Abstract
Herpesviruses are popular therapeutic gene delivery vector due to its capacity for packaging large amounts of heterologous DNA and long-term persistence in transduced cells. Its viral genome is capable of persisting in host cell as a non-integrated episome; minimising insertional mutagenesis.
Herpesvirus Saimiri (HVS), a prototype γ-2 herpesvirus, has been shown to infect a broad spectrum of cell types with high efficiencies. Persistence in high virus titer and the ability to transduce stably in dividing cells makes it a promising gene delivery vector. Researches are ongoing for the insertion of Bacterial Artificial Chromosome (BAC) into HVS to ease the manipulation of the viral genome. The development of amplicon system for HVS has also made significant progress to minimise cytotoxcity and immunogenicity.
An introductory overview of viral-based gene delivery vectors and a few delivery systems currently used to enhance delivery efficiency will be briefed. Herpesviruses, with particular emphasis on Herpesvirus Saimiri, will be evaluated on their potential as gene delivery vectors and applications in today's treatment for diseases.
Introduction
Viruses, although known for their potential to cause disease, have been engineered as tools for the treatment of disease. In the past two decades, extensive research has been ongoing to learn more about the infection and replication of viruses and how this can be manipulated to deliver gene and/or drugs to targeted sites in patients suffering from a range of diseases. Being able to infect cells and integrate/express their genomes, viruses represent excellent delivery vectors for gene therapy (Walther & Stein, 2000). Retrovirus was the first virus used in 1990 to transduce genes for the treatment of adenosine deaminase (ADA) deficiency (Blease et al., 1990). This breakthrough has led to an explosion in virus-based gene therapy.
Gene therapy protocols can be widely classified into viral and non-viral systems. Viral systems utilise the infectivity of viruses, to express exogenous transgene in host cell (Thomas et al., 2003, Walter & Stein, 2000). Although non-viral based systems have minimal issue with immunogenicity and pathogenicity, viral-based systems have shown to achieve more advances (Mah et al., 2002). There are many factors that determine the overall success of viral-based gene therapy. Different delivery systems deployed to introduce the transduced viruses into the host, such as microencapsulation, systemic delivery and vector targeting, affects the success of treatment. Different families of viruses will be discussed in short, with regards to their suitability for treatment. The potential of herpesviruses as promising gene delivery vectors will be evaluated with respect to its transduction efficacy, long-term persistence and its immunological effects on host.
Features of Viral-based Vectors for therapeutic considerations
There are many viruses used today as gene delivery vectors. The choice of virus vector for treatment is dependent of the nature of disease, the suitability of virus to targeted cells, location of treatment, transduction efficiency and pathogenicity potential of viral vectors. Table 1 summarises the features of Parvoviruses, Adenoviruses, and Retroviruses for gene therapy.
Viral Vector
Sequence
Transgenes Capacity
Cell Type Infectivity
Advantages
Disadvantages
AAV
SS DNA
~ 4kb
DD and ND
Broad cell tropism
Potential of targeted integration
Non-pathogenic
Low immunogenicity
Limited transgene capacity
Requires helper-virus to regulate lytic and latent phase
Difficulty in generating high virus titers
Expensive
Labour intensive engineering
Ad
DS DNA
Up to 35kb
DD and ND
Large transgene capacity
High level of gene expression
Episomal persistence
Immunogenic viral proteins
Transient gene expression
Retrovirus
SS RNA
~ 8kb
DD
Stable host DNA integration
Relatively high virus titers
Easy engineering of viral genome
Difficult targeting of infection
Inability to infect ND
Instability of vectors
Random host DNA integration
Herpesvirus
DS DNA
Up to 150kb
DD and ND
Large transgene capacity
Long term persistence
High virus titers
Episomal persistence
Infects wide spectrum of cell type
Natural tropism to neuronal cells
Immunogenic viral products
SS = Single-Stranded; DS = Double-stranded, DD = Dividing; ND = Non-Dividing
AAV = Adeno-Associated Virus, Ad = Adenovirus
Table 1. Features of viral-based vectors for gene therapy (Walter & Stein, 2000, Mah et al., 2002)
Being able to infect both dividing and non-dividing cells is a desirable trait. For instance, retroviral vector is unable to transduce non-dividing cells effectively (Miller et al., 1984); limiting its use in gene therapy. Specific integration of vector into host is also critical, to avoid insertional mutagenesis. The integration of recombinant AAV genome into host DNA is not site-specific; hence it poses risks nonsense mutation (Kotin et al., 1992). Recombinant Ad and herpesvirus vectors are able to persist in cells as episomal DNA instead of integrating with host DNA. This can prevent insertational mutagenesis and lessens modifications of the vectors. Retroviral vector has a limited transgenes capacity of 8 kb (Cepko et al., 1984). Despite so, its ability to transduce a wide range of cell types, high persistence and low immunogenicity, makes it a favourite vector for research.
