Genotoxicity in gene therapy

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NOT FOR CIRCULATION Current Opinion in Molecular Therapeutics 2008 10(3):214-223 © The Thomson Corporation ISSN 1464-8431

Genotoxicity in gene therapy: An account of vector integration and designer nucleases Jens Bohne1* & Toni Cathomen2 Address 1 Hannover Medical School, Department of Virology OE 5230, Carl-Neuberg-Str 1, D-30625 Hannover, Germany Email: [email protected] Charité Medical School, Institute of Virology (CBF) Hindenburgdamm 27, D-12203 Berlin, Germany

2

*To whom correspondence should be addressed

Genotoxicity is a collective term that includes any process which affects the integrity of genomic DNA. With regard to gene therapy, the insertion of genetic elements and the expression of DNA-modifying proteins are the main mediators of genotoxic side effects. The practicability of gene-addition-type gene therapy protocols has been demonstrated in several successful clinical trials, but the risk of insertional mutagenesis remains a major obstacle. Targeted strategies aimed at correcting a mutation directly in the genome can preserve temporal and tissue-specific expression of the afflicted gene. However, sufficient gene targeting frequency can only be achieved upon expression of tailor-made nucleases. Partly because of insufficient specificity, such designer nucleases can challenge genome integrity. Here, the origin and the consequences of genotoxicity are reviewed, with a focus on assays that have been developed to assess genotoxicity. In addition, approaches to reduce toxicity associated with the major gene therapy strategies are discussed. Keywords: Cytotoxicity, gene correction, gene repair, insertional mutagenesis, targeted integration, viral vector, zinc-finger nuclease

Introduction

Genotoxicity: A definition Anything that affects DNA integrity or leads to DNA or chromosomal damage, or both, is likely to exert genotoxicity. A classical view of genotoxicity is that it is caused by chemicals or radiation that modify DNA [1]. However, insertion of genetic elements or expression of proteins that act on DNA can also cause genotoxicity. It is clear that based on Paracelsus' law, which states "All substances are poisons; there is none which is not a poison. The right dose differentiates a poison….", the insertion of transgenes and the expression of designer nucleases lead to genotoxicity in a dose-dependent manner. Gene therapy protocols for inherited disorders can be generally classified into strategies that add a non-mutated copy of the defective gene or correct a disease-triggering mutation within the genome. The concept of gene-additiontype gene therapy is simple (Figure 1a) and successful initial clinical trials have demonstrated the feasibility of this strategy [2,3]. Despite, or because of, its apparent simplicity, this strategy has some drawbacks including the risk of insertional mutagenesis [4]. Targeted approaches aim to restore the genetic function by integration of a therapeutic expression cassette into a yet to be defined 'safe harbor' (Figure 1b), or by directly editing the mutated gene to restore the wildtype sequence (Figure 1c). The latter strategy preserves the temporal and tissue-specific control of the afflicted gene, but

the targeting frequency and off-target effects still pose a challenge. In this review, the origin and the consequences of genotoxicity and the progress that has been made to assess and reduce the toxicity associated with various gene therapy protocols are described and discussed.

The main strategies in gene therapy The conventional and, to date, most successful gene therapy for inherited disorders is gene addition [2,3,5,6]. The insertion and expression of a wild-type copy of the diseasecausing gene, at least in recessively inherited disorders, leads to correction of the phenotype at the cellular level. The therapeutic transgene is embedded into a full transcription unit, including promoter and termination signals, which ensure a certain level of transgene expression depending on the site of insertion (Figure 1a) [7]. Precise manipulations directly at the chromosomal locus represent a potential alternative. However, molecular surgery is challenging and clinical validation is still lacking. Historically, strategies in which a transgene cassette was inserted into a specific locus have been based on the function of specific viral proteins. For example: the Rep protein of adeno-associated virus (AAV) type 2 for integration into the AAVS1 locus on chromosome 19 [8-11]; the phage φC31 integrase to mediate integration into genomic pseudo attachment sites [12,13]; or a modified retroviral integrase or transposase [14,15]. In addition, insertion can occur by

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Genotoxicity in gene therapy: Vector integration and designer nucleases Bohne & Cathomen 215

Figure 1. Main strategies in gene therapy.

