HSV-1 Amplicon Vectors—Simplicity and Versatility
Authors: Miguel Sena-Esteves, Yoshinaga Saeki, Cornel Fraefel, Xandra O. Breakefield
Journal: Molecular Therapy (2000)

A large number of viral vector systems, including those based on herpes simplex virus type 1 (HSV-1), retrovirus, adenovirus, and adeno-associated virus (AAV), have been developed over the past two decades. Reducing or eliminating toxic components, while conserving the efficiency of gene transfer, has been the primary goal of these efforts. All currently available vector systems have their advantages and disadvantages (see Table 1). HSV-1-based vectors, both recombinant HSV-1 vectors and HSV-1 amplicons, exploit most of the advantages of the parent virus, including (i) a broad host range and the ability to transduce both dividing and nondividing cells; (ii) a transgene capacity of theoretically up to 150 kb, which allows insertion of multiple transcription units and/or elements from other viruses to create hybrid vectors; (iii) high stability of the virion; and (iv) relatively high titers (for reviews, see 1Breakefield X.O. Kramm C.M. Chiocca E.A. Pechan P.A. Herpes simplex virus vectors for tumor therapy.in: Sobol R.E. Scanlon K.J. In The Internet Book of Gene Therapy: Cancer Gene Therapeutics. Appleton and Lange, Stamford, CT1995: 41-56Crossref PubMed Scopus (91) Google Scholar, 2Glorioso J.C. DeLuca N.A. Fink D.J. Development and application of herpes simplex virus vectors for human gene therapy.Annu. Rev. Microbiol. 1995; 49: 675-710Abstract Full Text PDF PubMed Scopus (347) Google Scholar, 3Jacobs A. Breakefield X.O. Fraefel C. HSV-1 based vectors for gene therapy of neurological diseases and brain tumors: Part II.Neoplasia. 1999; 1: 402-416Crossref PubMed Google Scholar). HSV-1 amplicons were first described as contaminating wild-type-sized (~150 kb) defective genomes composed of multiple repetitions of a partial HSV sequence organized in a head-to-tail fashion (concatemers). The monomers were shown to consistently include at least one origin of viral DNA replication (oriS or oriL) and a DNA cleavage/packaging sequence (pac). Cloning of these two elements in bacterial plasmids was shown to be sufficient to direct concatemeric packaging of the plasmid sequence in HSV-1 virions in the presence of complementing packaging functions (4Spaete R.R. Frenkel N. The herpes simplex virus amplicon: Analyses of cis-acting replication functions.Proc. Natl. Acad. Sci. USA. 1985; 82: 694-698Crossref PubMed Scopus (92) Google Scholar, 5Spaete R.R. Frenkel N. The herpes simplex virus amplicon: A new eukaryotic defective-virus cloning-amplifying vector.Cell. 1982; 30: 295-304Crossref PubMed Scopus (70) Google Scholar). These plasmid-based vectors are easy to construct and, when packaged with a helper virus-free system, induce very limited cytopathic effects and immune responses (6Fraefel C. et al.Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors.Mol. Med. 1997; 3: 813-825Crossref PubMed Scopus (352) Google Scholar, 7Costantini L.C. Jacoby D.R. Wang S. Fraefel C. Breakefield X.O. Isacson O. Gene transfer to the nigrostriatal system by hybrid herpes simplex virus/adeno-associated virus amplicon vectors.Hum. Gene Ther. 1999; 10: 2481-2494Crossref PubMed Scopus (52) Google Scholar, 8Suter M. et al.BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1.Proc. Natl. Acad. Sci. USA. 1999; 96: 12697-12702PubMed Google Scholar).TABLE 1PROPERTIES OF SELECTED VIRUS-BASED VECTOR SYSTEMSVIRIONVECTOR TYPEADVANTAGES3DISADVANTAGESHSV-1RecombinantRecombinantTransduces dividing and nondividing cells, large transgene capacity (~30kb)Immunogenic, some toxicity, instability of transgene expression (except possibly with LAT promoter)Amplicon (no virus genes)helper virus dependentAs above (transgene capacity theoretically up to 150 kb)Similar to recombinant HSV-1 due to helper virushelper virus-freeAs above, low toxicity and low immunogenicityVector production depends on transfection of cells, instability of transgene expressionRetrovirusMoMLV-based (no virus genes)Integration into genome of dividing cellsRandom