Gene Delivery Systems
Several delivery systems are developed to facilitate the insertion of therapeutic gene/vector into the host. There are mainly 2 modes gene deliveries: In vivo and Ex vivo. In vivo delivery refers to the direct gene delivery into the host. Ex vivo delivery involves the transfection of host cell culture with the recombinant virus followed by insertion back into the host. Ex vivo delivery has an edge over in vivo such that host cells are given a more conducive condition for proliferation (Mah et al., 2002). The duration of vector contact with host cells can also be controlled, increasing the chances successful transfection. Furthermore, host cells that are used for transfection may give minimal immunoantigenity when implanted back into host (Muruve et al., 1997). Ex vivo delivery also minimise immunogenic response. Hence, ex vivo delivery systems are more extensively used in gene therapy research today. The objectives of developing various delivery systems are to overcome physical and extracellular barriers such as site and surface proteins expressed by target cells. Different delivery systems are ultilised with respect to the location of tissue targeted. Recent delivery systems such as microencapsulation, systemic delivery and vector targeting are invented to increase the vector concentration on-site and prolonging the duration of contact between vector and cell surface.
Microencapsulation
Ex vivo technique has been extensively used to treat blood diseases such as Beta-thalassemia and Severe Combined Immunodeficiency (SCID) (Mah et al., 2002). Host haemopoietic stem cells are transfected with the desired transgenes. In microencapsulation, the positively expressed clones will be cultured and encapsulated with alginate polymers, which are then implanted back into the host subcutaneously (Lohr et al., 2001). The capsule allows lateral nutrient exchange and protects the transduced cells from cytokines and immune cells.
Systemic Delivery and Vector Targeting
Viral vectors have been suggested in recent years to be directly injected to target tissues. Cathryn Mah et al. in 2002 suggested that modifications to the structure of viruses might increase the specificity to target cells. Systemic delivery methods such as intravenous injection would then become a less invasive way of introducing the viral vector. However, vectors may adhere to cell surfaces via non-specific interactions, delaying effective administration to targets. Transduction to non-target cell can also lead to cytotoxicity, triggering immune or apoptotic responses. Further considering the layers of tissues the vectors have to migrate across, less than many would have successfully reached the targeted site. Increasing the quantity of vectors to offset the above issue is not favourable as there is a possibility of generating immunogenic response. Although she has also suggested enhancing the viral specificity via promoters, to improve transgene expression and eliminate possibility of transduction of non-target cells, there would be too many architectural aspects for consideration before such factors can be fulfilled.
Herpesvirus as Gene Delivery Vectors
Herpesviruses are large, enveloped, double-stranded DNA viruses. The large genome of herpesviruses allows the insertion of large heterologous DNA. They are also capable of infecting many cell types including neurons. The long-term persistence nature of herpesviruses infection also increases the rate of successful transduction (White et al., 2002, White et al., 2003). Furthermore, the viral genome is capable of persisting as a non-integrated episome in latent infection. Herpesviruses are divided into 3 subgroups based on their biological and genetic properties, namely alpha (α), beta (β) and gamma (γ) (Roizman et al., 1981).
Alpha - Herpesvirus Vectors
Herpes Simplex Virus-1 (HSV-1) is the most established herpesvirus vector, which is a prototype of alpha-herpes virus. Although its genome is made up of 152kb of double-stranded DNA (84 coding genes), only 50% are essential for viral replication (Glorioso & Fink, 2004). HSV-1 has been mutated and developed into replication competent, oncolytic vector for treating tumours (Post et al., 2004, Han et al., 2007). For instance, G207 virus has its γ134.5 loci deleted, resulting in a significant decrease in neurovirulence, rendering it a suitable vector for treating neurological diseases. An insertion of a Lac Z gene into ICP6 gene disrupts the formation of ribonucleotide reductase in G207 virus. The accumulation of endogenous ribonucleotide reductase allows HSV to replicate more selectively in rapidly dividing cells. (Advani et al., 2002). When transduced to non-dividing cells, the virus becomes highly attenuated. This was proposed to be used in treatment for recurring malignant glioma as the G207 becomes more specific to targeted cells (Markert et al., 2000). It is shown to have expressed viral gene products in non-malignant cells which will trigger host immune response (Markovitz & Roizman, 2000). Hence, improvements have to be made in prolonging the attenuation of virus. In 2007, HSV neurovirulence was shown attenuated with picornavirus cis-acting genetic elements (Campbell et al., 2007).