B Targeted integration into 'safe harbor'

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(A) Gene addition. In order to compensate for a genetic defect (gene A – ), a complementary (c)DNA expression cassette embedded in a gene transfer vector is transduced into the target cell. For sustained expression in mitotic cells, the transgene (gene A + ) is integrated in a semi-random manner into the host genome, which can lead to insertional mutagenesis, such as dysregulation or gene disruption, of a different locus (in this example, gene B is mutated to gene Bmut ). (B) Targeted integration into a 'safe harbor'. To compensate for a genetic defect in gene A (gene A – ), a transgene expression cassette (gene A + ) flanked by sequences homologous to the 'safe harbor' (gene C) is embedded in a donor vector. A double-strand break (DSB) stimulates homologous recombination (HR) between the donor DNA and the 'safe harbor'. Integration into the gene C locus does not lead to genotoxic side effects. (C) Gene correction. In order to fully correct a genetic defect (gene A – ), a gene fragment encompassing wild-type sequences homologous to the mutant gene is used to transduce the target cell. A DSB stimulates HR between the donor DNA and the defect gene to generate an entirely refurbished locus (gene AWT ).

homologous recombination (HR) between the endogenous target locus and an exogenous donor DNA fragment (Figure 1b, 1c). The underlying method is called 'gene targeting' and was originally applied to investigate gene function in vivo by generating knockout and knockin animals [16•]. Because the HR frequency in mammalian cells is low, the generation of such animals relies on tedious positive and negative selection strategies [16•]. The low HR frequency and dependence on selection has prevented the application

of gene targeting in a therapeutic context. However, data from various laboratories established that the creation of a targeted DNA double-strand break (DSB) stimulates HR by activating the cellular DNA repair machinery. Initial experiments using the yeast homing endonuclease I-SceI demonstrated that the insertion of a DSB can increase the frequency of HR by several 100-fold in various cells [17-19], suggesting that DSB-induced stimulation of HR is a universal cellular phenomenon.

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216 Current Opinion in Molecular Therapeutics 2008 Vol 10 No 3

To date various DNA templates have been utilized for DSB-induced gene targeting. Although plasmid-DNA has been the most commonly used HR substrate, it might not be the first choice in a therapeutic setting. Single-stranded oligonucleotides [20] and viral vectors based on AAV [21] may be more efficacious. Because of their superior transduction record, integrase-deficient lentiviral vectors (IDLVs) are expected to be a valuable option for gene targeting and, in initial experiments, gene editing frequencies of up to 29% were reached in the absence of selection [22,23].

Gene transfer Viral vectors are the preferred vehicles for gene transfer [24]. The basic principles are similar for each virus family. The viral genome is deleted of any dispensable genetic information and plasmids that encode only genes required for essential functions in trans are generated. Ideally, a vector encodes only the transgene and the cis-regulatory elements necessary for packaging and transduction. Because of their integrative nature, transduction of target cells with retroviral vectors is inevitably associated with the risk of genotoxicity (Figure 2a) [25]. However, insertion is not a prerequisite for sustained transgene expression

in post-mitotic cells, as exemplified by the use of vectors based on AAV [26,27], HSV [28], high-capacity adenoviral vectors [29], or IDLVs [30]. As outlined below, however, such 'episomal' vectors still bear a genotoxic potential because they can be incorporated into the cellular genome by DNA repair pathways (Figure 2a) [16•]. Because it is important for retroviruses to ensure high expression of their genome independent of the integration locus, all retroviral promoters consist of condensed enhancer/promoter sequences [31,32], which induce high transgene expression, and a duplication of the enhancer/promoter region in the long terminal repeat (LTR), which may affect expression of neighboring genes by a process called insertional mutagenesis (Figure 2b; [33]).

