT for repression of VEGF-A, their TAT−ATF alone reduced tumor volume in mice only to 65% of that of PBS-treated control mice, and parallel use of the anticancer drug 5-flurouracil was required to reduce the blood vessel formation to ∼ 35% of that of PBS-treated control. We are interested in the efficacy of the most effective CPP−ATF of our group (i.e., the R9 conjugate) alone in mice. In 2008, we also demonstrated that cell-permeable artificial zincfinger proteins (CPP−AZPs) that prevented a viral replication protein from binding to its replication origin inhibited the DNA replication of human papillomavirus (HPV) in living cells [43]. Among the CPP−AZPs, the R9 conjugate showed the highest performance as well: 250 nM AZP−R9 reduced HPV-18 DNA replication to 3% of that of a control, and the 50% effective concentration (EC50) was b31 nM. Furthermore, a cytotoxicity assay revealed that the 50% inhibitory concentration (IC50) of AZP−R9 was N10 μM. Therefore, the selectivity index (SI), defined as IC50/EC50, was N300, which is better than that (i.e., SI=15–42) of the antiviral cidofovir for HPVs [105]. Thus, CPP−AZPs are expected to be a novel class of antiviral drugs.

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Finally, I will introduce the application of ATFs and ZFPs to antiviral therapies in chronological order.

type PBS sequence; the remaining 18 consisted of three different sequences with one or two mutations in the PBS. Although the virus replication rates of these HIV-1 mutants were decreased, they reduced the effectiveness of the ATF.

4.6.1. RNA virus In 2003, Klug and Choo's group reported inhibition of human immunodeficiency virus type 1 (HIV-1) by repressing the HIV-1 5′ long terminal repeat (LTR) promoter [106]. They generated 3- and 6-finger ZFPs, fused a KRAB repressor domain to them, and evaluated these ATFs in transient reporter assays using a Jurkat human T cell line. Among them, a 6-finger ATF reduced the Tat (a HIV-1 regulatory protein)-activated expression of a reporter gene under the control of the 5′ LTR promoter to 2–11% of that of a control. Furthermore, they confirmed the efficacy of the HIV-1 replication inhibition. To enrich the ATF-positive cells within the transiently transfected pool, they used a human glioma NP2 cell line variant, which requires cotransfection of the expression plasmid of the HIV-1 coreceptor CXCR4 for infection. In the replication assay, the ATF reduced the virus replication to 25% of that of a control. In 2004, Barbas's group reported inhibition of HIV-1 replication in primary human cells by repressing the 5′ LTR promoter [107]. They generated three 6-finger ZFPs (apparent Kds of 1–10 nM) and fused each to a KRAB repressor domain. They extensively analyzed the efficacies and specificities of their ATFs for the promoter repression in transient report assays. They found that the most effective ATF specifically repressed the promoter activity to 1–8% of that of a control. Then, they demonstrated that HIV-1 titers dropped 524-fold in the PM1 T-cell line that stably expresses the most effective ATF. Furthermore, in primary human peripheral blood mononuclear cells, retrovirally transduced ATF reduced the viral titers by 10 - to 100-fold. The repressive effect was maintained even at 18 days post-HIV-1 infection. The most effective ATF targeted an 18-bp sequence overlapping the site recognized by Klug's most effective 6-finger ATF that targets the region containing the three consecutive SP1-binding sites. Therefore, these results suggest that targeting cis element(s) critical for the gene expression by an ATF is effective for the transcriptional repression. In 2005, Hur's group reported ZFP variants that inhibit HIV-1 replication [108]. They designed several 3-finger ZFPs that target the consecutive SP1-binding sites and fused each to the pox virus zincfinger (POZ) domain from FBI-1 and a Tat mutant, rather than a repressor domain, to repress the transcription from the 5′ LTR promoter. The POZ domain prevents Sp1 from binding the SP1binding site, and the Tat mutant binds to its DNA target but does not interact with other proteins necessary for the promoter activation. Transient transfection with the ZFP variant resulted in reduction of the number of HIV-1-positive colonies to 0.02% of that of a control in a single-round HIV-1 replication inhibition assay. Soon after, Barbas's group reported the inhibition of HIV-1 replication by targeting the tRNA primer-binding site (PBS) [109]. The PBS is most highly conserved site in the HIV-1 genome. Human tRNALys binds to the PBS and is used as a primer for reverse transcription. The mutations in the PBS negatively affect virus production and infectivity. They designed four 6-finger ZFPs (apparent Kds of 0.8–2.4 nM)and fused each to a KRAB repressor domain. These ATF reduced the HIV-1 replication to 10–65% of that of a control in transient replication assays. However, only one of the ATFs inhibited virus production (N90% reduction) when transduced into primary human peripheral blood mononuclear cells by the lentiviral vector. Because HIVs develop resistance to antiviral drugs by rapid mutation of their genomes, Barbas's group examined whether repeated exposure of HIV-1 to the most effective ATF resulted in mutations that would allow the virus to escape regulation. After several rounds of infection, the resulting viruses were cloned and the PBS regions of 20 clones were sequenced. Of the 20 clones, only 2 contained the wild-