Beta-Herpesvirus Vectors
The Cytomegalovirus (CMV) belongs to the beta-herpesvirus family, which contains 230kbp of DNA (Roizman et al., 1981). This family of herpesvirus is generally known to possess large quantities heterologous DNA, providing the capacity to contain long transgene sequences. They are ideal for insertion of multiple genes which, in most cases of hereditary diseases, involves multiple types of proteins. In addition, its prolonged persistence in haematopoietic cells magnified its potential to be a gene delivery vector (Borst & Messerle, 2003). Despite being notorious for difficult genome manipulation, the use of Bacterial Artificial Chromosomes (BACs) to manipulate CMV genome has successfully fulfilled the construction of a CMV vector (Borst et al., 2004). The CMV amplicon have also been modified successfully and used to deliver about 210kbp of DNA into haemopoietic cells (Borst & Messerle, 2003).
Gamma-Herpesvirus Vectors
Gamma-Herpesvirus is able to persist as a non-integrated latent episome in dividing cells via episomal maintenance. The human gamma-1-herpesvirus Epstein-Barr virus (EBV) and the primate gamma-2-herpesvirus Herpesvirus saimiri (HVS) are instances. EBV is known to replicate, in sync with the host chromosome, due to the presence of EBV Nuclear Antigen-1 (EBNA-1) (Yates & Guan, 1991, Yates et al., 1985). EBNA-1 binds within the virus latent origin of replication oriP (Yates et al., 1985), where the cis-acting oriP contains multiple binding sites for EBNA-1 (Koons et al., 2001). oriP and trans-acting EBNA-1 protein have been inserted into amplicon systems and has shown to be stably maintained within mouse cell lines (Huertas et al., 2000). By deleting all oncogenic sequences and replacing with terminal repeats, the lytic origin of replication (oriLyt) for DNA amplification, and EGFP can be packaged to produce high-titer vector preparation. Hence, this EBV-based vector offers many advantages over other viral-based vectors (Conese et al., 2004). Further improvement to this vector via incorporating HSV-1 amplicons, prolongs persistence and increase larger transgene packaging capability, increasing the probability of effective gene delivery (Inoue et al., 2004). On the hind side, EBV is still an oncogenic virus which scientists question its potential of oncogenic virulence reactivation after recombination with modified vector within the host.
Herpes Simplex Virus - 1
Herpes Simplex Virus -1 (HSV-1) is the most commonly used for gene therapy studies. HSV vectors can exist in two major forms: namely the amplicon and the recombinant vector. An amplicon is an amplified DNA plasmid packaged to deliver a transgene of interest to the host cell (Macnab et al., 2008). HSV amplicons contain the origin of replication, therapeutic gene and packaging signals in a plasmid form (Spaete & Frenkel, 1982). These are produced in cells which have been infected with an HSV helper virus. Whereas, recombinant vectors are synthesized by direct insertion of therapeutic gene through homologous recombination. (Glorioso et al., 1994) HSV vectors are immunologically toxic in nature. Hence, HSV has to be modified to reduce is immunotoxicity and improve its gene expression (Glorioso et al., 1997). Viral proteins such as infected cell proteins (ICP) ICP4, ICP22 and ICP27 are found to mediate the cytotoxic effects in HSV infection, and its removal has shown to reduce the toxicity of vectors (Wu et al., 1996). Amplicons have been developed to reduce the cytotoxicity and immunoantigenicity against HSV-1 vectors. A helper virus is required to pack plasmids containing HSV-1 lytic replication origin and terminal packaging sequences along with the desired transgene in an infectious particle (Geller & Breakefield, 1988). Despite so, contamination with helper virus is still significant in recent studies. Researchers have moved towards developing helper-free amplicon systems in bacterial artificial chromosome (BAC). Stuffer DNA has also been inserted into HSV-1 genome, prohibiting its packaging into virion (Saeki et al., 2001). Helper-free amplicons has shown reduced inflammation when used to infect neurons (Olschowka et al., 2003).
