Current Gene Therapy - Volume 6, Issue 3, 2006

The study by Richard R. Spaete and Niza Frenkel [Spaete et al., 1982] that can be considered as the birth of the concept of herpes simplex virus type 1 (HSV-1) amplicons was published almost 25 years ago, giving us a good opportunity to celebrate this event. That paper was actually the climax of a series of very elegant and clever fundamental studies aimed to identify and characterize the origins of virus DNA replication, the packaging signals, and the packaging mechanisms of the HSV-1 genome, as developed in the introductory review to this special issue of Current Gene Therapy, written by Niza Frenkel. At the same time, that paper represented the beginning of one of the most interesting, useful, and powerful systems of viral vectors for gene transfer and gene therapy. After the initial demonstration, by Kwong and Frenkel in 1985, that amplicons could be used to efficiently deliver foreign DNA into cultured cells [Kwong et al., 1985], and the publication, by Geller and Breakefield in 1987, of the first study showing that these vectors could be used to express beta-galactosidase in rat cultured peripheral neurons (Geller and Breakefield, 1988), more than 200 papers have reported advances in amplicon technology or applications to gene transfer using these vectors. A first group of studies, too large to be referred to in detail in the scope of this short overview, aimed to improve the production of amplicon vectors both in terms of amount of infectious particles and of purity, in regard to the contamination with helper virus particles [Fraefel et al., 1996, Saeki et al., 2001, Zaupa et al., 2003], as exemplified in particular by the review of K. Kasai and Y. Saeki. Simultaneously, other studies focused on the possibility of expanding the host range of amplicon application by introducing viral or cellular genetic elements allowing vegetative replication or maintenance of the amplicon genome in proliferating cells [Wang et al.,1996], or by improving the stability of the transduced transgene via its integration into targeted loci of the cellular genome, using the adenovirus-associated vector system (Fraefel et al., 1997, Johnston, et al., 1997], as illustrated by the review of D. Glauser et al., Also in the same technological chapter, we should refer to the outstanding work developed by A. Chiocca, Y. Saeki, R. Wade-Martins and colleagues [Wade-Martins et al., 2001, Wade- Martins et al., 2003] to demonstrate that it was possible to use amplicons to transfer entire genomic loci, as developed in the review by Hibbit and Wade-Martins, opening the way to study how the use of long native regulatory sequences could confer physiological regulation of expression to the transgenic sequences. About half of the papers reporting applications of amplicon vectors to particular experimental systems, relate to the nervous system. Many studies have confirmed the strength of these vectors to protect neurons against a variety of natural or experimental injuries via expression of neurotrophins, antiapoptotic or antioxidant molecules, heat-shock proteins or proteins affecting neuronal metabolism. Other studies have shown that amplicons offer a way to study and modify behavioral traits, like anxiety, sexual behavior, learning, and memory, while still others have focused on the possibility of using amplicons to study and treat brain cancers or neurodegenerative diseases, in particular Parkinson disease. It is impossible to quote all these studies in this short introduction, but the reviews of Tyler et al., Shah and Breakefield, and Jerusalinsky and Epstein are here to illustrate and summarize the particular interest of amplicons in neurobiology and neurology. Although to a lesser extent, other works have used amplicons as gene delivery tools to other cells or tissues, including hepatocytes, dendritic cells, skeletal and cardiac muscle cells, etc. The review by Y. Wang illustrates more particularly the relevance of amplicons as tools for introducing genes into muscle cells. Some studies are currently exploring the ability of amplicons to behave as heterologous vector vaccines [Hocknell et al. 2002] and, in this regard, the ability of amplicons to simultaneously express many different antigens, immunomodulators, or even the whole set of structural viral proteins that could generate empty virus-like particles (VLPs) [Savard et al., 1997, Sena-Esteves et al., 1999] constitutes another outstanding illustration of the significance of these vectors as gene transfer tools. The reviews by Santos et al., and by Tsitoura et al., exemplify these aspects of the potentiality of amplicons. Finally, it is clear that the amplicon concept can be extended to other members of the herpesviridae. This is currently being done by a small number of teams and is illustrated in this issue by the review on HHV-6 and HHV-7 amplicons by Borenstein and Frenkel. The aim of this issue of Current Gene Therapy was to present a picture of the current state-of-the-art technology of amplicon vectors and to illustrate some of the most interesting applications that are currently being developed using these outstanding tools. It is clear that by adopting this point of view, and taking into account the limited size of this journal, it was not possible to invite other researchers for additional contributions other reviews.....

