Drug discovery with engineered zinc-finger proteins

© 2003 Nature Publishing Group

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | MAY 2003 | 361

Zinc fingers: versatile DNA-recognition domainsC

2H

2 zinc fingers are found in 2% of all human genes,

and they are by far the most abundant class of DNA-binding domains found in human transcriptionfactors1. The prevalence of these modules reflectstheir remarkable versatility for recognizing differentsequences of DNA, and variations in the amino acidsequence of the C

2H

2 domains allow them to be targeted

to different locations in the genome. Each zinc fingeris a short stretch of 30 amino acids, containing twoconserved cysteines and two conserved histidines2.The conserved residues coordinate a zinc ion thatallows the finger to fold into a compact structure con-taining a β-turn (which includes the conserved cys-teines) and an α-helix (which includes the conserved histidines)3,4. C

2H

2 zinc fingers typically occur in tan-

dem arrays, and many transcription factors have threeor more fingers working in concert to recognizeDNA, thereby targeting the transcription factor to itsappropriate promoter.

Several zinc-finger–DNA structures have beensolved, including Zif268,YYI, TFIIIA, Tramtrak and anartificial domain containing a consensus backbone5–8.

The determination of the Zif268–DNA crystal structuregave the first detailed view of how ZFPs interact withDNA. This structure shows how three C

2H

2 fingers can

recognize a nine-base-pair site in such a way that theymight be mixed and matched to target novel DNAsequences. In this complex, the fingers bind DNA atregularly spaced three-base-pair intervals, and each fingeruses a few key residues on its recognition helix to makethe crucial base contacts in the DNA major groove(FIG. 1). Many experiments have shown that changes atthese key residues are sufficient to alter the DNA-bindingspecificity of a finger, and this greatly simplifies theproblem of adapting the fingers to recognize novelDNA sequences9–12.

Achieving universal DNA recognitionA principal goal in zinc-finger design has been to pro-duce proteins that can recognize any pre-determinedDNA sequence. An important technology for achiev-ing this has been phage display13. This technique hasgenerated thousands of selected zinc fingers and hashelped uncover many of the underlying principles ofzinc-finger–DNA recognition12,14–20.

DRUG DISCOVERY WITHENGINEERED ZINC-FINGERPROTEINSAndrew C. Jamieson, Jeffrey C. Miller and Carl O. Pabo

Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerfultechnology platform with many uses in drug discovery and therapeutics. These proteins havebeen used as the DNA-binding domains of novel transcription factors (ZFP TFs), which are usefulfor validating genes as drug targets and for engineering cell lines for small-molecule screeningand protein production. Recently, they have also been used as a basis for novel humantherapeutics. Most of our advances in the design and application of these ZFP TFs rely on ourability to engineer ZFPs that bind short stretches of DNA (typically 9–18 base pairs) located withinthe promoters of target genes. Here, we summarize the methods used to design these DNA-binding domains, explain how they are incorporated into novel transcription factors (and otheruseful molecules) and describe some key applications in drug discovery.

Sangamo Biosciences, Inc.,Point Richmond TechnicalCenter, 501 CanalBoulevard, Suite A100,Richmond, CA 98404, USA.Correspondence to C. O. P.e-mail: [email protected]:10.1038/nrd1087


362 | MAY 2003 | VOLUME 2 www.nature.com/reviews/drugdisc

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sequences. As shown by extensive site-selection studiesusing a representative set of ZFPs, this ‘mix and match’approach successfully targets G-rich sequences, espe-cially sequences containing either 5′-GXXGXXGXX or5′-XXGKXGKXX (where X is one of the four bases, A,C, G or T and K is either G or T)17,22,23.

Even with this restriction on the nucleotide contentof the target site, GXXGXXGXX or XXGKXGKXXsequences occur approximately every 32 base pairs,which allows one to make suitable regulators for manyendogenous genes24–26.