integration, limited transgene capacity (~8 kb)HIV-based (no virus genes)Integration into genome of dividing and nondividing cellsHIV-1 elements, random integration, limited transgene capacity (~8 kb)AdenovirusRecombinantTransduces dividing and nondividing cellsImmunogenic, some toxicity, instability of transgene expression, limited transgene capacity (~8 kb)"gutless" or "high capacity" (no virus genes)As above, large transgene capapcity (~30 kb), low toxicity, and low immunogenicityVector production depends on helper virus or transfectionAdeno-associated virus(no virus genes)Integration into genome of dividing and nondividing cells, low toxicity, and low immunogenicityLimited transgene capacity (~4.5 kb)All vectors listed have a broad host range. Open table in a new tab All vectors listed have a broad host range. Current HSV-1-based vectors still retain some drawbacks: recombinant HSV-1 vectors, although replication-defective in noncomplementing cells, can express viral genes that induce cytopathic effects and immune responses. Moreover, transgene expression is usually transient (days) or short term (weeks). HSV-1 amplicons, on the other hand, do not express any viral genes but depend on helper functions for replication and packaging into virions (5Spaete R.R. Frenkel N. The herpes simplex virus amplicon: A new eukaryotic defective-virus cloning-amplifying vector.Cell. 1982; 30: 295-304Crossref PubMed Scopus (70) Google Scholar, 9Geller A.I. Breakefield X.O. A defective HSV-1 vector expresses Escherichia coli beta-galactosidase in cultured peripheral neurons.Science. 1988; 241: 1667-1669PubMed Google Scholar). If these helper functions are provided by replication-defective mutants of HSV-1, the resulting vector stocks are contaminated with helper virus, which can induce cytopathic effects and immune responses (10Ho D.Y. et al.Herpes simplex virus vector system: Analysis of its in vivo and in vitro cytopathic effects.J. Neurosci. Methods. 1995; 57: 205-215PubMed Google Scholar, 11Johnson P.A. Miyanohara A. Levine F. Cahill T. Friedmann T. Cytotoxicity of a replication-defective mutant of herpes simplex virus type 1.J. Virol. 1992; 66: 2952-2965Crossref PubMed Scopus (189) Google Scholar, 12Wood M.J. Byrnes A.P. Pfaff D.W. Rabkin S.D. Charlton H.M. In flammatory effects of gene transfer into the CNS with defective HSV-1 vectors.Gene Ther. 1994; 1: 283-291PubMed Google Scholar). The problems of cytotoxicity and antigenicity are largely reduced when the helper functions are provided by a packaging-defective HSV-1 genome (6Fraefel C. et al.Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors.Mol. Med. 1997; 3: 813-825Crossref PubMed Scopus (352) Google Scholar), which results in vector stocks that are essentially free of contaminating helper virus. Still the problem remains with HSV-1-based vectors, as with other virus vectors, that it is difficult to achieve stable transgene expression (6Fraefel C. et al.Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors.Mol. Med. 1997; 3: 813-825Crossref PubMed Scopus (352) Google Scholar, 13Fraefel C. et al.Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.J. Virol. 1996; 70: 7190-7197Crossref PubMed Scopus (113) Google Scholar, 14Saeki Y. et al.Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: Rescue of replication-competent virus progeny and packaging of amplicon vectors.Hum. Gene Ther. 1998; 9: 2787-2794Crossref PubMed Scopus (145) Google Scholar). Furthermore, as the current helper virus-free packaging systems depend on transfection of replication-competent, packaging-defective HSV-1 genomes cloned as sets of overlapping cosmids (6Fraefel C. et al.Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors.Mol. Med. 1997; 3: 813-825Crossref PubMed Scopus (352) Google Scholar) or as bacterial artificial chromosomes (14Saeki Y. et al.Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: Rescue of replication-competent virus progeny and packaging of amplicon vectors.Hum. Gene Ther. 1998; 9: 2787-2794Crossref PubMed Scopus (145) Google Scholar, 15Stavropoulos T.A. Strathdee C.A. An enhanced packaging system for helper-dependent herpes simplex virus vectors.J. Virol. 