HUMAN GENE THERAPY METHODS 24:329–332 (December 2013) ª Mary Ann Liebert, Inc. DOI: 10.1089/hgtb.2013.166
Commentary
Optimizing Delivery and Expression of Designer Nucleases for Genome Engineering Marinee K. Chuah1,2 and Thierry VandenDriessche1,2
Abstract
Genome engineering can be accomplished by designer nucleases. They are specifically designed to cleave double-stranded DNA at the desired target locus. This double-strand break subsequently engages the DNA repair pathway through nonhomologous end-joining (NHEJ), resulting in either gene disruption or gene repair. Alternatively, the presence of homologous donor DNA allows for targeted integration of this exogenous donor DNA in this target locus through homology-directed DNA repair. The key bottleneck in genome engineering relates to the delivery and expression of the designer nucleases. One of the most attractive vector platforms for genome engineering is based on integration-defective lentiviral vectors (IDLVs). The intrinsic episomal nature of IDLVs is well suited to ensure transient expression of designer nucleases and minimize potential risks associated with their sustained expression. Unfortunately, their expression is compromised because of epigenetic silencing that interferes with the transcriptional competence of IDLVs. In this issue, Pelascini and colleagues now showed that this bottleneck could be overcome by interfering with chromatin remodeling using histone deacetylase (HDAC) inhibitors. HDAC inhibition restored expression of designer nucleases from IDLVs and rescued their ability to achieve efficient targeted gene disruption by NHEJ comparable with that achieved with bona fide integrating lentiviral vectors. This study has implications for the ex vivo use of IDLVs for gene repair and gene targeting.
T
he ability to precisely and efficiently modify the human genome could lead to the development of novel and safer ways of treating human genetic and complex diseases. This type of ‘‘genome engineering’’ typically requires designer nucleases that can be redirected to cleave DNA at a specific nucleotide sequence (reviewed by Gaj et al., 2013). Such a double-strand DNA break (DSB) can be edited using the cellular DNA repair machinery by nonhomologous endjoining (NHEJ) (Fig. 1). NHEJ is an error-prone mechanism that typically results in the insertion or deletion of nucleotides (the so-called indels) at the target locus that results in gene inactivation. Alternatively, NHEJ could actually also result in gene correction by, for instance, restoring the reading frame of a mutant allele or replacing the mutated nucleotide with its wild-type equivalent. When a donor DNA template is provided that is homologous to the target locus, it allows for the targeted genomic integration into that locus by homologydirected repair (HDR). The presence of a DSB in the target locus increases the efficiency of homologous recombination with several orders of magnitude compared with conventional homologous recombination in the absence of DSB.
Consequently, this enables genome engineering in primary cells for potential clinical applications. Targeted integration can result in the repair or replacement of a defective gene by its wild-type counterpart. Alternatively, it can be exploited to achieve targeted integration of therapeutic genes into the socalled validated ‘‘safe harbor’’ loci (i.e., CCR5 and AAVS1 loci) (Lombardo et al., 2011). These loci exhibit an intrinsic reduced risk of insertional oncogenesis since they are distal from any known oncogenes or tumor suppressor genes. Several complementary designer nuclease technologies have been developed that allow for precise site-specific DNA cleavage (reviewed by Gaj et al., 2013). They include proteinbased genome engineering technologies such as zinc-finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), homing endonucleases or meganucleases (MNs), chemical endonucleases, and the more recently developed RNA-based clustered regularly interspaced small palindromic repeats (CRISPR)/Cas9 system (Gaj et al., 2013; Mali et al., 2013). Ideally, these designer nucleases allow for targeted gene editing or integration, though the so-called off-target effects have also been reported. These off-target
1 Department of Gene Therapy & Regenerative Medicine, Faculty of Medicine & Pharmacy, Free University of Brussels, Brussels B-1090, Belgium. 2 Department of Cardiovascular Sciences, Center for Molecular & Vascular Biology, University of Leuven, B-3000 Leuven, Belgium.