Phage display is most powerful when it is used in away that allows for the optimization of interactionsbetween neighbouring fingers (and the optimization ofcontacts that a finger might make with a neighbouringfinger binding site)15,16,27. An example is the ‘bipartiteselection’ strategy, in which the two halves of the Zif268three-finger DNA-binding domain are selected sepa-rately, and then recombined in vitro to make three-fingerdomains28. Two separate (non-overlapping) libraries areused to perform the bipartite selections for up to 2 × 45

= 2,048 different binding sites. In one library, the tip offinger 2 can add to the way finger 1 binds DNA, and inthe other library, the tip of finger 3 can add to the wayfinger 2 binds DNA. When the two halves are combined,the result is that the three fingers work in concert torecognize the entire nine-base-pair site. Recently, thisstrategy was applied commercially to obtain a largearchive of ZFPs that can be recombined into three-fingermodules that will recognize a vast array of nine-base-pair sequences. Two-finger modules that recognize sitesof the form 5′-GXXXXX or 5′-XXXXXG can also beobtained from the archive, and these can be used tomake six-finger ZFPs as discussed below.

Other strategies for selecting ZFPs have used a yeastone-hybrid29 and a bacterial two-hybrid selectionsystem30. Two advantages of these systems are that theZFPs are selected in the context of living cells and thebest proteins can be obtained in a single round of selec-tion, rather than the multiple rounds required byphage display. Very recently, a selection system usingmammalian cells has been described31.

The ZFP archives that have been obtained by designand selection make it possible to target virtually anyDNA sequence using pre-made zinc-finger modules.This can be achieved in a number of ways: by mixingand matching individual modules, by recombining thehalves of different three-finger proteins, by linkingtogether a set of two-finger modules (to make four- orsix-finger proteins) or by linking two three-finger unitsto make six-finger proteins.

Targeting unique DNA sequences in the genomeWhen controlling transcription, a ZFP TF should opti-mally regulate only its intended gene. One possible wayto achieve this is to use sets of six fingers to target aneighteen-base-pair site, which, if highly repetitivesequences are avoided, should occur only once in thehuman genome. A straightforward approach for con-structing such six-finger proteins is to append addi-tional fingers onto a smaller domain (such as Zif268)

In phage display, pools of ZFPs with fingers contain-ing different amino acid residues in their recognitionhelices are expressed on the surface of the M13 filamen-tous bacteriophage. Phage that bind to the desired DNAsequence are selected from the pool and, after severalrounds of amplification and selection, the DNA fromindividual clones is sequenced to reveal the identity ofthe amino acids involved in DNA binding.

Phage display reveals that in many instances theZFPs use a particular amino acid to recognize a particu-lar base in a given DNA triplet. For example, when aguanine base is selected for at the 5′ end of a DNAtriplet, the ZFPs frequently contain an arginine residueat position 6 on the recognition helix (FIG. 1). Choo et al.proposed a set of recognition ‘rules’ to summarize theseinteractions21. Although these rules do not have absolutepredictive power, they can be a useful aid in designingZFPs that will recognize a desired target site.

Data obtained from phage selection, from crystalstructures of other DNA complexes and from theknown binding specificities of naturally occurring zincfingers have been used to make ‘rationally designed’ zincfingers. A commercial archive containing several hun-dred of these individual zinc fingers with differentrecognition helices has been prepared by grafting themonto the Sp-1 backbone (Sp-1 is a human transcriptionfactor containing three zinc fingers). These fingers werecombined to make thousands of novel ZFPs by mixingand matching the fingers to recognize different DNA

C

G

C

G

C

E3D2

R-1

T

G

G

A

C

C

D2

R-1

G

C

G

C

G

C

T N A

5′ C 3′

5′3′

G R6

T6

H3

D2

R-1

R6

E3

a b

Figure 1 | Modular interactions between zinc fingers and DNA. a | The Zif268–DNA complexshowing the three zinc fingers bound in the major groove of DNA5. The DNA is blue and fingers 1,2, and 3 are red, yellow, and violet respectively. Zinc ions are shown as grey spheres. b | A diagramshowing the sequence-specific protein–DNA interactions between Zif268 and its DNA-bindingsite. The recognition helices of the three fingers are represented in the centre of the panel and thebases on the two strands of the DNA site are shown on either side. The identity of key residueson the recognition helices (positions –1, 2, 3 and 6 with respect to the start of the helix) are alsoshown using the single-letter code. Contacts observed in the crystal structure are represented asdashed lines. The fingers and bases that they contact are colour-coded using the same schemeas in a. The fingers are spaced at three-base-pair intervals and tend to contact three adjacentbases on one strand of DNA and one base on the other strand.