Genotoxicity associated with gene transfer vectors The term insertional mutagenesis describes the mutagenic effects of the insertion of any piece of DNA [33]. The effects include: (i) transcriptional upregulation or temporal deregulation of nearby genes [34]; (ii) generation of fusion or read-through transcripts from the inserted promoter into cellular genes [35]; and (iii) gene disruption by the destruction of open reading frames. The latter event is thought to be

Figure 2. Genotoxic effects of episomal vectors.

A Active integration

B Double-strand breaks and passive integration

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(A) Active integration. For sustained transgene expression in mitotic cells, a therapeutic gene expression cassette must be integrated into the host genome. In the case of retroviral vectors, integration is an active process catalyzed by the viral integrase. The strong enhancer/promoter elements (E/P) contained within the long terminal repeats may affect the activity of adjacent cellular promoters (large block arrow), leading, for example, to a strong upregulation of expression of a cellular gene (gene Bup ). (B) Double-strand breaks and passive integration. A zinc finger nuclease (ZFN) designed to create double-strand breaks (DSBs) in the target locus (gene A – ) is composed of two subunits. Each subunit encompasses three zinc finger domains (1-2-3) that recognize 9 base pairs of the target site and an endonuclease domain (endo). After dimerization, the nuclease is activated and cuts the DNA in between the two half-sites. In addition to a DSB in the target site, unspecific ZFN activity may create DSBs at off-target sites, which can lead to secondary mutations (gene Cmut ) or promote the integration of gene transfer vectors (gene Bmut ). cDNA complimentary DNA, pA polyadenylation signal.

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frequent, but its phenotypic effects are predicted to be small because of the diploid nature of the mammalian genome [36]. Therefore, the major side effect of retroviral vectors is transcriptional deregulation (Figure 2b) as the insertion of an enhancer/promoter can lead to a drastic upregulation of neighboring genes [37•]. Using a murine bone marrow transplantation (BMT) protocol, Li and coworkers were the first to observe a malignant transformation of gene-modified cells by insertional mutagenesis, which was caused by a replication-incompetent vector [4]. The leukemic clone was characterized by insertion of the vector into the ecotropic viral insertion site-1 (Evi-1) gene and strong upregulation. In fact, most of the common insertion sites observed in preclinical gene therapy models were previously identified using replication-competent murine leukemia virus. This information has been summarized in the retroviral tagged cancer gene database [38]. Long-term observational studies of primates that received BMT and chemotherapy revealed leukemic expansion caused by insertional mutagenesis [39]. Interestingly, a large fraction of the integration sites in these animal models were located in genes involved in proliferation and stem cell renewal [40], a phenomenon also observed in clinical gene therapy trials [41,42]. In successful gene therapy trials that restored the immune system in approximately 40 children suffering from X-linked severe combined immunodeficiency (SCID-X1) [3,5], four patients showed signs of T-cell leukemia in the Paris cohort [43,44]. All four cases appear to be causally linked to an insertion of the retroviral vector into the LMO-2 locus, a known proto-oncogene [45••]. Moreover, one case of malignant T-cell expansion was observed in the SCID-X1 trial in London, probably also caused by an insertion near the LMO-2 locus [42,46]. These cases hint at a complex interplay between vector insertion, the IL-2Rγc transgene and the proto-oncogene LMO-2 in this particular disease setting. In a clinical trial to treat chronic granulomatous disease, an expansion of gene-modified cells and clonal dominance was observed after transduction with a retroviral expression vector [47••]. Integration-site analysis revealed activating vector insertions into gene loci, which could have induced expanded myelopoiesis [47••]. However, the death of a patient 2.5 years after gene transfer was caused by severe sepsis rather than leukemia [48]. Preliminary analysis of transduced cells at the time of death revealed that, despite the fact that many hematopoietic cells contained the vector, expression of the transgene was almost undetectable, suggestive of gene silencing [48]. It is now well accepted that the insertion of enhancers next to oncogenes constitutes the first step toward leukemogenesis. Moreover, if the transgene is an oncogene itself, the insertion site may synergize with its oncogenic nature and lead to accelerated tumor development [49]. The IL-2Rγc transgene has been under constant scrutiny after the cases of leukemia observed in the SCID-X1 trials in Paris [43] and London [46]. This debate has been revived, as lymphomas were observed following lentiviral IL-2γc transfer into an X-SCID mouse model [50]. In two subsequent studies, transfer of IL-2γc was postulated to have only an indirect

