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Antiviral therapy of persistent viral infection using genome editing
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ScienceDirect Antiviral therapy of persistent viral infection using genome editing Frank Buchholz1 and Joachim Hauber2,3 Chronic viral infections are often incurable because current antiviral strategies do not target chromosomally integrated or non-replicating episomal viral genomes. The rapid development of technologies for genome editing may possibly soon allow for therapeutic targeting of viral genomes and, hence, for development of curative strategies for persistent viral infection. However, detailed investigation of different antiviral genome editing approaches recently revealed various undesired effects. In particular, the problem of frequent and swift development of resistant viruses has to be thoroughly analysed before genome editing approaches become an established option for antiviral treatment. Addresses 1 Medical Systems Biology, UCC, Medical Faculty Carl Gustav Carus, TU Dresden, Am Tatzberg 47/49, D-01307 Dresden, Germany 2 Heinrich Pette Institute – Leibniz Institute for Experimental Virology, Martinistrasse 52, D-20251 Hamburg, Germany 3 German Center for Infection Research (DZIF), Partner Site Hamburg, Germany Corresponding author: Hauber, Joachim ([email protected])
new therapeutic strategies primarily aiming at destroying virus genomes. In fact, these genome editing systems have already been successfully employed in various studies in cell cultures and small animal models to target several human pathogenic viruses, including human immunodeficiency virus (HIV) [1], hepatitis B and C virus (HBV and HCV) [2], herpesviruses (HSV, HCMV and EBV) [3–5], JC polyomavirus [6] and human papillomavirus (HPV) [7–10]; reviewed in [11–14]. Thus, genome editing represents the most direct anti-viral approach, and if proven to be safe in humans, may become a general antiviral strategy. Moreover, as a novel antiviral strategy, gene editing can also be employed to disrupt cellular genes that, for example, encode important virus receptor molecules [15,16], further expanding the possibilities for genome editing as an anti-viral approach. In this review, we contrast designer nuclease and designer recombinase genome editing technologies and provide an assessment of current prospects and challenges in the field, focussing on HIV and HBV genome editing as representative examples.
Current Opinion in Virology 2016, 20:85–91
The toolbox for gene editing
This review comes from a themed issue on Engineering for viral resistance
Current gene editing technologies differ considerably in their mode of action, namely error-prone versus error-free DNA modification (for comparison see Table 1). Obviously, this may affect the likelihood of developing resistance and subsequent viral escape.
Edited by Thomas Baumert
http://dx.doi.org/10.1016/j.coviro.2016.09.012 1879-6257/# 2016 Published by Elsevier B.V.
Introduction Once established, persistent, and particularly latent virus infection considerably hampers virus clearance from infected organisms. Consequently, although current antiviral therapies, commonly administering small molecular weight inhibitors, routinely suppress progeny formation, they may ultimately fail in virus eradication, and thus in achieving a cure. However, the advent of advanced genome editing methods, such as homing endonucleases (HE; i.e. meganucleases), zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), the CRISPR/Cas9 RNA-guided nuclease system, and engineered tyrosine recombinases (e.g. Cre variants), provide technologies for developing completely www.sciencedirect.com
Error-prone repair is characteristic of HE, ZFN, TALEN and CRISPR/Cas9 (for more detailed description of their molecular action see [17–19]). In contrast, error-free repair is typified by site-specific recombinase systems, such as Cre [20]. HE, ZFN, TALEN and CRISPR/Cas9, commonly referred to as designer nucleases [13], differ mainly in how these programmable enzymes are recruited to their DNA target site(s). HE, ZFN and TALEN interact with their particular DNA target sequences via intrinsic DNA binding domains. In the case of HE, which recognize target sites of 14–40 bp (commonly 18 bp), the DNA-binding and cleavage domains cannot be precisely separated [21]. Thus, in vitro engineering of such meganucleases to generate new binding specificities requires challenging, complex and tedious directed protein evolution technologies [22,23]. In contrast, ZFN and TALEN are characterized by a distinct modular structure, comprising artificial arrays of Current Opinion in Virology 2016, 20:85–91
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Table 1 Comparison of the main features of genome editing systems Editing system HE ZFN TALEN CRISPR/Cas T-SSR
Ease of DNA targeting
Precision of DNA repair
Dependence on host DNA repair
Multiplexing
Delivery
Typical target-site length (bp)
Typical protein size (kDa)
+ + +++
+++
+ + + +
+++
+++ ++ + + +++
18 18–36 30–40 20 34
40 45 105 164 42
HE: homing endonucleases; ZFN: zinc finger nucleases; TALEN: transcription activator-like effector nucleases; CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated; T-SSR: tyrosine-type site-specific recombinase.
