Modern Biopharmaceuticals (Recent Success Stories) || Precision Genome Surgery with Meganucleases: A Promising Biopharmaceutical for Gene Therapy

9Precision Genome Surgery with Meganucleases: A PromisingBiopharmaceutical for Gene TherapyAlfred Pingoud, George H. Silva, and Wolfgang Wende

9.1Introduction

The goal of gene therapy is to repair a genetic defect without additionallymodifying the genome. One of the most promising strategies is based on homo-logous recombination, in which the DNA sequence needed to correct the geneof interest is supplied in trans to stimulate site-specific recombination. A deficientgene is thus ideally replaced in its natural context with a functional copy, leavingthe genome intact elsewhere. This approach contrasts with gene “compensation”strategies currently in use that rely on random integration of a functional gene tosupplement the dysfunctional one (see, e.g., [1–3]), with the inadvertent conse-quence of detrimental genomic integration [4–9]. On top of that, the homologousrecombination method can be used for targeted gene inactivation, insertion, andalso deletion.A major obstacle for the homologous recombination-driven approach is the low

efficiency of natural recombination between an introduced DNA and the homolo-gous chromosomal target. Remarkably, making a specific double-strand break inthe target sequence increases the frequency of homologous recombination byseveral orders of magnitude [10–12]. It was soon realized that artificially stimulatinghomologous recombination via sequence-specific endonucleases could be used forspecific gene targeting and, eventually, gene therapy (Figure 9.1).

9.2Meganucleases

Meganucleases are defined as nucleases with extended recognition sites [13] thatin principle allow targeting a unique sequence in a complex genome. To facilitatedouble-strand break-induced recombination and thereby offer the possibility forhigh-precision genome surgery and gene therapy [14–17], several types of mega-nucleases have been created and successfully used in experimental systems: zinc-finger nucleases (ZFN)s, engineered homing endonucleases, and chimeric

Modern Biopharmaceuticals: Recent Success Stories, First Edition. Edited by Jörg Knäblein.# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

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restriction endonucleases (Figure 9.2). Whereas homing endonucleases [18],which recognize sequences of up to �40 base pairs in length, are so-callednaturally occurring meganucleases, artificially produced types can be generated by(i) recombining homing endonuclease domains [19–22]; (ii) reengineering theDNA binding site of homing endonucleases [23]; or (iii) fusing a nuclease domainto a DNA recognition module, such as zinc fingers [24] or triple helix-formingoligonucleotides (TFOs) [25,26].

9.2.1Zinc Finger Nucleases

ZFNs typically consist of the nonspecific catalytic domain of the Type IIS restrictionendonuclease FokI fused at its C-terminus to three to four zinc fingers [27–29]. Eachzinc finger is composed of �30 amino acid residues that fold into a characteristicbba structure and coordinates one Zn2þ ion using two cysteine and two histidineresidues. One zinc finger recognizes a three base-pair target site: each nucleotide iscontacted by a single amino acid projecting from the a-helix into the major groove ofthe DNA target. By altering these amino acids, the specificity of the zinc finger canbe changed. Zinc fingers have been designed that recognize almost all possible NNNtriplets and can be combined to recognize an extended sequence (Figure 9.3).

Figure 9.1 Gene targeting using ameganuclease. The target sequence consists ofa specific site flanked by regions used forhomology-based repair. For gene targeting, adesigned zinc finger nuclease, an engineeredhoming endonuclease, or a programmedrestriction enzyme is introduced into the celltogether with a plasmid-borne repair matrixcomprising the gene of interest flanked by the

homologous regions present in thechromosomal locus. Upon delivery to thenucleus, the chromosomal DNA is cleaved andthe gene of interest copied into thechromosome via a homology-directed double-strand break repair process. This leads to stableintegration that can be passed on to theprogeny.

166j 9 Precision Genome Surgery with Meganucleases: A Promising Biopharmaceutical for Gene Therapy

Protocols are available for “modular assembly” engineering of ZFNs directed atselected DNA sequences [30,31]. Because of cooperative and context-dependentcontacts that can affect DNA recognition, all zinc fingers to be incorporated in ZFNsneed to be optimized [32], in general by methods such as phage display [33] ordomain shuffling and cell-based selection [34–38].

Figure 9.2 Three approaches for cleaving DNAto increase the efficiency of homologousrecombination for gene correction. Byexploiting the host’s homology-directed repairmechanisms to fix double-strand breaks inDNA, precise changes can be made within agenome using highly specific nucleasestargeted to a sequence of interest. Cleavage canbe effected by a heterodimeric zinc finger

nuclease (a), a homing endonuclease (b), or aTFO linked to a restriction enzyme (c). Forefficient homology-directed repair, cleavageneed only be within 100 base pairs of thesequence to be changed. Mutated DNA regionshown in red and repaired DNA region in blue.Yellow arrow marks point of cleavage. Brokengray lines indicate specific protein DNA orTFO–DNA interactions.