1998; 72: 7137-7143Crossref PubMed Scopus (11) Google Scholar), the titers of amplicon stocks are limited [~108 transducing units (tu) per milliliter after concentration]. Although production of vector stocks for clinical trials is difficult at the present state of technology, HSV-1 amplicons have proven useful for studies in experimental biology, including establishing gene transfer/therapy protocols in animal models of inherited and acquired genetic disorders and evaluating the functions of proteins. Amplicon vectors contain four components (Fig. 1), each of which can be potentially engineered to improve gene delivery for different purposes. The envelope is a lipid bilayer bearing 10–12 glycoproteins which mediate virion binding and entry. The tegument is a space between the capsid and envelope which contains about 12 proteins involved in transactivation of viral genes (e.g., VP16), movement along microtubules, shut down of host macromolecular synthesis (VHS), and other, as yet undefined functions. The capsid is a tight icosahedral structure composed of seven proteins and contains within it the vector DNA genome. Since development of the first amplicon vector for gene transfer to mammalian cells (9Geller A.I. Breakefield X.O. A defective HSV-1 vector expresses Escherichia coli beta-galactosidase in cultured peripheral neurons.Science. 1988; 241: 1667-1669PubMed Google Scholar), changes in design have focused primarily on issues of gene expression: cell specificity, duration, and regulation. The use of tissue-specific promoters has been motivated not only by an attempt to specifically change the physiology of certain cell types, but also to achieve long-term gene expression. Although viral promoters have been shown to be very strong in the amplicon context, duration of gene expression has been limited, as these promoters tend to be inactivated within a relatively short period after infection (days to months) by mechanisms as yet undefined (13Fraefel C. et al.Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.J. Virol. 1996; 70: 7190-7197Crossref PubMed Scopus (113) Google Scholar, 16Ho D.Y. Mocarski E.S. Sapolsky R.M. Altering central nervous system physiology with a defective herpes simplex virus vector expressing the glucose transporter gene.Proc. Natl. Acad. Sci. USA. 1993; 90: 3655-3659Crossref PubMed Scopus (66) Google Scholar). Incorporation of neuronal specific promoters, such as those for preproenkephalin (17Kaplitt M.G. Kwong A.D. Kleopoulos S.P. Mobbs C.V. Rabkin S.D. Pfaff D.W. Preproenkephalin promoter yields region-specific and long-term expression in adult brain after direct in vivo gene transfer via a defective herpes simplex viral vector.Proc. Natl. Acad. Sci. USA. 1994; 91: 8979-8983Crossref PubMed Scopus (2207) Google Scholar) and tyrosine hydroxylase (18Oh Y.J. Moffat M. Wong S. Ullrey D. Geller A.I. O'Malley K.L. A herpes simplex virus-1 vector containing the rat tyrosine hydroxylase promoter directs cell type-specific expression of beta-galactosidase in cultured rat peripheral neurons.Brain Res. Mol. Brain Res. 1996; 35: 227-236Crossref PubMed Scopus (975) Google Scholar, 19Song S. et al.An HSV-1 vector containing the rat tyrosine hydroxylase promoter enhances both long-term and cell type-specific expression in the midbrain.J. Neurochem. 1997; 68: 1792-1803PubMed Google Scholar), has resulted in higher levels of transgene expression in appropriate cell types (10- to 40-fold) and a significant increase in the duration of transgene expression when compared to viral promoters (19Song S. et al.An HSV-1 vector containing the rat tyrosine hydroxylase promoter enhances both long-term and cell type-specific expression in the midbrain.J. Neurochem. 