Herpes simplex virus type 1 (HSV-1) is a member of the family Herpesviridae, subfamily Alphaherpesvirinae, genusSimplexvirus. It is a common human pathogen that causes infections of the orofacial mucosal surfaces and may rarely cause acute hepatitis, kerato-conjunctivitis or meningo-encephalitis.

The HSV-1 particle is composed of three different compartments, capsid, tegument, and envelope. The capsid proteins are involved in the formation and maturation of the icosahedral capsid [1] and packaging of the viral genome [2-6]. The tegument, which is located between capsid and envelope, is composed of viral proteins involved in transport of capsids to nuclear pores, attachment to the nuclear pore complex [7], release of the virus genome from the capsid into the nucleus [8], and remodeling the host cell environment to optimize replication [9-14]. The viral envelope is a lipid bilayer of host origin that contains 11 viral glycoproteins. These play important roles in viral attachment, entry, cell to cell spread, and egress [15, 16]. HSV-1 can enter the cells by receptor-mediated fusion between virus and cell membrane [17-20]. However, depending on the cell type and virus strain, HSV-1 can penetrate the host cell also by endocytosis [20-22] and phagocytosis [23]. In the cytoplasm, the capsid is transported to the nucleus via interactions with the minus-end-directed microtubule motor protein dynein [24-26] Capsids bind to the nuclear pore complex and release the DNA genome into the nucleus [7, 27, 28].

The HSV-1 genome is a double-stranded DNA (dsDNA) of 152 kbp. It is organized in two segments, unique long (UL) and unique short (US), both of which are flanked by inverted repeats (see Fig. 1). The essential cis elements for viral DNA replication and encapsidation include the origins of DNA replication, located in the UL (ori_L) and TR_s, (ori_S) regions [29, 30], and the packaging/cleavage signals (pac) that reside in the a sequences located at both termini of the genome as well as at the junction between the long and the short segments [31]. The viral genome circularizes after it reaches the nucleus [32] and serves as a template for DNA replication. However, there is also evidence that circularization is not required for replication [33]. The majority of the replicative intermediates are long concatemers that are thought to have been synthesized by a rolling-circle mechanism [34-36]. The concatemers are cleaved into unit-length genomes at the pac signals after filling pre-formed capsids [36, 37].

Fig. (1). Schematic representation of the HSV-1 genome. The HSV-1 genome is a linear double stranded DNA of approximately 152 kb in size, composed of two unique segments, UL and US, which are flanked by inverted repeats, TRL /IRL and IRS/TRS. The minimal cis elements required for HSV-1 DNA replication and packaging include the origin of DNA replication, ori, and the cleavage/packaging, pac, signals.

Fig. (2). Viral vectors. A) HSV-1 amplicon. The HSV-1 amplicon contains three types of genetic elements: i) sequences from bacteria (colE1 and ampR) that allow plasmid propagation in E. coli; ii) sequences from HSV-1, in particular an origin of DNA replication (ori) and a DNA packaging/cleavage signal (pac), which allow replication and packaging of the amplicon DNA into HSV-1 particles in the presence of HSV-1 helper functions in mammalian cells; and iii) a transgene cassette with the gene of interest. B) Recombinant AAV vector. Recombinant AAV vectors are bacterial plasmids that contain the AAV ITRs flanking a transgene of interest. Replication of the ITR cassette and packaging into AAV particles is achieved by supplying helpervirus functions and the rep and cap genes in cis or trans but outside the ITR cassette. C) HSV/AAV hybrid amplicon. In addition to the HSV-1 amplicon elements, HSV/AAV hybrid amplicon vectors contain the AAV rep gene and a transgene of interest flanked by AAV ITRs. D) HSV/EBV hybrid amplicon. In addition to the HSV-1 amplicon elements, HSV/EBV hybrid amplicon vectors contain the EBV origin of DNA replication (oriP) and the gene encoding EBNA-1 which together can support episomal retention and segregation of the vector in dividing cells. E) HSV/RV hybrid amplicon. In addition to the HSV-1 amplicon elements, HSV/RV hybrid amplicon vectors contain the retrovirus (MoMLV) gag, pol, and env genes, and the RV LTRs flanking a transgene of interest.