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FIG. 1. A pair of zinc finger nucleases (ZFN1 and ZFN2) can be delivered to the target cells using integration-defective lentiviral vectors (IDLVs). Their expression from the episomal IDLVs can be increased using epigenetic modifiers, particularly histone deacetylase (HDAC) inhibitors (e.g., trichostatin: TSA; vorinostat: SAHA). These HDAC inhibitors likely induce chromatin remodeling and relief the transcriptional repression. The ZFN1 and ZFN2 bind as heterodimers on the specific target DNA sequence and catalyze a double-strand break (DSB) because of the Fok1 nuclease that is linked to the ZF domains. Subsequently, the cellular machinery attempts to repair this DSB through NHEJ. This is an error-prone process that typically results in insertions and deletion of nucleotides at the DSB site. The increased ZFN expression induced by HDAC inhibitors ultimately results in an increased gene targeting efficiency at this target locus. integrations depend, at least in part, on the type of designer nuclease and the target locus (Cradick et al., 2013; Fu et al., 2013; Grau et al., 2013; Sander et al., 2013). The efficiency of targeted mutagenesis by NHEJ or HDR by homologous recombination also depends on epigenetic factors such as the chromatin configuration and/or methylation status of the target site (Daboussi et al., 2012; van Rensburg et al., 2013). One of the main challenges related to the use of these designer nucleases is their delivery and expression into the appropriate clinically relevant target cells. Both nonviral transfection and viral vectors have been employed to deliver these designer nucleases into cells. In vitro transfection of cell lines allows for a rapid validation of these designer nucleases. Electroporation has been considered to deliver designer nucleases into primary cells such as T cells or hematopoietic stem/progenitor cells (HSCs) (Holt et al., 2010; Daboussi et al., 2012). Unfortunately, this approach often results in high mortality caused primarily by DNA-related toxicity. However, toxicity could be substantially reduced by delivering the corresponding designer nucleases as mRNA instead (Carbery et al., 2010; Torikai et al., 2012). Nevertheless, for HDR, a DNA donor template would still need to be provided. One additional advantage of mRNA delivery is that it ensures that the corresponding designer nuclease is only expressed short-term, which overcomes concerns associated with their stable expression. In particular, prolonged expression of a given designer nuclease may increase the
CHUAH AND VANDENDRIESSCHE likelihood of off-target integrations, which in turn increases the risk of insertional oncogenesis. To overcome some of the limitations of nonviral transfection, adenoviral (Ad), adeno-associated viral (AAV), and lentiviral vectors (LV) have been employed to deliver designer nucleases. In particular, Ad vectors have been used to deliver ZFNs into various primary cells, including human T cells, HSCs, and primary epithelial stem cells (Perez et al., 2008; Coluccio et al., 2013; Hofer et al., 2013; Li et al., 2013). Ad vectors are also well suited for TALEN gene delivery in transformed and nontransformed cells (Holkers et al., 2013). Most importantly, the Ad platform is currently being explored in clinical trials to specifically inactivate the CCR5 receptor in T cells in order to render them resistant to HIV infection (Perez et al., 2008; Maier et al., 2013). AAV vectors have been employed for both genome editing in vitro and for liver-directed delivery of ZFNs (Li et al., 2011; Ha¨ndel et al., 2012; Anguela et al., 2013). ZFNs were able to efficiently induce DSBs when delivered directly to the mouse liver. When codelivered with an appropriately designed gene-targeting vector, they could stimulate gene replacement through both homology-directed and homologyindependent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to achieve phenotypic correction of the bleeding phenotype in hemophilia B mice. This raised the possibility of in vivo genome editing as a viable strategy for the treatment of genetic disease. However, in this case the long-term consequences of prolonged overexpression of ZFNs in vivo after AAV gene transfer are not fully understood as they may potentially evoke immune reactions and/or result in off-target integrations. Ultimately a transient expression platform may still be preferred to overcome some of these potential safety concerns. One of the most promising vectors for delivery of designer nucleases is the LV platform, particularly integrationdefective LVs (IDLVs). These IDLVs can be generated using conventional LV production methods, whereby the gag-pol helper construct encodes a defective integrase harboring a D64V substitution (Ya´n˜ez-Mun˜oz et al., 2006; Ma´trai et al., 2010, 2011). The class I D64V mutation in the integrase catalytic site substantially reduces integration (up to 1,000-fold) without