review © The American Society of Gene Therapy

Zinc-finger Nucleases: The Next Generation

Emerges
Toni Cathomen1 and J Keith Joung2,3
1
Institute of Virology (CBF), Charité Medical School, Berlin, Germany; 2Molecular Pathology Unit and Center for Cancer Research and Center for
Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA; 3Department of Pathology, Harvard
Medical School, Boston, Massachusetts, USA

Methods of modifying the human genome precisely and efficiently hold great promise for revolutionizing the gene
therapy arena. One particularly promising technology is based on the homologous recombination (HR) pathway and
is known as gene targeting. Until recently, the low frequency of HR in mammalian cells, and the resulting depen-
dence on selection to identify these rare events, has prevented gene targeting from being applied in a therapeutic
context. However, recent advances in generating customized zinc-finger nucleases (ZFNs) that can create a DNA
double-strand break (DSB) at preselected sites in the human genome have paved the way for HR-based strategies in
gene therapy. By introducing a DSB into a target locus of interest, ZFNs stimulate gene targeting by several orders
of magnitude through activation of cellular DNA repair pathways. The capability of this technology to achieve gene
conversion frequencies of up to 29% in the absence of selection demonstrates its potential power. In this paper we
review recent advances in, and upcoming challenges for, this emerging technology and discuss future experimental
work that will be needed to bring ZFNs safely into a clinical setting.
Received 9 October 2007; accepted 29 April 2008; published online 10 June 2008. doi:10.1038/mt.2008.114

Introduction Such engineered endonucleases basically fall into two classes:

The ability to modify a complex genome with precision transformed
biology in the late 1980s and early 1990s.1 The underlying technol-
ogy is known as gene targeting and is based on the cellular homol-
ogous recombination (HR) pathway, which has evolved mainly to
promote genetic recombination during meiosis and the repair of
DNA double-strand breaks (DSBs) before mitosis. Until recently,
the low frequency of HR in mammalian cells (~1 HR event per 106
cells), and the resulting dependence on elaborate selection strategies
to identify rare recombinants, has prevented gene targeting from
being applied in a therapeutic context. A recent technological break-
through, however, has changed this dreary perspective. By fusing
engineered zinc-finger (ZF) DNA-binding domains to a nonspecific
nuclease domain, so-called ZF nucleases (ZFNs) were generated.
These ZFNs can be designed to introduce a DSB into a desired target
locus and, as a consequence, stimulate gene targeting 100- to 10,000-
fold by activating cellular DNA repair pathways.2,3 Given recently
published gene modification efficiencies of 29% or higher,4 it can
now be envisioned that this technology could be used for correct-
ing inborn mutations in adult stem cells derived from patients with
genetic disorders. These corrected cells could subsequently be used
for repopulating an affected organ, thereby reversing the disorder.
Targeted Genome Modifications
With Tailored Nucleases
In order to exploit DSBs for therapeutic genome modifications,
researchers first had to develop novel customizable nucleases.
­redesigned homing endonucleases, which have been recently
reviewed elsewhere,5 and ZFNs.2,3 ZFNs consist of an engineered
Cys2-His2 ZF domain fused to the nuclease domain of the type
IIS restriction enzyme FokI.6 In this configuration, the DNA-
­binding ZF domain directs the nonspecific FokI cleavage domain
to a specific DNA target site. Because the FokI cleavage domain is
enzymatically active only as a dimer,7 the introduction of a DSB
is dependent upon dimerization of ZFNs. Accordingly, two ZFN
subunits are typically designed to recognize the target sequence in
a tail-to-tail conformation, with each monomer binding to “half-
sites” that are separated by a “spacer” sequence8 (Figure 1a).
Repair of a DSB induced by a pair of ZFNs can occur by one
of two potential pathways: nonhomologous end-joining (NHEJ)
or HR. If repair is mediated by NHEJ, an error-prone pathway
that can lead to insertions or deletions,9 the target gene can be
disrupted by frameshift mutations which, in many instances,
will lead to the expression of a truncated and/or nonfunctional
protein (Figure 1b). The feasibility of such an approach was ini-
tially demonstrated in Drosophila. Endogenous expression of
ZFNs targeting the y, ry, and bw loci was controlled by the hsp70
heat-shock promoter, which allowed transient expression of the
ZFNs in developing larvae.10,11 Between 0 and 78% of the result-
ing flies carried the targeted mutation in the germline, depend-
ing on the target locus and the temperature used for inducing the
hsp70 promoter. This suggests that ZFN expression levels and the
chromosomal context of the target locus are crucial parameters

Correspondence: Toni Cathomen, Charité Medical School, Institute of Virology (CBF), Hindenburgdamm 27, D-12203 Berlin, Germany.
E-mail: toni.cathomen@charite.de

1200 www.moleculartherapy.org vol. 16 no. 7, 1200–1207 july 2008

© The American Society of Gene Therapy Next Generation ZFNs

in DHFR and ~1% of cell clones revealed a biallelic modification.12

a Zinc-finger nuclease
Fokl Zinc-finger The observed range of insertion and deletions caused by muta-