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It is important to bear in mind that smaller three-finger proteins can also function relatively specificallyinside cells, as NUCLEOSOME formation and CHROMATIN con-densation will occlude the vast majority of potentialbinding sites. In fact, we expect that less than 1% of theDNA in a differentiated human cell will be readily avail-able for zinc-finger binding39. Given that so much of theDNA is occluded, and the limited range over which theregulatory domain acts, three-finger proteins (such asthe KRAB repressors found in nature) can be morespecific than they might first seem.

Regulating genes using ZFP TFsZFPs can be used to carry out a variety of cellular activi-ties by combining them with different functionaldomains. In many applications, activation or repressiondomains are added to make ZFP TFs, but useful pro-teins can also be made by combining the ZFPs with thefunctional domains from integrases40, methylases41 andnucleases42 (see Future directions). A nuclear localiza-tion sequence is also typically incorporated in the novelproteins to ensure that they are efficiently transportedto the cell nucleus43.

ZFP TFs can contain several kinds of activation orrepression domains. In many applications, ZFP TFscontaining the VP16 domain from herpes simplexvirus44 or the p65 domain from the cellular trans-cription factor NF-κB45 are used for activating transcription. The same ZFP DNA-binding domaincan be used for repressing transcription by adding the KRAB domain, which creates a ZFP TF that mimics the many naturally occurring zinc-fingerKRAB proteins46. Attaching nuclear hormone LBDsor chromatin-modifying domains (such as HISTONE

ACETYLTRANSFERASES OR HISTONE DEACETYLASES) generatesmolecules that can be used for regulating gene expression directly, or that can be used in high-throughput screening (HTS) for drug discovery (seebelow) (FIG. 3).

An example demonstrating how ZFP TFs can beused to control endogenous gene expression is theregulation of the ERBB2 and ERBB3 genes26. Thesegenes encode growth factor receptors that are impli-cated in human breast cancer, and the levels of proteinfrom transcription of these genes can be detectedusing labelled antibodies and flow cytometry. Barbasand colleagues designed six-finger ZFPs targeted tothe 5′-untranslated region of each gene. The designedZFPs, combined with either a VP64 activation domain(containing four repeats of the VP16 domain) or witha KRAB repression domain, were delivered to cul-tured breast cells using a retroviral vector. Not onlydid the ZFP TFs activate or repress their target genesas intended, but they also discriminated between thegenes even though the ZFP-binding sites were identi-cal at 15 out of 18 base pairs. The extent to whichother, unrelated, genes were co-regulated by the ZFPTFs was not, however, reported in these studies.

Recently, our laboratory used a six-finger protein(constructed by linking three two-finger modules) torepress the CHK2 gene (P. Gregory and S. Tan, personal

using the conserved five-residue (TGEKP) linkers thatjoin the fingers in many naturally occurring multi-fingerdomains32. Other designs have added a longer, moreflexible linker between different pre-existing three-finger domains33 (FIG. 2b). This approach might betteraccommodate the strain that seems to accumulate whenmore than three fingers bind DNA34. The introductionof a flexible linker has also been reported to improve thespecificity of DNA-binding inside cells29.

Moore et al. recently introduced a general methodfor assembling zinc fingers that involves concatenatingtwo or more two-finger modules using longer variantsof the conserved TGEKP linkers35 (FIG. 2c). The linkerscontain one or two extra glycine residues, and a bio-chemical analysis indicated that this novel way ofjoining the ZFPs also improves their DNA-bindingspecificity. A related strategy using even longer linkersallows adjacent fingers to skip over a base, and thisaffords additional flexibility in targeting an immensevariety of DNA sequences.

Another promising approach when targeting ZFPsto longer DNA sequences involves the use of dimeriza-tion domains that bring together two separate sets ofZFPs. ZFPs fused to the appropriate Gal4 domains36 orfused to leucine zipper domains can dimerize and bindto longer, appropriately spaced DNA to form multi-fingercomplexes37. Dimeric complexes can also form whenZFPs are fused to steroid hormone receptor ligand-binding domains (LBDs)38. The LBDs are inactive untilligand is added externally, and this allows the activity ofthe ZFP TFs to be chemically controlled (see below).