compromising other steps during LV transduction. IDLVs can support expression of the gene of interest from the nonintegrated episomal forms. This episomal DNA is progressively lost in actively dividing cells, resulting in transient expression. Given their episomal characteristics, IDLVs are well suited for delivery of designer nucleases. IDLVs have been validated for delivery of ZFNs and MNs and have been used for targeted mutagenesis by NHEJ and targeted integration into ‘‘safe harbor’’ loci in cell lines and in primary cells (Lombardo et al., 2007; Izmiryan et al., 2011; Provasi et al., 2012; Coluccio et al., 2013). Nevertheless, IDLVs containing TALENs undergo intragenic recombination because of the repetitive nature of the TALE repeats, which compromises their integrity and stability (Holkers et al., 2013). Although IDLVs can be used to express designer nucleases, their transcription competence is compromised compared with bona fide integrating LVs. This typically results in a lower expression of the gene of interest. This is likely caused by an epigenetic effect that influence chromatin
OPTIMIZING DESIGNER NUCLEASES FOR GENOME ENGINEERING remodeling of the episomal IDLV genomes. Episomal DNA can adopt a chromatin-like configuration that is prone to epigenetic regulation involving histone modifications (Pelascini et al., 2013a). In this issue, Pelascini et al. (2013b) restored the expression of ZFNs from IDLVs by interfering with this chromatin remodeling process and inhibiting histone deacetylases (HDACs) (Fig. 1). HDACs contribute to chromatin condensation and gene repression by catalyzing the removal of acetyl groups from specific lysine residues in the core histones. In particular, using broad-spectrum HDAC inhibitors that were approved by the U.S. Food and Drug Administration, such as trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA or vorinostat), they demonstrated that ZFN expression levels driven from IDLVs could be induced. Subsequently, this resulted in efficient targeted mutagenesis at the desired locus by NHEJ at levels that were comparable to those obtained by bona fide integrating LVs. Proof of concept was established at an endogenous gene locus (i.e., HPRT) or a synthetic exogenously administered transgene (i.e., GFP) in human myoblasts or HeLa cells, respectively. This is a particularly relevant finding that could be useful for basic studies that rely on gene inactivation or gene repair. Moreover, it may pave the way toward more efficient ex vivo targeted gene inactivation approaches in patient-derived progenitor/stem cells to derive autologous cellular grafts for treating acquired or dominant disorders. However, future studies are required to validate this in a clinically relevant disease model. The findings presented by Pelascini et al. (2013b) describe a new approach to improve the performance of IDLV for genome engineering. Consequently, this study may also have broad implications to enhance expression from the IDLV vector platform of other designer nucleases (i.e., TALENs, MNs, or CRISPR/ Cas9), transposases (Ma´te´s et al., 2009; Staunstrup et al., 2009; Vink et al., 2009; Moldt et al., 2011), or recombinases (Moldt et al., 2008). Could the HDAC inhibitors have resulted in an increased gene disruption by altering the accessibility of the target locus itself through chromatin remodeling? This hypothesis would be consistent with the recent demonstration that epigenetic modulation and accessibility of the target site affects the efficiency of targeted mutagenesis by NHEJ (Daboussi et al., 2012; van Rensburg et al., 2013). However, the frequencies of gene disruption by bona fide integrating LVs were similar in cells that were treated with HDAC inhibitors compared with nontreated controls. Hence, it is unlikely that the epigenetic effect mediated by SAHA or TSA could be attributed to a direct effect on the target locus itself (i.e., HPRT, GFP). This suggests that chromatin remodeling did not play a significant role in modulating the accessibility of these selected target loci to the corresponding ZFNs. Presumably, the HPRT and GFP target loci are already located in regions with a relatively open chromatin configuration, lessening a possible direct impact of HDAC inhibitors on the accessibility of these loci. It is conceivable, however, that HDAC inhibitors may enhance targeting efficiency in genes located within condensed heterochromatic regions not only by increasing ZFN expression levels from the IDLV templates but also by directly modulating the accessibility of target loci themselves, which requires further studies. There are still several outstanding questions that would need to be addressed. It is known that the efficiency of HDR