Linker
nuclease DNA-binding domain
domain genic DNA repair of the cleavage event was from +2 to –302 base
3 2 1 N pair (bp). Future therapeutic applications of such a strategy may
5� 3�
include the targeted knockout of dominant mutant alleles or the
3� 5� disruption of receptors used by pathogens, e.g., the knockout of
N
1 2 3
the CCR5 (chemokine receptor 5) locus to render human T cells
Target half-site (L) Spacer sequence Target half-site (R)
of HIV-infected individuals resistant to infection with the virus.13
b Gene disruption Gene targeting, on the other hand, relies on HR between the
endogenous target locus and an exogenously introduced homolo-
geneA
dn
1 2 3 4 5 6 gous DNA fragment, which we refer to as the “donor DNA.” In the
pA
absence of a DSB at the target locus, typically fewer than 1 in 105
of targeted cells will contain the desired genetic modification, a
geneA– 1 2 3 4 5 6
pA frequency too low to be useful for gene therapy.14 However, proof-
of-principle experiments involving the meganuclease I-SceI,
c Gene correction
which binds to an 18-bp recognition site, demonstrated that the
4 Donor DNA
insertion of a DSB in the target locus stimulates recombination
with an exogenous donor DNA by several orders of magnitude.15,16
geneAmut 1 2 3 4 5 6 Importantly, stimulation of HR by I-SceI has been accomplished
pA
in various cell lines, including mouse ES cells,17 thereby indicat-
ing that DSB-induced stimulation of HR may be operative in a
1 2 3 4 5 6
geneAWT
wide variety of different cell types. A concern is that NHEJ will
pA
compete with HR to seal the broken chromosome. Recent experi-
d Gene addition ments in two different human cell lines indicated that HR-medi-
cDNA fragment
Donor DNA
ated gene targeting accounted for 60 to 70% of all DSB repair
2 3 4 5 6
pA events at an endogenous locus,4 suggesting that, in the presence
of large amounts of a donor DNA, HR is the preferred pathway to
geneAmut 1 2 3 4 5 6 repair DSBs.
pA The architecture of the donor DNA determines the outcome
of the gene targeting event. For example, donor DNA can be
geneA+ 1 2 3 4 5 6 4 5 6
pA pA
designed to either create or correct a mutation in a specific gene
locus ­(Figure 1c). In the first study in which an endogenous mam-
Figure 1  Zinc-finger nuclease (ZFN)-mediated genome editing. malian locus was targeted by ZFNs, gene editing at the human
(a) Architecture and application of ZFNs. A ZFN designed to create IL2Rγ (interleukin-2 receptor gamma chain) locus was achieved
a DNA double-strand break (DSB) in the target locus is composed of
in up to 18% of transiently transfected K562 cells, a human eryth-
two monomer subunits. Each subunit encompasses three zinc-fingers
(orange, 1-2-3), which recognize 9 base pairs within the full target site, roleukemia cell line, and in ~5% of primary human T cells.18
and the FokI endonuclease domain (green). A short linker (grey) con- When integrase-deficient lentiviral vectors were used for ZFN
nects the two domains. After dimerization the nuclease is activated and gene transfer, the gene targeting frequency at the IL2Rγ locus in
cuts the DNA in the spacer sequence, separating the two target half-
sites (L) and (R). (b) Gene disruption. A DSB (yellow flash) introduced
K562 cells could be increased up to 29%,4 suggesting that gene
by the ZFN into a dominant mutant allele (geneAdn) is repaired by the transfer efficiency and ZFN expression levels are crucial factors
error-prone nonhomologous end-joining pathway. Deletions and inser- for the success of such an approach.
tions that can occur disrupt the coding sequence (geneA–) and render Alternatively, the donor DNA can contain either an entire
the expressed protein nonfunctional. (c) Gene correction. In order to
restore a genetic defect directly in the genome (geneAmut), a targeting
expression cassette or a complementary DNA fragment of the gene
vector (donor DNA) encompassing wild-type sequences homologous to to be corrected. In the latter case, the main benefit of gene addi-
the mutant gene (grey areas) is transduced into the target cell. A ZFN- tion is that a single pair of ZFNs, combined with a single donor
induced DSB stimulates homologous recombination (HR) between the DNA, can be used for “correcting” all mutations located down-
donor DNA and the defective gene (geneAmut) to generate a corrected
locus (geneAWT). (d) Gene addition. In order to restore the phenotype of
stream of the DSB in a given gene, while still keeping gene expres-
a cell harboring a genetic defect (geneAmut), a partial cDNA flanked by sion under the control of the endogenous promoter (­Figure 1d).
sequences homologous to the mutant gene, is embedded in a targeting Targeted insertions of entire expression cassettes, on the other
vector. A ZFN-induced DSB stimulates HR between the donor DNA and hand, aim at restoring the cellular phenotype by integration of a
the mutant gene. Expression of the gene is reconstituted (geneA+) and
remains under the control of the endogenous promoter. therapeutic expression cassette into a yet to be defined “safe har-
bor.” Such a “safe harbor” should include the support of high and
that determine the level of NHEJ-mediated mutagenesis. Recently, sustained transgene expression levels without any signs of geno-
ZFN-mediated gene knockout has also been achieved in mamma- toxic side effects. Therefore, at least in theory, a single pair of ZFNs
lian cells. After transfection of a Chinese hamster ovary cell line, combined with a customized donor DNA can be used for the safe
the transient expression of ZFNs targeting the DHFR (dihydrofo- “correction” of any given monogenetic inherited disorder that is
late reductase) locus resulted in ~15% of cells carrying a mutation amenable to combined gene/stem cell therapy protocols. Because

Molecular Therapy vol. 16 no. 7 july 2008 1201

Next Generation ZFNs
individuals who harbor a homozygous deletion in the CCR5 gene
are healthy,19 this locus represents a candidate site for targeted
transgene insertion. In proof-of-principle studies, targeted inte-
gration of an enhanced green fluorescent protein expression cas-
sette into the human CCR5 locus was achieved in 39% of Jurkat
cells, 3.5% of embryonic stem cells, and 0.1% of hematopoietic
stem cells.4 Moreover, it has been shown that ZFNs can mediate
targeted insertion of a 7.8-kb-long DNA sequence into an endog-
enous locus in 6% of transfected K562 cells.20 Surprisingly, two
homology arms of 750 bp were sufficient for efficient gene target-
ing. These results demonstrate that the addition of large DNA
fragments is feasible, but that the HR frequency is highly cell-type
specific, an observation made also in another study.21 Although
these experiments in immortalized cell lines and in embryonic
stem cells appear to be promising, it remains to be determined in
long-term follow-up studies whether the human CCR5 locus will
ultimately prove to be a “safe harbor.”
Engineering Platforms For Zf Domains
Cys2-His2 ZF domains are the most abundant DNA-binding motif
in eukaryotes and consist of ~30 residues that fold into a ββα-
structure coordinated by a zinc ion.22 A number of investigators
have demonstrated that changing one or more of the six critical
residues located within or adjacent to the structurally conserved
α-helix (also referred to as the “recognition helix”) can alter the
DNA-binding specificity of a single ZF.23–30 Many individual fin-
gers with new specificities have been created by rational design
or by selection from randomized libraries, using either phage dis-
play or a bacterial cell-based two-hybrid (B2H) system.31–39 ZFNs
described in the literature to date contain three or four ZF domains
arranged in tandem arrays. Because a single ZF recognizes ~3 bp of
a DNA sequence,40 a ZFN subunit binds to 9- or 12-bp-long target
sites, depending on the number of ZF domains present. Assuming
perfect specificity by each ZFN monomer, dimers of three-finger
ZFNs would therefore be expected to bind to an 18-bp target DNA
site while dimers of four-finger proteins recognize a 24-bp target
site. Recognition sites of 18 or 24 bp are long enough to define a
statistically unique sequence in a human genome.
As mentioned earlier, various methods of engineering multi-ZF
domains have been described in the literature. In order to simplify
our review we have grouped these systems into three categories:
(i) “modular assembly” methods (Figure 2a), (ii) “­context-­sensitive
selection” methods (Figure 2b), and (iii) “2 + 2” method of the
company Sangamo BioSciences (Richmond, CA) (Figure 2c).
(i) “Modular assembly” has been described by several
groups,25,26,31,41,42 but the efficacy of this approach as a general
method for making ZFNs is controversial. As implied by its name,
the process of modular assembly involves the joining of single ZF
domains of known DNA-binding specificities to create the DNA-
binding domain of a ZFN (Figure 2a).43–45 Different groups of
investigators have described large archives of single-finger mod-
ules31,36–38,41,46 and two laboratories provide free web-based software
tools47,48 that facilitate the identification of potential ZFN sites and
the design of multi-finger domains. Although modular ­assembly
is conceptually appealing in its simplicity, and some modularly
assembled domains revealed excellent DNA-binding activity
in vitro49 and at endogenous loci in Drosophila and ­Caenorhabditis
1202
© The American Society of Gene Therapy
elegans cells,11,50,51 other reports have suggested that the efficacy
rate for producing active three-finger proteins for use in mamma-
lian cells may be far less than 100%.41,52 Motivated by these find-
ings, we recently performed a systematic large-scale evaluation of
168 three-finger domains designed to recognize 104 diverse target
sites using a B2H assay as a screening method. This B2H assay is
rapid and accurately identifies three-finger domains that will fail
to function as ZFNs in human cells. For 79 of the 104 target sites
that we targeted by modular assembly, we failed to obtain even a
single three-finger array that showed evidence of DNA-binding
activity in the B2H assay.53 The apparently low efficacy of modular
assembly is particularly problematic for the purpose of making
ZFNs because of the need to engineer two different ZF domains
for each target site of interest. Assuming a success rate of ~24%
(25/104) for making three-finger domains by modular assembly,
the theoretical success rate for generating a dimeric ZFN com-
plex with this method would be ~6%. However, even these values
are likely to be overestimates of the true success rate of modular
assembly because we have found that not all ZF arrays that scored
positively in the B2H will be active in human cells.53
The low success rate of modular assembly is most likely
because neighboring ZF domains are not truly independent in
their DNA-binding activities (i.e., that ZFs are not always truly
modular in their behavior). Structural studies of ZFs bound to
their cognate DNA-binding sites have demonstrated that fingers
can “reach over” to make contacts in the target sites of neighbor-
ing fingers, and that residues in the recognition helix of one finger
can influence the orientation of recognition helix side-chains in an
adjacent finger.32,40,54–61 The fact that modular assembly disregards
these context-dependent effects may explain why this method
frequently produces ZF domains with suboptimal DNA-binding
affinities and specificities, and thereby likely fails to produce func-
tional ZFNs for use in human cells.
(ii) A number of “context-sensitive selection strategies” that
attempt to account for context-dependent effects have been
described in the literature, including methods known as bi-partite
selection, sequential optimization, and context-sensitive parallel
optimization (Figure 2b).3,62–64 These approaches attempt to take
into account the relative position of an individual ZF module in
the final ZF array (i.e., position 1, 2, or 3) and the impact of the
neighboring finger(s); or, more simply stated, they try to identify
combinations of fingers that work well together. A significant
disadvantage of all of these approaches is that they remain inac-
cessible to most research groups because they require specialized
expertise in the construction of large randomized ZF libraries and
the use of labor-intensive selection methods (e.g., phage display
or B2H system) for interrogating them. However, published and
unpublished experience with these methods suggests that they are
robust and that they each yield multi-finger domains with high
DNA-binding affinities and specificities, as measured in vitro with
purified proteins.62–64 Moreover, when fused to the catalytic FokI
domain, the resulting ZFNs showed higher activity and lower
­toxicity in human cells as compared to their modularly assembled
cousins.65
(iii) The proprietary ZF engineering platform of Sangamo
BioSciences has been shown to yield ZFNs capable of editing
endogenous mammalian genes.4,12,18 Although we do not know
www.moleculartherapy.org vol. 16 no. 7 july 2008
© The American Society of Gene Therapy Next Generation ZFNs