effect on T-cell lymphomas by restoration of IL-7 signaling, which allowed progression into T-cell development [51,52].

Progress to assess genotoxicity of viral vectors The safety of gene therapy vectors for treating hematopoietic disorders can be assayed in in vivo animal models or via in vitro cultivation and selection of bone marrow (BM) cells [53]. An important parameter for the assessment of genotoxicity is the average transgene copy number per cell after transduction. It serves as an indicator of the total number of possible integrations per genome and follows the Poisson distribution for vector-dose dependence [54•,55]. For example, when a vector dose of five transducing units per cell is used, 7% of the clones acquire more than eight insertions [54•]. Most in vivo models utilize lethal or sub-lethal conditioning to eradicate the host's hematopoietic system [56]. These models select stem cells that undergo massive expansion to reconstitute hematopoiesis. The animals are then monitored for clonal aberrations and signs of leukemia, that is, direct evidence that insertional mutagenesis is linked to tumor formation. For example, a dose-escalation study using vectors encoding a fluorescent marker protein revealed leukemic expansion caused by insertional mutagenesis in a murine BMT model [57]. However, a side-by-side comparison of different vector types and architecture was lacking. For example, analogous to lentiviral vectors, the 3' promoter/ enhancer sequences in retroviral vectors can be deleted to generate self-inactivating vectors (SIN) [58]. After reverse transcription, this deletion is copied to the 5'-end, so that transcription of the transgene can only be directed from an internal promoter. In a tumor-prone knockout mouse model deficient in both p53 and retinoblastoma pathways [59], Naldini and coworkers showed that retroviral BM transduction using a murine leukemia virus (MLV)-based vector with two intact LTRs led to an earlier onset of tumors when compared with a lentiviral SIN vector with deleted LTRs and an internal promoter [60]. This was the first study to suggest that deletion of promoter/enhancer sequences decreased tumorigenic potential. Ongoing studies using lentiviral vectors that harbor strong retroviral LTRs revealed that these vectors are as prone to induce oncogenesis as MLV-based retroviral vectors, suggesting that the enhancer/ promoter sequences have a major impact on oncogenesis [E Montini and L Naldini, personal communication]. Furthermore, Kamijo et al developed a combined IL-2γc/ Arf knockout model. Arf is a tumor suppressor gene and its knockout leads to T-cell malignancies with a high frequency [61]. This genetic set up resembles a worst-case scenario for SCID-X1 [62•]. Hematopoietic cells were enriched for immature progenitor cells and transduced with a retroviral vector encoding IL-2γc and showed a high incidence of insertion-related T-cell malignancies [62•]. Interestingly, the SCID-X1 background is necessary for tumor development, suggesting either a lack of immune surveillance or massive expansion supporting leukemogenesis. Such animal models are invaluable to dissect the influences of both transgene expression and vector design.