The HSV amplicon vector was derived in 1981/1982 after elaborate experience with "defective viruses", arising spontaneously in viral stocks propagated at high multiplicities of infection (m.o.i.). The defective viruses were found to contain large concatemeric genomes with repeat units of limited complexity. We employed cloned defective genome repeats to generate the "amplicon" vectors, which in the presence of helper virus replicate to produce packaged large concatemeric genomes, transmissible to uninfected cells. The cloned amplicons were then employed to fine map and analyze the signals essential for amplicon propagation: (i) A DNA replication origin, producing concatemeric genomes by rolling circle replication. Three DNA replication origins were identified in the HSV genome. (ii) Signals termed pac-1 and pac-2, directing a measuring function for coordinate cleavage of the concatemeric genomes and their packaging as full-size (150 kb) genomes. Using amplicons, foreign genes of large sizes could be linked to less than 1 kb of the cis-acting HSV DNA sequences and become amplified in packaged defective genomes, transmissible to new cells. The transgenes are expressed efficiently, due to sequence reiterations. Large quantities of vectors can be produced in vitro. The amplicons are attractive vectors for use as non-integrating gene delivery vectors. The packaging signals pac-1 and pac-2 are well conserved in different herpesviruses and amplicons with a DNA replication origin and cleavage and packaging signals have been produced in additional herpesviruses. Depending on amplicon-host cell combination, the vectors can be employed with and without mutated helper virus(es) to obtain high gene expression, and desired effect on the target cell. In the absence of helper virus, the defective virus produced is limited for spread in the targeted cells. We expect that new vectors employing state of the art transgenes, will be developed to generate amplicon based concatemeric defective viruses capable of efficient expression of these genes.

The herpes simplex virus (HSV) amplicon vector is a versatile plasmid-based gene delivery vehicle with a large transgene capacity (up to 150 kb) and the ability to infect a broad range of cell types. The vector system was originally developed by Frenkel and her colleagues in 1980. Ever since, a great deal of effort by various investigators has been directed at minimizing the toxicity associated with the inevitable contamination by helper virus. In 1996, Fraefel and his colleagues successfully devised a cosmid-based packaging system that was free of contamination by helper virus (socalled helper virus-free packaging), which utilized as helper a set of 5 overlapping cosmid clones that covered the entire HSV genome, which lacked the DNA packaging/cleavage signals. With the helper virus-free system, broader applications of the vector became possible. Cloning of the entire HSV genome in bacteria artificial chromosome (BAC) plasmids enabled stable maintenance and propagation of the helper HSV genome in bacteria. It also allowed for the development of BAC-based helper virus-free packaging systems. In this article, we review various versions of DNA-based methods to prepare HSV amplicon vectors free of helper virus contamination. We also examine recent advances in vector design, including methods of vector construction, hybrid amplicon vectors, and the infectious BAC system. Future directions in improving packaging systems and vector designs are discussed.

Chimeric or hybrid herpes simplex virus type 1/adeno-associated virus amplicon vectors combine the large transgene capacity of HSV-1 with the potential for site-specific genomic integration and stable transgene expression of AAV. These chimeric vectors have been demonstrated to support transgene expression for significantly longer periods than standard HSV-1 amplicons. Moreover, HSV/AAV hybrid vectors can mediate integration at the AAVS1 preintegration site on human chromosome 19 at a relatively high rate, although random integration has also been observed. One major remaining hurdle of HSV/AAV hybrid vectors is the low packaging efficiency and titers when AAV rep sequences are included in the amplicon vector. In the conditions prevalent during the replication/packaging of HSV/AAV hybrid amplicons into HSV-1 virions, in particular the presence of HSV-1 replication factors and AAV Rep protein, at least three different viral origins of DNA replication are active: the HSV-1 ori, the AAV inverted terminal repeats (ITRs), and the p5 promoter/ori driving expression of the AAV rep gene. A detailed understanding of the properties of these origins of DNA replication and the molecular mechanisms of interactions between them, may allow designing novel hybrid vectors that allow the efficient and precise integration of large transgenes in the human genome.