Fig. (3). Schematic map of the wild type AAV genome. A) Secondary structure formed by the inverted terminal repeat, ITR. Depicted are the Rep binding sites, RBEs, and the terminal resolution site, TRS. B) The AAV genome expresses two clusters of genes, rep and cap, from three different promoters, p5, p19, p40, by alternative splicing.

Fig. (4). The life cycle of AAV. Co-infection of AAV and helpervirus, adenovirus or HSV-1, leads to viral gene expression, viral DNA replication, and production of progeny virus. In the absence of helpervirus, the genome of AAV can integrate into a specific site on human chromosome 19. In the presence of helpervirus, integrated AAV genomes are rescued and enter the lytic replication cycle.

Fig. (5). Comparison between self-complementary AAV (scAAV) and rAAV vectors. scAAV delivers a dimeric inverted repeated DNA molecule thereby bypassing the rate-limiting second-strand DNA synthesis of rAAV.

HSV-1 encodes approximately 89 genes [38], which are expressed in a cascade of three temporal phases: immediate-early, early, and late. The late genes can be subdivided in leaky-late (expression is not dependent on viral DNA synthesis) and true-late (expression depends on viral DNA synthesis) [31, 39, 40].

There are several hypotheses on the mechanisms of envelopment of the nucleocapsid. The generally accepted view suggests a two-step envelopment process in which the capsid acquires a primary envelope by budding at the inner nuclear membrane and then is de-enveloped by fusion with the outer nuclear membrane [41, 42]. The secondary envelope is acquired when the capsid buds into the Golgi or cytoplasmic vesicles [43-49]. The alternative pathways described include (i) budding at the inner nuclear membrane followed by intraluminal transport via ER and Golgi and (ii) exit via impaired nuclear pores and envelopment at the outer nuclear membrane or ER membrane [50-52]. Regardless of the mechanism of envelopment, mature virions seem to exit the cell by exocytosis via intraluminal transport to Golgi cisternae and formation of transport vacuoles [53-55].

An important aspect of HSV-1 biology is the capability of this virus to establish latent infections in sensory neurons of the trigeminal ganglia [56]. The latent HSV-1 genome is a circular, condensed episome, and viral gene expression is limited to the non-coding, latency-associated transcripts (LATs) [57]. Expression of LATs was demonstrated to increase the number of neurons in which latency is established [58] and to affect the efficiency of reactivation [59, 60]. Recent findings that LAT encodes several micro RNAs (miRNA) in HSV-1 infected cells corroborates with the proposed hypothesis that the exonic regions of LAT might function as primary miRNA precursors [61]. At least two of the identified miRNA precursors in latently infected neurons may facilitate the establishment and maintenance of viral latency by post-transcriptionally regulating viral gene expression [62-65].

Latent HSV-1 can periodically reactivate in response to a variety of stimuli, including fever, UV light, hormonal imbalance, malignancy or immune suppression, and enter a new lytic cycle, usually at the site of the primary infection. Recently, the requirement of ICP0 for viral DNA replication [66-68] and for exit from latency has been reconsidered, as in vivo studies showed that reactivation of HSV-1 genomes does not depend on viral DNA amplification [69] nor functional ICP0 [70]. Upon stress conditions, and in the absence of other viral proteins, VP16 was demonstrated to be activated [71], supporting the hypothesis that de novo expression of VP16 regulates entry into the lytic program in neurons. Repeated reactivation does not appear to kill the neurons, indicating that the extent of virus replication must be limited.

The understanding of the biological properties of HSV-1 and the molecular mechanisms of virus replication have allowed the design of specialized vector systems for somatic gene therapy, oncolytic virotherapy, and vaccination.