Herpesvirus Saimiri
Herpesvirus saimiri (HVS) is a prototype γ-2 herpesvirus, also known as rhadinovirus, originated from the squirrel monkey, Saimiri sciureus. It possesses significant similarities with other herpesviruses such as EBV, bovine herpesvirus 4 (BHV-4), Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) (Virgin et al., 1997). The genome of HVS (strain A11) consists of a unique internal low G+C content DNA segment (LDNA) flanked by a high G+C content tandem repetitions (H-DNA). These genes encode products which may be involved in transformation, immune evasion and long-term persistence of the viral episome. HVS strains are classified into A, B and C, based on their oncogenic properties. However, only strains A and C are capable of immortalising marmoset T lymphocytes to interleukin-2 independent proliferation (Szomolanyi et al., 1987). Specifically, only subgroup C is capable of transforming human, rabbit and rhesus monkey lymphocytes in vitro (Fickenscher & Fleckenstein, 2001). These strains encode oncogenes known as saimiri transforming proteins which associate with Ras resulting in immortalizing the lymphocytes Ras (Jung & Desrosiers, 1995). Therefore, all HVS-based vectors contain deletions within the transforming Saimiri Transforming Proteins and Tip genes to eliminate oncogenicity.
HVS Amplicon System
The objective of an amplicon system in viral-based gene delivery is to eliminate all the pathogenic and immunogenic factors while retaining the natural infectivity and transgene expression ability of the vector (Macnab et al., 2008). This is an important biosafety aspect in consideration for the type of virus used for gene therapy. An amplicon system utilises a non-viral vector plasmid to couple with the natural infectivity of the wild type (WT) virus and desired transgene to generate an amplicon containing virus-like particle (AmpVLP). The amplicon plasmid must hence contain appropriate viral packaging sequences. The HVS genome of 112 kb provides a transgene packaging potential via the generation of a gutless system. Gutless herpesvirus vectors are excellent for synthesizing amplicon plasmids as the inserted transgenes can be equivalent in size, or slightly larger (White et al., 2002) than the size of the WT viral genome. Hence these factors make HVS a very attractive virus to be manipulated for gene delivery.
HVS Episomal Maintenance
Although the episomal persistence of herpesvirus made them a popular vector for research, little has been discovered on the mechanism of HVS episomal maintenance. During latency, the viral genome persists as non-integrated episomes in the nuclei of the host cells. Using KSHV as a model to evaluate the episomal persistence of γ-herpesviruses, the gene responsible is encoded by ORF73, which is also commonly known as latency associated nuclear antigen (LANA). LANA was discovered and suggested to be one of the only three genes responsible for the viral latent phase (Dittmer et al., 1998). It is said that LANA functions to maintain the extra chromosomal viral genome during KSHV latent infection, when it binds to the TRs within KSHV genome (Ballestas et al., 1999, Hu et al., 2002). The amino and carboxyl terminals of LANA are able to bind with the mitotic chromosomes domain of the host cell (Piolot et al., 2001). As histone H1, methyl CpG binding protein 2 and DEK are also associated with the domains, it may be an indication that LANA binds functionally, analogous to the EBNA-1 (Leight & Sugden, 2000). Since EBNA-1 is known to be essential for the viral maintenance of EBV, LANA could be involved in episomal maintenance simultaneously (Lee et al., 1999).
HVS ORF 73 has been demonstrated to colocalise with HVS genomic DNA on host mitotic chromosomes (Calderwood et al., 2004), to maintain the stability of HVS TR-containing plasmids (Verma & Robertson, 2003). This result shows the mechanism for episomal maintenance in HVS has close resemblance as of KSHV, as described. Moreover, an experiment on deleted ORF 73 and TRs in HVS mutants showed viral episome does not persist (White et al., 2003 and Calderwood et al., 2005). These results suggest that ORF 73 and TR are essential for the episomal maintenance for HVS.
The viral DNA is also found to replicate once per cell cycle during the S-phase together with the DNA of the host cell (Yates and Guan, 1991). The genes responsible for latent replication are also found to have been actively transcribed from the euchromatin. (Alberter and Ensser, 2007, Stedman et al., 2004 and Stedman et al., 2008). Recent work also shows that the replication of HVS episomes occur in early S-phase together with cellular euchromatin (Vogel et al., 2010). Hence, it suggests that the S-phase of host cell is required for the persistence of HVS episomes. Otherwise, current researches have yet to establish the definite mechanism of HVS episomal maintenance.