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ZFNs, like homing endonucleases, stimulate homologous recombinationthrough targeted double-strand cleavage [39]. DNA catalysis requires that theZFNs dimerize via the catalytic domain of FokI and thus double-strand cleavageoccurs between two closely oriented (usually separated by six nucleotides) bindingsites. For example, a ZFN having four “fingers” necessitates two copies of the 12base pair recognition site in a tail-to-tail orientation in order to dimerize and producea double-strand break. This effectively generates a 24 base pair recognition site,which is long enough to specify a unique locus in the human genome. In principle,the binding sites need not be identical, but this requires that two different ZFNs bepresent in the cell. One of the more remarkable gene targeting results with ZFNswas reported by scientists from Sangamo BioSciences, Inc. [40,41], who usedtwo four-finger ZFNs in human cells to cut a 24 base-pair site in the IL2Rcgene (Figure 9.4), which is mutated in severe combined immune deficiency(SCID). They achieved a permanent correction of a mutant IL2Rc gene with anefficiency of 18% modified cells without selection.When two different ZFNs are used as heterodimers to cleave a nonsymmetrical

target site, an unwanted and potentially toxic side reaction may occur involvingcleavage of the two symmetrical target sites recognized by the inherent

Figure 9.3 A transcription factor that contains three zinc fingers. (a) The crystal structure (PDB:1aay) of the protein bound to its ten base pair recognition sequence is shown. (b) A schematicdepicting the interaction of the individual zinc fingers with the target sequence.


homodimers. This can be circumvented by re-engineering the dimer interface of theFokI cleavage domain such that heterodimerization is strongly preferred overhomodimerization. This has been achieved recently and led to a large reductionin cell toxicity, a major problem associated with ZFNs [15,42–44].

9.2.2Homing Endonucleases

All efforts to create homing endonucleases (HEs) of altered specificity have beendevoted toHEsof theLAGLIDADGfamily,which is the largest andbest studied familyin both structural and functional terms. The familymembers are characterized by theLAGLIDADG sequence that has both a structural role at the subunit/subunit ordomain/domain interface and a catalytic role by supplying a carboxylate for thecoordination of the divalent metal ion cofactor Mg2þ. Whereas monomeric enzymesof this family (e.g., I-DmoI, an intron-encoded HE, and PI-SceI, an intein-encodedHE) have two LAGLIDADG sequences, homodimeric enzymes have only one persubunit (e.g., I-CreI). Crystal structure information is available for ten HEs of thisfamily (Figure 9.5) and for most of them also co-crystal structure information ofenzyme–DNA complexes [18]: I-AniI, I-CeuI [45], I-CreI, I-DmoI, I-MsoI, I-SceI, PI-PfuI, PI-SceI, PI-TkoII [46], and I-Tsp0611 [47]. LAGLIDADG HEs have a uniqueconserved core structure characterized by an abbabba fold. Two such a/b units (twodomains in the monomeric enzyme and two subunits in the homodimeric enzymes)are arranged about a pseudo twofold axis, with the LAGLIDADG helices (the firsta-helix of the abbabba core structure) at the domain–domain or subunit–subunitinterface. Theb-strands of the twodomains or the two subunits each areorganized in a

Figure 9.4 Schematic of the zinc fingernuclease developed by Sangamo BioSciencesto target the IL2Rc gene. This meganucleasewas developed to target a gene that isdefective in severe combined immunedeficiency (SCID). It consists of two proteins(ZF1234 and ZF5678), each having four zinc

fingers fused to the nonspecific DNAcleavage domain of the restrictionendonuclease FokI [40]. The two proteinsdimerize on the DNA and cleave bothstrands of the 24 base-pair site indicated(Courtesy of J. Wu, K. Kandavelou, andS. Chandrasegaran: [29]).


four-stranded antiparallel b-sheet that together with two loops connecting theb-strands form the DNA binding site. The intein-encoded homing endonucleases(PI-PfuI, PI-SceI, PI-TkoII) have an additional domain that extends the DNA bindingsite. Unlike restriction endonucleases, HEs tolerate base substitutions in mostpositions of their recognition sequence, as has been established for I-CreI [48], PI-SceI [49], and I-SceI [50]. HEs therefore seem to have a greater plasticity of target siterecognition than restriction endonucleases, a finding that has fueled efforts toengineer HEs as gene-specific reagents for genomic applications [51].

