HSV-1-Based Amplicon Vectors: Design and Applications

Home , Amplicon

Alberto L Epstein Centre de Ge´ne´tique Mole´culaire et Cellulaire, CNRS – UMR 5534, Universite´ Claude Bernard Lyon, Villeurbanne, France

Amplicons are defective, helper-dependent herpes simplex to produce relatively large amounts of essentially helper-free virus type 1 (HSV-1)-based vectors able to convey more than amplicons, as well as to expand the host range of these 100 kbp of foreign DNA to the nucleus of mammalian cells. vectors. In this review, we will update the current know-how This unique feature make amplicons very appealing for concerning design, construction, and recent applications, as preventive or therapeutic gene transfer requiring the trans- well as the potential and current limitations, of this interesting duction of very large pieces of DNA, as well as for upstream and promising class of vectors. fundamental studies, such as functional genomics. Several Gene Therapy (2005) 12, S154–S158. doi:10.1038/ recent achievements in amplicon technology have allowed sj.gt.3302617

Keywords: HSV-1; amplicons; gene transfer

Introduction unattainable dream. Some major recent improvements in amplicon technology have now brought these potential- Amplicon vectors1 are HSV-1 particles identical to wild- ities into reality. These improvements are the develop- type HSV-1 from the structural, immunological and host- ment of production methods that generate significant range points of view, but which carry a concatemeric amounts of nontoxic-amplicon vector stocks essentially form of a DNA plasmid, named the amplicon plasmid, free of contaminant helper particles, and the advent of instead of the viral genome. An amplicon plasmid is a DNA recombination techniques allowing to produce standard Escherichia coli plasmid carrying one origin of amplicon plasmids conveying more than 100 kbp of replication (generally ori-S) and one packaging signal foreign DNA. (pac or ‘a’) from HSV-1, in addition to the transgenic sequences of interest. The major interest of amplicons as gene transfer tools stems from the fact that they carry no Generation of helper-free amplicon vectors virus genes and consequently do not induce synthesis of Classically, amplicon vectors were prepared in cells virus proteins. Therefore, these vectors are fully nontoxic transfected with the amplicon plasmid and superinfected for the infected cells and nonpathogenic for the with helper HSV-1, which supplied the full set of inoculated organisms. A second and major advantage proteins required to amplify and package the amplicon that arises from the lack of virus genes is that most of the DNA into HSV-1 particles. Figure 1 shows pA-EUA1, a 153 kbp capacity of the HSV-1 particle can be used to typical EGFP-expressing amplicon plasmid carrying a accommodate very large pieces of foreign DNA. This is multiple cloning site for introducing any transgene of undoubtedly the most outstanding property of ampli- interest. As the helper virus used was generally a cons, as there is no other viral vector system available replication-defective mutant of HSV-1, the amplicon displaying the capacity to deliver such a large amount of stocks were produced in transcomplementing cell lines.2 foreign DNA to the nuclear environment of mammalian However, the use of defective HSV-1 as helper resulted in cells. In addition to possessing a larger delivering capa- the production of helper-contaminated vector stocks, and city, amplicons can be considered as safer than defective the contaminant helper particles, even if defective, recombinant HSV-1 vectors, as much as the absence of induced significant cytotoxicity and inflammatory re- virus genes in the amplicon genome strongly reduces the sponses,3 therefore preventing their use in gene therapy risk of reactivation, complementation or recombination of vaccination protocols. To overcome these obstacles, with latent HSV-1 genomes. Contrarily, high-titre ampli- different helper systems that produce essentially helper- cons are considerably more difficult to prepare in high free vector stocks have been recently developed. The first amounts than recombinant-defective vectors. of these systems was based on the cotransfection of The possibility of using amplicons to transduce large amplicon plasmids with a set of five overlapping amounts of foreign DNA to both quiescent and dividing cosmids that supplied the full-set of transacting HSV-1 cells appeared however, and for a long-time, as an functions required to build amplicon vectors. These cosmids however lacked the pac sequences, thus avoid- Correspondence: Dr AL Epstein, Centre de Geńe´tique Molećulaire et ing packaging of the helper virus genomes that could Cellulaire, CNRS UMR 5534 – Universite´ Claude Bernard Lyon 1, Baˆt. emerge following homologous recombination between G. Mendel, 16 rue Raphae¨l Dubois, 69100 Villeurbanne, France the cosmids.4 As a consequence, these cosmids allowed HSV-1-based amplicon vectors AL Epstein S155 the other encodes the g34.5 protein. This helper virus is thus defective and can be produced only in ICP4 transcomplementing cells. The g34.5 protein is not essential in most cultured cells, but its absence renders the virus completely nonneurovirulent. Therefore, upon transfection of the amplicon plasmid to cells simulta- neously expressing Cre and ICP4, infection with HSV-1 LaLdeltaJ helper