DNA-binding motifs linked to the non-specific catalytic domain of the restriction enzyme FokI [17–19]. Importantly, the FokI nuclease domain must dimerize to cut DNA, therefore two ZFN or TALEN molecules are required to target a single site [24]. The respective DNA binding domains of the proteins are customized using in vitro platforms to assemble three to six zinc finger modules (one module/3 bp of target DNA) or 15–20 TALE modules (one module/1 bp of target DNA) [25]. Thus, target sites typically vary from 18–36 bp for ZFN and 30–40 bp for TALEN. Notably, targeting of longer sites generally improves the specificity of these programmable nucleases (i.e. minimizes off-target effects). Unlike HE, ZFN and TALEN, the CRISPR-associated Cas9 nuclease is recruited to a DNA site via its association with a short guide RNA (gRNA) that hybridizes to a target DNA site of about 20 bp [17,19,25]. Thus, the DNA binding specificity solely depends on RNA-DNA base pairing. This is a huge advantage for easily targeting the Cas9 nuclease to sites of interest, since it only requires constructing a specific gRNA. Furthermore, this feature allows multiplexing and the use of Cas9 nickase dimer mutants to improve specificity, and hence prevent offtarget effects [26,27]. However, upon target site recognition, all of these nucleases, HE, ZFN, TALEN and CRISPR/Cas9 alike, introduce free DNA double-strand breaks (DSBs) at the target locus as the first step in gene correction/inactivation. Since DSBs represent one of the most dangerous lesions for a cell [28], these breaks activate the intrinsic cellular error-prone non-homologous end-joining (NHEJ) repair mechanism, typically inducing an abundance of random insertions and deletions (indels) at the target locus, which usually inactivate the target gene [17,19,24]. Since indel formation cannot be controlled, NHEJ has a considerable negative impact on genomic fidelity, and therefore, particularly when targeting highly active replicating systems such as viruses, facilitates the loss of target sites and development of resistance. Tyrosine-type site-specific recombinase (T-SSR) systems, such as Cre recombinase, enable highly predictable Current Opinion in Virology 2016, 20:85–91
and accurate genome editing, since they act in an errorfree manner independently of endogenous DNA repair pathways (e.g. NHEJ) [20]. Thus, T-SSRs mediate precise DNA cleavage and ligation without the gain or loss of nucleotides. Drawbacks include the fact that recombination requires the presence of two identical target site sequences of 34 bp. When in the same orientation, their recombination results in accurate removal of the intervening sequence. Such an arrangement exists naturally in the case of integrated retroviruses (i.e. proviruses), which are flanked by identical long terminal repeat (LTR) sequences. Furthermore, similar to HE, engineering T-SSRs to have new binding specificities necessitates rather complex directed molecular evolution technologies [29–31].
Targeting episomal HBV cccDNA Hepatitis B virus (HBV) infection is a global health problem, putting 350 million infected people at risk of developing cirrhosis of the liver or hepatocellular carcinoma [32]. The HBV genome persists in infected hepatocytes as an episomal cccDNA (covalently closed circular DNA) [12]. Since cccDNA is not addressed by current therapy principles, a cure of HBV infection would require developing novel therapeutic approaches to eradicate the viral cccDNA. Obviously, genome editing technologies could be perfectly suited to provide therapeutic options targeting HBV cccDNA [12]. A series of studies have investigated targeting various cccDNA-derived sequences using ZFN [33,34], TALEN [35,36] and the CRISPR/Cas9 wildtype [37–45] or ‘nickase’ systems [46], a mutated version of Cas9 that generates a single-strand DNA break (nick) at the specific target site [26]. These studies demonstrated HBV inactivation in cultured cell lines and HBV mouse models in the short term (up to a maximum of 10 days of follow-up) and provided proof of principle for HBV treatment by genome editing technologies. To further develop these approaches towards clinical application, long-term analysis of potential viral escape is imperative. This is particularly important, since HBV genomes are characterized by considerable sequence variability. For example, at least www.sciencedirect.com
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eight distinct HBV genotypes (A–H), each differing in more than 8% of their nucleotides, are globally distributed [47].
Inactivation of the HIV co-receptor CCR5 Currently, the clinically most advanced antiviral genome editing strategy focuses on inactivating the cellular gene encoding the chemokine receptor 5 (CCR5), the major HIV-1 co-receptor for cell entry [15]. Individuals homozygous for the naturally occurring but rare CCR5 inactivating D32 allele (CCR5D32) are resistant to infection by CCR5-tropic HIV-1; this trait could be emulated in larger HIV patient cohorts by CCR5-specific gene editing [15,16]. Preclinical studies using HIV-infected humanized mice, engrafted with either human CD4+ T cells or human CD34+ hematopoietic stem cells (HSC), treated with CCR5 gene-inactivating ZFN prior to transplantation, indeed demonstrated robust antiviral responses [48,49]. These studies paved the way for a first clinical trial involving a small cohort of HIV patients (n = 12) receiving CCR5-specific ZFN-modified autologous CD4+ T cells, which demonstrated that the infusion of CCR5modified autologous CD4+ T cells is safe and feasible [50]. To develop this treatment strategy further, several clinical trials using either CCR5-gene modified CD4+ T cells or CD34+ HSC are currently being designed (summarized in [16]). Nevertheless, CCR5-specific ZFNs are not optimal for human application due to frequently observed off-target effects, resulting in potential cleavage of various unrelated gene sequences [48,51,52]. To reduce this off-target problem, CCR5-specific TALENs have recently been developed which, as opposed to ZFN, display higher target site specificity (due to recognizing larger gene sequences), and in turn, lower toxicity [53–55]. However, CCR5-specific TALENs have so far not been translated into clinical applications. Irrespective of