The principal aim of gene therapy for recessive genetic diseases is to supplement the loss of function of an endogenous gene. For the treatment of many diseases regulation of transgene expression at physiological levels, expression of multiple splice variants, and correct tissue specificity are of utmost importance for effective therapy. We therefore believe the use of a complete genomic locus, in which the native promoter and regulatory regions drive and control expression, is an elegant and effective alternative to traditional complementary DNA (cDNA) vectors utilising heterologous promoters. Viral vectors have proved, over the years, to be an effective means of gene delivery in vitro and in vivo, but the size of complete genomic loci precludes their use in most viral systems. One notable exception comprises the amplicontype vectors based on human herpesviruses, such as the herpes simplex virus type I (HSV-1) amplicon vector. The large genome of HSV-1 (152 kb) confers upon HSV-1 amplicons a very large transgene capacity sufficient to accommodate approximately 95% of human genomic loci. The combination of the large transgene capacity, a broad range of cell tropism, and the ability to infect dividing and non-dividing cells makes HSV-1 amplicons an excellent vector system to develop for the delivery of large genomic loci. Here we review recent work which has shown that HSV-1 amplicons can be used for the delivery and expression of large genomic inserts >100 kb to cells in culture to rescue phenotypes in cellular models of genetic disease. We then discuss applications for high capacity HSV-1 amplicons in vivo and their potential to facilitate the use of large genomic inserts in gene therapy treatment regimes.

Strategies that employ HSV amplicon vectors in the prevention and/or amelioration of pathogenic states afflicting the central nervous system (CNS) have been extensively documented in preclinical disease models. The versatility of the HSV amplicon platform allows for the implementation of therapeutic approaches that require expression of genes exhibiting neuroprotective or neuroplastic activities, or even applications that necessitate the elaboration of antigenspecific immune responses to pathogenic proteins/structures harbored within the CNS. This discourse highlights the successes and challenges encountered using HSV amplicon vectors as tools for the dissection of neural network function and as therapeutics directed against a variety of neurologic disorders.

This review summarizes recent data on the use of HSV-1-based amplicon vectors for in vivo gene delivery to the brains of rats and mice to study and modify behaviour. Here we describe studies that have focused on cognitive functions like learning and memory. In addition, the use of amplicons in other behavioural studies, like addiction, social interaction, anxiety and stress, will be briefly updated. Several remarkable findings have been achieved, thanks to the use of these very efficient and non-toxic naturally neurotropic vectors, most particularly the consistent observation that genetic manipulation of a rather limited number of neurons in restricted regions of the brain, could result in significant behavioural changes, a notion that is therefore emerging as a common unifying hypothesis, thanks to these works.

HSV amplicon vectors provide a unique tool in the armamentarium of weapons for treatment of cancer. Their large capacity (up to 150 kb) allows incorporation of multiple and large transgenes, including whole gene loci, as well as components of other viruses to control the fate of transgenes in the host cells. Means have been developed to achieve heritable transmission of transgenes in tumor cells by episomal replication or genomic integration. Therapeutic transgenes incorporated into amplicon vectors have included anti-angiogenic agents, immune enhancing proteins, prodrug activating enzymes, and apoptosis-inducing factors, as well as inhibitory RNAs for tumor-associated messages. Perks of this vector system include the ability to combine amplicon vectors with oncolytic HSV recombinant vectors to extend the therapeutic range and to target non-dividing as well as dividing tumor cells. Tumor vaccination is favored by the high infectivity of dendritic antigen-presenting cells with HSV vectors, and the vectors themselves appear to have intrinsic immune enhancing properties. Promoter manipulation can be used to target therapeutic gene expression to specific tumor cell types and to achieve drug regulated transgene expression. Further, amplicon vectors can be used to convert tumor cells into packaging cells for retrovirus and adeno-associated virus vectors, thus generating vectors on site. Amplicon vectors have also proven to be a versatile tool to explore imaging modalities to monitor gene delivery and tumor responses to therapeutic intervention.

HSV-1 amplicon vectors have been considered as a promising gene delivery system for gene therapy of skeletal muscle diseases, due to the ability to infect non-dividing cells such as differentiated muscle cells, and to accommodate large transgenes such as the 14-kb dystrophin cDNA. Studies revealed that HSV-1 amplicons can transduce cultured differentiated and undifferentiated muscle cells with high efficiency. Studies also revealed that HSV-1 amplicons are capable of delivering at least 23-kb transgene DNA, including the full-length dystrophin cDNA into muscle cells. The combination of high transduction efficiency, the ability to accommodate large constructs and ease of manipulation makes HSV-1 amplicons an ideal gene delivery tool for the study of muscle ion channels in which gene transduction is frequently employed in cultured muscle cells that are resistant to all the transfecting reagents. However, intramuscular injection of HSV-1 amplicons has been proven inefficient in mature muscles. Evidence has shown that this is mainly because the basal membrane that sheaths each myofibers blocks HSV-1 virions from myofiber cell surface receptors. This result led to the conclusion that HSV-1 amplicons are more suitable for ex vivo manipulation of diseased muscle progenitors or stem cells for autologous cell therapy than in vivo intramuscular injection. Efforts to confer stable transduction ability on amplicons have made progress. A new generation of HSV/AAV hybrid amplicons has been shown to be capable of integrating large transgenes into the AAVS1 site of the human genome, thus, holding potential to achieve a safe and lasting gene transduction in human muscle cells.