Engineering of HVS-Based Vectors
Genomic loci usually consist of transgenes, distal regulatory elements and introns, which are essentially for the production of splice variants (Li et al., 1999, Rowntree et al., 2001). Since HVS-based vectors are capable of accommodating large DNA fragments, they are more favourable for delivering the genome loci into the targeted host cells. However, the insertion of the heterologous genes into the HVS genome via a single region of TR, was the only approach to achieve homologous recombination. This approach is time consuming and an additional replication-competent virus is also required to prevent the modifications of key viral genes (Grassman & Fleckenstein, 1989). Hence, the engineering of HVS-based vector became a limiting factor for success.
In the 1990s, a breakthrough in developing F-factor derived BACs created the ease in engineering large viral DNA. This BAC allows a stabilised insertion of about 300kb of foreign DNA via the regulation of F-factor replicon in Escherichia Coli (Shizuya et al., 1992). A BAC construct comprising of essential BAC elements and a selective marker is inserted into the viral genome via homologous recombination. The recombinant viral BAC enables the shuttling of viral genome between eukaryotic and prokaryotic cells. This tool is very useful as HVS genome can be easily manipulated with the use of prokaryotic molecular cloning techniques.
Subsequently, many successful cloning of herpesviruses such as HSV and EBV using viral BACs were reported (Griffiths et al., 2006). However, the problem of HVS-BAC not being able to establish latent infection has surfaced. This may be due to the site in which the BAC is inserted, resulting in the loss of episomal maintenance mechanism of the cloned HVS. There are a few researches that suggested different site of BAC insertion. For instance, the insertion of BAC elements into ORF15, a non-essential homologue of CD59, showed promising result of producing mature lytic HVS with latent infection ability (Albrecht et al., 1992, Rother et al., 1994, White et al., 2003).
In addition to enhancing the convenience of manipulating genome of herpesvirus, a unique restriction site known as I-Ppo-I, has been inserted into HVS-BAC genome (Fig 1). This restriction site allows direct cloning of heterologous genes into the HVS-based vector (White et al., 2003); saving time.
Fig 1. The I-Ppo-I restriction site in A strain 4 HVS allows direct insertion of genes. This figure illustrates the insertion of a transgene expression cassette containing transgene of interest and kanamycin resistance gene.
HVS Persistence in Human Cells in vitro
The potential of HVS-based vectors to persist in human cell has been assessed since 1990s. Neomycin and hygromycin are genetic markers used to assess the infectivity and persistence of HVS. These markers were inserted into HVS genome using homologous recombination (Grassmann & Fleckenstein, 1989, Simmer et al., 1991). Positive result was observed as the modified HVS vectors were able to infect and persist episomally in a variety of human cultured cell lines. Human carcinoma cell lines have also been shown to be infected by HVS of up to 100% efficacy (Stevenson et al., 1999, Stevenson et al., 2000b). The virus DNA replicates and persists as latent episome even after cell division (Hall et al., 2000). No significant increase in replication time within the carcinoma cell lines indicates that the HVS episome does not interupt cell growth (Smith et al., 2001). HVS-based vectors have also shown to have high efficacy in infecting haematopoietic cell lineage. Human bone marrow stromal cells are shown to be susceptible to HVS infection with increasing dose (Frolova-Jones et al., 2000). However, controversial results are observed in stem cells of different lineage and animal tested in vitro. Stevenson et al., infected totipotent mouse embryonic stem (ES) cells with HVS vectors in 2000. Result has shown that the infected ES cells have differentiated into macrophages terminally with no apparent change in cell morphology. In contrary, Doody et al., have demonstrated otherwise in 2005, that HVS episome have no effect on human erythroid lineage differentiation. This renders the infectivity and persistence of HVS in human stem cell lineages, a room for further research. The lymphoid lineage can be used in place of erythroid lineage since nuclear content of lymphoid cells remains relatively unchanged compared to cells of erythroid lineage. The result observed above may be due to discontinued erythropoiesis in in vitro culture. Erythroid differentiation may be halt at Proerythroblast stage due the lack of suitable environmental conditions. Infectivity may have possibly occurred but discontinued persistence of HVS vector due to the limitation of in vitro condition.
HVS Persistence in Human Cells in vivo
Fig 2. (a).GFP is expressed in mice bearing SW480 HVS-GFP tumours under light and UV (488nm) for GFP identification. (b) GFP is also observed in cross-section of tumour xenograft (Smith et al., 2001).
