Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases Erica A Moehle Jeremy M Rock Ya Li Lee Yann Jouvenot Russell C DeKelver Philip D Gregory Fyodor D Urnov and Michael C Holmes Sangamo BioSciences Inc Point Richmond Technology Center 501 Canal Boulevard Suite A100 Richmond CA 94804 Communicated by Carl O Pabo Harvard Medical School Boston MA December 27 2006 received for review November 14 2006 gene therapy protein production somatic cell genetics T he C2H2 zinc finger 1 the most abundant DNA recognition motif in eukarya 2 3 is highly amenable to engineering for the recognition of virtually any DNA sequence 4 6 These properties have been successfully exploited to enable the modulation of gene expression via their application as designed transcription factors ZFP TFs 7 as well as direct modification of the DNA itself via engineered zinc finger nucleases ZFNs for human gene correction 8 The latter process based on work from several laboratories including our own 9 16 overcomes the exceedingly low frequency of spontaneous homologous recombination in mammalian cells which until recently has made the targeted modification of human genome sequence in vivo impractical 17 18 Although this limitation has been addressed in settings where drug based selection schemes can be applied 19 20 it is restricted to particular cell types e g fibroblasts and mouse embryonic stem cells Such traditional gene targeting requires the construction of elaborate vectors a 6 to 8 week regimen of treatment with two distinct selective agents and the isolation of individual cell clones by limiting dilution only a subset of which carries the desired targeting event 18 ZFN mediated gene correction 8 in contrast occurs at high frequency without selection is applicable to a broad range of www pnas org cgi doi 10 1073 pnas 0611478104 primary and transformed cells and does not require cell cloning because it invokes a natural process of genetic information transfer via a double strand break DSB A DSB evoked by a stalled DNA replication fork or by an environmental insult is normally eliminated via end joining 21 or homology directed repair HDR The latter is a specialized form of homologous recombination that transfers genetic information to the broken chromosome from a DNA molecule of related sequence 22 25 Indeed we have earlier shown that targeting a DSB to a specific site in the genome with engineered ZFNs Fig 1A transfers single base pair changes from a donor plasmid into the chromosome with efficiencies that can exceed 20 16 However rapid and efficient gene correction is a localized event and a single DSB whether induced by a homing endonuclease 26 or by a ZFN M C H Y L L and F D U unpublished data can allow efficient correction of mutations only within an 200 bp window surrounding the break The complex mutational spectrum underlying many human monogenic diseases would therefore require tailoring ZFNs to each cluster of mutations This requirement has prompted us to investigate the feasibility of using ZFNs to drive site specific gene addition specifically the integration of long DNA segments into a predetermined locus Both medical gene therapy and industrial e g engineering cell lines for protein production gene addition is currently achieved via random integration of the transgene into the genome a process that presents safety concerns from a clinical perspective 27 and is costly and time consuming in industrial applications because of chromatin based effects on expression of a randomly integrated transgene 28 A considerable effort notwithstanding 29 30 only limited progress has been made so far in controlling the location of gene insertion and extensive screening or selection for the desired event is almost invariably a prerequisite The present work shows that efficient site specific gene addition into a predetermined endogenous locus in human cells can occur in the absence of selection We show that if a ZFN cleaved locus is provided with an engineered template that consists of novel genetic information flanked by appropriate regions of target site homology then break repair occurs via Author 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 Sangamo BioSciences 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 time employees of Sangamo BioSciences Inc Freely available online through the PNAS open access option Abbreviations ZFN zinc finger nuclease DSB double strand break HDR homologydirected repair SDSA synthesis dependent strand annealing To whom correspondence should be addressed E mail furnov sangamo com This article contains supporting information online at www pnas org cgi content full 0611478104 DC1 2007 by The National Academy of Sciences of the USA PNAS February 27 2007 vol 104 no 9 3055 3060 APPLIED BIOLOGICAL SCIENCES Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine biotechnology and basic research We find that a precisely placed double strand break induced by engineered zinc finger nucleases ZFNs can stimulate integration of long DNA stretches into a predetermined genomic location resulting in high efficiency site specific gene addition Using an extrachromosomal DNA donor carrying a 12 bp tag a 900 bp ORF or a 1 5 kb promoter transcription unit flanked by locus specific homology arms we find targeted integration frequencies of 15 6 and 5 respectively within 72 h of treatment and with no selection for the desired event Importantly we find that the integration event occurs in a homology directed manner and leads to the accurate reconstruction of the donorspecified genotype at the endogenous chromosomal locus and hence presumably results from synthesis dependent strand annealing repair of the break using the donor DNA as a template This site specific gene addition occurs with no measurable increase in the rate of random integration Remarkably we also find that ZFNs can drive the addition of an 8 kb sequence carrying three distinct promoter transcription units into an endogenous locus at a frequency of 6 also in the absence of any
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