The viral genome is a linear double stranded DNA molecule of 152 kb. There are two unique regions, long & short (termed UL & US) which are linked in either orientation by internal repeat sequences (IRL & IRS). At the non-linker end of the unique regions are terminal repeats (TRL & TRS). There are up to 81 genes (Marconi et al, 1996), of which about half are not essential for growth in cell culture. Once these non essential genes have been deleted, 40-50 kb of foreign DNA can be accommodated within the virus (Glorioso et al, 1995). Three main classes of HSV-1 genes have been identified, namely the immediate-early (IE or alpha) genes, early (E or beta) genes & late (L or gamma) genes.
Following infection in susceptible cells, lytic replication is regulated by a temporally co-ordinated sequence of gene transcription. Vmw65 (a tegument structural protein) activates the immediate early genes (IP0, ICP4, ICP22, ICP27 & ICP477), which are transactivating factors allowing the production of early genes. The early genes encode genes for nucleotide metabolism & DNA replication. Late genes are activated by the early genes & code for structural proteins. The entire cycle takes less than 10h & invariably results in cell death.
The molecular events leading to the establishment of latency have not been fully determined. Gene expression during latency is driven by the latency associated transcripts (LATs) located in the IRL region of the genome. Two LATs (2.0 & 1.5kb) are transcribed in the opposite direction to the IE gene ICP0. LATs have a role in HSV-1 reactivation from latency (Steiner et al, 1989) & the establishment of latency (Sawtell & Thompson, 1992). Two latency active promoters which drive expression of the LATs have been identified (Marconi et al, 1996) & may prove useful for vector transgene expression.
Two basic approaches have been used for production of HSV-1 vectors, namely amplicons & recombinant HSV-1 viruses. Amplicons are bacterially produced plasmids containing col E1 ori (an Escherishia coli origin of replication), OriS (the HSV-1 origin of replication), HSV-1 packaging sequence, the transgene under control of an immediate-early promoter & a selectable marker (Federoff et al, 1992). The amplicon is transfected into a cell line containing a helper virus (a temperature sensitive mutant) which provides all the missing structural & regulatory genes in trans. Both the helper & amplicon containing viral particles are delivered to the recipient. More recent amplicons include an Epstein-Barr virus derived sequence for plasmid episomal maintenance (Wang & Vos, 1996).
Recombinant viruses are made replication deficient by deletion of one the immediate-early genes e.g. ICP4, which is provided in trans. Though they are less pathogenic & can direct transgene expression in brain tissue, they are toxic to neurones in culture (Marconi et al, 1996). Deletion of a number of immediate-early genes substantially reduces cytotoxicity & also allows expression from promoters that would be silenced in the wild type latent virus. These promoters may be of use in directing long term gene expression.
Replication-conditional mutants are only able to replicate in certain cell lines. Permissive cell lines are all proliferating & supply a cellular enzyme to complement for a viral deficiency. Mutants include thymidine kinase (During et al, 1994), ribonuclease reductase (Kramm et al, 1997), UTPase, or the neurovirulence factor g34.5 (Kesari et al, 1995). These mutants are particularly useful for the treatment of cancers, killing the neoplastic cells which proliferate faster than other cell types (Andreansky et al, 1996, 1997).
A number of neurological diseases could be amenable to gene therapy by HSV-1 vectors (Kennedy, 1997). Though most attention has focused on cancers, there has been some success in Parkinsons disease by expressing tyrosine hydroxylase in striatal cells (Geller et al, 1995, During et al, 1994), thus replacing the supply of L-dopa. Federoff et al (1992) induced nerve repair following axotomy of the superior cervical ganglion, by injection of a vector expression nerve growth factor. This resulted in restored levels of tyrosine hydroxylase.
However, Wood et al (1994) observed strong inflammatory responses to HSV-1 amplicon vectors, both at the primary site of the injection & at secondary sites supplied by nerve fibres originating from area of the injection. In addition up to 20% of experimental animals may die shortly after injection with HSV-1 vector (Kucharizuk et al, 1997), though the reason is unknown. A viral protein, ICP47, has been identified, which reduces viral antigen presentation & may be employed in future HSV-1 vectors to reduce cytotoxicity (York et al, 1994).
Because of its tropism for neuronal tissue issues of cellular targeting have been largely overlooked. However, wild type HSV-1 can infect & lyse other non-neuronal cell types, such as skin (Al-Saadi et al, 1983), & it would be advantageous to target a specific subset of neurones. As HSV-1 will travel down the length of nerve efficient cellular targeting would improve its safety profile when used as a vector. Indeed a replication-restricted HSV-1 vector has been used to treat human malignant mesothelioma (Kucharizuk et al, 1997).
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