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is correlated with that of targeted mutagenesis by NHEJ at a given locus (Daboussi et al., 2012). It is therefore likely that these HDAC modifiers may also enhance targeted gene replacement or gene integration into ‘‘safe harbor’’ loci, which warrants further investigation. One potential concern associated with the use of HDAC inhibitors relates to their potential toxicity. Although HDAC inhibitors are entering phase I and II clinical trials, they target primarily life-threatening diseases like cancer. However, it is somewhat reassuring that these broad-spectrum HDAC inhibitors did not increase apoptosis nor did they impair targeted mutagenesis by NHEJ (Pelascini et al., 2013b). Nevertheless, these pan-HDAC inhibitors may induce more subtle pleiotropic effects that may interfere with normal cell function without necessarily causing apoptosis. This is particularly relevant in the context of IDLVmediated gene targeting of stem/progenitor cells. It is therefore important to exclude that the selected HDAC inhibitors would interfere with the proliferation and/or differentiation of the targeted stem/progenitor cell population. Ultimately, it may be desirable to use more specific inhibitors that selectively inhibit specific HDAC isoforms instead of using pan-HDAC inhibitors (Di Micco et al., 2013). A multipronged approach will likely be required to augment the efficiency of genome engineering by modifying the intrinsic properties of the designer nucleases themselves and optimizing the extrinsic conditions that favor their expression and stability. References Anguela, X.M., Sharma, R., Doyon, Y., et al. (2013). Robust ZFNmediated genome editing in adult hemophilic mice. Blood 122, 3283–3287. Carbery, I.D., Ji, D., Harrington, A., et al. (2010). Targeted genome modification in mice using zinc-finger nucleases. Genetics 186, 451–459. Coluccio, A., Miselli, F., Lombardo, A., et al. (2013). Targeted gene addition in human epithelial stem cells by zinc-finger nuclease-mediated homologous recombination. Mol. Ther. 21, 1695–1704. Cradick, T.J., Fine, E.J., Antico, C.J., and Bao, G. (2013). CRISPR/ Cas9 systems targeting b-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592. Daboussi, F., Zaslavskiy, M., Poirot, L., et al. (2012). Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases. Nucleic Acids Res 40, 6367–6379. Di Micco, S., Chini, M.G., Terracciano, S., et al. (2013). Structural basis for the design and synthesis of selective HDAC inhibitors. Bioorg. Med. Chem. 21, 3795–3807. Fu, Y., Foden, J.A., Khayter, C., et al. (2013). High-frequency offtarget mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826. Gaj, T., Gersbach, C.A., and Barbas, C.F., 3rd. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405. Grau, J., Boch, J., and Posch, S. (2013). TALENoffer: genomewide TALEN off-target prediction. Bioinformatics 29, 2931– 2932. Ha¨ndel, E.M., Gellhaus, K., Khan, K., et al. (2012). Versatile and efficient genome editing in human cells by combining zincfinger nucleases with adeno-associated viral vectors. Hum. Gene Ther. 23, 321–329.
332 Hofer, U., Henley, J.E., Exline, C.M., et al. (2013). Pre-clinical modeling of CCR5 knockout in human hematopoietic stem cells by zinc finger nucleases using humanized mice. J. Infect. Dis. 208 Suppl 2, S160–S164. Holkers, M., Maggio, I., Liu, J., et al. (2013). Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells. Nucleic Acids Res. 41, e63. Holt, N., Wang, J., Kim, K., et al. (2010). Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat. Biotechnol. 28, 839–847. Izmiryan, A., Basmaciogullari, S., Henry, A., et al. (2011). Efficient gene targeting mediated by a lentiviral vector-associated meganuclease. Nucleic Acids Res. 39, 7610–7619. Li, H., Haurigot, V., Doyon, Y., et al. (2011). In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221. Li, L., Krymskaya, L., Wang, J., et al. (2013). Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases. Mol. Ther. 21, 1259–1269. Lombardo, A., Genovese, P., Beausejour, C.M., et al. (2007). Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat. Biotechnol. 25, 1298–1306. Lombardo, A., Cesana, D., Genovese, P., et al. (2011). Sitespecific integration and tailoring of cassette design for sustainable gene transfer. Nat. Methods 8, 861–869. Maier, D.A., Brennan, A.L., Jiang, S., et al. (2013). Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5. Hum. Gene Ther. 24, 245–258. Mali, P., Yang, L., Esvelt, K.M., et al. (2013). RNA-guided human genome engineering via Cas9. Science 339, 823–826. Ma´te´s, L., Chuah, M.K., Belay, E., et al. (2009). Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 41, 753–761. Ma´trai, J., Chuah, M.K., and VandenDriessche, T. (2010). Recent advances in lentiviral vector development and applications. Mol. Ther. 18, 477–490. Ma´trai, J., Cantore, A., Bartholomae, C.C., et al. (2011). Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk. Hepatology 53, 1696–1707. Moldt, B., Staunstrup, N.H., Jakobsen, M., et al. (2008). Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase. BMC Biotechnol. 8, e60. Moldt, B., Miskey, C., Staunstrup, N.H., et al. (2011). Comparative genomic integration profiling of Sleeping Beauty transposons