Figure 9.5 Compilation of the available crystalstructures of homing endonucleases of theLAGLIDADG family. For the homodimericenzymes, one subunit is colored with thea-helices in red and the �-sheets in blue; theother subunit is shown in gray. For the intein-encoded enzymes, only the endonucleasedomain is shown with the a-helices in red andthe �-sheets in blue; the protein-splicingdomain, which harbors an additional DNArecognition region (DRR), is shown in gray.

The subunit/subunit or domain/domaininterface is mainly formed by the LAGLIDADGhelices that contain the active site residues (inyellow). Engineering efforts have been devotedmainly to I-CreI, I-MsoI, and I-DmoI. Shown arethe crystal structures of 10 homingendonucleases [PDB entries: I-AniI (1P8K), I-CeuI (2EX5), I-CreI (1T9I), I-DmoI (1B24), I-MsoI (2FLD9, I-SceI (1R7M), I-Tsp061I(2DCH), PI-PfuI (1DQ3),PI-SceI (1VDE), PI-TkoII (2CW7)].


Three different strategies have been pursued to alter HE specificity:

i) Computational redesign using an in silico screen [23]: although the studywas impressive from a methodological point of view, the change in specificitywas moderate – the I-MsoI variant obtained differs in two amino acidresidues from the wild-type enzyme (subunit) and recognizes a DNAsequence that differs in only one position from the wild-type sequence(half-site) that is cleaved by the variant 1000-fold more quickly than thewild-type sequence.

ii) Screening for variants from a library of HE variants, followed by “specificity andactivity maturation“ by random mutagenesis (see also Chapter 6). Thisapproach has been pioneered by Paques and colleagues from Cellectis SA(Romainville, France). They succeeded in generating I-CreI-derived HEs[20,52–55] that can cleave non-palindromic sequences that differ in up to17 of the 22 positions of the original target with an efficiency comparable towild-type enzyme cleaving the wild-type target (Figure 9.6) [53]. It is noteworthythat using this approach, I-CreI variants have been successfully produced to

Figure 9.6 General strategy for the generationof I-CreI variants of new specificity. A largecollection of I-CreI variants with locally alteredspecificities produced from the wild-typeenzyme by random mutagenesis and screeningfor active enzymes is generated and “stored”(OMEGABASE). A combinatorial approach isthen used to assemble suitable variants intohomodimeric proteins, which recognize

palindromic sequences (“Half megas”), andsubsequently into heterodimeric proteins(“Custom meganucleases”). In a final step thecustom meganucleases are subjected torandom mutagenesis and further screening,resulting in a highly active heterodimeric I-CreIvariant with redesigned specificity (“refinedmeagnuclease”). (Courtesy of F. Paques).


target in CHO cells the clinically relevant human XPC gene, which is mutatedin a rare autosomal recessive genetic disease, Xeroderma pigmentosum. As forZNFs, heterodimeric I-CreI variants, needed for the recognition of non-palindromic sequences, can recombine to form the corresponding homo-dimers that may lead to unwanted side reactions when used for targetedgenome engineering. As demonstrated recently, by appropriate engineering ofthe dimer interface of I-CreI, homodimer formation can be suppressed [56].

iii) Recombining domains or subunits of HEs of the LAGLIDADG family via theLAGLIDADG helix, which is the principal structural element responsible fordomain–domain or subunit–subunit interaction. Using this “mixing-and-matching” strategy, hybrid HEs with hybrid specificity can be obtained. Todate, this has been achieved for I-CreI/I-DmoI [19,20] and for domain A ofI-DmoI [21].

It is likely that in the future we will see a combination of the three approaches:starting with different protein scaffolds (naturally occurring homing endonu-cleases, homing endonuclease variants, and hybrids of homing endonucleases),libraries will be generated and screened, and promising variants will be improvedin their specificity and activity by random and/or site-directed mutagenesis.