HSV-1 amplicon vectors efficiently transduce cultured antigen-presenting cells (APC), including both human and murine dendritic cells as well as primary human chronic lymphocytic leukemia (CLL) B cells. Helper-free amplicons have been shown to be especially well-suited for this purpose, since they do not impair the antigen-presenting functions of these target cells. In vivo, amplicon vectors have been used in preclinical studies aimed at the development of therapeutic cancer vaccines, as well as vaccines for Alzheimer's disease, and selected microbial pathogens. Studies in small animal model systems have shown that ex vivo transduction of irradiated tumor cells with amplicon vectors encoding immunomodulatory cytokines such as IL-2 or GM-CSF can elicit protective responses against a tumor challenge. In an experimental model for cancer immunotherapy, direct transduction of preformed tumors with vectors encoding CD40L resulted in slowed tumor growth or tumor eradication. Other studies have examined the ability of amplicons to elicit immune responses against encoded antigens, and have shown that strong cellular immune responses can be generated against amplicon encoded HIV-1 antigens in mice. Thus, amplicon vectors have shown significant promise as vaccine vectors in a range of settings. These promising initial findings highlight the need to perform additional studies, including experiments to evaluate the immunogenicity of amplicon vectors in additional animal models, possibly including nonhuman primates. Overall, amplicon vectors offer compelling advantages when compared to other vaccine-delivery platforms, which include the capacity to incorporate a very large transgene payload and the potential to efficiently transduce mucosal surfaces. It will be important to design future studies to directly test and exploit these features of the amplicon system. The next few years therefore promise to be an exciting and important period in the development of amplicons as vaccine vectors.

The lack of efficient systems for the propagation of the hepatitis C virus in vitro, in the past decade, led to the development of several heterologous expression systems for the study of the HCV proteins and the HCV life cycle. HSV- 1 amplicon vectors encoding the HCV structural and some of the non structural proteins were generated initially for the expression of high levels of these proteins into mammalian cells. The recent developments in the production of amplicon vectors, allowing the elimination of the contaminating helper HSV-1 virus have given a novel impulse in the study of these vectors as possible vaccine candidates. In this review, an extensive list of the existing amplicon vectors expressing HCV proteins is provided, together with a brief overview of the results obtained by these studies.

Amplicon-6 and Tamplicon-7 are novel non-integrating vectors derived from the lymphotropic Human Herpesviruses 6 and 7 (HHV-6 and HHV-7). In the presence of helper viruses the amplicon vectors replicate to yield packaged defective genomes of size approximately 150 kb and consisting of multiple repeat units containing (i) the oriLyt DNA replication origin (ii) the pac-1 and pac-2 cleavage and packaging signals (iii) bacterial plasmid DNA sequences (iv) the chosen transgene(s). Employing CD46 as a receptor HHV-6 gains entry into varied cells, including lymphocytes and dendritic cells, whereas HHV-7 employs the CD4 receptor to target CD4+ cells. The amplicon-based vectors have facilitated the characterization of viral DNA replication and packaging. Following electroporation and helper virus superinfection, the vectors can be transmitted as cell associated and as cell-free virions secreted into the medium. Analyses by flow cytometry have shown good cell spread and efficient gene expression. Exemplary transgenes have included: (i) The Green Fluorescence Protein (GFP) (ii) Genes for potential use in anti-viral vaccination e.g., the HSV-1 glycoprotein D (gD) with and without the trans-membrane region, expressed intracellularly, at the cell membrane or as secreted proteins. (iii) Tumor cell antigens. (iv) Apoptotic genes for development of oncolytic vectors. Due to their cell tropism, their structure as concatemeric genomes, with less than 1.5 kb of viral DNA sequences, the HHV-6 and 7 amplicons have the potential to become unique vectors for immunization and lymphotropic gene therapy.