the designer nuclease used, viral escape is likely to be a major limitation of CCR5 knockout strategies [15]. HIV infected individuals homozygous for the CCR5D32 allele can be infected with viruses using CXCR4 or alternative co-receptors for cell entry [56,57]. Moreover, following treatment with a CCR5 antagonist, CXCR4-tropic viruses may arise from a preexisting virus reservoir in some HIV infected patients [58]. In fact, allogeneic transplantation into an HIV patient using HSC from a donor homozygous for the CCR5D32 allele, upon discontinuation of antiretroviral therapy (cART), indeed resulted in a shift from predominantly CCR5-tropic HIV towards CXCR4-tropic virus, highlighting the risk of viral escape in CCR5 gene editing strategies [59]. Last but not least, ethical issues have to be considered, because the inactivation of genes in humans via designer nucleases, even in somatic cells, is seen critically by some stakeholders [60]. www.sciencedirect.com
Disruption and excision of HIV proviral DNA Stable integration of proviral DNA into the host cell genome is considered to be the major hurdle so far preventing the development of a cure for HIV/AIDS. Highly desirable would be technologies that can specifically inactivate, or even better, remove integrated viral genomes (i.e. proviruses), thereby reversing an established infection at the cellular level. A first approach that succeeded in quantitatively removing HIV proviral DNA from infected cell cultures was based on engineering an HIV-1 LTR-specific variant of Cre recombinase, called Tre, by molecular evolution [31]. Tre mediates recombination between 34 bp LTR sequences of a primary HIV-1 subtype A isolate with high specificity [61]. Moreover, Tre recombinase displayed pronounced and significant antiviral activity in humanized mice engrafted with either human CD4+ T cells or human CD34+ HSC [62]. While the Tre approach demonstrated proof of principle, it has limited application in a therapeutic setting because the Tre target sequence is not found in most HIV-1 clinical isolates. This necessitated developing another T-SSR variant (broad-range recombinase 1; Brec1) that is active against the majority of HIV-1 primary isolates (e.g. >94% of HIV-1 subtype B) [63]. Lentiviral vectormediated delivery of Brec1 into HIV patient-derived CD4+ T cell cultures led to complete inhibition of viral replication due to Brec1-mediated provirus excision. Likewise, when patient-derived CD4+ T cells were transduced with Brec1 and subsequently transplanted into immunodeficient mice, at weeks 13–18 post-transplantation plasma viral loads declined in a Brec1-dependent manner, to levels below the limit of detection (<20 HIV1 RNA copies/ml). Finally, elaborate and long-term toxicity experiments demonstrated that Brec1 does not cause any apparent cytopathic or genotoxic effects, and importantly, removes HIV-1 proviral DNA with unsurpassed precision [63]. As already discussed, in contrast to T-SSRs or meganucleases, designer nucleases (i.e. ZFN, TALEN and CRISPR/Cas) can be more easily customized to recognize novel target sites, including HIV-specific sequences. Therefore, as might be expected, a series of studies rushed into testing designer nucleases that could inactivate HIV proviral DNA in various tissue culture models. In principle, individual viral gene sequences (e.g. pol) can be targeted for provirus inactivation. Alternatively, targeting LTR-specific sites could result in excising the intervening proviral DNA. However, with endonuclease-mediated DNA cleavage, the subsequent NHEJ reaction usually favours error-prone repair at the proximal DNA lesion, as opposed to recombination between distal target sites (i.e. LTR-specific sites located at the 50 and 30 terminus of the targeted provirus). It therefore remains to Current Opinion in Virology 2016, 20:85–91
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be seen whether quantitative provirus excision by designer nucleases can actually be achieved. So far, delivery of LTR-specific ZFN or TALEN into HIV-infected cell lines has resulted in provirus excision frequencies ranging from 30% to 53% [64–66]. Also worth noting, recent testing of HIV pol-specific ZFNs, upon disruption of reverse transcriptase (RT) sequences, revealed treatment-resistant but replicationcompetent HIV [67]. Replication of such mutant virus could then be abolished by introducing additional (secondary) mutations, suggesting that multiple sequences must be targeted by endonuclease-based gene editing to prevent the emergence of resistant virus [67]. Comparably, the potential of CRISPR/Cas9 to mediate significant HIV inactivation/excision was demonstrated in HIV-1 reporter cell lines, primary CD4+ T cells, including patient-derived lymphocytes, and a transgenic mouse model [68–73]. These studies suggested that the CRISPR/Cas9 system represents a promising therapy strategy for HIV eradication. However, this notion was challenged recently by several independent studies that questioned the feasibility of this approach [74,75,76,77]. Together, the CRISPR/Cas9 studies tested >20 HIVspecific gRNAs, including gRNAs previously shown in transient transfection experiments to inhibit HIV-1 progeny formation [71]. By extending the experimental time frame, the studies demonstrated that indeed many CRISPR/Cas9-induced mutations (indels) efficiently inhibited virus replication during the initial days of Cas9/gRNA expression. Subsequently, however, frequent and accelerated viral escape was observed, even when LTR-specific gRNAs were tested [74,75,76,77]. Detailed analysis of these escape mutants revealed that the rapid development of resistant viruses is caused by CRISPR/Cas9’s mode of action, since its gene disruption function depends on error-prone NHEJ DNA repair. Thus, indels formed around the Cas9/gRNA cleavage site frequently contain subtly altered target sequences, which can no longer be recognized by the same gRNA [78]. Therefore, it is hypothesized that multiple viral DNA regions must be simultaneously targeted to overcome accelerated development of resistant HIV. Clearly, such an approach may be difficult to implement in the clinic, since it would require the simultaneous delivery of multiple gRNAs together with Cas9 endonuclease into the same HIV infected cell.