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In contrast to the in vivo models, in vitro assays cannot be linked directly to a leukemic phenotype, unless the cell clones produced are transplanted back into mice. However, in vitro experiments can assess the influences of vector architecture in a high-throughput manner. The selection forces in such assays are either prolonged cultivation in the presence of reduced cytokine levels or replating BM cells in limiting dilution, conditions in which mock-treated cells do not survive [63]. Both cultivation schemes select for a partly transformed phenotype. The first in vitro assay was established by Copeland and coworkers [64]. During prolonged cultivation, clones arose that were characterized by insertions next to proto-oncogenes, such as Evi-1 and PRDM16. These clones showed myeloid characteristics and resembled those produced by insertional mutagenesis and clonal selection. In the first side-by-side comparison, Baum and coworkers compared the genotoxicity of LTR versus SIN vectors [65]. Purified lineage negative cells from murine BM were transduced with either vector type, expanded and replated in limiting dilution; a significant decrease in the transformation frequency by SIN vectors was observed. This assay was utilized to determine the genotoxicity of the short version of the EF1α promoter in a SIN vector backbone. This vector fell below the detection limits in the in vitro assay and showed only low levels of transactivation capacity in a reporter system [66]. In summary, various assays to test the long-term effects of genotoxic events have been established and the preclinical models were shown to be predictive for clinical trials. There is a large overlap of insertion sites between animal models and patients [41,42,47••] and, depending on disease-specific factors, both the vector type and the nature of the transgene may influence the extent of clonal dominance or malignant transformation. Global insertion site analysis provided additional data on how vector architecture alters the insertion pattern and may increase safety [67]; however, the results must be taken with caution since these high-throughput analyses are not closely linked to a phenotype.

Customized nucleases To harness the stimulatory potential of DSBs for therapeutic gene targeting, novel customizable artificial nucleases were developed. Such engineered endonucleases fall into two general classes: zinc finger nucleases (ZFNs) and homing endonucleases (HEs) [68••,69•,70]. ZFNs are artificial nucleases composed of an engineered Cys2-His2 zinc finger (ZF) domain linked to the cleavage domain of the restriction endonuclease FokI [71]. ZFs are small protein domains of approximately 30 residues, which typically contact three bases of DNA [72,73]. By varying a small number of residues, artificial ZF domains able to recognize most of the 64 possible triplets have been generated. These ZFs can then be assembled into tandem arrays to recognize a wide variety of novel target sites [74,75•,76]. Because the active form is a dimer [77], two ZFN subunits are typically designed to recognize the target sequence in a tail-to-tail conformation (Figure 2a). Since ZFN subunits usually include three or four ZFs, the full target site encompasses 18 or 24 base pairs (bp), which

is statistically large enough to define a unique site in a human genome. Studies in different cells and organisms have observed gene targeting frequencies between 1 and 39% when the DSB was introduced by engineered ZFNs [78-86]. These high targeting efficiencies in unselected cells clearly demonstrate the potential of this technology to achieve targeted integration (Figure 1b) or gene correction (Figure 1c). A second scaffold for tailored nucleases is based on HEs, such as I-SceI. HEs are sequence-specific meganucleases that recognize large target sites (12 to 24 bp). Although several hundred natural HEs have been identified [69•], the repertoire of cleavable sequences is still limited. Progress in computational modeling of the protein-DNA interface and novel efficient screening methods have established that some HE scaffolds have sufficient plasticity to allow the engineering of tailored DNA-binding domains with novel specificity [87-89]. As progress is made, custom-designed HEs are expected to represent an efficient alternative to ZFNs.