a Modular assembly b Context-sensitive selection c 2+2 strategy

Zinc-finger archive Customized libraries Two-finger archive
2 2 2 2 3’ 2’ 1 3’ 2 1’ 3 2’ 1’ 2 1 2 1 2 1 2 1
gaa gac gag gat 5�-xxxxxxgct 5�-xxxgtaxxx 5�-ggaxxxxxx gaagaa gacgaa gaggaa gatgaa
2 2 2 2 Anchor Anchor Anchor 2 1 2 1 2 1 2 1
gca gcc gcg gct
Selections
2 2 2 2 2 1 2 1 2 1 2 1
gga ggc ggg ggt 3’ 2’ 1 3’ 2 1’ 3 2’ 1’
3’ 2’ 1 3’ 2 1’ 3 2’ 1’
2 2 2 2 3’ 2’ 1 3’ 2 1’ 3 2’ 1’
gta gtc gtg gtt
Shuffling

3 2 1
3 2 1
3 2 1 2b 1b 2a 1a

Selection Improvement

2c 2b 2a 3 2 1 2b 1b 2b 1a
5�-ggagtagct 5�-ggagtagct 5�-gccggagtagct

Figure 2  Zinc-finger engineering platforms. (a) “Modular assembly” involves the joining of single zinc-finger domains of known DNA-binding
specificities. Large archives of single-finger modules have been created by selection from randomized libraries using phage display. This approach
is conceptually simple but neglects positional and context-dependent effects. (b) “Context-sensitive selection” strategies attempt to identify com-
binations of zinc-fingers that work well together. One particular strategy for performing such a selection (among several described in the literature)
is shown in the figure. The first selection step takes into account the relative position of the finger (1, 2, or 3), while the second step factors in the
impact of the respective neighbor(s). (c) The “2 + 2 strategy” is a proprietary platform and details are not known. It is likely that the four-finger
domains are assembled from pre-existing archives of two-finger units with known DNA-binding specificities, followed by further optimization using
an algorithm-based approach.

precisely how these four-finger domains are generated, published
papers and presentations at meetings suggest a two-step proce-
dure:18,24,66–68 in the first step, four-finger domains are assembled
from large pre-existing archives of two-finger units with known
DNA-binding specificities; in the second step, promising “lead”
proteins may be optimized using a proprietary algorithm-based
approach (Figure 2c). Because it requires access to two proprie-
tary resources (the archive and the algorithm), this method is thus
far accessible only to academic researchers who are collaborating
with Sangamo.66,69
Zfn-associated Toxicity
ZFN-induced cytotoxicity is a major potential issue, and has been
reported in several studies.10,11,52,70–72 Cell death and apoptosis
associated with ZFN expression are most likely the result of exces-
sive cleavage at off-target sites, which, in turn, suggests imperfect
­target-site recognition by the ZF DNA-binding domains. Given
that therapeutic gene targeting will strongly depend on creation of
a DSB at a specific target site, the implementation of quantitative
assays to assess immediate and long-term genotoxicity of artifi-
cial nucleases is of paramount importance.73 In some studies, the
extent of cytotoxicity associated with ZFN expression was quanti-
fied by measuring cell survival65,70,74 or apoptosis;52 however, these
assays are very coarse measures of toxicity and provide little infor-
mation about the contributing mechanisms. In order to address
this problem, assays that directly document the number of off-
target cleavage events have been developed. Using antibodies spe-
cific for phosphorylated histone H2AX (γ-H2AX) or p53 tumor
suppressor-binding protein 1 (53BP1), we as well as others have
quantified the relative number of ZFN-induced repair foci formed
after the creation of a DSB.71,72 These quantitative assays can be
used to characterize the specificity and immediate genotoxicity of
any artificial nuclease of interest, but they do not provide informa-
tion about the sites at which off-target DSBs occur.
Assessment of the in vivo cleavage specificity of ZFNs, i.e., the
ratio of on-target versus off-target cleavage events in a complex
genome, remains a significant and thus far unsolved challenge.
Although a recently published bacteria-based in vivo specific-
ity profiling system for ZF DNA-binding domains can provide
information about the DNA-binding profile of monomeric ZF
domains,75,76 it cannot predict actual cleavage sites of ZFN dimers
in the human genome. A possible approach to identify ZFN
­cleavage sites directly might be to exploit the fact that DSBs in
a cellular genome serve as efficient integration sites for episomal
DNA, such as vectors based on adeno-associated virus.77 Sequenc-
ing of the adeno-associated virus vector integration sites after
ectopic ZFN expression could offer direct information about the
locations of off-target DSBs in a cell.
In this context it is important to mention that, at least in
theory, only one donor DNA molecule will be used as a ­template
for HR with a target allele, while the remaining donor mole-
cules could potentially integrate into other naturally occurring
or ZFN-induced off-target DSBs. Therefore an additional criti-
cal para­meter for therapeutic gene targeting approaches will be
to assess the ratio of targeted vs. untargeted donor integration
events. Both the immediate risk of ZFN-induced mutagenesis
and the untargeted integration of the donor DNA to induce
unpredictable oncogenicity can be assessed by soft agar transfor-
mation studies78 or in vitro transformation assays using purified
lineage-negative cells from murine bone marrow.79 Moreover,
cytogenetic analyses, like spectral karyotyping,80 can provide
information about whether ZFN activity induces chromosomal
abnormalities and/or translocations. We emphasize however
that the long-term consequences of ZFN-induced DSBs can be
studied only in vivo. Assays developed earlier to evaluate the
genotoxicity of retroviral vectors in gene therapy protocols81,82
should prove useful in studying the malignant potential of cells
after overexpression of ZFNs.