Conclusion Advanced genome editing technologies, such as HE, ZFN, TALEN, CRISPR/Cas or T-SSR, facilitate novel antiviral strategies by directly targeting viral genomes. Initial studies with these systems have indeed shown pronounced antiviral effects in various model systems. Current Opinion in Virology 2016, 20:85–91
However, in addition to more general technology-related obstacles such as potential strategy-related cytotoxicities [79], and insufficient efficiency or poor delivery into target cells [80], specifically the issue of developing resistant virus has to be addressed. Thus, prior to clinical application of antiviral gene editing, long-term in vitro and in vivo (i.e. in appropriate animal systems) analyses of the potential to trigger viral escape seem to be mandatory.
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50. Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G, Spratt SK, Surosky RT, Giedlin MA, Nichol G, Holmes MC, Gregory PD, Ando DG, Kalos M, Collman RG, Binder-Scholl G, Plesa G, Hwang WT, Levine BL, June CH: Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 2014, 370:901-910. First-in-human genome editing trial treating CD4+ T cells of HIV-infected subjects with CCR5-specific ZFN. 51. Pattanayak V, Ramirez CL, Joung JK, Liu DR: Revealing off target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods 2011, 8:765-770. Report describing the development of an in vitro selection method for revealing off-target cleavage specificities of (CCR5-specific) ZFN. 52. Gabriel R, Lombardo A, Arens A, Miller JC, Genovese P, Kaeppel C, Nowrouzi A, Bartholomae CC, Wang J, Friedman G, Holmes MC, Gregory PD, Glimm H, Schmidt M, Naldini L, von Kalle C: An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol 2011, 29:816-823. This study presents an in vivo assay for unbiased genome-wide analysis of zinc-finger nuclease off-target effects, including the analysis of CCR5ZFN specificity. 53. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ: A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 2011, 29:143-148. 54. Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T: A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 2011, 39:9283-9293. 55. Mock U, Machowicz R, Hauber I, Horn S, Abramowski P, Berdien B, Hauber J, Fehse B: mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5. Nucleic Acids Res 2015, 43:5560-5571. 56. Gray L, Churchill MJ, Keane N, Sterjovski J, Ellett AM, Purcell DF, Poumbourios P, Kol C, Wang B, Saksena NK, Wesselingh SL, Price P, French M, Gabuzda D, Gorry PR: Genetic and functional analysis of R5X4 human immunodeficiency virus type 1 envelope glycoproteins derived from two individuals homozygous for the CCR5delta32 allele. J Virol 2006, 80:36843691. 57. Henrich TJ, Hanhauser E, Hu Z, Stellbrink HJ, Noah C, Martin JN, Deeks SG, Kuritzkes DR, Pereyra F: Viremic control and viral coreceptor usage in two HIV-1-infected persons homozygous for CCR5 Delta32. AIDS 2015, 29:867-876. 58. Westby M, Lewis M, Whitcomb J, Youle M, Pozniak AL, James IT, Jenkins TM, Perros M, van der Ryst E: Emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants in a minority of HIV-1-infected patients following treatment with the CCR5 antagonist maraviroc is from a pretreatment CXCR4-using virus reservoir. J Virol 2006, 80:4909-4920. 59. Kordelas L, Verheyen J, Beelen DW, Horn PA, Heinold A, Kaiser R, Trenschel R, Schadendorf D, Dittmer U, Esser S: Shift of HIV tropism in stem-cell transplantation with CCR5 Delta32 mutation. N Engl J Med 2014, 371:880-882. A clinical case study that reports shift of HIV tropism upon transplantation with CCR5-inactivated stem-cells. 60. Kohn DB, Porteus MH, Scharenberg AM: Ethical and regulatory aspects of genome editing. Blood 2016, 127:2553-2560. 61. Sarkar I, Hauber I, Hauber J, Buchholz F: HIV-1 proviral DNA excision using an evolved recombinase. Science 2007, 316:1912-1915. First report on a tailored site-specific recombinase that quantitatively excises the HIV proviral DNA from infected cell cultures. 62. Hauber I, Hofmann-Sieber H, Chemnitz J, Dubrau D, Chusainow J, Stucka R, Hartjen P, Schambach A, Ziegler P, Hackmann K, Schrock E, Schumacher U, Lindner C, Grundhoff A, Baum C, Manz MG, Buchholz F, Hauber J: Highly significant antiviral activity of HIV-1 LTR-specific Tre-recombinase in humanized mice. PLoS Pathog 2013, 9:e1003587.