NUCLEOSOME

The basic structural subunit ofchromatin, which consists of~200 base pairs of DNA and an octamer of histones (a family of small, highly conserved basic proteins).

CHROMATIN

The compact form that DNA is organized into in eukaryoticcells, which contains genomicDNA, histones and non-histone proteins.

HISTONE ACETYLTRANSFERASES

AND DEACETYLASES

Enzymes that modify histonesby adding and removing acetylgroups, a chemical modification that can affectchromatin structure.

3-finger ZFPrecognizes 9 base pairs

a

2 × 3-finger ZFPrecognizes 18 base pairs

b

3 × 2-finger ZFPrecognizes 18 base pairs

c

N

C

Figure 2 | Incorporation of zinc-finger modules into multi-finger arrays. a | A tandemarray of three zinc fingers. A single zinc finger is depicted as a ribbon drawing, with an orangesphere representing a zinc ion. Each of the rectangular units represents a zinc finger in atandem array. The fingers are linked together by canonical (TGEKP) linkers, shown as shorthorizontal bars. b | A six-finger zinc-finger protein (ZFP) consisting of two three-finger domains(2 x 3) linked by a non-canonical flexible linker (shown as a longer horizontal bar). c | A six-finger(3 x 2) ZFP obtained by concatenating three two-finger domains. The linkers between fingers 2and 3 and between fingers 4 and 5 are longer than the canonical linker.



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Assembling ZFP TFs for gene regulationNow that large archives of zinc-finger modules are avail-able, ZFP TFs can be readily assembled using a similarprotocol for each target gene (FIG. 4). In outline, a com-puter program searches the promoter sequence for bind-ing sites that can easily be targeted by combining ZFPmodules from the archive. The search typically reveals alarge number of matches, and the precise target sites arechosen after considering their proximity to the transcrip-tion start site, and the location of known regulatory siteswithin the promoter or the location of DNase hypersensi-tive sites that have been mapped to the locus. (Knowledgeabout these hypersensitive sites is often useful for target-ing ZFPs as they represent areas of the DNA that are easilyaccessible to ZFP binding24,39.) The appropriate zinc-finger modules are then assembled into a multi-fingerDNA-binding domain using standard recombinant DNAprocedures. Next, the assembled ZFPs are cloned intoplasmids that encode the regulatory domains, to create aZFP TF. These constructs are transfected into cells, andtechniques such as reverse transcription of RNA followedby the polymerase chain reaction can be employed tomonitor changes in the transcriptional levels of the targetgene. In the majority of cases, several ZFP TFs that regu-late the target gene can be obtained within two weeks.

communication). An analysis of its biological speci-ficity using Affymetrix expression arrays shows thatthis repressor acts with singular specificity inside cells.Of several thousand active genes that were measuredusing the expression array, the ZFP TF repressed onlythe CHK2 gene. Further studies will be needed to see ifsix-finger proteins typically give such a remarkablelevel of specificity in human cells. Recently, encourag-ing results showing the specific behaviour of ZFPshave also been obtained in transgenic plants47.

Other published examples of ZFP TFs that regulateendogenous genes include the three-finger ZFPs usedto activate the human EPO25 and VEGF genes24, six-finger ZFPs used to repress peroxisome proliferatoractivated receptor-γ (PPARγ) gene expression in mice48

and a five-finger ZFP that represses the human ABCB1gene (also known as MDR1)29. This last example usesZFPs that were selected from a yeast one-hybrid sys-tem. Recently, ZFPs derived from yeast have also beenused to activate the BAX gene and so drive apoptosis incultured human cells49. The growing body of literaturereflects the increasingly routine use of ZFP TFs forcontrolling gene expression, and there are innumerableother unpublished examples of genes that have beenregulated with ZFP TFs.