The progression from in vitro to in vivo analysis is carried out to confirm the persistence of HVS in the host. Smith et al., has performed tumour xenograft studies in nude mice using uninfected or HVS-infected human carcinoma cell lines in 2001. The tumour xenografts were grown for 3 months and the persistence of HVS were assessed via the expression of GFP. Fig 2. shows the persistence of HVS in the tumour xenografts. A PCR assay performed on the tumour xenograft shows that HVS infection is localised. However, subsequent RT-PCR assay detected a low level of lytic gene expression in the xenograft. Despite so, viral DNA and RNA is absent in the host tissue, suggesting that the lytic gene products may be produced due to poor replication in murine cells. Moreover, the product is not found to spread into the host. GFP expression is detected in the whole 3-month analysis, proving that there is a sustained transgene expression.
Fig 3. Sustained GFP expression in vivo analysis via direct intra-tumoral injection (Smith et al, 2005a) into i) A637s, ii) MiaPaCa, iii) MCF7s and iv) xenografts.
A similar experiment conducted in 2005 via direct injection of HVS-based vector into tumour xenograft also shows its long-term persistence (Smith et al., 2005b). In this analysis, various carcinoma cell lines are used (Fig 3). HVS-based vector has also shown to persist better compared to other herpesvirus such as EBV. The specific duration of HVS episomal persistence in vivo cannot be determined due to the limitation of tumour size used in these experiments. Otherwise, Hoggarth et al., has demonstrated HVS-based vector is able to persist for more than 40 divisions without selection, in the expression of HSV-1 TK gene, in 2004.
Fig 4. (a) HVS-Luc has infected mouse tissue, identified by ORF73 HVS DNA detection, after 10 weeks of IV and IP administration. GAPDH DNA was also detected in all tissues, indicating sufficient DNA samples have been extracted for analyses. (b) Assay for Luc expression from tissues harvested 10 weeks post infection. Tissues were frozen in liquid nitrogen and grinded into fine powder (Smith et al., 2001).
The dissemination of HVS-based vector following intravenous (IV) or intraperitoneal (IP) administration is assessed after acknowledging that it can persist in tumour xenograft. Non-invasive optical bio-imaging has been ultilised to detect the dissemination of luciferase-expressing HVS-based vector. The high degree of bioluminescence sensitivity allows detection of luciferase expressing cells as few as 102 to 103 (Edinger et al., 2002). In a 10-week assessment, both IV and IP administration of HVS, consistently showed luciferase bioluminescence (HVS-Luc) in the liver (Smith et al., 2005b) (Fig 4). PCR assay was also performed to detect viral DNA in different tissues. A large amount of viral DNA was detected in liver tissue accounting for the infection of liver; supporting the imaging data. Trace amounts of viral DNA was also detected in spleen, kidney and heart. The imaging data did not detect bioluminescence in the heart. This might be due to insufficient cardiocytes infected, hence undetected. Although virus dissemination experiments have not been performed in other animal models besides mice, the above result makes HVS a highly potential vector to infect and persist in vivo.
In viral-based gene therapy, transgenic viruses are engineered to infect only the targeted cells. Infection of non-targeted tissues may lead to an over-expression of transgene. An accumulation of viral products in other tissues may trigger the immune response of host that can lead to fatality rather than intended treatment. From the above result, though the objective was to observe infectivity and persistence of HVS in vivo, it also indicate that HVS can effectively infect various tissue types via IV and IP, creating a concern for over-expression of transgene and viral products that may be detrimental to the host.
Applications of Herpesvirus Vectors
The use of HVS as a gene delivery vector was first trialed to express bovine growth hormone (bGH) in New World primates. The primates were able to produce bGH. However immunoglobulin G was also developed against bGH. This suggests that HVS can be used to transduce functional genes into patients with hereditary diseases as a result missing gene products. (Desrosiers et al., 1985). HVS was also shown to transfect primate haematopoietic cells (Doody et al., 2005), revealing the potential to for treatment in erythroid lineage diseases.
Treatment for Hereditary Diseases
Recently, HSV-1 amplicon vector is used to treat Friedreich's ataxia (FA), in mice models in vivo (Lim et al., 2007). FA is the most common hereditary ataxia characterized by the loss of muscle coordination ability. It is caused by a reduced expression of the mitochondrial protein frataxin, due to a mutation in the frataxin gene (FRDA). HSV-1 amplicon vectors expressing human frataxin complementary DNA, were injected into localized gene knockout mice. These mice, initially behavioural deficit, exhibit behavioural recovery, 4 weeks after injection. This breakthrough has thus become the first proof of principle in the recovering neurological function via a viral vector by recovering frataxin deficiency (Lim et al., 2007).