Molecular Therapy vol. 16 no. 7 july 2008 1203

Next Generation ZFNs © The American Society of Gene Therapy

The Design Of Safer Nucleases a Insufficient specificity of DNA binding

In principle, at least three general strategies could be employed 3 2 1 N
5� 3�
to increase the specificity of engineered ZFNs: (i) improving the
DNA-binding specificities of the ZF domains, (ii) optimizing the 3� 1 2 3 5�
N
linker sequence that connects the ZF domain with the FokI cleav-
age domain, and (iii) regulating DNA-cleavage activity of the FokI Ambiguity of interdomain linker
nuclease domain. b
3 2 1 N
5� 3�
Although recent work in the field has highlighted the
­tremendous power and broad applicability of ZFNs for biologi- 3� 1 2 3 5�
N
cal studies and gene therapy, the low numbers of natural loci suc-
cessfully targeted by ZFNs in mammalian genomes testifies how c Cleavage at target half-sites N
1
difficult it is to develop effective ZFNs. To our knowledge, the sole 3 2
published examples of endogenous mammalian loci that were 5� 3�

­successfully altered using ZFNs are the IL2Rγ and CCR5 genes 3� 5�
in human cells4,18 and the DHFR locus in Chinese hamster ovary
1 2 3
N

cells.12 The Zinc Finger Consortium (http://www.zincfingers. Cleavage by homodimers

org), an international group of 14 academic laboratories, is dedi- d
3 2 1 N
5�
cated to the development of a robust and effective “open-source” 3�

ZF engineering platform. Our current method of choice for engi- 3� 5�

neering multi-finger domains is based on a previously described
context-sensitive optimization approach.64 Although laborious, it
is a robust method known to yield three-finger ZFNs that function
with higher efficiencies and lower toxicities in human cells when
compared with analogous ZFNs made by modular ­assembly.65 This
finding is consistent with the hypothesis that suboptimal DNA-
binding activity—frequently observed with modularly assembled
ZF domains—can be a primary component of ZFN-associated
toxicity (Figure 3a). The development of a robust and publicly
available ZF engineering platform remains a high priority for the
Zinc Finger Consortium and such a platform will become avail-
able in the near future.
Although interdomain ZFN linkers of various lengths have
been described and tested,8 to our knowledge no linker has yet
been described that provides perfect specificity for a “spacer”
DNA of a single length. The consequence of this ambiguity is that
a pair of ZFNs can cut not only at sites with a spacer length of
e.g., 6 bp, but also at sites with 5-bp and 7-bp spacers (Händel and
Cathomen, unpublished results), thereby increasing the num-
ber of potential off-target sites and cleavage events (Figure 3b).
Systematic analysis of candidate-based linkers or, alternatively, a
combinatorial library approach in which both linker composi-
tion and length are randomized, followed by iterative selection
and counter-selections, may help to identify linker variants that
enforce ZFN-mediated DNA cleavage at target sites of a single
spacer length.
In contrast with many natural endonucleases, ZFNs do not
contain an allosteric mechanism that regulates DNA-cleavage.
In vitro experiments with the natural FokI restriction enzyme or
ZFNs showed that a FokI monomer or a ZFN subunit bound to
its target site can associate through protein–protein interaction
with a second monomer/subunit that remains detached from
the recognition sequence, in order to catalyze DNA cleavage.7,83
It is therefore conceivable that at high intranuclear concentra-
tions a ZFN subunit binds to its canonical 9-bp target site as a
monomer, but then becomes incorrectly activated once it forms
a dimer with a second ZFN subunit, which is not properly bound
to DNA (Figure 3c). Given that a 9-bp half-site ­ statistically
N
1 2 3
e Ideal zinc-finger nuclease
−
3 2 1 N
5� 3�
− +
3� + 5�
N
1 2 3
Figure 3  Various sources for zinc-finger nuclease (ZFN) off-target
activity: (a) insufficient specificity of DNA-binding, permitting ZFN bind-
ing to unintended DNA sites, (b) ambiguity of the interdomain linker,
allowing cleavage at noncanonical spacer lengths (e.g., at a 7-base-pair
(bp) spacer instead of the intended 6-bp spacer), (c) cleavage at isolated
target half-sites, and (d) cleavage by homodimeric ZFNs. (e) An ideal
ZFN architecture consists of an affinity-matured DNA-­binding domain,
an optimized linker sequence, and a destabilized and asymmetric dimer
interface that regulates the FokI cleavage activity.
occurs >10,000 times in the human genome, such a scenario
would generate many additional potential cleavage sites. Apply-
ing an in silico protein engineering technology, we have recently
shown that the attenuation of dimerization by reducing the num-
ber of hydrophobic interactions in the dimer interface decreased
ZFN-associated toxicity significantly.72 We speculate that weak-
ening the dimer interface prevents ZFN dimers from forming in
solution, thereby making endonuclease activity more dependent
on DNA binding. This helps to ensure that two ZFN subunits
can dimerize only after both subunits are properly bound to the
DNA target site.
Cleavage of a target locus requires that two different ZFN sub-
units bind as a heterodimer at the desired cleavage site. However,
symmetry at the FokI dimerization interface also permits homodi-
mers to form, thus enabling cleavage at additional sites (Figure 3d).
By altering interacting residues in the protein–protein interface of
the FokI dimerization domain, we as well as others have recently
engineered asymmetric ZFN variants that prevent the undesirable
homodimerization of ZFN subunits.71,72 Although the actual mech-
anism by which the dimerization variants overcome toxicity is open
to speculation because of lack of biochemical in vitro data, these
studies show that ZFN dimerization variants harboring an asym-
metric dimer interface revealed significantly reduced toxicity with-
out compromising on performance.71,72 An ideal ZFN architecture