Adding functional domains to ZFPsenables diverse applications

a High-throughput screening

d DNA modification

CH3

b Gene activation

p65

c Targeted DNA cleavage

FokI

g Chemically controlledgene regulation

p65

HDAC

h Chromatin modification

f Gene repression

KRAB

e Targetedintegration

IN IN

LBD

DNMT

Figure 3 | Combining zinc-finger proteins with different functional domains. An engineered zinc-finger protein (ZFP) can becombined with different functional domains for many different applications. The ZFPs are depicted as horizontal arrays of greyrectangles containing either three or six fingers. Pictorial representations of functional domains are attached to the ZFPs by shortlines, representing linkers. a | A ZFP combined with a ligand-binding domain (LBD), for use in high-throughput screening. b | A ZFPcombined with the p65 domain for activating transcription. c | A ZFP combined with a nuclease domain from a Type II restrictionenzyme, such as FokI, for targeted DNA cleavage. d | A ZFP combined with a DNA methyltransferase (DNMT) domain for DNAmodification. e | A ZFP combined with a retroviral integrase (IN), for inserting exogenous sequences into DNA. f | A ZFP combinedwith a repression domain, such as the KRAB domain of the KOX protein, for repressing transcription. g | A ZFP transcription factorthat only fully assembles in the presence of a small-molecule drug. h | A ZFP combined with a chromatin-modifying domain, such asa histone deacetylase (HDAC), for small-molecule screening.



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Chimaeric transcription factors in HTSUsing a ZFP TF to activate an endogenous gene providesan alternative way to perform HTS. Traditionally, HTSrequires either the use of a cDNA to overexpress thetarget protein, or requires searching for a cell line thatoverproduces the protein naturally, or as a result ofgene amplification. Using ZFP TFs to activate endo-genous genes can produce sufficient amounts of pro-tein for most cell-based assays, and this approach couldbe especially useful if use of the cDNA is restricted byintellectual property considerations (BOX 1).

As an example of this strategy, zinc fingers haverecently been used to upregulate the gastrin/cholecysto-kinin B G-protein-coupled receptor (GPCR) gene(CCKBR) at a level that allows the cell line to be used forscreening ligands (P.-Q. Liu, personal communication).In particular, a ZFP TF targeted to the gene promoterresulted in a 200-fold increase in transcription of thereceptor gene. Furthermore, we found that these cellshave levels of receptor protein that allow a calciumflux assay to be used in testing for CCKBR antago-nists. Cell lines of this general type could have broadapplicability, as GPCRs mediate cellular responses toan enormous variety of signalling molecules, and asGPCRs are one of the most important classes ofreceptor proteins for drug targeting50.

An interesting and completely different use of ZFPsin drug screening involves attaching a ZFP to the regu-latory domain of a transcription factor that mediatessignal transduction. Compounds that affect this signal-transduction pathway then modulate the endogenousreporter gene targeted by the ZFP (in this case, onetypically targets the ZFP to a gene that is easy to assay).ZFPs fused to the LBDs of nuclear hormone receptorsillustrate how this procedure can be applied.

When a ligand binds to a nuclear hormone receptor,the receptor translocates to the nucleus, binds to anuclear response element (NRE) and stimulates trans-cription of one or more genes. These nuclear hormonereceptors control a diverse set of biological functions,but often are difficult to screen in drug assays due tothe structural similarity between various isotypes, andbecause several receptors can act at the same (or verysimilar) NREs51. This ZFP-based strategy enables bothseparation of the signal, and multiplexing of theassays, as several ZFPs targeted to different reportergenes can be fused to different LBDs and co-expressedin a single cell line. The bioactivity of compounds canthen be assayed by monitoring the expression levels ofthe reporter genes, providing a unique approach toanalysing ligand potency and specificity in a multiplexedassay using a single cell line.

New assays have also been developed by linkingZFPs to a set of other functional domains, includingthose involved in chromatin modification52. Many ofthese original proteins were notoriously difficult toassay in vitro, especially in a format suitable for high-throughput drug screening. We find, however, thattheir activities can be localized (and readily assayed) ifthe relevant functional domain is linked to a ZFP andthus targeted to an easily assayed endogenous reporter

Regulating genes for target validationAnalysing the functions of human genes is an integralpart of the modern drug discovery process. The comple-tion of the human genome project has identified morethan 30,000 human genes, and thousands of these mayrepresent potential new drug targets50. ZFP TFs can beapplied to this analysis in two ways. One direct wayinvolves designing ZFPs to validate the phenotypiceffects of activating or repressing a gene. Alternatively,libraries of ZFP TFs might be used to screen cells fordesired phenotypic changes.