CHUAH AND VANDENDRIESSCHE mobilized with high efficacy from integrase-defective lentiviral vectors in primary human cells. Mol. Ther. 19, 1499–1510. Pelascini, L.P., Janssen, J.M., and Gonc¸alves, M.A.F.V. (2013a). Histone deacetylase inhibition activates transgene expression from integration-defective lentiviral vectors in dividing and non-dividing cells. Hum. Gene Ther. 24, 78–96. Pelascini, L., Maggio, I., Liu, J., et al. (2013b) Histone deacetylase inhibition rescues gene knockout levels achieved with integrase-defective lentiviral vectors encoding zinc-finger nucleases. Hum. Gene Ther. [Epub ahead of print] Perez, E.E., Wang, J., Miller, J.C., et al. (2008). Establishment of HIV-1 resistance in CD4 + T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816. Provasi, E., Genovese, P., Lombardo, A., et al. (2012). Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat. Med. 18, 807–815. Sander, J.D., Ramirez, C.L., Linder, S.J., et al. (2013). In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites. Nucleic Acids Res. 41, e181. Staunstrup, N.H., Moldt, B., Ma´te´s, L., et al. (2009). Hybrid lentivirus-transposon vectors with a random integration profile in human cells. Mol. Ther. 17, 1205–1214. Torikai, H., Reik, A., Liu, P.Q., et al. (2012). A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood 119, 5697–5705. van Rensburg, R., Beyer, I., Yao, X.Y., et al. (2013). Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells. Gene Ther. 20, 201–214. Vink, C.A., Gaspar, H.B., Gabriel, R., et al. (2009). Sleeping beauty transposition from nonintegrating lentivirus. Mol. Ther. 17, 1197–1204. Ya´n˜ez-Mun˜oz, R.J., Balaggan, K.S., MacNeil, A., et al. (2006). Effective gene therapy with nonintegrating lentiviral vectors. Nat. Med. 12, 348–353.
Address correspondence to: Dr. Marinee K. Chuah Department of Gene Therapy & Regenerative Medicine Faculty of Medicine & Pharmacy Free University of Brussels (VUB) Building D, Room D306 Laarbeeklaan 103 B-1090 Brussels Belgium E-mail: [email protected]
Commentary
Optimizing Delivery and Expression of Designer Nucleases for Genome Engineering Marinee K. Chuah1,2 and Thierry VandenDriessche1,2
Abstract
Genome engineering can be accomplished by designer nucleases. They are specifically designed to cleave double-stranded DNA at the desired target locus. This double-strand break subsequently engages the DNA repair pathway through nonhomologous end-joining (NHEJ), resulting in either gene disruption or gene repair. Alternatively, the presence of homologous donor DNA allows for targeted integration of this exogenous donor DNA in this target locus through homology-directed DNA repair. The key bottleneck in genome engineering relates to the delivery and expression of the designer nucleases. One of the most attractive vector platforms for genome engineering is based on integration-defective lentiviral vectors (IDLVs). The intrinsic episomal nature of IDLVs is well suited to ensure transient expression of designer nucleases and minimize potential risks associated with their sustained expression. Unfortunately, their expression is compromised because of epigenetic silencing that interferes with the transcriptional competence of IDLVs. In this issue, Pelascini and colleagues now showed that this bottleneck could be overcome by interfering with chromatin remodeling using histone deacetylase (HDAC) inhibitors. HDAC inhibition restored expression of designer nucleases from IDLVs and rescued their ability to achieve efficient targeted gene disruption by NHEJ comparable with that achieved with bona fide integrating lentiviral vectors. This study has implications for the ex vivo use of IDLVs for gene repair and gene targeting.
T
he ability to precisely and efficiently modify the human genome could lead to the development of novel and safer ways of treating human genetic and complex diseases. This type of ‘‘genome engineering’’ typically requires designer nucleases that can be redirected to cleave DNA at a specific nucleotide sequence (reviewed by Gaj et al., 2013). Such a double-strand DNA break (DSB) can be edited using the cellular DNA repair machinery by nonhomologous endjoining (NHEJ) (Fig. 1). NHEJ is an error-prone mechanism that typically results in the insertion or deletion of nucleotides (the so-called indels) at the target locus that results in gene inactivation. Alternatively, NHEJ could actually also result in gene correction by, for instance, restoring the reading frame of a mutant allele or replacing the mutated nucleotide with its wild-type equivalent. When a donor DNA template is provided that is homologous to the target locus, it allows for the targeted genomic integration into that locus by homologydirected repair (HDR). The presence of a DSB in the target locus increases the efficiency of homologous recombination with several orders of magnitude compared with conventional homologous recombination in the absence of DSB.