1997; 68: 1792-1803PubMed Google Scholar). Stereotaxic injection of those amplicons into specific regions of the central nervous system yields gene expression restricted to neurons that normally express those proteins (17Kaplitt M.G. Kwong A.D. Kleopoulos S.P. Mobbs C.V. Rabkin S.D. Pfaff D.W. Preproenkephalin promoter yields region-specific and long-term expression in adult brain after direct in vivo gene transfer via a defective herpes simplex viral vector.Proc. Natl. Acad. Sci. USA. 1994; 91: 8979-8983Crossref PubMed Scopus (2207) Google Scholar, 18Oh Y.J. Moffat M. Wong S. Ullrey D. Geller A.I. O'Malley K.L. A herpes simplex virus-1 vector containing the rat tyrosine hydroxylase promoter directs cell type-specific expression of beta-galactosidase in cultured rat peripheral neurons.Brain Res. Mol. Brain Res. 1996; 35: 227-236Crossref PubMed Scopus (975) Google Scholar, 19Song S. et al.An HSV-1 vector containing the rat tyrosine hydroxylase promoter enhances both long-term and cell type-specific expression in the midbrain.J. Neurochem. 1997; 68: 1792-1803PubMed Google Scholar). Replacement of viral promoters by liver-specific promoters and enhancers also resulted in transcriptional targeting of amplicons to hepatocytes (6Fraefel C. et al.Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors.Mol. Med. 1997; 3: 813-825Crossref PubMed Scopus (352) Google Scholar). Since there appears to be no difference in retention of vector genomes in these cells, promoter downregulation or differences in transactivator availability are the most likely explanations for the differences in length of transgene expression (13Fraefel C. et al.Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.J. Virol. 1996; 70: 7190-7197Crossref PubMed Scopus (113) Google Scholar, 19Song S. et al.An HSV-1 vector containing the rat tyrosine hydroxylase promoter enhances both long-term and cell type-specific expression in the midbrain.J. Neurochem. 1997; 68: 1792-1803PubMed Google Scholar). Another aspect that may influence the duration of amplicon-mediated gene expression is the type of transgene used, which in most cases has been the bacterial β-galactosidase gene (lacZ). This gene may be subject to methylation mechanisms that contribute to the short duration of gene expression widely observed (20Jones P.L et al.Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.Nat. Genet. 1998; 19: 187-191PubMed Google Scholar). Loss of transgene expression can also result from loss of transgene DNA over time. The amplicon DNA is believed to be delivered to the host nucleus as a 150-kb circular element, which is incapable of replicating with the genome of host cells, and thus is lost over time from a dividing cell population and probably also over a longer time interval in nondividing cells by degradation. Recently, two different hybrid amplicon systems have been developed to mediate retention of transgene cassettes, which incorporate elements from either Epstein–Barr virus (EBV) or AAV. Two elements from the EBV genome, the latent origin of DNA replication (oriP) and the gene encoding the Epstein–Barr nuclear-associated antigen 1 (EBNA-1), have been shown to confer long-term episomal replication and retention during mitotic segregation to bacterial plasmids in dividing nonrodent mammalian cells (21Yates J.L. Warren N. Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells.Nature. 1985; 313: 812-815Crossref PubMed Scopus (48) Google Scholar). When oriP and EBNA-1 are incorporated into amplicons they serve to extend the length of transgene expression in dividing cells compared to standard amplicons (22Wang S. Vos J.M. A hybrid herpesvirus infectious vector based on Epstein-Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo.J. Virol. 1996; 70: 8422-8430Crossref PubMed Scopus (118) Google Scholar). Transfection of a plasmid(s) containing an AAV-inverted terminal repeat (ITR)-flanked transgene and the AAV rep gene can result in a high overall number of integration events, as well as in site-specific integration of the transgene cassette (23Balague C. Kalla M. Zhang W.W. Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.J. Virol. 1997; 71: 3299-3306PubMed Google Scholar, 24Pieroni L. et al.Targeted integration of adeno-associated virus-derived plasmids in transfected human cells.Virology. 