HSV-1 is an attractive vector for gene therapy due to its (i) large transgene capacity, (ii) high transduction efficiency and broad cell tropism that includes both dividing and non-dividing cells, and (iii) ability to establish latency while maintaining at least some transcriptional activity. However, as HSV-1 is a human pathogen, its use as a vector can result in host immune responses and cytopathogenic effects in patients, and possibly reactivation of and recombination with latent wild-type HSV-1. Taking these aspects into consideration, two different HSV-1-based vector systems, recombinant and amplicon, have been developed.

Initially, recombinant herpesviruses were constructed for functional studies of viral genes and development of vaccines. However, advances in site-specific modification of the viral genome facilitated the use of HSV-1 as a gene transfer vehicle [72]. Different approaches for the construction of recombinant HSV-1 vectors are based on the target tissue and purpose of gene delivery e.g. replication-conditional recombinant HSV-1 vectors are suitable for therapeutic treatment of tumors; replication-defective recombinant HSV-1 vectors are applied for gene replacement therapy [73]. Although some preclinical studies show promising results, several obstacles have to be overcome: (i) replication-defective mutants of HSV-1 can cause cytopathic effects in primary cultures of neuronal cells and inflammatory responses in neural tissue in vivo; (ii) most viral and nonviral promoters are silenced after injection into the brain. Therefore, the main focus in the development of new HSV-1-based vectors has been directed at achieving nontoxic, long-term gene expression in neurons.

The second type of HSV-1-based vector system, the HSV-1 amplicon vector, originated about three decades ago. Spaete and Frenkel analyzed the nature of defective virus genomes generated during the passage of standard HSV-1 stocks at high multiplicity of infection [74, 75]. Their investigations revealed that an ori and a pac signal were the only two cis-acting sequences required for the replication and packaging of defective virus genomes in the presence of trans-acting HSV-1 helpervirus (Fig. 2A). The word amplicon was used to delineate the fact that multiple copies of a DNA sequence of interest can be amplified in a head-to-tail arrangement in concatemeric defective virus genomes and packaged into HSV virions [76]. HSV-1 amplicon vectors share similar structural and immunological properties with the wild type HSV-1 particle, which can trigger cell signaling and cellular responses that may have a transient impact on cell homeostasis or gene expression. However, the lack of virus genes and protein synthesis reduces the risk of reactivation, complementation and recombination with latent or resident HSV-1 genomes.

HSV-1 amplicon vectors have been used to infect efficiently a number of different cell types, including epidermal cells and dendritic cells in the skin [77, 78], some cell types in the cochlea [79, 80], hepatocytes [81], skeletal muscle [82], neurons [83], glioblastoma, and other tumor cells [84-86]. Despite the promising features of the HSV-1 amplicon vector as a gene delivery system, further developments concerning vector production, stability of transgene expression, and interaction with target cells are essential. Recently, the presence of bacterial sequences in the amplicon genome was shown to be responsible for the formation of inactive chromatin, leading to a rapid transgene silencing [87]. Strategies to increase the stability of transgene expression included the use of: (i) cell type-specific promoters [88, 89] and (ii) genetic elements from other viruses that confer genetic stability, such as integration of the transgene into host chromosomes [90, 91] or conversion of the amplicon genome into a replication-competent extrachromosomal element [92-95].

One of these strategies, the combination of genetic elements from HSV-1 amplicons and adeno-associated virus (AAV) to achieve site-specific integration of the transgene into the host genome and long-term transgene expression is described in detail below, after a short introduction into AAV biology.

Adeno-associated virus (AAV) belongs to the genus Dependovirus within the subfamily Parvovirinae, family Parvoviridae [96]. Different AAV serotypes have been identified that can infect a broad range of species; about 11 serotypes and more than 100 variants of AAV infect primates. Based on serological studies, AAV serotypes 2, 3, and 5 most probably have a human origin [97, 98], while AAV4 appears to have originated in monkeys [99]. AAV6 shares some genomic similarities with AAV2 and AAV1, raising the hypothesis that a recombination event could have occurred in vivo or in cell culture [100, 101]. The AAV serotypes 1 to 6 were isolated as contaminants in laboratory adenovirus stocks, while AAV 7, 8, 9, 10, and 11 were isolated as DNA molecules using a “signature PCR”, a screening-based strategy [102]. Despite being widespread among species and infecting different tissues, AAV infections have not been associated with any pathology. Primate AAV serotypes share significant sequence similarities, and the occurrence of cross-reaction of neutralizing antibodies may be species specific or depend on tissue type or route of administration [101, 103, 104].