Genotoxicity associated with engineered nucleases ZFN-induced cytotoxicity is a major issue and was observed in several studies [79,80,84,90-93]. Cell death and apoptosis upon ZFN expression is most likely caused by excessive cleavage at off-target sites, suggesting imperfect target site recognition by the respective DNA-binding domains [91]. Although the full target site encompasses a total of 18 bp, a ZFN subunit is only able to recognize a 9 bp half-site, which is statistically found more than 10,000 times in the human genome. Analogous to the natural FokI enzyme [94], it is conceivable that, at high concentrations, a ZFN subunit binds to a canonical 9-bp target site as a monomer, which then becomes incorrectly activated when it forms a dimer with a second subunit that is not properly bound to the DNA. In consequence, both poor specificity of DNA-binding and unregulated DNA-cleavage at 9 bp half-sites may contribute to genotoxicity [93]. The creation of DSBs leads to the activation of a multitude of different pathways, including cell cycle checkpoint responses and appropriate DNA repair pathways [95]. In addition to the HR-based mechanism, DSBs can be repaired by non-homologous end-joining, an error-prone pathway that can lead to insertions or deletions [96]. It is increasingly clear that an inability to respond accurately to DSBs leads to genomic instability, including translocations, which have been implicated in carcinogenesis because of their ability to activate oncogenes [97]. Furthermore, integration of episomal DNA into cellular genomes is a well-known and well-documented occurrence [16•]. In particular, it has been shown that DSBs are hotspots for integration of 'episomal' DNA viruses [78,98]. If integration of episomal DNA into naturally occurring DSBs is a general phenomenon, it is conceivable that the DSB repair machinery may utilize all types of postulated non-integrating DNA vectors, including plasmid-DNA, AAV or IDLVs, as patches to seal broken chromosomes. Numerous off-target DSBs created

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by an unspecific ZFN would accordingly enhance the genotoxic potential of these vectors (Figure 2a).

Progress to assess genotoxicity of designer nucleases Since therapeutic gene targeting depends on the insertion of a DSB at a specific target site, the implementation of quantitative assays to assess genotoxicity of artificial nucleases is paramount [99]. In some studies the extent of cytotoxicity upon ZFN expression was quantified, but measuring cell death [83,84] or apoptosis [79] as global toxicity assessments provides few data on the contributing mechanisms. Urnov and coworkers demonstrated that ZFN variants with improved in vitro binding and cleavage parameters reduced cytotoxicity [85]. Unfortunately, this study did not specify the nature of the optimization and was not designed to detect surplus DSBs at sites other than the target locus, which is the major issue in ZFN-induced genotoxicity. To address this problem, assays that directly measure the levels of off-target cleavage events have been developed. Using antibodies specific for phosphorylated histone H2AX (γ-H2AX) or p53 tumor suppressor-binding protein 1, the number of ZFN-induced repair foci formed after formation of DSBs were quantified [92,93]. This quantitative assay can be used to initially characterize the specificity and the immediate genotoxicity of any artificial nuclease of interest. ZFN function should be additionally examined by taking advantage of the fact that DSBs in a cellular genome attract episomal DNA vectors, such as AAV [78]. Assessment of the AAV vector integration sites upon ectopic ZFN expression should directly provide data on DNA-cleavage specificity. However, the long-term consequences of ZFN-induced DSBs can only be investigated in vivo, and the assays developed to assess genotoxicity of retroviral vectors may become useful tools to investigate the malignant potential of cells that express ZFNs in gene correction protocols.

Safer nucleases Two general strategies can be pursued to reduce toxicity associated with engineered ZFNs: increasing the specificity of DNA-binding and improving regulation of DNA cleavage. In contrast to many natural endonucleases, ZFNs do not contain an allosteric mechanism that regulates DNA cleavage. Nonetheless, because of the dimeric nature of ZFNs, protein-engineering strategies may compensate for the missing regulation. Through substitution of interacting residues in the protein–protein interface of the ZFN dimerization domain, asymmetric dimerization variants can be created that prevent the undesirable homodimerization of two subunits [78,93]. Moreover, lowering the interaction energy between the two subunits attenuates dimerization, thus preventing ZFN dimers from forming in solution and consequently ensuring that two ZFN subunits only dimerize after both subunits have properly bound the DNA target site [93]. Both strategies have been pursued and such ZFN variants revealed significantly reduced toxicity without compromising on function [92,93]. DNA-binding specificity of the DNA-binding domain is another critical parameter. ZF-based DNA-binding domains