1204 www.moleculartherapy.org vol. 16 no. 7 july 2008

© The American Society of Gene Therapy
therefore includes a highly specific DNA-binding domain, a rigid
interdomain linker, and a weak, asymmetric dimerization interface
in the FokI cleavage domain (Figure 3e).
Toward Clinical Application
It has long been envisaged that by applying the ZFN technology
to stem cells, inherited mutations could be repaired ex vivo and
functionally corrected stem cells could be transplanted back into
patients to repopulate the affected tissues and cure the disease.
Importantly, gene correction would restore the functionality of
the affected gene product and, at the same time, retain its normal
endogenous expression pattern, thereby overcoming a major limi-
tation of conventional gene therapy approaches. Gene correction
might work even more efficiently if the repaired gene provides the
modified stem cell with a growth advantage. For example, in the
case of X-linked severe combined immunodeficiency, which is
caused by mutations in the IL2Rγ locus, correction of only a small
number of genetically corrected HSCs will be sufficient to restore
proper function of the immune system.84
Several obstacles continue to limit the exploitation of ZFNs in
a therapeutic setting. We believe the following criteria should be
met in order for ZFNs to be successfully applied in a clinical set-
ting: (i) high DNA-binding specificity of the ZF domain; (ii) reg-
ulated cleavage by the ZFN; (iii) efficient delivery; (iv) transient
ZFN expression; (v) comprehensive evaluation of treated cells for
potential ZFN-induced side effects; and (vi) assessment of the
potential immune reactivity against ZFNs, especially against the
bacterial FokI domain.
(i) High specificity of DNA-binding is the single most impor-
tant parameter associated with the use of artificial nucleases. As
expected, higher DNA-binding specificity correlates with better
performance and less toxicity of ZFNs in human cells.53,65 Engi-
neering platforms that take into account positional and context-
dependent effects are more likely to yield reliably multi-ZF arrays
with higher specificity of DNA-binding. Ideally, ZFN pairs must
be sufficiently specific so as to bind and cleave only a single DNA
site in a cellular genome.
(ii) The FokI cleavage domain constitutes the catalytically
active part of ZFNs. As noted earlier in this paper, in contrast to
many natural endonucleases, ZFNs do not contain an allosteric
mechanism that regulates DNA-cleavage; however, structure-
based redesign of the dimer interface has been used to partially
compensate for this lack of regulation.71,72 As a result, ZFNs
containing such variant FokI domains reveal a superior overall
performance by significantly reducing the number of off-target
DSBs.4,12,71,72 Additional redesign of the ZFN architecture to enable
allosteric activation of cleavage activity subsequent to binding to
DNA would further improve the specificity of ZFN action.
(iii) Ensuring the simultaneous delivery of two different ZFN
subunits and a donor DNA in primary cells represents a major
challenge for current gene transfer technologies. To date, four
different systems have been reported to be suitable for mediating
DSB-stimulated targeted genome editing in human cells: plasmid-
DNA introduced by transfection,20,52,65,70-72,74 adeno-associated
virus vectors,85,86 integrase-deficient lentiviral vectors4,65 and modi-
fied adenoviral vectors.67,68,87 Although plasmid-DNA has been the
most commonly used expression vector thus far, it might not be the
Molecular Therapy vol. 16 no. 7 july 2008
Next Generation ZFNs
first choice in a therapeutic setting. Because of their superior trans-
duction record, adeno-associated virus vectors, integrase-deficient
lentiviral vectors, and adenovirus type 5 vectors substituted with
a type 35 fiber structure (Ad5/35) are promising tools to deliver
high numbers of ZFN expression cassettes into stem cells, such as
hematopoietic4 and mesenchymal human stem cells.87 However,
independent of the nature of the expression vector, the delivery of
DNA expression cassettes containing strong promoters—and this
can include donor DNAs for targeted gene addition—is associated
with the potential risk of insertional mutagenesis, as reported for
both integrating and “episomal” ­vectors.88–90
(iv) Because of expected off-target effects, transient expression
of ZFNs is strongly preferred over permanent expression of the
nucleases. In order to achieve this goal and to reduce the risk of
vector integration, ZFNs might be delivered in the future directly
as proteins or by transfection of ZFN-encoding mRNAs. However,
in view of the fact that a high intranuclear concentration of ZFNs
may be required for efficient cleavage, it remains to be determined
whether protein transduction or mRNA transfer will be suffi-
ciently efficient to mediate ZFN-based genome modifications.
(v) Therapeutic gene targeting or gene knockout depends on
the insertion of a specific DSB at the target site. A major safety
concern when using ZFNs for genome editing is the possible
genotoxicity associated with high-level expression of ZFNs. It
will therefore be absolutely ­ necessary to thoroughly verify the
safety of each pair of ZFNs before moving them into the clini-
cal setting. The global toxicity of ZFN overexpression can be
­measured by assessing cell survival65,70,74 or apoptosis,52 while
surplus DSBs can be quantified by determining the number of
ZFN-induced repair foci.71,72 On the other hand, extensive cyto-
genetic analysis of ZFN-treated ­target cells must be carried out
in order to verify that chromosomal breaks and/or transloca-
tions have not occurred. Finally, in vitro and in vivo transfor-
mation assays in susceptible fibroblast cell lines and pluripotent
stem cells should be carried out to ascertain that overexpression
of the ZFNs or untargeted integration of the donor DNA does
not induce a malignant cell phenotype.
(vi) Because of its bacterial origin, the FokI cleavage domain
of the ZFNs may be highly immunogenic. Even if ZFNs are only
transient expressed ex vivo, animals (or patients in a clinical trial)
should be closely monitored, as an added precaution, for the
development of any immune reactivity towards FokI. An immune