Consequently, this enables genome engineering in primary cells for potential clinical applications. Targeted integration can result in the repair or replacement of a defective gene by its wild-type counterpart. Alternatively, it can be exploited to achieve targeted integration of therapeutic genes into the socalled validated ‘‘safe harbor’’ loci (i.e., CCR5 and AAVS1 loci) (Lombardo et al., 2011). These loci exhibit an intrinsic reduced risk of insertional oncogenesis since they are distal from any known oncogenes or tumor suppressor genes. Several complementary designer nuclease technologies have been developed that allow for precise site-specific DNA cleavage (reviewed by Gaj et al., 2013). They include proteinbased genome engineering technologies such as zinc-finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), homing endonucleases or meganucleases (MNs), chemical endonucleases, and the more recently developed RNA-based clustered regularly interspaced small palindromic repeats (CRISPR)/Cas9 system (Gaj et al., 2013; Mali et al., 2013). Ideally, these designer nucleases allow for targeted gene editing or integration, though the so-called off-target effects have also been reported. These off-target
1 Department of Gene Therapy & Regenerative Medicine, Faculty of Medicine & Pharmacy, Free University of Brussels, Brussels B-1090, Belgium. 2 Department of Cardiovascular Sciences, Center for Molecular & Vascular Biology, University of Leuven, B-3000 Leuven, Belgium.
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FIG. 1. A pair of zinc finger nucleases (ZFN1 and ZFN2) can be delivered to the target cells using integration-defective lentiviral vectors (IDLVs). Their expression from the episomal IDLVs can be increased using epigenetic modifiers, particularly histone deacetylase (HDAC) inhibitors (e.g., trichostatin: TSA; vorinostat: SAHA). These HDAC inhibitors likely induce chromatin remodeling and relief the transcriptional repression. The ZFN1 and ZFN2 bind as heterodimers on the specific target DNA sequence and catalyze a double-strand break (DSB) because of the Fok1 nuclease that is linked to the ZF domains. Subsequently, the cellular machinery attempts to repair this DSB through NHEJ. This is an error-prone process that typically results in insertions and deletion of nucleotides at the DSB site. The increased ZFN expression induced by HDAC inhibitors ultimately results in an increased gene targeting efficiency at this target locus. integrations depend, at least in part, on the type of designer nuclease and the target locus (Cradick et al., 2013; Fu et al., 2013; Grau et al., 2013; Sander et al., 2013). The efficiency of targeted mutagenesis by NHEJ or HDR by homologous recombination also depends on epigenetic factors such as the chromatin configuration and/or methylation status of the target site (Daboussi et al., 2012; van Rensburg et al., 2013). One of the main challenges related to the use of these designer nucleases is their delivery and expression into the appropriate clinically relevant target cells. Both nonviral transfection and viral vectors have been employed to deliver these designer nucleases into cells. In vitro transfection of cell lines allows for a rapid validation of these designer nucleases. Electroporation has been considered to deliver designer nucleases into primary cells such as T cells or hematopoietic stem/progenitor cells (HSCs) (Holt et al., 2010; Daboussi et al., 2012). Unfortunately, this approach often results in high mortality caused primarily by DNA-related toxicity. However, toxicity could be substantially reduced by delivering the corresponding designer nucleases as mRNA instead (Carbery et al., 2010; Torikai et al., 2012). Nevertheless, for HDR, a DNA donor template would still need to be provided. One additional advantage of mRNA delivery is that it ensures that the corresponding designer nuclease is only expressed short-term, which overcomes concerns associated with their stable expression. In particular, prolonged expression of a given designer nuclease may increase the
CHUAH AND VANDENDRIESSCHE likelihood of off-target integrations, which in turn increases the risk of insertional oncogenesis. To overcome some of the limitations of nonviral transfection, adenoviral (Ad), adeno-associated viral (AAV), and lentiviral vectors (LV) have been employed to deliver designer nucleases. In particular, Ad vectors have been used to deliver ZFNs into various primary cells, including human T cells, HSCs, and primary epithelial stem cells (Perez et al., 2008; Coluccio et al., 2013; Hofer et al., 2013; Li et al., 2013). Ad vectors are also well suited for TALEN gene delivery in transformed and nontransformed cells (Holkers et al., 2013). Most importantly, the Ad platform is currently being explored in clinical trials to specifically inactivate the CCR5 receptor in T cells