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63. Karpinski J, Hauber I, Chemnitz J, Schafer C, Paszkowski-Rogacz M, Chakraborty D, Beschorner N, Hofmann-Sieber H, Lange UC, Grundhoff A, Hackmann K, Schrock E, Abi-Ghanem J, Pisabarro MT, Surendranath V, Schambach A, Lindner C, van LJ, Hauber J, Buchholz F: Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nat Biotechnol 2016, 34:401-409. Study describing the molecular evolution and in vitro and in vivo analysis of LTR-specific broad-range recombinase 1, recognizing the vast majority of the clinically relevant HIV-1 strains and subtypes. 64. Qu X, Wang P, Ding D, Li L, Wang H, Ma L, Zhou X, Liu S, Lin S, Wang X, Zhang G, Liu S, Liu L, Wang J, Zhang F, Lu D, Zhu H: Zincfinger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells. Nucleic Acids Res 2013, 41:7771-7782. 65. Qu X, Wang P, Ding D, Wang X, Zhang G, Zhou X, Liu L, Zhu X, Zeng H, Zhu H: Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells. Mol Biol Rep 2014, 41:5819-5827. 66. Ebina H, Kanemura Y, Misawa N, Sakuma T, Kobayashi T, Yamamoto T, Koyanagi Y: A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector. PLOS ONE 2015, 10:e0120047. 67. De Silva Feelixge HS, Stone D, Pietz HL, Roychoudhury P, Greninger AL, Schiffer JT, Aubert M, Jerome KR: Detection of treatment-resistant infectious HIV after genome-directed antiviral endonuclease therapy. Antiviral Res 2016, 126:90-98. 68. Ebina H, Misawa N, Kanemura Y, Koyanagi Y: Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep 2013, 3:2510. 69. Hu W, Kaminski R, Yang F, Zhang Y, Cosentino L, Li F, Luo B, Alvarez-Carbonell D, Garcia-Mesa Y, Karn J, Mo X, Khalili K: RNAdirected gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc Natl Acad Sci USA 2014, 111:11461-11466. 70. Liao HK, Gu Y, Diaz A, Marlett J, Takahashi Y, Li M, Suzuki K, Xu R, Hishida T, Chang CJ, Esteban CR, Young J, Izpisua Belmonte JC: Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. Nat Commun 2015, 6:6413. 71. Zhu W, Lei R, Le DY, Li J, Guo F, Wainberg MA, Liang C: The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA. Retrovirology 2015, 12:22. 72. Kaminski R, Chen Y, Fischer T, Tedaldi E, Napoli A, Zhang Y, Karn J, Hu W, Khalili K: Elimination of HIV-1 genomes from human T-lymphoid cells by CRISPR/Cas9 gene editing. Sci Rep 2016, 6:22555. 73. Kaminski R, Bella R, Yin C, Otte J, Ferrante P, Gendelman HE, Li H, Booze R, Gordon J, Hu W, Khalili K: Excision of HIV-1 DNA by gene editing: a proof-of-concept in vivo study. Gene Ther 2016, 23:690-695. 74. Wang G, Zhao N, Berkhout B, Das AT: CRISPR-Cas9 can inhibit HIV-1 replication but NHEJ repair facilitates virus escape. Mol Ther 2016, 24:522-526. This study demonstrates rapid and consistent viral escape upon CRISPR/ Cas9-mediated targeting of HIV-1. 75. Wang Z, Pan Q, Gendron P, Zhu W, Guo F, Cen S, Wainberg MA, Liang C: CRISPR/Cas9-Derived mutations both inhibit HIV-1 replication and accelerate viral escape. Cell Rep 2016, 15:481489. Study reporting that HIV-1 can escape CRISPR/Cas9-mediated inhibition. 76. Ueda S, Ebina H, Kanemura Y, Misawa N, Koyanagi Y: Anti-HIV-1 potency of the CRISPR/Cas9 system insufficient to fully inhibit viral replication. Microbiol Immunol 2016, 60:483-496. This study reports the failure of CRISPR/Cas9 to quantitatively inhibit HIV1 replication due to generation of resistant virus. 77. Yoder KE, Bundschuh R: Host double strand break repair generates HIV-1 strains resistant to CRISPR/Cas9. Sci Rep 2016, 6:29530. This study demonstrates that targeting HIV-1 coding regions by CRISPR/ Cas9 frequently leads to viral escape. www.sciencedirect.com
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78. Liang C, Wainberg MA, Das AT, Berkhout B: CRISPR/Cas9: a double-edged sword when used to combat HIV infection. Retrovirology 2016, 13:37. 79. Aguirre AJ, Meyers RM, Weir BA, Vazquez F, Zhang CZ, Ben-David U, Cook A, Ha G, Harrington WF, Doshi MB, Kost-Alimova M, Gill S, Xu H, Ali LD, Jiang G, Pantel S, Lee Y, Goodale A, Cherniack AD, Oh C, Kryukov G, Cowley GS,
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Current Opinion in Virology 2016, 20:85–91
ScienceDirect Antiviral therapy of persistent viral infection using genome editing Frank Buchholz1 and Joachim Hauber2,3 Chronic viral infections are often incurable because current antiviral strategies do not target chromosomally integrated or non-replicating episomal viral genomes. The rapid development of technologies for genome editing may possibly soon allow for therapeutic targeting of viral genomes and, hence, for development of curative strategies for persistent viral infection. However, detailed investigation of different antiviral genome editing approaches recently revealed various undesired effects. In particular, the problem of frequent and swift development of resistant viruses has to be thoroughly analysed before genome editing approaches become an established option for antiviral treatment. Addresses 1 Medical Systems Biology, UCC, Medical Faculty Carl Gustav Carus, TU Dresden, Am Tatzberg 47/49, D-01307 Dresden, Germany 2 Heinrich Pette Institute – Leibniz Institute for Experimental Virology, Martinistrasse 52, D-20251 Hamburg, Germany 3 German Center for Infection Research (DZIF), Partner Site Hamburg, Germany Corresponding author: Hauber, Joachim ([email protected])
new therapeutic strategies primarily aiming at destroying virus genomes. In fact, these genome editing systems have already been successfully employed in various studies in cell cultures and small animal models to target several human pathogenic viruses, including human immunodeficiency virus (HIV) [1], hepatitis B and C virus (HBV and HCV) [2], herpesviruses (HSV, HCMV and EBV) [3–5], JC polyomavirus [6] and human papillomavirus (HPV) [7–10]; reviewed in [11–14]. Thus, genome editing represents the most direct anti-viral approach, and if proven to be safe in humans, may become a general antiviral strategy. Moreover, as a novel antiviral strategy, gene editing can also be employed to disrupt cellular genes that, for example, encode important virus receptor molecules [15,16], further expanding the possibilities for genome editing as an anti-viral approach. In this review, we contrast designer nuclease and designer recombinase genome editing technologies and provide an assessment of current prospects and challenges in the field, focussing on HIV and HBV genome editing as representative examples.