response may also limit the ability of clinicians to perform ­multiple
rounds of ZFN-based treatments.
Conclusions
The main advantage of using ZFN-stimulated gene targeting as
compared to conventional gene-addition-type gene therapy is the
potential to preserve temporal and tissue-specific gene expression.
Gene knockout through NHEJ-mediated repair of ZFN-induced
DSBs is another promising application of this technology. Impor-
tantly, gene disruption may also allow the treatment of hereditary
disorders with dominant inheritance if the mutated allele can
be disrupted specifically by ZFN-induced DSBs. Because both
approaches depend on the activity of custom-made ZFNs, the
reduction of off-target DSBs and the development of appropri-
ate delivery tools are vital. Nevertheless, the overall efficiency of
1205
Next Generation ZFNs
ZFN-induced genome editing may depend on additional param-
eters, such as the apoptotic threshold of a cell and the proficiency
in activating the appropriate DNA repair pathways, both of which
may vary significantly among different cell types.
Despite the tremendous progress that has been made recently,
the development of methods to improve our understanding of
ZFN-associated genotoxicity and to quantify it remains an impor-
tant priority and challenge for future research. Cleavage at unin-
tended, off-target DNA sites is likely to be the decisive factor in
ZFN-associated toxicity. Examination of the ZFN architecture has
suggested three parameters that are likely to affect the frequency
of off-target cleavage events: the DNA-binding specificity of each
ZFN subunit, the stability and specificity of the ZFN dimeriza-
tion interface, and the promiscuity of the ZFN dimer for “spacer”
sequences of different lengths. Although the development of a
malignant phenotype is hypothesized to require multiple genetic
insults, every genetic manipulation poses a risk, especially in stem
and progenitor cells with their high proliferative potential. In view
of recent adverse events in gene therapy trials to treat X-linked
severe combined immunodeficiency, the development of both
“safer” ZFNs and “safer” delivery methods to minimize genotoxic
side effects will be important in order to bring the next generation
of gene therapy tools into the clinical setting.
Acknowledgments
We thank Janine Büchel for help with the illustrations, and the
­members of our laboratories for suggestions and critical reading of
the ­ manuscript. Grants CA311/2–SPP1230 of the German Research
Foundation (to T.C.), ZNIP–037783 of the European Commission
(to T.C.), NIH R21 RR024189 (to J.K.J.), the Cystic Fibrosis Founda-
tion ­MCCRAY07G0 (to J.K.J.), and the Massachusetts General Hospital
­Department of Pathology (to J.K.J.) have supported our own ongoing
work mentioned throughout this review.
References
1. Capecchi, MR (2005). Gene targeting in mice: functional analysis of the mammalian
genome for the twenty-first century. Nat Rev Genet 6: 507–512.
2. Porteus, MH and Carroll, D (2005). Gene targeting using zinc finger nucleases.
Nat Biotechnol 23: 967–973.
3. Durai, S, Mani, M, Kandavelou, K, Wu, J, Porteus, MH and Chandrasegaran, S (2005).
Zinc finger nucleases: custom-designed molecular scissors for genome engineering of
plant and mammalian cells. Nucleic Acids Res 33: 5978–5990.
4. Lombardo, A, Genovese, P, Beausejour, CM, Colleoni, S, Lee, YL, Kim, KA et al. (2007).
Gene editing in human stem cells using zinc finger nucleases and integrase-defective
lentiviral vector delivery. Nat Biotechnol 25: 1298–1306.
5. Paques, F and Duchateau, P (2007). Meganucleases and DNA double-strand
break-induced recombination: perspectives for gene therapy. Curr Gene Ther 7:
49–66.
6. Kim, YG, Cha, J and Chandrasegaran, S (1996). Hybrid restriction enzymes: zinc finger
fusions to Fok I cleavage domain. Proc Natl Acad Sci USA 93: 1156–1160.
7. Smith, J, Bibikova, M, Whitby, FG, Reddy, AR, Chandrasegaran, S and Carroll, D
(2000). Requirements for double-strand cleavage by chimeric restriction enzymes with
zinc finger DNA-recognition domains. Nucleic Acids Res 28: 3361–3369.
8. Bibikova, M, Carroll, D, Segal, DJ, Trautman, JK, Smith, J, Kim, YG et al. (2001).
Stimulation of homologous recombination through targeted cleavage by chimeric
nucleases. Mol Cell Biol 21: 289–297.
9. Lieber, MR, Ma, Y, Pannicke, U and Schwarz, K (2003). Mechanism and regulation of
human non-homologous DNA end-joining. Nat Rev Mol Cell Biol 4: 712–720.
10. Bibikova, M, Golic, M, Golic, KG and Carroll, D (2002). Targeted chromosomal
cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics
161: 1169–1175.
11. Beumer, K, Bhattacharyya, G, Bibikova, M, Trautman, JK and Carroll, D (2006).
Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics 172:
2391–2403.
12. Santiago, Y, Chan, E, Liu, PQ, Orlando, S, Zhang, L, Urnov, FD et al. (2008). Targeted
gene knockout in mammalian cells by using engineered zinc-finger nucleases.
Proc Natl Acad Sci USA 105: 5809–5814.
13. Rossi, JJ, June, CH and Kohn, DB (2007). Genetic therapies against HIV Nat Biotechnol
25: 1444–1454.
14. Vasquez, KM, Marburger, K, Intody, Z and Wilson, JH (2001). Manipulating the
mammalian genome by homologous recombination. Proc Natl Acad Sci USA 98:
8403–8410.
1206
© The American Society of Gene Therapy
15. Choulika, A, Perrin, A, Dujon, B and Nicolas, JF (1995). Induction of homologous
recombination in mammalian chromosomes by using the I-SceI system of
Saccharomyces cerevisiae. Mol Cell Biol 15: 1968–1973.
16. Rouet, P, Smih, F and Jasin, M (1994). Expression of a site-specific endonuclease
stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci USA
91: 6064–6068.
17. Smih, F, Rouet, P, Romanienko, PJ and Jasin, M (1995). Double-strand breaks at the
target locus stimulate gene targeting in embryonic stem cells. Nucleic Acids Res
23: 5012–5019.
18. Urnov, FD, Miller, JC, Lee, YL, Beausejour, CM, Rock, JM, Augustus, S et al. (2005).
Highly efficient endogenous human gene correction using designed zinc-finger
nucleases. Nature 435: 646–651.
19. Lusso, P (2006). HIV and the chemokine system: 10 years later. EMBO J 25:
447–456.
20. Moehle, EA, Rock, JM, Lee, YL, Jouvenot, Y, DeKelver, RC, Gregory, PD et al. (2007).
Targeted gene addition into a specified location in the human genome using