in order to render them resistant to HIV infection (Perez et al., 2008; Maier et al., 2013). AAV vectors have been employed for both genome editing in vitro and for liver-directed delivery of ZFNs (Li et al., 2011; Ha¨ndel et al., 2012; Anguela et al., 2013). ZFNs were able to efficiently induce DSBs when delivered directly to the mouse liver. When codelivered with an appropriately designed gene-targeting vector, they could stimulate gene replacement through both homology-directed and homologyindependent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to achieve phenotypic correction of the bleeding phenotype in hemophilia B mice. This raised the possibility of in vivo genome editing as a viable strategy for the treatment of genetic disease. However, in this case the long-term consequences of prolonged overexpression of ZFNs in vivo after AAV gene transfer are not fully understood as they may potentially evoke immune reactions and/or result in off-target integrations. Ultimately a transient expression platform may still be preferred to overcome some of these potential safety concerns. One of the most promising vectors for delivery of designer nucleases is the LV platform, particularly integrationdefective LVs (IDLVs). These IDLVs can be generated using conventional LV production methods, whereby the gag-pol helper construct encodes a defective integrase harboring a D64V substitution (Ya´n˜ez-Mun˜oz et al., 2006; Ma´trai et al., 2010, 2011). The class I D64V mutation in the integrase catalytic site substantially reduces integration (up to 1,000-fold) without compromising other steps during LV transduction. IDLVs can support expression of the gene of interest from the nonintegrated episomal forms. This episomal DNA is progressively lost in actively dividing cells, resulting in transient expression. Given their episomal characteristics, IDLVs are well suited for delivery of designer nucleases. IDLVs have been validated for delivery of ZFNs and MNs and have been used for targeted mutagenesis by NHEJ and targeted integration into ‘‘safe harbor’’ loci in cell lines and in primary cells (Lombardo et al., 2007; Izmiryan et al., 2011; Provasi et al., 2012; Coluccio et al., 2013). Nevertheless, IDLVs containing TALENs undergo intragenic recombination because of the repetitive nature of the TALE repeats, which compromises their integrity and stability (Holkers et al., 2013). Although IDLVs can be used to express designer nucleases, their transcription competence is compromised compared with bona fide integrating LVs. This typically results in a lower expression of the gene of interest. This is likely caused by an epigenetic effect that influence chromatin
OPTIMIZING DESIGNER NUCLEASES FOR GENOME ENGINEERING remodeling of the episomal IDLV genomes. Episomal DNA can adopt a chromatin-like configuration that is prone to epigenetic regulation involving histone modifications (Pelascini et al., 2013a). In this issue, Pelascini et al. (2013b) restored the expression of ZFNs from IDLVs by interfering with this chromatin remodeling process and inhibiting histone deacetylases (HDACs) (Fig. 1). HDACs contribute to chromatin condensation and gene repression by catalyzing the removal of acetyl groups from specific lysine residues in the core histones. In particular, using broad-spectrum HDAC inhibitors that were approved by the U.S. Food and Drug Administration, such as trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA or vorinostat), they demonstrated that ZFN expression levels driven from IDLVs could be induced. Subsequently, this resulted in efficient targeted mutagenesis at the desired locus by NHEJ at levels that were comparable to those obtained by bona fide integrating LVs. Proof of concept was established at an endogenous gene locus (i.e., HPRT) or a synthetic exogenously administered transgene (i.e., GFP) in human myoblasts or HeLa cells, respectively. This is a particularly relevant finding that could be useful for basic studies that rely on gene inactivation or gene repair. Moreover, it may pave the way toward more efficient ex vivo targeted gene inactivation approaches in patient-derived progenitor/stem cells to derive autologous cellular grafts for treating acquired or dominant disorders. However, future studies are required to validate this in a clinically relevant disease model. The findings presented by Pelascini et al. (2013b) describe a new approach to improve the performance of IDLV for genome engineering. Consequently, this study may also have broad implications to enhance expression from the IDLV vector platform of other designer nucleases (i.e., TALENs, MNs, or CRISPR/ Cas9), transposases (Ma´te´s et al., 2009; Staunstrup et al., 2009; Vink et al., 2009; Moldt et al., 2011), or recombinases (Moldt et al., 2008). Could the HDAC inhibitors have resulted in an increased gene disruption by altering