virus will supply the required virus proteins to generate amplicon stocks. As most helper genomes will lose their packaging signals in these cells, the vector stocks will be only marginally contaminated (o0.2%) with helper particles, which are otherwise defective and nonpathogenic. Furthermore, since the Cre/loxP helper system is based on infection, instead of transfection procedures, it allows serial passages of the vector stocks, and a three-step protocol has been set-up that allows to prepare as much amplicons as required for a particular purpose.7–9 Although this system is therefore well suited for large-scale production of amplicon vectors it needs further improvements as the presence Figure 1 Structure of pA-EUA1, a typical amplicon plasmid. AmpR and of contaminant helper virus, even if marginal, could be a ColE1: bacterial sequences. Ori-S: origin of HSV-1 replication. ‘a’: HSV-1 problem for some applications. packaging signal. prIE4: immediate-early 4 promoter from HSV-1. EGFP: enhanced green fluorescent protein. pA(BGH): bobine growth hormone polyadenylation site. KpnI, XhoI, SalI, ClaI, HindIII and PstI: unique Infectious transfer of very large foreign DNA restriction sites of the multiple cloning site. Thanks to their ability to package very large transgenic sequences, amplicons can be used to deliver complete genomic DNA loci, or DNA sequences that regulate chromatin structure and function or subnuclear localiza- producing amplicon vectors with only very low levels of tion. This may prove useful to design improved gene contamination with helper particles. This system was therapy vectors having advantages such as prolonged, further improved by using a safer and more efficient physiological and tissue-specific expression, generation bacterial artificial chromosome (BAC) as the source of of multiple splice variants from primary genomic HSV-1 functions, instead of the set of cosmids.5 The HSV- transcripts, or synthesis of the full set of proteins 1 helper genome carried by the BAC does not contain the required to assemble complex structures as well as viral packaging signals, it lacks at least one essential gene metabolic or signalling pathways. (encoding ICP27) and its size largely exceeds that of the Almost the entire human genome is covered by fully wild-type HSV-1 genome. These helper genomes, there- sequenced BAC contigs that can be converted into fore, cannot be packaged into newly assembled HSV-1 amplicon plasmids by introducing one copy of HSV-1 particles, and any eventual recombinant genome that ori-S and ‘a’ sequences, either by site-specific recombina- could nevertheless arise in the vector stocks will be tion or by homologous recombination in bacteria. This defective due to the absence of the gene encoding ICP27.6 very important goal has been recently achieved by Wade- Although the more recent version of these systems does Martins et al,10 who used amplicons to transduce a allow production of entirely helper-free amplicon stocks 115 kbp insert containing the complete human hypox- with titres exceeding 107 transducing units/ml, the total anthine phosphoribosyl transferase (HPRT) locus into a amount of amplicon vectors produced with this method human HPRT-deficient fibroblast cell line. The same is somewhat limited since it is based on DNA cotransfec- team also used amplicons to deliver a 135 kbp fragment tion procedures and the vector stocks produced in this carrying the human low-density lipoprotein receptor way cannot be further amplified. Therefore, while this (LDLR) into human fibroblasts derived from patients method is very well suited for producing amplicons for with familial hypercholesterolemia (FH). The infectious fundamental research or experimental gene transfer to LDLR locus was shown to express at physiological levels small laboratory animals, it appears hardly suitable for in these cells.11 Furthermore, the LDLR locus was large-scale production of amplicon vectors, as required maintained within a replicating episomal amplicon for vaccination or gene therapy approaches. vector (carrying the Epstein–Barr virus (EBV) latent A very different helper system recently developed is replicon, composed of EBNA-1 and ori-P sequences, see based on the deletion, by Cre/loxP site-specific recombi- below) and expressed for 3 months at physiological nation, of the packaging signals of the helper virus in the levels following infectious delivery to CHO cells.11 Still cells that are producing the amplicons. This system uses more recently, this group used amplicons to deliver a as helper a novel recombinant HSV-1 (named HSV-1 132 kbp sequence of genomic DNA comprising the LaLdeltaJ) that carries a unique and ectopic packaging ‘a’ CDKN2A/CDKN2B region, and demonstrated that this signal flanked by two loxP sites in parallel orientation.7 locus was correctly expressed, allowing the synthesis of This virus is Cre-sensitive and cannot be packaged in three cell-cycle regulatory proteins that are expressed Cre-expressing cells, due to efficient deletion of the from overlapping genes, utilizing alternative splicing packaging signals. In addition, HSV-1 LaLdeltaJ lacks the and promoter usage.12 These exciting experiments two genes surrounding the virus packaging signals. One illustrate the outstanding potential of amplicons as of these genes encodes the essential protein ICP4, while genomic transfer vectors.

