Oncolytic Herpes Simplex Virus Engineering and Preparation
Herpes simplex virus-1 (HSV-1) is an enveloped, double-stranded DNA virus that has been used with modification as an oncolytic virus against a number of tumor types. Modifications that make HSV-1 replication-conditional, i.e., selectively divide in repli
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n Herpes simplex virus-1 (HSV-1) is an enveloped, double-stranded DNA virus with several advantages for use as an oncolytic virus: a large genome suitable for insertion of foreign genes; tropism for neural cells; a safety mechanism in its sensitivity to agents, such as ganciclovir; high titers can be generated; and it does not integrate into the host genome, so it is unlikely to be oncogenic (1, 2). These advantages have led to the use of HSV-1s genetically engineered to be replication-conditional, i.e., selectively dividing in replicating cells, as oncolytic viruses in the treatment of numerous cancer types (2–4). Here, we describe the basic structure of wildtype HSV-1 and the protocols for modification, purification, amplification, and titration of HSV-1 for use as an oncolytic virus. We also describe several specific examples of oncolytic HSVs modified with the methods presented here.
David H. Kirn et al. (eds.), Oncolytic Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 797, DOI 10.1007/978-1-61779-340-0_1, © Springer Science+Business Media, LLC 2012
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P.K. Agarwalla and M.K. Aghi
HSV-1 has four main components: a core that contains double-stranded DNA (dsDNA), an icosadeltahedral capsid, an amorphous tegument, and an outer lipid bilayer envelope with glycoprotein spikes (3, 4). The DNA is arranged as two unique sequences – Unique Long (UL) and Unique Short (US) – that are each flanked by repeat sequences. Homologous recombination at these repeat sequences results in four possible permutations of linear and inverted UL and US sequences. During infection and after cell entry mediated by surface glycoproteins, the viral genome circularizes and begins transcription of immediate early (IE) genes, which are involved with transcription regulation of the host cell and virus. The IE genes also contribute to transcription regulation of early (E) and late (L) genes (2–4). The first engineered HSV-1 oncolytic virus had a mutation in the viral thymidine kinase (TK) gene, and showed killing of glioma cells in vitro and in models of glioma in vivo (5, 6). This mutant is replication-conditional because the mutant virus can only replicate in dividing cells because only dividing cells like tumor cells express sufficient amounts of mammalian TK to complement the lack of viral TK. Unfortunately, viral expression of TK is essential for the antiviral efficacy of nucleoside analogues like ganciclovir or acyclovir. Viral thymidine kinase monophosphorylates these nucleoside analogues (viral TK is much more efficient than human nucleoside kinases at monophosphorylating antiviral nucleoside analogues), which are then further phosphorylated by cellular kinases, ultimately producing ganciclovir or acyclovir triphosphate, which are incorporated into elongating DNA chains, after which they interrupt DNA synthesis. Because of the inability to use nucleoside analogues as a safety mechanism for this first-generation, viral-TK mutated vector, significant safety concerns were raised and, in fact, neurotoxicity was seen at high doses
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