1998; 249: 249-259Crossref PubMed Scopus (56) Google Scholar). Incorporation of these elements into the amplicon backbone has been shown to result in longer retention of amplicon DNA and gene expression in dividing and nondividing cells (6Fraefel C. et al.Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors.Mol. Med. 1997; 3: 813-825Crossref PubMed Scopus (352) Google Scholar, 7Costantini L.C. Jacoby D.R. Wang S. Fraefel C. Breakefield X.O. Isacson O. Gene transfer to the nigrostriatal system by hybrid herpes simplex virus/adeno-associated virus amplicon vectors.Hum. Gene Ther. 1999; 10: 2481-2494Crossref PubMed Scopus (52) Google Scholar, 25Johnston K.M. et al.HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.Hum. Gene Ther. 1997; 8: 359-370Crossref PubMed Scopus (62) Google Scholar). Another way to achieve stable integration of transgenes via amplicon vectors is to use them as a platform to convert cells to retrovirus producer cells, with the retrovirus vectors mediating secondary transgene delivery and integration (26Sena-Esteves M. Saeki Y. Camp S.M. Chiocca E.A. Breakefield X.O. Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons.J. Virol. 1999; 73: 10426-10439Crossref PubMed Scopus (36) Google Scholar). Inducible gene expression in the amplicon context has been achieved by insertion of glucocorticoid- (27Lu B. Federoff H.J. Herpes simplex virus type 1 amplicon vectors with glucocorticoid-inducible gene expression.Hum. Gene Ther. 1995; 6: 419-428Crossref PubMed Scopus (96) Google Scholar) or tetracycline-responsive elements (28Ho D.Y. McLaughlin J.R. Sapolsky R.M. In ducible gene expression from defective herpes simplex virus vectors using the tetracycline-responsive promoter system.Brain Res. Mol. Brain Res. 1996; 41: 200-209Crossref PubMed Scopus (221) Google Scholar, 29Fotaki M.E. Pink J.R. Mous J. Tetracycline-responsive gene expression in mouse brain after amplicon-mediated gene transfer.Gene Ther. 1997; 4: 901-908Crossref PubMed Scopus (21) Google Scholar). A major difficulty in regulating gene expression in amplicons relates to the fact that the most commonly used origin of viral DNA replication contains multiple transactivation elements (TAATGARATT sequences) recognized by the potent HSV-1 VP16 transactivating protein, which is brought in by the infecting virion, leading to unspecific activation of promoters in the amplicon, as well as in the host genome. This results in elevated levels of basal transcription with loss of regulated gene expression in some cell types. Replacement of the origin of DNA replication with a smaller version without the VP16-binding sequences but with comparable amplicon packaging efficiency resulted in a much lower basal expression and a significant degree of inducibility (27Lu B. Federoff H.J. Herpes simplex virus type 1 amplicon vectors with glucocorticoid-inducible gene expression.Hum. Gene Ther. 1995; 6: 419-428Crossref PubMed Scopus (96) Google Scholar). The presence of multiple transcriptional units in close proximity can also cause nonspecific activation of drug-regulated promoters. Minimizing this effect is particularly important when genes for toxic proteins are present, since their expression may prevent or reduce the efficiency of virion packaging. Active repression of the regulated promoters in the off-state may be necessary to package such amplicons (30Rossi F.M. et al.Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16.Nat. Genet. 1998; 20: 389-393Crossref PubMed Scopus (42) Google Scholar, 31Freundlieb S. Schirra-Muller C. Bujard H. A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells.J. Gene Med. 1999; 1: 4-12Crossref PubMed Scopus (150) Google Scholar). Through the rolling circle mode of virus DNA replication and the genome packaging capacity of the HSV-1 capsid (~150 kb), amplicon virions typically carry multiple copies of the transgene expression cassette (number of copies per virion ~150 ÷ size of amplicon plasmid). This results in very high levels of gene expression from a single transducing unit, thus contributing to a low ratio of foreign antigen input (from virion) to transgene-expressing unit. Recently, the possibility of inducing decatenation of amplicon genomes into plasmid size units was demonstrated by including a loxP site in the amplicon and infecting cells expressing Cre recombinase (32Logvinoff C. Epstein A.L. Genetic engineering of herpes simplex virus and vector genomes carrying loxP sites in cells expressing Cre recombinase.Virology. 