Current Opinion in Virology 2016, 20:85–91
The toolbox for gene editing
This review comes from a themed issue on Engineering for viral resistance
Current gene editing technologies differ considerably in their mode of action, namely error-prone versus error-free DNA modification (for comparison see Table 1). Obviously, this may affect the likelihood of developing resistance and subsequent viral escape.
Edited by Thomas Baumert
http://dx.doi.org/10.1016/j.coviro.2016.09.012 1879-6257/# 2016 Published by Elsevier B.V.
Introduction Once established, persistent, and particularly latent virus infection considerably hampers virus clearance from infected organisms. Consequently, although current antiviral therapies, commonly administering small molecular weight inhibitors, routinely suppress progeny formation, they may ultimately fail in virus eradication, and thus in achieving a cure. However, the advent of advanced genome editing methods, such as homing endonucleases (HE; i.e. meganucleases), zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), the CRISPR/Cas9 RNA-guided nuclease system, and engineered tyrosine recombinases (e.g. Cre variants), provide technologies for developing completely www.sciencedirect.com
Error-prone repair is characteristic of HE, ZFN, TALEN and CRISPR/Cas9 (for more detailed description of their molecular action see [17–19]). In contrast, error-free repair is typified by site-specific recombinase systems, such as Cre [20]. HE, ZFN, TALEN and CRISPR/Cas9, commonly referred to as designer nucleases [13], differ mainly in how these programmable enzymes are recruited to their DNA target site(s). HE, ZFN and TALEN interact with their particular DNA target sequences via intrinsic DNA binding domains. In the case of HE, which recognize target sites of 14–40 bp (commonly 18 bp), the DNA-binding and cleavage domains cannot be precisely separated [21]. Thus, in vitro engineering of such meganucleases to generate new binding specificities requires challenging, complex and tedious directed protein evolution technologies [22,23]. In contrast, ZFN and TALEN are characterized by a distinct modular structure, comprising artificial arrays of Current Opinion in Virology 2016, 20:85–91
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Table 1 Comparison of the main features of genome editing systems Editing system HE ZFN TALEN CRISPR/Cas T-SSR
Ease of DNA targeting
Precision of DNA repair
Dependence on host DNA repair
Multiplexing
Delivery
Typical target-site length (bp)
Typical protein size (kDa)
+ + +++
+++
+ + + +
+++
+++ ++ + + +++
18 18–36 30–40 20 34
40 45 105 164 42
HE: homing endonucleases; ZFN: zinc finger nucleases; TALEN: transcription activator-like effector nucleases; CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated; T-SSR: tyrosine-type site-specific recombinase.
DNA-binding motifs linked to the non-specific catalytic domain of the restriction enzyme FokI [17–19]. Importantly, the FokI nuclease domain must dimerize to cut DNA, therefore two ZFN or TALEN molecules are required to target a single site [24]. The respective DNA binding domains of the proteins are customized using in vitro platforms to assemble three to six zinc finger modules (one module/3 bp of target DNA) or 15–20 TALE modules (one module/1 bp of target DNA) [25]. Thus, target sites typically vary from 18–36 bp for ZFN and 30–40 bp for TALEN. Notably, targeting of longer sites generally improves the specificity of these programmable nucleases (i.e. minimizes off-target effects). Unlike HE, ZFN and TALEN, the CRISPR-associated Cas9 nuclease is recruited to a DNA site via its association with a short guide RNA (gRNA) that hybridizes to a target DNA site of about 20 bp [17,19,25]. Thus, the DNA binding specificity solely depends on RNA-DNA base pairing. This is a huge advantage for easily targeting the Cas9 nuclease to sites of interest, since it only requires constructing a specific gRNA. Furthermore, this feature allows multiplexing and the use of Cas9 nickase dimer mutants to improve specificity, and hence prevent offtarget effects [26,27]. However, upon target site recognition, all of these nucleases, HE, ZFN, TALEN and CRISPR/Cas9 alike, introduce free DNA double-strand breaks (DSBs) at the target locus as the first step in gene correction/inactivation. Since DSBs represent one of the most dangerous lesions for a cell [28], these breaks activate the intrinsic cellular error-prone non-homologous end-joining (NHEJ) repair mechanism, typically inducing an abundance of random insertions and deletions (indels) at the target locus, which usually inactivate the target gene [17,19,24]. Since indel formation cannot be controlled, NHEJ has a considerable negative impact on genomic fidelity, and therefore, particularly when targeting highly active replicating systems such as viruses, facilitates the loss of target sites and development of resistance. Tyrosine-type site-specific recombinase (T-SSR) systems, such as Cre recombinase, enable highly predictable Current Opinion in Virology 2016, 20:85–91
and accurate genome editing, since they act in an errorfree manner independently of endogenous DNA repair pathways (e.g. NHEJ) [20]. Thus, T-SSRs mediate precise DNA cleavage and ligation without the gain or loss of nucleotides. Drawbacks include the fact that recombination requires the presence of two identical target site sequences of 34 bp. When in the same orientation, their recombination results in accurate removal of the intervening sequence. Such an arrangement exists naturally in the case of integrated retroviruses (i.e. proviruses), which are flanked by identical long terminal repeat (LTR) sequences. Furthermore, similar to HE, engineering T-SSRs to have new binding specificities necessitates rather complex directed molecular evolution technologies [29–31].