Gene Therapy HSV-1-based amplicon vectors AL Epstein S156 Expanding the amplicon host range The studies described in the previous paragraphs were possible thanks to a third major achievement of amplicon technology: the possibility to avoid dilution of the episomic amplicon genomes in proliferating cells. As the amplicon genome does not express HSV-1 replication functions and does not integrate into the host chromosomes, it will be diluted upon cell proliferation. For this reason, amplicons were thought to be particularly well suited only for transduction into quiescent cells, like neurons, muscle cells or hepatocytes. However, it has been possible to construct amplicons that persist in proliferating cells, thanks to the introduction of ori-P and EBNA-1 genes from EBV, another member of the herpesvirus family, into the amplicon genome.13 These EBV genetic elements, which ensure vegetative maintain and segregation of EBV latent genomes in human cells, also allow maintain and segregation of episomic amplicon genomes.

Figure 2 Cultured primary rat cardiomyocytes (a), primary rat hepato- Applications cytes (b), and human glioblastoma Gli36 cells (c) were infected with a After the initial demonstration that they could be used to GFP-expressing amplicon vector (generated using pA-EUA1 as the deliver foreign DNA,14 amplicons have been widely and amplicon plasmid), at a multiplicity of infection of 5 TU/cell. (d) In vivo successfully used to express genes of neurobiological,15 labelling of rat hippocampal pyramidal neurons infected with the same immunological16 or therapeutical17 interest in cultured amplicon vector (green). Noninfected glial cells (red) were labelled with glial-specific antibodies (done in collaboration with Dr Diana Jerusalinsky, cells or living organisms. Most applications of amplicons University of Buenos Aires, Argentine). are of neurobiological interest and these studies can be grouped into four main categories. Firstly, amplicons have been used to express toxic,18 proapoptotic19 or immunostimulatory20 genes into experimental gliomas rat primary cardiomyocytes (a), rat primary hepatocytes or glioma cell lines. They have been used to deliver (b), human glioma cells (c), as well as to rat hippocampal neurotrophins, like NGF21 or BDNF,22 antiapoptotic pyramidal neurons (d) in vivo. genes,23,24 heat-shock proteins25 or antioxidant enzymes,26 in attempts to protect neurons against a variety Control of expression from amplicon vectors of neurological insults. Amplicons have also been used Using helper-free vector stocks it is now possible to to deliver tyrosine hydroxylase and other genes to the investigate how transgenic expression from amplicons nigro-striatal system or to cultured striatal cells, in is regulated in the absence of helper particles. Although studies aimed to study or treat Parkinson’s disease.17,27,28 the factors that control expression from the amplicon Finally, amplicons expressing genes affecting neuro- genome remain poorly understood, several teams, includ- transmitter action or neuroreceptor synthesis have been ing ourselves, observed that parameters like the intensity, used to study behavioral traits like learning and the length and the host range of transgene expression are memory.29–32 remarkably different in helper-free and in helper-con- Other studies have shown that amplicons are also well taminated amplicon stocks. More precisely, using helper- suited for delivering genes to skeletal muscle myoblasts free stocks, transgene expression becomes strikingly cell- and myotubes,33,34 to cultured cardiomyocytes and heart type, cell cycle and multiplicity of infection dependent. slices,35 to primary hepatocytes,36 dendritic cells,37 and These restrictions, which have also been described with many cell lines derived from human or murine cancers. polydeleted recombinant vectors expressing no viral In addition, the large transgenic capacity of amplicons immediate-early proteins,45 are usually not observed with has been exploited to efficiently transduce the full set of helper-contaminated amplicons, indicating that both, proteins of MoMLV retrovirus, thus rescuing integrated cellular and viral functions, can modulate