The genome of AAV is a linear single stranded DNA of 4.7 kb, and either the positive or negative strand can be packaged with equal efficiency. The genome is flanked by inverted terminal repeats (ITR) of 145 bp, containing a palindromic sequence that forms a hairpin as a T-shaped secondary structure. The Rep binding site (RBS) and the terminal resolution site (TRS) are regions within the ITRs that play important roles in the replication and packaging of the AAV genome [105] (Fig. 3A). Two open reading frames (ORFs), Rep and Cap, are responsible for encoding overlapping proteins through alternative splicing (Fig. 3B). The Rep proteins, Rep78/68, and Rep52/40 are transcribed from two different promoters, p5 and p19, respectively, and are involved in DNA replication, transcription, and chromosomal integration. The p5 promoter contains a TATA box, a RBS, a TRS, the Yin Yang 1 (YY1) binding site, and a downstream sequence that can form a hairpin structure. The RBS is involved in Rep-mediated promoter regulation activity [106, 107], thus in the absence of helper functions small amounts of Rep are expressed that bind to the p5 promoter inhibiting transcription [108, 109]. The regulatory activity of Rep seems to be involved in the maintenance of a constant ratio of Rep and Cap proteins during infection in order to keep the balance between AAV genome replication and packaging. The Cap ORF encodes three overlapping proteins, VP1, VP2 and VP3, from a single promoter, p40. These structural proteins compose the AAV icosahedral capsid whose diameter ranges from 18 to 26 nm [110].

The replication of the AAV genome is based on a “rolling hairpin model”. The hairpin structure at the ITR acts as a primer that converts the DNA into a double-stranded template, and together with the essential cis-acting elements RBS and TRS, and helpervirus functions, the replication and transcription of the AAV genome is initiated [111-113]. The Rep78/68 proteins play major roles in the replication process due to DNA-binding, endonuclease, and helicase activities. After binding to the RBS, Rep induces a site- and strand-specific nick at the TRS, creating a new genome end allowing the reinitiation of the synthesis and formation of a monomer extended form that can be packaged [114]. If the hairpin structure in the monomer turnaround form is not resolved before reinitiation on the other genome end, continued synthesis leads to double stranded dimer molecules, in which two genomes in the inverted orientations (head-to-head or tail-to-tail) are covalently joined by a single ITR [115]. Interestingly, the RBS and TRS signals located within the p5 promoter sequence have been demonstrated to act together as an alternative origin of DNA replication in the presence of adenovirus [116, 117] or HSV-1 helpervirus functions [108]. Replication from a plasmid cloned p5 replication origin led to the accumulation of large, head-to-tail linked concatameric replication products, which could readily be packaged into HSV-1 virions if the HSV-1 packaging/cleavage signal was included on the plasmid [108]. These findings indicate that the AAV p5 replication origin could substitute for the HSV-1 origin of DNA replication on HSV/AAV hybrid vectors (see below). AAV is a replication defective virus as it depends on a helper virus, such as adenoviruses [118], a herpesvirus [119-121], or vaccinia virus [122] for productive replication (Fig. 4). Helper viruses are also responsible for inducing a cell cycle arrest in late S or G2 phase, as in the case of adenoviruses [123] or for down regulating host cell functions as in the case of HSV-1 as the helpervirus [124, 125].

Many studies have assessed the different elements from the helperviruses required for AAV replication. A model has been proposed where the HSV-1 helicase/primase proteins constitute a scaffold that recruits ICP8, Rep and cellular replication (RPA) proteins to the self-primed AAV DNA into replication compartments [126-129]. The HSV-1 polymerase complex is preferentially used for AAV replication instead of the cellular machinery [113, 130]. Interestingly, an inhibitory effect of Rep78/68 proteins has been described on HSV-1 replication [131, 132], suggesting a regulatory effect of AAV over HSV-1, thereby limiting expression of HSV-1 early genes [113, 129].