Targeting episomal HBV cccDNA Hepatitis B virus (HBV) infection is a global health problem, putting 350 million infected people at risk of developing cirrhosis of the liver or hepatocellular carcinoma [32]. The HBV genome persists in infected hepatocytes as an episomal cccDNA (covalently closed circular DNA) [12]. Since cccDNA is not addressed by current therapy principles, a cure of HBV infection would require developing novel therapeutic approaches to eradicate the viral cccDNA. Obviously, genome editing technologies could be perfectly suited to provide therapeutic options targeting HBV cccDNA [12]. A series of studies have investigated targeting various cccDNA-derived sequences using ZFN [33,34], TALEN [35,36] and the CRISPR/Cas9 wildtype [37–45] or ‘nickase’ systems [46], a mutated version of Cas9 that generates a single-strand DNA break (nick) at the specific target site [26]. These studies demonstrated HBV inactivation in cultured cell lines and HBV mouse models in the short term (up to a maximum of 10 days of follow-up) and provided proof of principle for HBV treatment by genome editing technologies. To further develop these approaches towards clinical application, long-term analysis of potential viral escape is imperative. This is particularly important, since HBV genomes are characterized by considerable sequence variability. For example, at least www.sciencedirect.com
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eight distinct HBV genotypes (A–H), each differing in more than 8% of their nucleotides, are globally distributed [47].
Inactivation of the HIV co-receptor CCR5 Currently, the clinically most advanced antiviral genome editing strategy focuses on inactivating the cellular gene encoding the chemokine receptor 5 (CCR5), the major HIV-1 co-receptor for cell entry [15]. Individuals homozygous for the naturally occurring but rare CCR5 inactivating D32 allele (CCR5D32) are resistant to infection by CCR5-tropic HIV-1; this trait could be emulated in larger HIV patient cohorts by CCR5-specific gene editing [15,16]. Preclinical studies using HIV-infected humanized mice, engrafted with either human CD4+ T cells or human CD34+ hematopoietic stem cells (HSC), treated with CCR5 gene-inactivating ZFN prior to transplantation, indeed demonstrated robust antiviral responses [48,49]. These studies paved the way for a first clinical trial involving a small cohort of HIV patients (n = 12) receiving CCR5-specific ZFN-modified autologous CD4+ T cells, which demonstrated that the infusion of CCR5modified autologous CD4+ T cells is safe and feasible [50]. To develop this treatment strategy further, several clinical trials using either CCR5-gene modified CD4+ T cells or CD34+ HSC are currently being designed (summarized in [16]). Nevertheless, CCR5-specific ZFNs are not optimal for human application due to frequently observed off-target effects, resulting in potential cleavage of various unrelated gene sequences [48,51,52]. To reduce this off-target problem, CCR5-specific TALENs have recently been developed which, as opposed to ZFN, display higher target site specificity (due to recognizing larger gene sequences), and in turn, lower toxicity [53–55]. However, CCR5-specific TALENs have so far not been translated into clinical applications. Irrespective of the designer nuclease used, viral escape is likely to be a major limitation of CCR5 knockout strategies [15]. HIV infected individuals homozygous for the CCR5D32 allele can be infected with viruses using CXCR4 or alternative co-receptors for cell entry [56,57]. Moreover, following treatment with a CCR5 antagonist, CXCR4-tropic viruses may arise from a preexisting virus reservoir in some HIV infected patients [58]. In fact, allogeneic transplantation into an HIV patient using HSC from a donor homozygous for the CCR5D32 allele, upon discontinuation of antiretroviral therapy (cART), indeed resulted in a shift from predominantly CCR5-tropic HIV towards CXCR4-tropic virus, highlighting the risk of viral escape in CCR5 gene editing strategies [59]. Last but not least, ethical issues have to be considered, because the inactivation of genes in humans via designer nucleases, even in somatic cells, is seen critically by some stakeholders [60]. www.sciencedirect.com
Disruption and excision of HIV proviral DNA Stable integration of proviral DNA into the host cell genome is considered to be the major hurdle so far preventing the development of a cure for HIV/AIDS. Highly desirable would be technologies that can specifically inactivate, or even better, remove integrated viral genomes (i.e. proviruses), thereby reversing an established infection at the cellular level. A first approach that succeeded in quantitatively removing HIV proviral DNA from infected cell cultures was based on engineering an HIV-1 LTR-specific variant of Cre recombinase, called Tre, by molecular evolution [31]. Tre mediates recombination between 34 bp LTR sequences of a primary HIV-1 subtype A isolate with high specificity [61]. Moreover, Tre recombinase displayed pronounced and significant antiviral activity in humanized mice engrafted with either human CD4+ T cells or human CD34+ HSC [62]. While the Tre approach demonstrated