Treatment for Polygenic Diseases
The ability of herpesvirus to accommodate large heterologous DNA sequences makes it viable for treatment of diseases such as cancer, which is usually polygenic. The large capacity allows the packaging of complete eukaryotic genetic materials such as the cis-regulatory elements or a complete gene loci; enhancing the specificity of gene expression in the targeted tissue. For instance, EBV has been shown to deliver over 100kb of human DNA (Kelleher et al., 1998, Wade-Martins et al., 2003, White et al., 2002). Replication-competent, oncolytic HSV-1 vector has also recently been demonstrated to be effective against gastroesophageal cancer cell lines. Transgenes encoding for a fusogenic membrane glycoprotein and Fcy::Fur, are inserted into HSV-1 vector and tested on the cell lines at sequential multiplicities of infection (MOI). All cell lines were susceptible to viral infection and demonstrated a dose-dependent effect, with greater and faster cytotoxicity at higher MOIs (Wong et al., 2010).
Immunotherapy and anti-tumour agent
As discussed earlier, the ability of HVS to transform T lymphocytes, has led to its use in immunotherapy against cancers (Hiller et al., 2000a, Hiller et al., 2000b, Knappe et al., 2000). A prodrug activating HVS thymidine kinase (TK) gene has been inserted into HVS vectors to enhance its biological safety. This allows the highly pathogenic virus to be cleared by the administration of Ganciclovir (at low concentrations) after the therapy. Moreover, the persistence of HVS vectors has been unaffected after the insertion and its replication unexpectedly enhanced (Knappe et al., 2000). Hence this novel combination of virus and drug therapy is likely to become widely established for T-cell dependent immunotherapy of resistant residual leukemia; minimising the risk of graft versus host disease (Hiller et al., 2000a, Hiller et al., 2000b, Knappe et al., 2000). Therefore, this combination of treatment can be administered readily with minimal worry of mortality due to pathogenicity.
Hoggarth et al. in 2004, has transduced cells using HVS-TK vector and successfully transduced cells have shown to become susceptible to the administration of Ganciclovir and (E)-5-(2-bromovinyl)-2'deoxyuridine. This discovered the potential of the vector to be developed into an antitumour agent. Griffiths et al. also suggested to insert therapeutic gene that can control the removal of these modified cells when they are no longer necessary, in 2006. However, the gene that is considered for insertion has to be unique to the cancer cells; to avoid killing normal cells that express the same gene.
A recent research proposed the use expanded natural killer (NK) cells to treat advanced malignancies. The experiment expanded patient NK cells in vitro with IL-2 and an irradiated Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (EBV-TM-LCL) to produce 14 NK-cell products. The gene products are analysed and shown to be identical to those produced by patient normal NK cells (Park et al., 2010). The expanded NK cells also over-expressed genes responsible for dendritic cell activation, resulting in the up-regulation of immunogenic products (NK cell-activating receptor, natural cytotoxicity triggering receptor 3, myxovirus restistance 1, lymphotoxin b, and BCL2-associated X protein) that are likely boost the immune response of patients against malignant cells.
The approach of boosting the host immune response to tumour cells is promising. It can be further developed into an ex vivo gene therapy to target a wide spectrum malignancies. Targeting of specific malignant tissues is also highly possible. Activated lymphocytes against the specific tumour can be isolated and transduced to over-express genes for antibody production. This would facilitate the immune response of the host to focus on destroying the specific tumour cells via antigen recognition. Since antigen targeting within the body is not site or tissue specific, it will be able to recognise the metastases at secondary sites, further improving the effectiveness of such immunotherapy.
Treatment for autoimmune diseases
Wieser et al., demonstrated the use of HVS-based vector for the treatment of chronic inflammatory disease such as rheumatoid arthritis (RA). Although the cause of RA is still not completely understood, a possible trigger is known to be an excessive production of pro-inflammatory cytokines such as TNF-α and IL-1β. These cytokines are constantly produced; resulting in progressive inflammation, primarily in synovial joints and may affect other tissues. Cartilage-degrading matrix metalloproteases (MMPs) such as MMP-1 (Collagenase-1) or MMP-3 (Stromelysin-1) are also activated, resulting in the wearing off of the synovial joints. Anti-inflammatory factors such as IL-1 receptor antagonist (IL-1 RA) and IL-10 can be used to counter the effects of these pro-inflammatory cytokines. Mifepristone-antiprogestin-inducible expression system by GeneSwitchTM was inserted into a HVS-based vector to express the anti-inflammatory transgenes IL-1 RA and IL-10. Primary human fibroblasts and rheumatoid arthritis (RA) fibroblast-like cells (RASF) were then transduced with the HVS-based vector. Although the global patterns of cytokine secretion was not shown to have changed significantly after transduction, regulated expression of IL-1 RA and IL-10 was observed to block IL-1 β-stimulated MMP-3 expression from RASF cells (Wieser et al., 2005).