designed zinc finger nucleases. Proc Natl Acad Sci USA 104: 3055–3060.
21. Cornu, TI and Cathomen, T (2007). Targeted genome modifications using
integrase-deficient lentiviral vectors. Mol Ther 15: 2107–2113.
22. Wolfe, SA, Nekludova, L and Pabo, CO (2000). DNA recognition by Cys2His2 zinc
finger proteins. Annu Rev Biophys Biomol Struct 29: 183–212.
23. Pabo, CO, Peisach, E and Grant, RA (2001). Design and selection of novel Cys2His2
zinc finger proteins. Annu Rev Biochem 70: 313–340.
24. Jamieson, AC, Miller, JC and Pabo, CO (2003). Drug discovery with engineered
zinc-finger proteins. Nat Rev Drug Discov 2: 361–368.
25. Segal, DJ and Barbas, CF 3rd (2001). Custom DNA-binding proteins come of age:
polydactyl zinc-finger proteins. Curr Opin Biotechnol 12: 632–637.
26. Beerli, RR and Barbas, CF 3rd (2002). Engineering polydactyl zinc-finger transcription
factors. Nat Biotechnol 20: 135–141.
27. Klug, A (2005). Towards therapeutic applications of engineered zinc finger proteins.
FEBS Lett 579: 892–894.
28. Lee, DK, Seol, W and Kim, JS (2003). Custom DNA-binding proteins and artificial
transcription factors. Curr Top Med Chem 3: 645–657.
29. Falke, D and Juliano, RL (2003). Selective gene regulation with designed transcription
factors: implications for therapy. Curr Opin Mol Ther 5: 161–166.
30. Jantz, D, Amann, BT, Gatto, GJ Jr. and Berg, JM (2004). The design of functional
DNA-binding proteins based on zinc finger domains. Chem Rev 104: 789–799.
31. Liu, Q, Xia, Z, Zhong, X and Case, CC (2002). Validated zinc finger protein designs for
all 16 GNN DNA triplet targets. J Biol Chem 277: 3850–3856.
32. Jamieson, AC, Wang, H and Kim, SH (1996). A zinc finger directory for high-affinity
DNA recognition. Proc Natl Acad Sci USA 93: 12834–12839.
33. Rebar, EJ and Pabo, CO (1994). Zinc finger phage: affinity selection of fingers with
new DNA-binding specificities. Science 263: 671–673.
34. Choo, Y and Klug, A (1994). Selection of DNA binding sites for zinc fingers using
rationally randomized DNA reveals coded interactions. Proc Natl Acad Sci USA 91:
11168–11172.
35. Wu, H, Yang, WP and Barbas, CF 3rd (1995). Building zinc fingers by selection:
toward a therapeutic application. Proc Natl Acad Sci USA 92: 344–348.
36. Segal, DJ, Dreier, B, Beerli, RR and Barbas, CF 3rd (1999). Toward controlling
gene expression at will: selection and design of zinc finger domains recognizing
each of the 5′-GNN-3′ DNA target sequences. Proc Natl Acad Sci USA 96:
2758–2763.
37. Dreier, B, Beerli, RR, Segal, DJ, Flippin, JD and Barbas, CF 3rd (2001). Development
of zinc finger domains for recognition of the 5′-ANN-3′ family of DNA sequences
and their use in the construction of artificial transcription factors. J Biol Chem 276:
29466–29478.
38. Dreier, B, Fuller, RP, Segal, DJ, Lund, CV, Blancafort, P, Huber, A et al. (2005).
Development of zinc finger domains for recognition of the 5′-CNN-3′ family DNA
sequences and their use in the construction of artificial transcription factors.
J Biol Chem 280: 35588–35597.
39. Desjarlais, JR and Berg, JM (1992). Toward rules relating zinc finger protein sequences
and DNA binding site preferences. Proc Natl Acad Sci USA 89: 7345–7349.
40. Pavletich, NP and Pabo, CO (1991). Zinc finger-DNA recognition: crystal structure of
a Zif268-DNA complex at 2.1 A. Science 252: 809–817.
41. Bae, KH, Kwon, YD, Shin, HC, Hwang, MS, Ryu, EH, Park, KS et al. (2003). Human
zinc fingers as building blocks in the construction of artificial transcription factors.
Nat Biotechnol 21: 275–280.
42. Blancafort, P, Segal, DJ and Barbas, CF 3rd (2004). Designing transcription factor
architectures for drug discovery. Mol Pharmacol 66: 1361–1371.
43. Cathomen, T, Segal, DJ, Brondani, V and Müller-Lerch, F (2008). Generation and
functional analysis of zinc finger nucleases. Methods Mol Biol 434: 277–290.
44. Carroll, D, Morton, JJ, Beumer, KJ and Segal, DJ (2006). Design, construction and in
vitro testing of zinc finger nucleases. Nat Protoc 1: 1329–1341.
45. Mani, M, Kandavelou, K, Dy, FJ, Durai, S and Chandrasegaran, S (2005). Design,
engineering, and characterization of zinc finger nucleases. Biochem Biophys Res
Commun 335: 447–457.
46. Wright, DA, Thibodeau-Beganny, S, Sander, JD, Winfrey, RJ, Hirsh, AS, Eichtinger, M
et al. (2006). Standardized reagents and protocols for engineering zinc finger
nucleases by modular assembly. Nat Protoc 1: 1637–1652.
47. Mandell, JG and Barbas, CF 3rd (2006). Zinc finger tools: custom DNA-binding
domains for transcription factors and nucleases. Nucleic Acids Res 34:
W516–W523.
48. Sander, JD, Zaback, P, Joung, JK, Voytas, DF and Dobbs, D (2007). Zinc Finger
Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res
35: W599–W605.
49. Segal, DJ, Beerli, RR, Blancafort, P, Dreier, B, Effertz, K, Huber, A et al. (2003).
Evaluation of a modular strategy for the construction of novel polydactyl zinc finger
DNA-binding proteins. Biochemistry 42: 2137–2148.
50. Bibikova, M, Beumer, K, Trautman, JK and Carroll, D (2003). Enhancing gene
targeting with designed zinc finger nucleases. Science 300: 764.
www.moleculartherapy.org vol. 16 no. 7 july 2008
© The American Society of Gene Therapy
51. Morton, J, Davis, MW, Jorgensen, EM and Carroll, D (2006). Induction and repair of
zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic
cells. Proc Natl Acad Sci USA 103: 16370–16375.
52. Alwin, S, Gere, MB, Guhl, E, Effertz, K, Barbas, CF 3rd, Segal, DJ et al. (2005). Custom
zinc-finger nucleases for use in human cells. Mol Ther 12: 610–617.
53. Ramirez, CL, Foley, JE, Wright, DA, Muller-Lerch, F, Rahman, SH, Cornu, TI et al.
(2008). Unexpected failure rates for modular assembly of engineered zinc fingers. Nat
Methods 5: 374–375.
54. Isalan, M, Choo, Y and Klug, A (1997). Synergy between adjacent zinc fingers in
sequence-specific DNA recognition. Proc Natl Acad Sci USA 94: 5617–5621.
55. Desjarlais, JR and Berg, JM (1993). Use of a zinc-finger consensus sequence framework
and specificity rules to design specific DNA binding proteins. Proc Natl Acad Sci USA
90: 2256–2260.
56. Elrod-Erickson, M, Rould, MA, Nekludova, L and Pabo, CO (1996). Zif268
protein-DNA complex refined at 1.6 A: a model system for understanding zinc
finger-DNA interactions. Structure 4: 1171–1180.