An interesting example of how a ZFP TF can be usedfor target validation involves the problem of distinguish-ing the roles of different receptor isoforms in fat-celldifferentiation (adipogenesis)48. During adipogenesis,the nuclear hormone receptor PPARγ exists as twoisoforms, γ1 and γ2. When a ZFP TF was used torepress transcription of both isoforms in mouse cells,adipogenesis was completely blocked. Adipogenesiswas restored when a cDNA encoding the γ2 isoformwas expressed in the cells, but not when a cDNAencoding the γ1 isoform was expressed. In this case,the ZFP repressor caused a discernible phenotypicchange in the cells, and subsequent experimentsallowed a precise distinction to be made between theroles of the two isoforms. This type of study could beuseful in drug design, as one could then focus onmaking a drug that only affects the desired isoform.

Scan target promoter for ZFP-binding sites

Additional bioinformatics orDNA hypersensitivity dataSelect target binding sites

Assemble ZFPs from an archiveof pre-made modules

Screen ZFPs for function in cells

Figure 4 | The zinc-finger protein transcription factor assembly process. A computerprogram is used to scan the promoter of the target gene and identify all possible zinc-fingerprotein (ZFP) binding sites from a database. Target binding sites are then chosen according totheir distribution across the promoter. Additional data, such as DNA hypersite mapping or othertypes of bioinformatics, can also be used to guide the selection of target sites. ZFPs that bindthe selected target sites are assembled from an archive of pre-made modules, and are incorporated into ZFP transcription factors (TFs). The ZFP TFs are then screened for efficacyin cell-based assays.



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involved in cancer, cardiovascular disease, viral infec-tions, muscular dystrophy, cystic fibrosis, diabetes,glaucoma and chronic pain.

ZFP TFs can produce physiologically desirable effectsin whole animal models, as was recently demonstratedby the production of blood vessels in a mouse earmodel55. A ZFP TF that was designed to activate theendogenous Vegfa gene was delivered into the mouseear by an adenovirus, and activation of the Vegfa geneled to the expression of all its major splice variants. Theproduction of these splice variants is a normal conse-quence of Vegfa activation in the body, and a number ofreports have indicated that the resulting Vegfa isoformsare required to establish a signalling gradient duringANGIOGENESIS56. Importantly, the blood vessels that wereproduced when the ZFP TF activated the Vegfa geneshowed all the visible qualities of normal blood vessels.By contrast, when a cDNA encoding a single isoformwas introduced into the mouse ear, the vasculature hadthe abnormal appearance associated with ‘leaky’ bloodvessels (FIG. 5).

It is not yet known precisely how important the issueof splice variants will be in gene therapy, but currentestimates are that approximately half of human geneshave alternative splice forms57. For some genes, thenumber of different splice variants is extremely large58,making cDNA treatments impractical if multiple iso-forms are needed. Even in the case of Vegfa activation,which requires only three principal isoforms to establishan extracellular signalling gradient, it should be fareasier to add a single ZFP rather than to directly add themultiple Vegfa splice variants in their proper ratios.

When using ZFPs for gene therapy, it might be useful(as with gene therapy in general) to ensure that theeffective dose and timing of the therapeutic regime canbe regulated with a small molecule. For ZFP TFs thismight be achieved either by controlling expression ofthe transcription factor (using a drug-inducible pro-moter), or by expressing the ZFP TF in an inactive formthat can be activated as desired by dosing with a small-molecule drug. One approach has been to fuse the ZFPto steroid hormone receptor LBDs38. In the absence ofhormone, the ZFP TF remains inactive in the cyto-plasm, where it is believed to be complexed with cellularfactors, such as heat-shock proteins59. After ligand bind-ing, the ZFP TF can dissociate from the complex andtranslocate to the cell nucleus in an active form.Modified LBDs that place the ZFP TF under the controlof drugs such as 4-hydroxytamoxifen or RU-486 couldprove especially useful for clinical applications38.

