Targeted gene addition into a specified locationin the human genome using designed zincfinger nucleasesErica A. Moehle, Jeremy M. Rock, Ya-Li Lee, Yann Jouvenot, Russell C. DeKelver, Philip D. Gregory,Fyodor D. Urnov*, and Michael C. HolmesSangamo BioSciences, Inc., Point Richmond Technology Center, 501 Canal Boulevard, Suite A100, Richmond, CA 94804Communicated by Carl O. Pabo, Harvard Medical School, Boston, MA, December 27, 2006 (received for review November 14, 2006)Efficient incorporation of novel DNA sequences into a specific sitein the genome of living human cells remains a challenge despite itspotential utility to genetic medicine, biotechnology, and basicresearch. We find that a precisely placed double-strand breakinduced by engineered zinc finger nucleases (ZFNs) can stimulateintegration of long DNA stretches into a predetermined genomiclocation, resulting in high-efficiency site-specific gene addition.Using an extrachromosomal DNA donor carrying a 12-bp tag, a900-bp ORF, or a 1.5-kb promoter-transcription unit flanked bylocus-specific homology arms, we find targeted integration fre-quencies of 15%, 6%, and 5%, respectively, within 72 h of treat-ment, and with no selection for the desired event. Importantly, wefind that the integration event occurs in a homology-directedmanner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, andhence presumably results from synthesis-dependent strand an-nealing repair of the break using the donor DNA as a template. Thissite-specific gene addition occurs with no measurable increase inthe rate of random integration. Remarkably, we also find that ZFNscan drive the addition of an 8-kb sequence carrying three distinctpromoter-transcription units into an endogenous locus at a fre-quency of 6%, also in the absence of any selection. These datareveal the surprising versatility of the specialized polymerasemachinery involved in double-strand break repair, illuminate apowerful approach to mammalian cell engineering, and open thepossibility of ZFN-driven gene addition therapy for human geneticdisease.gene therapy 兩 protein production 兩 somatic cell geneticsThe C2H2zinc finger (1), the most abundant DNA recogn itionmotif in eukarya (2, 3), is highly amenable to engineering forthe recognition of virtually any DNA sequence (4–6). Theseproperties have been successfully exploited to enable the mod-ulation of gene expression via their application as designedtranscription factors (ZFP-TFs) (7), as well as direct modifica-tion of the DNA itself via engineered zinc finger nucleases(ZFNs) for human gene correction (8). The latter process, basedon work from several laboratories including our own (9–16),overc omes the exceedingly low frequency of spontaneous ho-mologous recombination in mammalian cells, which until re-cently has made the targeted modification of human genomesequence in vivo impractical (17, 18). A lthough this limitationhas been addressed in settings where drug-based selectionschemes can be applied (19, 20), it is restricted to particular cellt ypes, e.g., fibroblasts and mouse embryonic stem cells. Suchtraditional ‘‘gene targeting’’ requires the construction of elabo-rate vectors, a 6- to 8-week regimen of treatment with twodistinct selective agents, and the isolation of individual cellclones by limiting dilution, only a subset of which carries thedesired targeting event (18).ZFN-mediated gene correction (8), in contrast, oc curs at highf requency without selection, is applicable to a broad range ofprimary and transformed cells, and does not require cell cloningbecause it invokes a natural process of genetic informationtransfer via a double-strand break (DSB). A DSB evoked by ast alled DNA replication fork or by an environment al insult isnor mally eliminated via end-joining (21) or homolog y-directedrepair (HDR). The latter is a specialized for m of homologousrec ombination that transfers genetic information to the brokenchromosome from a DNA molecule of related sequence (22–25).Indeed, we have earlier shown that t argeting a DSB to a specificsite in the genome with engineered ZFNs (Fig. 1A) transferssingle-base-pair changes from a donor plasmid into the chro-mosome with efficiencies that can exceed 20% (16).However rapid and ef ficient, gene correction is a localizedevent, and a single DSB, whether induced by a homing endo-nuclease (26) or by a ZFN (M.C.H., Y.-L.L., and F.D.U.,unpublished data), can allow efficient correction of mutationsonly w ithin an ⬇200-bp window surrounding the break. Thec omplex mut ational spectr um underlying many human mono-gen ic diseases would therefore require tailoring ZFNs to eachcluster of mutations. This requirement has prompted us toinvestigate the feasibility of using ZFNs to drive site-specific‘‘gene addition,’’ specifically, the integ ration of long DNAsegments into a predeter mined locus. Both medical (gene ther-apy) and industrial (e.g., engineering cell lines for proteinproduction) gene addition is currently achieved via randomintegration of the transgene into the genome, a process thatpresents safety concerns from a clinical perspective (27) and isc ostly and time-c onsuming in industrial applications because ofchromatin-based effects on expression of a randomly integratedtransgene (28). A c onsiderable effort notwithstanding (29, 30),only limited progress has been made so far in controlling thelocation of gene insertion, and extensive screen ing or selectionfor the desired event is almost invariably a prerequisite.The present work shows that efficient, site-specific geneaddition into a predetermined endogenous locus in human cellscan occur in the absence of selection. We show that if aZFN-cleaved locus is provided w ith an engineered template thatc onsists of novel genetic information flanked by appropriateregions of target site homology, then break repair occurs v iaAuthor contributions: E.A.M. and J.M.R. contributed equally to this work; E.A.M., J.M.R.,P.D.G., F.D.U., and M.C.H. designed research; E.A.M., J.M.R., Y.-L.L., Y.J., R.C.D., and F.D.U.performed research; E.A.M., J.M.R., F.D.U., and M.C.H. analyzed data; and P.D.G., F.D.U.,and M.C.H. wrote the paper.Conflict of interest statement: C.O.P. is chair of the Scientific Advisory Board for SangamoBioSciences, Inc. E.A.M., J.M.R., Y.-L.L., Y.J., R.C.D., P.D.G., F.D.U., and M.C.H. are full-timeemployees of Sangamo BioSciences, Inc.Freely available online
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