4.6.2. DNA virus In 2003, Klug's group reported inhibition of the herpes simplex virus 1(HSV-1) replication cycle, which is closely related to expression of the immediate-early (IE) genes [110]. To repress the expression of one IE gene (IE175k), they generated a 6-finger ZFP that targets the promoter and fused it to a KRAB repressor domain. Because they examined the efficacy of the ATF in transient transcription assays and the transfection of the ATF-expression plasmid was 30–35%, they did not repress the expression of IE175k in whole COS-1 cells. However, analysis of the virus replication by using COS-1 cells that were sorted based on a cotransfected maker gene (i.e., GFP) showed that the ATF reduced the production of the HSV-1 infectious particles to 19% of that of a control. In 2006, we reported inhibition of the viral replication of the human papillomavirus (HPV) by using a 6-finger AZP [42]. HPV induces cervical cancer, which is the second most common malignancy in women worldwide. Previously, we had demonstrated that DNA replication of a plant geminivirus, beet severe curly top virus (BSCTV), was inhibited by an AZP that was designed to block binding of the replication protein (Rep) to its replication origin [16], and transgenic Arabidopsis thaliana plants expressing the AZP showed complete resistance to BSCTV infection [41]. The six-finger AZP, which binds to the 19-bp DNA containing the entire Rep-binding site, was designed using our nondegenerate recognition code table [16]. We applied the AZP technology to HPV type 18 (HPV-18), one of the high-risk HPVs. We designed two 6-finger AZPs (apparent Kd of 10 pM) to prevent the viral replication protein E2 from binding to its replication origin. Transient replication assays revealed that the genedelivered AZP reduced the viral replication in mammalian cells. In particular, one AZP reduced the replication level to ∼ 10% of that of a control. We confirmed that the replication was inhibited by the binding of the AZP to the target site by using mutant HPV-18 replication origins in transient replication assays. One of the advantages of our strategy is the very low or nonexistent risk of the emergence of resistant viruses. Conventional antiviral drugs are designed to inactivate one component (usually viral DNA polymerase) of a viral protein. Therefore, viruses can easily escape the attack of an antiviral drug by mutation of the viral protein without a significant loss of their original activity. In contrast, our strategy targets binding of viral replication proteins to their replication origins. In order to escape the AZP attack without sacrificing replication efficiency, viruses need to mutate a DNA base(s) in a region recognized by the AZP (so that the AZP cannot bind to the site) and additionally to mutate the viral protein at the same time so that the mutant protein can bind to the mutated origin. Mutation of an AZP binding site alone is unfavorable to DNA viruses because the native viral protein cannot bind to the mutated replication origin either. The probability of the synchronized double mutation, which is equal to the probability of mutation in the viral replication origin multiplied by that of mutation in the E2 protein, should be extremely low. Therefore, it is likely that viruses resistant to our approach will emerge less frequently than those resistant to conventional antiviral drugs. As described in Section 4.4.5, we recently reported cell-permeable AZPs that inhibited the HPV-18 replication as well when exogenously added to the medium [43]. In 2008, Tyrrell's group reported inhibition of the viral transcription of the duck hepatitis B virus (HBV), a model virus for human HBV [111]. They generated two 6-finger ZFPs (apparent Kds of 12.3 and 40.2 nM) to target the enhancer region of the duck HBV covalently closed circular DNA by using the program Zinc Finger Tools made available by Barbas's group [37]. They found that these ZFPs reduced

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