Another strategy for achieving small-molecule con-trol that was recently applied to Vegfa activation uses arapamycin analogue to control the assembly of the ZFPTF. In this experiment, the ZFP and the activationdomains are each fused to additional domains that canbind to separate halves of the small-molecule drug.Administration of the drug brings the DNA-bindingdomain and the activation domain together (therebyassembling a functional transcription factor), whichallows transcriptional activity to be controlled byadjusting the concentration of the drug60.

gene. (The alternative approach of changing the activityof the protein with a drug can have genome-wideeffects that are hard to predict or monitor.) So far,there is at least one example in the literature showinghow histone methyltransferase activity can be coupledto repression of the Vegfa gene53. The chromatin-modifying activities of histone deacetylases, which areoften associated with haematological disorders result-ing from chromosomal translocations54, have alsobeen linked to zinc fingers, and their activity can bemonitored in a similar way.

Engineered cell lines are also useful for producingpharmaceutically valuable proteins. Existing methodsfor protein overproduction typically use gene amplifica-tion (such as in the production of human erythropoeitin)or strong viral promoters to drive gene transcription.In any case in which the level of transcription limits theproduction of protein, ZFP TF activation can bedesigned to give further upregulation. Such increasesin protein production can be extremely valuable, asthey dramatically increase the output from a given cellculture facility and/or prevent the need for additionalcapital investment.

Zinc fingers as therapeutic agentsUltimately, the most important application of ZFPsmight involve their use as therapeutics that controlgene expression to cure or prevent disease. In thisregard, they might be used to activate or repress a widerange of genes, including ones that are not ‘druggable’with conventional pharmaceuticals. It is estimated thatonly 25–50% of the 3,000 human genes implicated indisease will provide appropriate targets for small-molecule drugs50. Using ZFP TFs to control geneexpression could provide an alternative strategy forcontrolling genes implicated in disease, including those

ANGIOGENESIS

Growth of new blood vessels.

a b c d

Figure 5 | Zinc-finger protein transcription factor-mediated regulation of the Vegfa genein a mouse ear model. a | An untreated control mouse ear. b | A mouse ear treated with acDNA expressing a single Vegfa isoform showing ‘leaky’ vessel formation, indicated by diffusered staining. c | An untreated control mouse ear. d | A mouse ear treated with a Vegfa-activatingzinc-finger protein transcription factor showing an increase in vasculature, which appearsnormal and ‘non-leaky’.

Box 1 | By-passing complementary DNA patents

Regulating endogenous genes with zinc-finger protein transcription factors (ZFP TFs)could make it unnecessary to access intellectual property on the use of cDNAs for drugscreening and the production of protein-based drugs. This use of ZFPs might beextremely important, as the US Patent and Trademark Office has granted more than20,000 patents on genes or gene-related molecules74. The ever increasing level ofintellectual property restrictions can make it difficult to obtain clear commercial rightsfor cDNA, but the naturally occurring components of living organisms — which can beupregulated by ZFPs — are not usually subject to these restrictions.



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ZFPs may also be applied to the treatment of epi-genetic diseases, which are often associated with defectsin the regulation of DNA methylation70. Xu et al. pro-posed the use of a ZFP to target a methyltransferase toDNA41 and targeted methylation of a sequence contain-ing a CpG dinucleotide (such as might occur in the CpGislands of gene promoters) has been demonstrated71.Once the proper methylation state has been imprinted, itshould be heritably propagated without the need tomaintain the zinc-finger fusion protein in cells. A publi-cation in press also shows that ZFP TFs can be used tooverride the methylation-associated repression of thehuman IGF2 and H19 alleles, which are associated withBeckwith–Wiedemann syndrome72. This strategy couldbe useful, as the changes are far more specific (becausethey are targeted to a specific gene by the ZFP) thanthose that might be achieved using drugs designed toalter the methylation state of genes.

Recent interest in stem cells has highlighted some oftheir potential applications in medicine, and ZFP TFscould also have applications in controlling stem-cell fate(V.V. Bartsevich, personal communication). One couldimagine a wide range of applications in cell replacementtherapy, perhaps in treating neurodegenerative condi-tions, such as Parkinson’s disease. In this case, a ZFP TFmight mimic the naturally occurring transcriptionfactor NR4A2 (also known as NURR1), which pro-motes the formation of dopaminergic neurons thatcould be used for cell-replacement therapy73.