the accessibility of the target locus itself through chromatin remodeling? This hypothesis would be consistent with the recent demonstration that epigenetic modulation and accessibility of the target site affects the efficiency of targeted mutagenesis by NHEJ (Daboussi et al., 2012; van Rensburg et al., 2013). However, the frequencies of gene disruption by bona fide integrating LVs were similar in cells that were treated with HDAC inhibitors compared with nontreated controls. Hence, it is unlikely that the epigenetic effect mediated by SAHA or TSA could be attributed to a direct effect on the target locus itself (i.e., HPRT, GFP). This suggests that chromatin remodeling did not play a significant role in modulating the accessibility of these selected target loci to the corresponding ZFNs. Presumably, the HPRT and GFP target loci are already located in regions with a relatively open chromatin configuration, lessening a possible direct impact of HDAC inhibitors on the accessibility of these loci. It is conceivable, however, that HDAC inhibitors may enhance targeting efficiency in genes located within condensed heterochromatic regions not only by increasing ZFN expression levels from the IDLV templates but also by directly modulating the accessibility of target loci themselves, which requires further studies. There are still several outstanding questions that would need to be addressed. It is known that the efficiency of HDR
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is correlated with that of targeted mutagenesis by NHEJ at a given locus (Daboussi et al., 2012). It is therefore likely that these HDAC modifiers may also enhance targeted gene replacement or gene integration into ‘‘safe harbor’’ loci, which warrants further investigation. One potential concern associated with the use of HDAC inhibitors relates to their potential toxicity. Although HDAC inhibitors are entering phase I and II clinical trials, they target primarily life-threatening diseases like cancer. However, it is somewhat reassuring that these broad-spectrum HDAC inhibitors did not increase apoptosis nor did they impair targeted mutagenesis by NHEJ (Pelascini et al., 2013b). Nevertheless, these pan-HDAC inhibitors may induce more subtle pleiotropic effects that may interfere with normal cell function without necessarily causing apoptosis. This is particularly relevant in the context of IDLVmediated gene targeting of stem/progenitor cells. It is therefore important to exclude that the selected HDAC inhibitors would interfere with the proliferation and/or differentiation of the targeted stem/progenitor cell population. Ultimately, it may be desirable to use more specific inhibitors that selectively inhibit specific HDAC isoforms instead of using pan-HDAC inhibitors (Di Micco et al., 2013). A multipronged approach will likely be required to augment the efficiency of genome engineering by modifying the intrinsic properties of the designer nucleases themselves and optimizing the extrinsic conditions that favor their expression and stability. References Anguela, X.M., Sharma, R., Doyon, Y., et al. (2013). Robust ZFNmediated genome editing in adult hemophilic mice. Blood 122, 3283–3287. Carbery, I.D., Ji, D., Harrington, A., et al. (2010). Targeted genome modification in mice using zinc-finger nucleases. Genetics 186, 451–459. Coluccio, A., Miselli, F., Lombardo, A., et al. (2013). Targeted gene addition in human epithelial stem cells by zinc-finger nuclease-mediated homologous recombination. Mol. Ther. 21, 1695–1704. Cradick, T.J., Fine, E.J., Antico, C.J., and Bao, G. (2013). CRISPR/ Cas9 systems targeting b-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592. Daboussi, F., Zaslavskiy, M., Poirot, L., et al. (2012). Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases. Nucleic Acids Res 40, 6367–6379. Di Micco, S., Chini, M.G., Terracciano, S., et al. (2013). Structural basis for the design and synthesis of selective HDAC inhibitors. Bioorg. Med. Chem. 21, 3795–3807. Fu, Y., Foden, J.A., Khayter, C., et al. (2013). High-frequency offtarget mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826. Gaj, T., Gersbach, C.A., and Barbas, C.F., 3rd. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405. Grau, J., Boch, J., and Posch, S. (2013). TALENoffer: genomewide TALEN off-target prediction. Bioinformatics 29, 2931– 2932. Ha¨ndel, E.M., Gellhaus, K., Khan, K., et al. (2012). Versatile and efficient genome editing in human cells by combining zincfinger nucleases with adeno-associated viral vectors. Hum. Gene Ther. 23, 321–329.
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Address correspondence to: Dr. Marinee K. Chuah Department of Gene Therapy & Regenerative Medicine Faculty of Medicine & Pharmacy Free University of Brussels (VUB) Building D, Room D306 Laarbeeklaan 103 B-1090 Brussels Belgium E-mail: [email protected]