9.2.3Restriction Endonuclease-TFO Fusions

This approach depends on the high specificity of restriction enzymes and that oftriple helix formation. The target site of a restriction enzyme–oligonucleotidefusion is a composite of the recognition site of the restriction enzyme and thetriple helix-forming site (TFS). With such “programmable” restriction enzymes,any gene can in principle be targeted given the combinatorial flexibility this fusionoffers in addressing a short, yet precisely recognized restriction site next to adefined TFS. This approach has been demonstrated to work in vitro for therestriction endonuclease PvuII [26] (Figure 9.7). A drawback of this approach,however, is that triple-helix formation is slow, meaning that after delivery to thecell the restriction enzyme part of the conjugate would cleave DNA at multiplesites (unaddressed PvuII sites) before triple-helix formation has targeted theenzyme to the site of interest. This problem has been solved by producing a“caged” version of the enzyme that requires photoactivation to become catalyti-cally active [57,58] (Figure 9.8). Triple helix formation with oligonucleotides wasuntil recently restricted to oligo purine–oligo pyrimidine stretches. Using TFOsthat contain four different synthetic nucleotides, recognition of all four base pairsby triple helix formation at physiological pH is possible: 20-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine recognizes AT base pairs with high affinity; 3-methyl-2aminopyridine binds to GC at higher pHs than cytosine, while 6-(3-aminopropyl)-7-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one and N-(4-(3-acetamidophenyl)thiazol-2-yl-acetamide) bind to CG and TA base pairs, respectively [59]. Thissuggests that “programmable” restriction enzymes can be targeted to all


sequences close to a restriction site (to date tested only with PvuII sites). While ithas been established that programmed restriction endonucleases can be used asveritable meganucleases in vitro, it remains to be shown how specific these fusionsare on genomic DNA in vivo.

Figure 9.8 Generation of a restrictionendonuclease–triple helix-formingoligonucleotide conjugate. A parallel triple helixis formed by Hoogsteen base pairs between aWatson–Crick double helix and anoligonucleotide. To increase triple helixformation under physiological conditions, asynthetic oligonucleotide carrying modified

bases (M, P; see text) is used. It is covalentlylinked via a 50-amino link to a bifunctionalcross-linker. This activated oligonucleotide isthen coupled to a single cysteine residue insingle-chain PvuII (scPvuII). It has been shownthat this scPvuII–TFO conjugate specificallycleaves DNA at the composite target site (fordetails see [26]).

Figure 9.7 Schematic of a restrictionendonuclease–triple helix-formingoligonucleotide conjugate bound to DNA. Therestriction endonuclease (REase>)–triple helix-forming oligonucleotide (TFO) conjugate has abipartite recognition sequence, consisting of

the recognition site of the REase (e.g., CAGCTGfor PvuII) and the complementary sequence forthe TFO, that is, the TFS. With a TFO of 16 basepairs in length, a composite target site of 22base pairs results, likely to be unique in thehuman genome.


An alternative to using restriction endonuclease–oligonucleotide fusions is toemploy etoposide–oligonucleotide fusions [60]. Etoposide binds specifically totopoisomerase II and induces it to generate double-strand breaks. By attachingetoposide to TFOs that bind to DNA in a highly sequence-specific manner, DNAcleavage can be targeted to specific sites. Psoralen introduces cross-links into thetarget DNA; when fused to a TFO it stimulates, by an as yet unknown mechanism,homologous recombination manifold [61].The clear advantage of restriction enzyme– or etoposide–oligonucleotide

fusions over ZFNs or HEs is the realization of truly programmable enzymes,in a similar manner as DNA and RNApolymerases are programmable with a DNAtemplate.

9.3Prospects of Gene Therapy Using Meganucleases

Five monogenic diseases have been the target of recent gene therapy trials with viralvectors:

i) Ornithine transcarbamylase deficiency (OTC) [62,63]: the most common ureacycle disorder that leads to reduced ammonia detoxification by impairingcondensation of carbamyl phosphate and ornithine to form citrulline. Morbid-ity and mortality are high, especially in male patients (OTC is an X-linkeddisorder) with the neonatal form;

ii) Hemophilia (due to factor IX deficiency) [64,65]: factor IX deficiency (again anX-linked disease), or Christmas disease, causes hemophilia B, which in itssevere form causes frequent spontaneous bleeding episodes often into thejoints and muscles [66];

iii) SCID [67–69]: SCID is a genetic disorder in which both B- and T-cell develop-ment is impaired due to a defect in one of several possible genes, for example,the IL2Rc gene (X-linked), the adenosine deaminase gene, and therecombination activating genes RAG-1 and RAG-2 (X-linked);

iv) Chronic granulomatous disease (CGD) [70], an inherited X-linked disorder ofphagocytic cells that results from an inability of phagocytes to producebactericidal superoxide anions, leading to recurrent life-threatening bacterialand fungal infections in affected patients who invariably die in the first decadeof life;

v) Leber’s congenital amaurosis (LCA): LCA is a group of inherited blindingdiseases. One form (LCA2), caused by mutations in the gene encoding retinalpigment epithelium-specific 65-kD protein (RPE65), is associated with poorvision at birth and complete loss of vision in early adulthood [71,72].