proof of principle, it has limited application in a therapeutic setting because the Tre target sequence is not found in most HIV-1 clinical isolates. This necessitated developing another T-SSR variant (broad-range recombinase 1; Brec1) that is active against the majority of HIV-1 primary isolates (e.g. >94% of HIV-1 subtype B) [63]. Lentiviral vectormediated delivery of Brec1 into HIV patient-derived CD4+ T cell cultures led to complete inhibition of viral replication due to Brec1-mediated provirus excision. Likewise, when patient-derived CD4+ T cells were transduced with Brec1 and subsequently transplanted into immunodeficient mice, at weeks 13–18 post-transplantation plasma viral loads declined in a Brec1-dependent manner, to levels below the limit of detection (<20 HIV1 RNA copies/ml). Finally, elaborate and long-term toxicity experiments demonstrated that Brec1 does not cause any apparent cytopathic or genotoxic effects, and importantly, removes HIV-1 proviral DNA with unsurpassed precision [63]. As already discussed, in contrast to T-SSRs or meganucleases, designer nucleases (i.e. ZFN, TALEN and CRISPR/Cas) can be more easily customized to recognize novel target sites, including HIV-specific sequences. Therefore, as might be expected, a series of studies rushed into testing designer nucleases that could inactivate HIV proviral DNA in various tissue culture models. In principle, individual viral gene sequences (e.g. pol) can be targeted for provirus inactivation. Alternatively, targeting LTR-specific sites could result in excising the intervening proviral DNA. However, with endonuclease-mediated DNA cleavage, the subsequent NHEJ reaction usually favours error-prone repair at the proximal DNA lesion, as opposed to recombination between distal target sites (i.e. LTR-specific sites located at the 50 and 30 terminus of the targeted provirus). It therefore remains to Current Opinion in Virology 2016, 20:85–91
88 Engineering for viral resistance
be seen whether quantitative provirus excision by designer nucleases can actually be achieved. So far, delivery of LTR-specific ZFN or TALEN into HIV-infected cell lines has resulted in provirus excision frequencies ranging from 30% to 53% [64–66]. Also worth noting, recent testing of HIV pol-specific ZFNs, upon disruption of reverse transcriptase (RT) sequences, revealed treatment-resistant but replicationcompetent HIV [67]. Replication of such mutant virus could then be abolished by introducing additional (secondary) mutations, suggesting that multiple sequences must be targeted by endonuclease-based gene editing to prevent the emergence of resistant virus [67]. Comparably, the potential of CRISPR/Cas9 to mediate significant HIV inactivation/excision was demonstrated in HIV-1 reporter cell lines, primary CD4+ T cells, including patient-derived lymphocytes, and a transgenic mouse model [68–73]. These studies suggested that the CRISPR/Cas9 system represents a promising therapy strategy for HIV eradication. However, this notion was challenged recently by several independent studies that questioned the feasibility of this approach [74,75,76,77]. Together, the CRISPR/Cas9 studies tested >20 HIVspecific gRNAs, including gRNAs previously shown in transient transfection experiments to inhibit HIV-1 progeny formation [71]. By extending the experimental time frame, the studies demonstrated that indeed many CRISPR/Cas9-induced mutations (indels) efficiently inhibited virus replication during the initial days of Cas9/gRNA expression. Subsequently, however, frequent and accelerated viral escape was observed, even when LTR-specific gRNAs were tested [74,75,76,77]. Detailed analysis of these escape mutants revealed that the rapid development of resistant viruses is caused by CRISPR/Cas9’s mode of action, since its gene disruption function depends on error-prone NHEJ DNA repair. Thus, indels formed around the Cas9/gRNA cleavage site frequently contain subtly altered target sequences, which can no longer be recognized by the same gRNA [78]. Therefore, it is hypothesized that multiple viral DNA regions must be simultaneously targeted to overcome accelerated development of resistant HIV. Clearly, such an approach may be difficult to implement in the clinic, since it would require the simultaneous delivery of multiple gRNAs together with Cas9 endonuclease into the same HIV infected cell.
Conclusion Advanced genome editing technologies, such as HE, ZFN, TALEN, CRISPR/Cas or T-SSR, facilitate novel antiviral strategies by directly targeting viral genomes. Initial studies with these systems have indeed shown pronounced antiviral effects in various model systems. Current Opinion in Virology 2016, 20:85–91
However, in addition to more general technology-related obstacles such as potential strategy-related cytotoxicities [79], and insufficient efficiency or poor delivery into target cells [80], specifically the issue of developing resistant virus has to be addressed. Thus, prior to clinical application of antiviral gene editing, long-term in vitro and in vivo (i.e. in appropriate animal systems) analyses of the potential to trigger viral escape seem to be mandatory.
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