designed to recognize a chosen target site can be generated by modular assembly with the assistance of web-based software tools [100,101]. However, although modularly assembled DNA-binding domains can have excellent affinities [102], positional effects, cooperativity in ZF–DNA interactions, and recognition of a fourth base in the target sequence [72,73,103-107] lead to a considerable decrease in both DNA-binding affinity and specificity. These issues can only be addressed by sophisticated context-sensitive optimization methods [108-110]. As shown, ZF engineering strategies, which account for context-dependent DNAbinding effects, yield multi-finger domains that show higher activity and lower toxicity as ZFNs in human cells [91].

Safer vectors Despite the side effects observed, conventional gene therapy is a highly powerful strategy to treat monogenetic diseases. More sophisticated methods, such as gene correction or targeted insertion, may displace gene-addition-type gene therapy, but the efficacy in animal models and clinical trials of these systems must first be established. In the meantime it is important to increase the safety of the current protocols. Theoretical consideration for clinical trials suggested that each patient received one to ten stem cell clones with insertions in LMO2 [45••]. On a broader perspective, this means that the transduction of a patient's cells always leads to insertion of the vector next to a proto-oncogene and the clone formed would potentially be subjected to positive selection. Accordingly, the strategy for development must focus on the enhancer/promoter sequences within the vector. Although SIN vectors carrying a powerful retroviral enhancer can still activate neighboring genes [65], data clearly suggest that the SIN vector design provides the same efficacy as LTR-driven vectors, but demonstrates improved safety [60,65,111]. Importantly, promoter strength is not the only parameter that determines the expression level. Improving mRNA processing permits the usage of weaker promoters that are able to produce a sufficient amount of protein to correct a phenotype [112]. The use of cellular promoters is, therefore, under scrutiny as some of them can confer sufficient transgene expression, but may show less genotoxicity [66,113]. Moreover, transgene expression can be restricted to a certain target cell population when using tissue-specific promoters. This capability was realized in a clinical trial for thalassemic disease, where transgene expression was regulated by the β-globin locus control region [114]. In addition, the SIN vector design allows insertion of insulator elements that might reduce the interaction of the enhancer with neighboring genes. For example, Nienhuis and coworkers showed that incorporation of an insulator led to reduced clonal dominance [115].

Conclusions Although the development of a malignant phenotype requires multiple cooperating steps, every genetic manipulation poses a risk, especially in stem and progenitor cells, which have high proliferative potential. In view of the adverse events in the X-SCID gene therapy trial [43,46], prevention of genotoxic side effects is paramount

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for human gene therapy and safer vectors for clinical trials are under development. SIN vectors are expected to eventually replace LTR vectors because of their improved safety profile. The usage of cellular instead of viral promoters should increase safety and ensure an expression pattern that resembles the physiological expression characteristics more closely. Conservation of temporal and tissue-specific gene expression is expected to ultimately be achieved by targeted strategies. Therapeutic gene targeting represents the gold standard, but the technology is still in its infancy and refinements regarding delivery, efficacy, and safety must be made. Since this strategy depends on the stimulation of HR through the creation of a DSB by specifically designed nucleases, the reduction of off-target effects and the development of appropriate delivery tools are vital. A preclinical evaluation of this promising strategy in a relevant animal disease model should address both the safety and the efficacy of therapeutic gene targeting. Quantitative assays to assess these parameters are therefore indispensable in order to bring the next generation of gene therapy tools into the clinic.

Acknowledgments We thank Matthew D Weitzman, Boris Fehse, Ute Modlich, and Christopher Baum for helpful suggestions and critical reading of the manuscript. "TreatID" (JB), a grant of the German Federal Ministry of Education and Research, and grants CA311/1 and CA311/2 (TC) of the German Research Foundation, has supported our own work mentioned throughout this review.

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