Treatment for Liver diseases
The high persistence and infectivity of HVS in liver demonstrated in the study of HVS dissemination in mice, suggested its application for treatment of inherited and acquired liver disease (Prieto et al., 2003, Smith et al., 2005a). HVS-based vectors have also been demonstrated to infect hepatic myofibroblasts (HM). HM are critical repair and profibrogenic cells in the liver, produced in response to hepatic injury and inflammation (Friedman, 2000). There have been suggestions to develop gene delivery systems to attenuate the profibrogenic phenotype in chronic liver disease (Gao et al., 2002, Janoschek et al., 2004). However, it is limited by the availability of HM for in vivo study since they are only produced in a damaged liver. Despite so, Smith et al., using in vitro models of HM, hepatic stellate cells, determined high transduction and long-term gene expression (Fig 5). This result is not unexpected as herpesviruses are suspected causative infectious agents for Human herpesvirus 8 (HHV-8) DNA sequences and EBV has been reported in splenic and hepatic inflammatory myofibroblastic tumor suggesting the involvement of both viruses in HM (Mergan et al., 2005).
Fig 5. GFP-expressing cells were observed in transduced hepatic myofibroblasts using UV fluorescence microscopy and light microscope.
Development in HVS Amplicon System
In 2008, Macnab et al., has shown promising progress in the generation of HVS amplicons. A partially gutless HVS-based "amplicon" was constructed with BamHI restriction digest and re-ligation of HVS-GFP-BAC (White et al., 2003). Approximately 50 kb of the viral genome (ORFs19-62), was used for the construction of a recombinant HVS "amplicon" vector, termed HVS_Bam in Fig- 6 (Macnab et al., 2008).
However, this system is not a true amplicon as it still contains some viral genetic information. The virus titer is also relatively low even with the use of helper virus. Further research on the production of a fully gutless HVS amplicon can be directed at the increasing the virus titer and eliminating the viral products that are unnecessarily expressed, which may be immunogenic.
Fig 6. (A) A diagram HVS_Bam construct using BamHI digestion on HVS-GFP-BAC. BamHI digestion removes ORFs 19-62, and transcriptional activating genes, ORFs 50 and 57. Episomal maintenance elements on ORF 73 and TRs are retained. (B) PFGE of potential HVS_Bam clones. (Lane 1) Promega Lamda EcoRI/HindIII Marker, (Lane 2) NEB PFGE Marker II, (Lane 3) BamHI digest of WT HVS-GFP-BAC clones (Lane 4-15) BamHI digests of potential HVS_Bam clones. (*) Clones selected for further analysis (Macnab et al., 2008)
Conclusion
Herpesvirus can infect a broad spectrum of human cells. HVS-based vectors have also been shown to infect and persist in haematopoietic cells and carcinoma cells, as latent episome. Without the need to integrate with the chromosome of host cells, the issue with mutagenesis upon insertion does not pose a risk of using HVS-based vectors. This also saves researchers from the time and funding spent to locate a suitable position in which the viral genome can be integrated into host DNA. Herpesvirus, being a double-stranded DNA virus, does not require transcriptional genes and mechanisms to convert RNA into DNA, unlike retrovirus. Furthermore, its ability to accommodate large heterologous DNA fragment potentates its use to treat diseases that involve multiple genes, such as cancer and hereditary diseases. As of all viruses, being pathogenic in nature, biological safety aspects are to be incorporated for active elimination of vector when necessary. Herpesvirus strains are known to be oncogenic and hence viruses have to be affirmatively made replication-defective and with oncogenes removed. Research on the production of amplicon system for HVS has been progressing well in recent years. Besides the amplicon system, the breakthrough of HVS-BAC vector has also been characterised to create ease in manipulation of herpesvirus genomes. The novel approaches suggest that herpesviruses, particularly HVS, are developing to become gene delivery vectors of great medical importance.