ConclusionsThe control of gene expression with ZFP TFs can beapplied to a broad range of drug-discovery applications.ZFP TFs have been successfully applied to target valida-tion, HTS, cell-line engineering, and in an animal dis-ease model in preparation for use in human disease. Inthe latter case, controlling the endogenous gene has theadvantage that transcripts are made with the appropri-ate ratio of splice variants. Furthermore, ZFP TFs mightoffer ways to overexpress a gene product without resort-ing to the use of cDNAs, which might be covered asintellectual property (BOX 1).

The use of ZFP TFs is only one of several ways thatZFPs might be applied in drug discovery. In the future,we expect that ZFPs will be combined with other kinds offunctional domains to make proteins that perform usefulcellular tasks, such as gene correction and insertion oftransgenes.As research in molecular medicine advances,we expect that we will be able to find as yet unknownways to apply ZFPs to treat disease.

ZFP TFs that might be useful in the treatment ofcancer are also being developed, and these are beingtested with a viral vector that selectively replicates incells lacking a functional retinoblastoma (RB1)tumour suppressor gene61. The first ZFP to be testedusing this system is designed to activate the gene forgranulocyte-macrophage colony-stimulating factor(GM-CSF) in the target cells before viral replicationand cell destruction. It is expected that secretion ofGM-CSF and the release of cell antigens from trans-duced cells will not only provoke an antitumourimmune response at the immediate site, but couldalso charge the immune system to help screen theentire body for metastatic cells62.

ZFP TFs could also find therapeutic applications asrepressors, and there have been interesting tests withviral genes. One recent paper shows how ZFP TFs mightoffer a potential strategy for treating HIV infections. Inparticular, it has been shown that ZFPs targeted to theHIV 5′ long terminal repeat repressed HIV replicationin a cellular infection assay63, leading to the possibilitythat ZFP TFs can be added to a patient’s progenitor T cells and used to combat AIDS. In a companion paperstudying herpes simplex virus, a ZFP TF was also usedto inhibit replication of the virus, by repressing thepromoter of a gene that is normally activated early inthe viral replication cycle64.

Future directionsZFPs might have other important applications in genetherapy and medicine, including a proposed strategyfor gene correction. This strategy uses ZFPs that havebeen fused to the endonuclease domain of FokI, aType II restriction enzyme65 (FIG. 3). Such proteins canbe used to cleave DNA at a pre-determined target site,and it has been shown that the generation of double-stranded breaks by this method can promote homo-logous recombination of extrachromosomal DNA inamphibian oocytes and non-homologous end joiningin Drosphila66,67. Recent work in human cells on thistopic is awaiting publication68.

It is also possible to use ZFPs to target an integrase toparticular sites in DNA40, and this type of strategy couldbe used to insert exogenous DNA at a specific locationin the human genome. Targeted integration of a retro-virus might be useful in a gene therapy trial, as it couldreduce the risk of the retrovirus recombining withhuman DNA at a location that turns on an oncogene, asoccurred recently during treatment of children withsevere combined immune deficiency69.

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AcknowledgementsThe authors would like to express their indebtedness to all thosewho contributed to the critical reading of this manuscript, includingDr P. Gregory, D. E. Wolffe, Dr E. Rebar, Dr A. McNamara, Dr A. Reik,Dr F. Urnov, Dr M. Holmes and T. J. Cradick. The authors alsoapologize to those researchers whose many contributions were notincluded owing to space limitations.

Online links

DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ABCB1 | BAX | CCKBR | EPO | ERBB2 | ERBB3 | NR4A2 | PPARγ |RB1 | VEGF |Online Mendelian Inheritance in Man:http://www.ncbi.nlm.nih.gov/Omim/Beckwith–Wiedemann syndrome | Parkinson’s disease

FURTHER INFORMATIONEncyclopedia of Life Sciences: http://www.els.netAaron Klug | protein–DNA interactions | protein–nucleic acidinteraction: major groove recognition determinantsAccess to this interactive links box is free online.

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