57. Kim, CA and Berg, JM (1996). A 2.2 A resolution crystal structure of a designed zinc
finger protein bound to DNA. Nat Struct Biol 3: 940–945.
58. Isalan, M, Klug, A and Choo, Y (1998). Comprehensive DNA recognition through
concerted interactions from adjacent zinc fingers. Biochemistry 37: 12026–12033.
59. Dreier, B, Segal, DJ and Barbas, CF 3rd (2000). Insights into the molecular recognition
of the 5ʹ-GNN-3ʹ family of DNA sequences by zinc finger domains. J Mol Biol
303: 489–502.
60. Wolfe, SA, Grant, RA, Elrod-Erickson, M and Pabo, CO (2001). Beyond the
“recognition code”: structures of two Cys2His2 zinc finger/TATA box complexes.
Structure 9: 717–723.
61. Miller, JC and Pabo, CO (2001). Rearrangement of side-chains in a Zif268 mutant
highlights the complexities of zinc finger-DNA recognition. J Mol Biol 313: 309–315.
62. Greisman, HA and Pabo, CO (1997). A general strategy for selecting high-affinity zinc
finger proteins for diverse DNA target sites. Science 275: 657–661.
63. Isalan, M, Klug, A and Choo, Y (2001). A rapid, generally applicable method to
engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nat Biotechnol
19: 656–660.
64. Hurt, JA, Thibodeau, SA, Hirsh, AS, Pabo, CO and Joung, JK (2003). Highly specific
zinc finger proteins obtained by directed domain shuffling and cell-based selection.
Proc Natl Acad Sci USA 100: 12271–12276.
65. Cornu, TI, Thibodeau-Beganny, S, Guhl, E, Alwin, S, Eichtinger, M, Joung, JK et al.
(2008). DNA-binding specificity is a major determinant of the activity and toxicity of
zinc-finger nucleases. Mol Ther 16: 352–358.
66. Scott, CT (2005). The zinc finger nuclease monopoly. Nat Biotechnol 23: 915–918.
67. Reik, A, Holmes, MC, Zhou, Y, Mendel, M, Liu, P-Q, Lee, GK et al. (2007). [373]
Designer” cellular immunotherapy: zinc finger nucleases generate glucocorticoid receptor
negative il-13 zetakine expressing CD8+ T-cells. 10th Annual Meeting of the American
Society of Gene Therapy: Seattle, WA.
68. Perez, EE, Wang, J, Liu, O, Kim, K, Wang, N, Lee, G et al. (2007). [436] Targeted
disruption of CCR5 using engineered zinc finger protein nucleases provides in vivo
protection from HIV. 10th Annual Meeting of the American Society of Gene Therapy,
Seattle, WA.
69. Kaiser, J (2005). Gene therapy: putting the fingers on gene repair. Science 310:
1894–1896.
70. Porteus, MH (2006). Mammalian gene targeting with designed zinc finger nucleases.
Mol Ther 13: 438–446.
71. Miller, JC, Holmes, MC, Wang, J, Guschin, DY, Lee, YL, Rupniewski, I et al. (2007). An
improved zinc-finger nuclease architecture for highly specific genome editing.
Nat Biotechnol 25: 778–785.
Molecular Therapy vol. 16 no. 7 july 2008
Next Generation ZFNs
72. Szczepek, M, Brondani, V, Buchel, J, Serrano, L, Segal, DJ and Cathomen, T (2007).
Structure-based redesign of the dimerization interface reduces the toxicity of
zinc-finger nucleases. Nat Biotechnol 25: 786–793.
73. Cathomen, T and Weitzman, MD (2005). Gene repair: pointing the finger at genetic
disease. Gene Ther 12: 1415–1416.
74. Porteus, MH and Baltimore, D (2003). Chimeric nucleases stimulate gene targeting in
human cells. Science 300: 763.
75. Meng, X, Brodsky, MH and Wolfe, SA (2005). A bacterial one-hybrid system for
determining the DNA-binding specificity of transcription factors. Nat Biotechnol 23:
988–994.
76. Meng, X, Thibodeau-Beganny, S, Jiang, T, Joung, JK and Wolfe, SA (2007). Profiling
the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a
rapid cell-based method. Nucleic Acids Res 35: e81.
77. Miller, DG, Petek, LM and Russell, DW (2004). Adeno-associated virus vectors
integrate at chromosome breakage sites. Nat Genet 36: 767–773.
78. Casillas, MA, Brotherton, SL, Andrews, LG, Ruppert, JM and Tollefsbol, TO (2003).
Induction of endogenous telomerase (hTERT) by c-Myc in WI-38 fibroblasts
transformed with specific genetic elements. Gene 316: 57.
79. Modlich, U, Bohne, J, Schmidt, M, von Kalle, C, Knoss, S, Schambach, A et al. (2006).
Cell-culture assays reveal the importance of retroviral vector design for insertional
genotoxicity. Blood 108: 2545–2553.
80. Padilla-Nash, HM, Barenboim-Stapleton, L, Difilippantonio, MJ and Ried, T (2007).
Spectral karyotyping analysis of human and mouse chromosomes. Nat Protocols 1:
3129.
81. Modlich, U, Kustikova, OS, Schmidt, M, Rudolph, C, Meyer, J, Li, Z et al. (2005).
Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by
combinatorial insertional mutagenesis. Blood 105: 4235–4246.
82. Montini, E, Cesana, D, Schmidt, M, Sanvito, F, Ponzoni, M, Bartholomae, C et al.
(2006). Hematopoietic stem cell gene transfer in a tumor-prone mouse model
uncovers low genotoxicity of lentiviral vector integration. Nat Biotechnol 24:
687–696.
83. Catto, LE, Ganguly, S, Milsom, SE, Welsh, AJ and Halford, SE (2006). Protein assembly
and DNA looping by the FokI restriction endonuclease. Nucleic Acids Res 34:
1711–1720.
84. Hacein-Bey-Abina, S, Le Deist, F, Carlier, F, Bouneaud, C, Hue, C, De Villartay, JP et al.
(2002). Sustained correction of X-linked severe combined immunodeficiency by
ex vivo gene therapy. N Engl J Med 346: 1185–1193.
85. Porteus, MH, Cathomen, T, Weitzman, MD and Baltimore, D (2003). Efficient gene
targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol Cell
Biol 23: 3558–3565.
86. Miller, DG, Petek, LM and Russell, DW (2003). Human gene targeting by
adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol Cell Biol
23: 3550–3557.
87. Yao, S, Wang, J, Lee, G, Perez, A, Wang, N, Kim, K et al. (2007). [426] Highly-efficient
genome modification in human mesenchymal stem cells using engineered zinc
finger nucleases. 10th Annual Meeting of the American Society of Gene Therapy,
Seattle, WA.
88. Hacein-Bey-Abina, S, von Kalle, C, Schmidt, M, Le Deist, F, Wulffraat, N, McIntyre, E
et al. (2003). A serious adverse event after successful gene therapy for X-linked severe
combined immunodeficiency. N Engl J Med 348: 255–256.
89. Hacein-Bey-Abina, S, Von Kalle, C, Schmidt, M, McCormack, MP, Wulffraat, N,
Leboulch, P et al. (2003). LMO2-associated clonal T cell proliferation in two patients
after gene therapy for SCID-X1. Science 302: 415–419.
90. Donsante, A, Miller, DG, Li, Y, Vogler, C, Brunt, EM, Russell, DW et al. (2007). AAV
vector integration sites in mouse hepatocellular carcinoma. Science 317: 477.
1207