transgenic retrovirus vectors,38–40 the nonstructural41 or structural42 expression from the amplicon genome. A better under- proteins of hepatitis C virus (HCV), or the regulatory and standing of the cellular and viral factors that contribute to coding sequences from adeno-associated virus (AAV) the restrictions observed in transgenic expression from genome, in attempts to increase the stability of trans- helper-free amplicon vectors will allow the positive duced genes by amplification or integration.43 Lastly, modification of these vectors towards a highly improved amplicons have also been used as vaccines, for example and efficient gene therapy vector system. against HIV44 or intracellular bacteria (data not pub- lished). Taken together, these studies illustrate the broad Conclusion and perspectives potential of amplicons as gene transfer tools, able to To summarize, thanks to the major achievements efficiently deliver genes to many different target cells in a described in this review, it is becoming possible to variety of experimental situations, in vitro and in vivo, efficiently use amplicons to transduce more than a both for fundamental studies, for vaccination protocols 100 kbp of foreign DNA into quiescent or replicating or for gene therapy trials. Figure 2 illustrates the cells, using nontoxic vectors that can now be produced to transducing efficiency of amplicon vectors to cultured reasonable amounts. This is the current state of the art of

Gene Therapy HSV-1-based amplicon vectors AL Epstein S157 amplicon vector technology. For the near future, three 8 Logvinoff C, Epstein AL. Intracellular Cre-mediated deletion of major areas of research can be anticipated in this field, the unique packaging signal carried by a herpes simplex virus the outcome of which will be critical to go beyond the type 1 recombinant and its relationship to the cleavage- current technological frontiers and applications. The first packaging process. J Virol 2000; 74: 8402–8412. one relates to the possibility of scaling-up the production 9 Logvinoff C, Epstein AL. A novel approach for herpes simplex methods, thus producing still larger amounts of purified virus type 1 amplicon production, using the Cre-loxP recombi- vectors than those generated by current procedures. The nation system to remove helper virus. Hum Gene Ther 2001; 12: second major area relates, as mentioned in the preceding 161–167. paragraph, to the possibility of controlling, improving, or 10 Wade-Martins R et al. An infectious transfer and expression achieving long-term physiological transgenic expression system for genomic DNA loci in human and mouse cells. Nat 19 from amplicon vectors for therapeutic applications in Biotech 2001; : 1067–1070. 11 Wade-Martins R, Saeki Y, Chiocca EA. Infectious delivery of a different cell types. The third area, which is partially 135-kb LDLR genomic locus leads to regulated complementation related to the previous one, concerns the possibility of of low-density lipoprotein receptor deficiency in human cells. avoiding transgenic silencing by the innate immune Mol Ther 2003; 7: 604–612. response. Although this area of research is emerging only 12 Inoue R et al. Infectious delivery of the 132 kb CDKN2A/ now, it can be inferred from what is known about HSV-1 CDKN2B genomic DNA region results in correctly spliced gene immune biology that amplicons will induce an antiviral expression and growth suppression in glioma cells. Gene Therapy cellular response, at least in some cell types, and will 2004; 11: 1195–1204. stimulate both the innate and adaptive branches of the 13 Wang S, Voss J-MH. A hybrid herpesvirus infectious vector immune response in the infected organism. These based on Epstein–Barr virus and herpes simplex virus type 1 for responses can result in the elimination of the vector or gene transfer into human cells in vitro and in vivo. J Virol 1996; 70: in the silencing of the therapeutic transgenes. More work 8422–8430. should be done in order to better understand the vector/ 14 Kwong AD, Frenkel N. The herpes simplex virus amplicon. IV. host interactions, but it can be predicted that the large Efficient expression of a chimeric chicken ovalbumin gene ampli- size capacity of the amplicon genome, allowing to fied within defective virus genomes. Virology 1985; 142: 421–425. express several viral or cellular proteins well known to 15 Geller AI, Breakefield XO. A defective HSV-1 vector expresses downregulate or to inhibit the antiviral and immune Escherichia coli beta-galactosidase in cultured peripheral neu- responses, will be a major advantage of amplicons over rons. Science 1988; 241: 1667–1669. other vectors to fight against the silencing cellular forces. 