The outcome of these clinical trials has been variable [16]; of particular concernwas the occurrence in a few cases of fatal immunological complications and ofmonoclonal lymphoproliferation, which were due to expression of viral proteinsand integration of viral DNA into critical loci of the host DNA, respectively.


The principal alternative to gene therapy with viral vectors is gene therapy basedon DNA double-strand break-induced recombination [14,7–9]. To be effective, thedouble-strand break must be close (within a few hundred base pairs) to the genedefect. For specific double-strand cleavage, meganucleases are the nucleases ofchoice because they are sufficiently specific to cleave a unique site in a complexgenome. Of the three kinds ofmeganucleases that have been developed (ZFNs,HEs,and programmed restriction endonucleases), ZFNs and HEs have been shown tofunction in vivo, whereas for programmed restriction enzymes only in vitro data werereported.The first gene correction experiments with specifically designed ZFNs were

reported in 2003 [39,73] and 2005 [40,74,75]. While these studies showed thatgene targeting with ZFNs works in a variety of different cells, including human cells(293, K562, and CD4þ Tcells), toxicity, presumably due to nonspecific cleavage, wasfound to be a critical issue.Gene targeting experiments with the HE I-SceI began as early as the mid-1990s

[10,12,76], with mouse cells (NIH3T3, PCCC7, and ES cells) followed by similarexperiments with human cells (293 cells). The first gene targeting experiments withspecifically designed HEs were reported in 2006 by the Cellectis group, which hadproduced an I-CreI variant that specifically cuts a sequence in the human RAG1gene [55]. Mutations in the RAG1 (or RAG2) gene cause Omenn syndrome, anautosomal recessive form of SCID. More recently, the Cellectis group producedanother I-CreI variant that targets the human XPC gene, which is mutated in X.pigmentosum [53]. This skin disease, associated with extreme sensitivity to sunlightand early development of skin cancer, is transmitted in an autosomal recessivemanner. The RAG1 and XPC examples demonstrated that it is possible to modify achromosomal locus with engineered HEs. While it does seem likely that one cantarget many genes with specifically engineered I-CreI variants, it must be consideredthat engineering HE variants with new specificities is considerably more difficultthan producing ZFNs with new specificities. While HEs appear to be less toxic thanZFNs, as demonstrated for I-SceI [27,73,75], the toxicity issue must always beaddressed for every new engineered meganuclease.

9.4Summary and Outlook

The genes for ZFNs and HEs, along with a repair matrix that acts as template forhomologous recombination, must be introduced into cells ex vivo by transfection,a process characterized by poor yield. This hurdle could in principle be overcomeby using replication deficient, nonintegrative lentiviruses [77] to introduce thegene for the ZFN or HE together with the correction matrix into cells. In this case,only a transient infection by the lentivirus is required (see also Chapter 2). Oncethe gene coding for the meganuclease has been expressed and the host genomehas been cut at the target gene, followed by homologous recombination using therepair matrix, the presence of the lentivirus in the cell is no longer needed. Still,

9.4 Summary and Outlook j175

only a minority of cells will be “cured,” limited by the frequency of the homolo-gous recombination process.Programmed restriction enzymes, being artificial fusions of a protein and an

oligonucleotide, require “profection,” a process similar to transfection that allowsproteins and protein conjugates to be introduced into cells [78] together with therepair matrix. As with ZFNs and HEs, gene therapy using programmed restrictionendonucleases will be carried out ex vivo, preferably with hematopoietic or otherstem cells (see also Chapter 3) and [79–81]. In some cases (e.g., SCID), correctedcells have a selective growth advantage [67,82], meaning that transfection orprofection yield is not as critical as in other cases wherein other selective measuresmust be employed. The extent to which one must promote or select for (via, e.g.,extraneous markers) the proliferation of corrected cells remains to be seen.At the time of this writing, gene therapy using meganucleases was still at an

experimental stage and not yet ready for human clinical trials. Nevertheless,researchers are currently developing the necessary tools and know-how throughgene targeting experiments in animal models, along with economically relevantprojects involving transgenic plants, to make this approach feasible as a new andexciting class of modern biopharmaceuticals.

9. 9.5Note

After submission of our manuscript, a major development concerning the design ofnucleases suitable for genome engineering occurred. The so called TALE nucleasesare likely to replace engineered meganucleases and zinc finger nucleases. Theyconsist of a DNA binding module (derived from TAL effector proteins) and a DNAcleavage module (typically, the catalytic domain of FokI). Transcription activator likeeffector proteins have a simple DNA recognition code that allows designing proteinsthat would bind any DNA sequence. TALE nucleases, therefore, are fully program-mable Refs. [83–86].

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