In the absence of helpervirus, the AAV genome can integrate into a specific site termed AAVS1, on chromosome 19q13.3-qter of human cells [133-136] (Fig. 4). The integration is mediated by Rep78/68 and ITRs through a nonhomologous deletion/insertion recombination event [134, 136-143]. Also, an integration efficiency element (IEE) has been identified within the p5 promoter of AAV [144], and more specifically a 16-bp RBE was shown to be sufficient for AAV genome integration [145]. Rescue of the integrated AAV genome is possible by superinfection with helper virus [130]. Although HSV-1 ICP0 seems to contribute to the activation of the rep gene from latent AAV genomes [146], it is not sufficient to induce rep synthesis [130]. Some studies have demonstrated the autonomous replication of AAV under special conditions [147-149], however, the efficiency of replication is significantly lower than in presence of helpervirus functions.

AAV can infect different tissues and bind to unique cellular receptors, which can account for a serotype-specific tissue tropism. Several cellular receptors used by AAV for cell entry have been identified, including heparan sulfate [150], fibroblast growth factor receptor [151], and integrin alphaVbeta5 [152]. The initial steps of AAV infection, attachment to cellular glycosaminoglycan receptors and interactions with coreceptors seem to define the intracellular trafficking pathway of the capsid. Upon entry, AAV capsids are endocytosed via clathrin-coated pits [153], a process that requires dynamitin [154], and transported through both late and recycling endosomes. Trafficking in recycling endosomes appears to be favorable for efficient transgene expression [155]. The process of uncoating is still not well characterized [153, 156], however, AAV appears to enter the nucleus through a mechanism independent of the nuclear pore complex [157].

The broad cell tropism, lack of pathogenicity, and stable long-term gene expression make AAV an attractive vector for gene therapy [158]. AAV2 was the first AAV isolate to be developed into a recombinant vector for transgene delivery as it has been shown to infect a broad range of cell types in animal models [159], showing high efficiency in most of the tumor cells tested [160]. Recombinant AAV vectors are constructed by replacing the Rep and Cap ORFs with a transgene of interest flanked by the ITRs. Rep, Cap and helper functions can be supplied in trans in order to allow replication of the transgene in the host cell [135] (Fig. 2B). Different methods of delivering helper functions have evolved, from co-infection with Ad or HSV-1 [161, 162], to plasmid-based protocols [163], and stable-expression by cell lines [164-166]. The development of a baculovirus based vector production method in insect (SF9) cells has also shown promising results [167, 168].

Recombinant AAV vectors have been tested in preclinical studies for a variety of diseases such as hemophilia, α-1 anti-trypsin deficiency, cystic fibrosis, Duchenne muscular dystrophy, rheumatoid arthritis, prostate and melanoma cancer, Canavan disease [169], Alzheimer’s, and Parkinson’s [170].

Recombinant AAV have shown efficient transduction of different regions of the brain, and are currently used in several clinical trials for neurological disorders [171-173]. Increased interest in designing AAV vectors for the treatment of neurodegenerative diseases that require gene delivery to broad areas or very local and specific areas of the brain are now the focus of many studies [174]. AAV2 has been the most widely used AAV serotype for gene delivery to the CNS, transducing almost exclusively neurons in different brain structures [175-178], and supporting long-term transgene expression in the CNS [179-181] as well as in the dorsal root ganglia [182]. AAV2 has shown higher transduction efficiency in glioblastoma in vitro and in vivo when compared to serotypes 4 and 5 [183]. However, other studies have demonstrated a higher distribution and transduction in the CNS when using rAAV serotypes 1 and 5 [175, 184, 185]. The different AAV serotypes have been exploited on their ability to efficiently transduce distinct regions of the brain due to different cellular tropisms [174, 183, 186].