16 Tung C et al. Rapid production of interleukin-2-secreting tumor cells by herpes simplex virus-mediated gene transfer: implica- tions for autologous vaccine production. Hum Gene Ther 1996; 18: Acknowledgements 2217–2224. 17 During MJ, Naegele JR, O’Malley KL, Geller AI. Long-term AL Epstein acknowledges AFM (Association Française behavioural recovery in parkinsonian rats by an HSV vector contre les Myopathies) and ARC (Association pour la expressing tyrosine hydroxylase. Science 1994; 266: 1399–1403. Recherche sur le Cancer) for constant support to his 18 Rainov NG et al. A chimeric fusion protein of cytochrome laboratory. CYP4B1 and green fluorescent protein for detection of pro-drug activating gene delivery and for gene therapy in malignant glioma. Adv Exp Med Biol 1998; 451: 393–403. References 19 Shah K, Tang Y, Breakefield XO, Weissleder R. Real-time imaging of TRAIL-induced apoptosis of glioma tumors in vivo. Oncogene 1 Spaete RR, Frenkel N. The herpes simplex virus amplicon: a new 2003; 22: 6865–6872. eucaryotic defective-virus cloning-amplifying vector. Cell 1982; 20 Herrlinger U et al. Helper virus-free helper simplex virus type 1 30: 295–304. amplicon vectors for granulocyte-macrophage colony-stimulat- 2 Geller AI, Keyomarsi K, Bryan J, Pardee AB. An efficient ing factor-enhanced vaccination therapy for experimental deletion mutant packaging system for defective herpes simplex glioma. Hum Gene Ther 2000; 11: 1429–1438. virus vectors; potential applications to human gene therapy 21 Federoff HJ, Geschwind MD, Geller AI, Kessler JA. Expression and neuronal physiology. Proc Natl Acad Sci USA 1990; 87: of nerve growth factor in vivo from a defective herpes simplex 8950–8954. virus 1 vector prevents effects of axotomy on sympathetic 3 Johnson PA et al. Cytotoxicity of a replication-defective mutant ganglia. Proc Nat Acad Sci USA 1992; 89: 1636–1640. of herpes simplex virus type 1. J Virol 1992; 66: 2952–2965. 22 Geschwind MD et al. Defective HSV-1 vector expressing BDNF 4 Fraefel C et al. Helper virus-free transfer of herpes simplex in auditory ganglia elicits neurite outgrowth: model for virus type 1 plasmid vectors into neural cells. J Virol 1996; 70: treatment of neuron loss following cochlear degeneration. Hum 7190–7197. Gene Ther 1996; 7: 173–180. 5 Saeki Y et al. Herpes simplex virus type 1 DNA amplified as 23 Linnik MD, Zahos P, Gescwind MD, Federoff HJ. Expression of bcl- bacterial artificial chromosomes in Escherichia coli: Rescue of 2 from a defective herpes simplec virus-1 vector limits neuronal replication-competent virus progeny and packaging of amplicon death in focal cerebral ischemia. Stroke 1995; 26: 1670–1675. vectors. Hum Gene Ther 1998; 9: 2787–2794. 24 Lawrence MS, Ho DY, Steinberg GK, Sapolsky RM. Over- 6 Saeki Y et al. Improved helper virus-free packaging system for expression of Bcl-2 with herpes simplex virus vectors protects HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 CNS neurons against neurological insults in vitro and in vivo. DNA in bacterial artificial chromosome. Mol Ther 2001; 3: J Neurosci 1996; 16: 486–496. 591–601. 25 Hoehn B et al. Overexpression of HSP72 after induction of 7 Zaupa C, Revol-Guyot V, Epstein AL. Improved packaging experimental stroke protects neurons from ischemic damage. system for generation of high-level non-cytotoxic HSV-1 J Cereb Blood Flow Metab 2001; 21: 1303–1309. amplicon vectors using Cre-loxP site-specific recombination to 26 Wang H et al. Over-expression of antioxidant enzymes protects delete the packaging signals of defective helper genomes. Hum cultured hippocampal and cortical neurons from necrotic Gene Ther 2003; 14: 1049–1063. insults. J Neurochem 2003; 87: 1527–1534.