2000; 267: 102-110PubMed Google Scholar). It remains to be seen if this process will result in longer retention of the amplicon genome and/or transgene expression. Conventional packaging systems for amplicon vectors (Fig. 2A) utilize mutants of HSV-1 as helper viruses, which result in a number of problems associated with the inevitable coexistence of helper virus and amplicons in vector stocks (5Spaete R.R. Frenkel N. The herpes simplex virus amplicon: A new eukaryotic defective-virus cloning-amplifying vector.Cell. 1982; 30: 295-304Crossref PubMed Scopus (70) Google Scholar, 9Geller A.I. Breakefield X.O. A defective HSV-1 vector expresses Escherichia coli beta-galactosidase in cultured peripheral neurons.Science. 1988; 241: 1667-1669PubMed Google Scholar, 33Kwong A.D. Frenkel N. The herpes simplex virus amplicon. IV. Efficient expression of a chimeric chicken ovalbumin gene amplified within defective virus genomes.Virology. 1985; 142: 421-425Crossref PubMed Scopus (176) Google Scholar, 34Geller A.I. Keyomarsi K. Bryan J. Pardee A.B. An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology.Proc. Natl. Acad. Sci. USA. 1990; 87: 8950-8954PubMed Google Scholar, 35Lim F. et al.Generation of high-titer defective HSV-1 vectors using an IE 2 deletion mutant and quantitative study of expression in cultured cortical cells.Biotechniques. 1996; 20: 460-469Google Scholar). These problems include (i) cytopathic effects and immune responses caused by gene expression from the helper virus, (ii) possible interactions between the helper virus and endogenous viruses, and (iii) the risk of generating replication-competent helper virus. Therefore, recent efforts on improving the HSV-1 amplicon packaging systems have been directed primarily toward the elimination of contaminating helper viruses in vector preparations. A significant improvement in this regard was made by the development of a helper virus-free packaging system [(13Fraefel C. et al.Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.J. Virol. 1996; 70: 7190-7197Crossref PubMed Scopus (113) Google Scholar); Fig. 2B]. This system is based on transfection of a set of five overlapping cosmids that span the HSV-1 genome (36Cunningham C. Davison A.J. A cosmid-based system for constructing mutants of herpes simplex virus type 1.Virology. 1993; 197: 116-124PubMed Google Scholar) to provide the necessary functions for the replication and packaging of amplicon DNA into HSV-1 particles. The packaging of the HSV-1 genome itself is blocked by deletion of the DNA cleavage/packaging (pac) signals from the cosmids. Vector stocks produced by this method contain reasonable amplicon titers (106 to 107 tu/ml) and very small amounts of helper virus, thus causing minimal to no cytopathic effects (6Fraefel C. et al.Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors.Mol. Med. 1997; 3: 813-825Crossref PubMed Scopus (352) Google Scholar, 13Fraefel C. et al.Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.J. Virol. 1996; 70: 7190-7197Crossref PubMed Scopus (113) Google Scholar). Readministration of stocks of helper virus-free amplicon vector in vivo was shown to be as efficient at gene delivery as the first administration, even in immuno-competent mice (6Fraefel C. et al.Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors.Mol. Med. 1997; 3: 813-825Crossref PubMed Scopus (352) Google Scholar). Limitations to this amplicon packaging system include (i) the genetic instability of the cosmid clones, (ii) complicated procedure in preparing five HSV-1 DNA fragments from cosmids, and (iii) difficulty in large-scale production of vector stocks. To overcome the first two problems, the entire 152-kb genome of HSV-1, without pac signals, was cloned as a bacterial artificial chromosome (BAC) in Escherichia coli (14Saeki Y. et al.Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: Rescue of replication-competent virus progeny and packaging of amplicon vectors.Hum. Gene Ther. 1998; 9: 2787-2794Crossref PubMed Scopus (145) Google Scholar, 15Stavropoulos T.A. Strathdee C.A. An enhanced packaging system for helper-dependent herpes simplex virus vectors.J. Virol. 1998; 72: 7137-7143Crossref PubMed Scopus (11) Google Scholar). The clone designated fHSVΔpac (14Saeki Y. et al.Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: Rescue of replication-competent virus progeny and packaging of amplicon vectors.Hum. Gene Ther. 1998; 9: 2787-2794Crossref PubMed Scopus (145) Google Scholar) appears to be very stable, as restriction enzyme patterns are entirely conserved, even after serial passages in bacteria. Transfection of fHSVΔpac alone does not generate replication-competent virus but cotransfection with amplicon DNA results in efficient packaging of amplicon vectors. This BAC-based packaging system (Fig. 2C) achieves several improvements over the cosmid-based version, including (i) genetic stability of the helper HSV-1 genome in E. coli, (ii) a simplified packaging procedure, (iii) increased vector titers (approximately twofold; >107 tu/ml), and (iv) facilitation of further modification of the helper HSV-1 genome through homologous recombination in E. coli. Vector stocks prepared with fHSVΔpac were expected to be contaminated with replication-competent helper viruses, as these can be generated by a single recombination event between the oriS sequences present on both fHSVΔpac and amplicon, thus allowing reintroduction of pac into the helper genome. However, contamination with such viruses has proven surprisingly low, <100 plaque-forming units (PFU) per milliliter. Virtual elimination of contaminating replication-competent helper virus has now been achieved by deleting an essential HSV-1 gene, ICP27, from fHSVΔpac and providing ICP27 in trans from another plasmid (Saeki et al., manuscript in preparation). Amplicon vector stocks prepared with this system show a significant reduction in cytotoxicity after infection of cells in culture at high m.o.i. The current packaging system, which requires cotransfection of amplicon DNA and HSV-1 helper DNA into cells, is simple and quick to perform for multiple small-scale experiments. However, it remains quite difficult to prepare the large amounts of high-titer vector stocks which would be needed for human gene therapy applications. One of the best solutions for large-scale production would be the development of packaging cell lines; however, the cytotoxic nature of many HSV-1 genes makes this task quite difficult. Theoretically HSV-1 amplicons can be used to insert complete genes including regulatory regions up to a size of 150 kb. The largest amplicon packaged to date is 51 kb in size and was shown to have characteristics very similar to those of smaller amplicons in both packaging efficiency and duration of gene expression (37Wang X. Zhang G.R. Yang T. Zhang W. Geller A.I. Fifty-one kilobase HSV-1 plasmid vector can be packaged using a helper virus-free system and supports expression in the rat brain.Biotechniques. 2000; 28: 102-107Crossref PubMed Scopus (70) Google Scholar). The ability to manipulate amplicons with large genomic inserts in bacteria may require a new breed of amplicons, which are based on F-plasmid-based artificial chromosomes (famplicons) (38Saeki Y. Fraefel C. Gene transfer using helper virus-free HSV-1 amplicon and hybrid amplicon vectors.in: Cid-Arregui A. Carranca A.G. In Viral Vectors in Gene Therapy and Basic Research. Eaton Publishing, Natick, MA2000Crossref PubMed Google Scholar). The sequence complexity of these famplicons will also require a departure from more common recombinant DNA techniques toward recombination strategies in bacteria (38Saeki Y. Fraefel C. Gene transfer using helper virus-free HSV-1 amplicon and hybrid amplicon vectors.in: Cid-Arregui A. Carranca A.G. In Viral Vectors in Gene Therapy and Basic Research. Eaton Publishing, Natick, MA2000Crossref PubMed Google Scholar) and possibly also in yeast where large seg