Gene Therapy HSV-1-based amplicon vectors AL Epstein S158 27 Geller AI et al. An HSV-1 vector expressing tyrosine hydroxylase 37 Willis RA et al. Dendritic cells transduced with HSV-1 amplicons causes production and release of L-DOPA from cultured rat expressing prostate-specific antigen generate antitumor immu- striatal cells. J Neurochem 1995; 64: 487–496. nity in mice. Hum Gene Ther 2001; 12: 1867–1879. 28 Sun M et al. Correction of a rat model of Parkinson’s disease by 38 Savard N, Cosset F-L, Epstein AL. Use of defective HSV-1 vectors coepression of tyrosine hydroxylase and aromatic amino acid harbouring Gag, Pol and Env genes to induce the rescue of decarboxylase from a helper virus-free herpes simplex virus defective retroviral vectors. J Virol 1997; 71: 4111–4117. type 1 vector. Human Gene Ther 2003; 14: 415–424. 39 Parrish E et al. Cell engineering for muscle gene therapy: 29 Brooks AI et al. Enhanced learning in mice parallels vector- extemporaneous production of retroviral vector packaging macro- mediated nerve growth factor expression in hippocampus. Hum phages using defective herpes simplex virus type 1 vectors Gene Ther 2000; 11: 2341–2352. harbouring gag, pol, env genes. Cytotechnology 1999; 30: 173–180. 30 Neill JC et al. Enhanced auditory reversal learning by genetic 40 Sena-Esteves M et al. Single-step convesion of cells to retrovirus activation of protein kinase C in small groups of rat hippocam- vector producers with herpes simplex virus–Epstein–Barr virus pal neurons. Brain Res Mol Brain Res 2001; 93: 127–136. hybrid amplicons. J Virol 1999; 73: 10426–10439. 31 Adrover M et al. Hippocampal infection with HSV-1-derived 41 Hong Z et al. Enzymatic characterization of hepatitis C virus vectors expressing a NMDAR1 antisense modifies behavior. NS3/4A complexes expressed in mammalian cells by using Genes Brain Behav 2003; 2: 103–113. the herpes simplex virus amplicon system. J Virol 1996; 70: 32 Cheli VT et al. Gene transfer of NMDAR1 subunit sequences to 4261–4268. the rat CNS using herpes simplex virus vectors interfered with 42 Tsitoura R et al. Expression of hepatitis C virus envelope habituation. Cell Mol Neurobiol 2002; 3: 303–314. glycoproteins by herpes simplex virus-1 based amplicon vectors. 33 Wang Y et al. HSV-1 amplicon vectors are a highly efficient gene J Gen Virol 2002; 83: 561–566. delivery system for skeletal muscle myoblasts and myotubes. 43 Johnston KM et al. HSV-1/AAV hybrid amplicon vectors extend Am J Physiol Cell Physiol 2000; 278: 619–626. trransgene expression in human glioma cells. Hum Gene Ther 34 Wang Y et al. Herpes simplex virus type 1 amplicon vector- 1997; 10: 359–370. mediated gene transfer to muscle. Hum Gene Ther 2002; 13: 261–273. 44 Hocknell PK et al. Expression of human immunodeficiency virus 35 Ferrera R et al. Efficient and non-toxic gene transfer to type1 gp120 from herpes simplex virus type 1-derived amplicons cardiomyocytes using novel generation amplicon vectors result in potent, specific, and durable cellular and humoral derived from HSV-1. JMCC 2004; 3: 219–223. immune responses. J Virol 2002; 76: 5565–5580. 36 Muller L et al. Gene transfer into hepatocytes mediated by 45 Samaniego LA, Neiderhiser L, DeLuca NA. Persistence and herpes simplex virus–Epstein–Barr virus hybrid amplicons. expression of the herpes simplex virus genome in the absence of J Virol Methods 2005; 123: 65–72. immediate-early proteins. J Virol 1998; 72: 3307–3320.

Gene Therapy