Since a seminal study from 1991 by Martuza et al. (2), this virus has provided a leading role in applications of oncolytic viruses to cancer therapy. Challenges regarding use of HSV-1 in the treatment of brain tumors have been defined by the fact that the wild-type virus is neurovirulent and infects/replicates/kills both dividing and quiescent cells. Such challenges have been addressed through genetic manipulations with the objective of reducing/eliminating neurovirulence and growth/replication in quiescent normal cells. Viral genes can be deleted through homologous recombination, generating HSV-1 mutants that are less virulent and replication conditional, a term used to describe viruses that replicate selectively in dividing cells. The recombinant HSV-1 viruses that have been studied as anticancer agents fall into three categories: (1) viruses deleted in P-genes involved in nucleic acid metabolism, such as uracil DNA glycosylase or the large subunit of viral ribonucleotide reductase; (2) viruses lacking both copies of the y34.5 gene; and (3) viruses with mutations in two or more different genes.

Group 1 vectors share the desirable property of being replication conditional because they only replicate when a dividing cell complements the mutation they carry in an enzyme involved in DNA synthesis. HSV-1 vectors mutated in the large subunit of ribonucleotide reductase, such as hrR3, or in the thymidine kinase gene, such as dlptk, exhibit efficacy in experimental brain tumor models and limited neurotoxicity (6). Despite showing reduced neurovirulence and the ability to infect dividing cells selectively, uracil DNA glycosylase mutants have not been studied as a single mutant in brain tumor therapy, only as part of a double-mutant HSV-1 vector

Group 2 vectors harbor deletions in y34.5 and include the vectors R3616, R4009, and 1716. These vectors display low neurovirulence. The role of y34.5 in promoting DNA replication may render a y34.5 mutant replication conditional because only actively replicating cells express the DNA repair enzymes that perform the same function for the virus as y34.5 (7). The other reason for possible tumor selectivity resides within the finding that these mutants grow better in cells with an overactive ras pathway, similar to reovirus targeting of this pathway.

The risks of using single mutant HSV-1 vectors (groups 1 and 2) include recombination with latent host virus to restore wild-type phenotype, reactivation of latent virus in the host, and suppressor mutations to restore wild-type phenotype. The risk of recombination with latent host HSV-1 has not yet been investigated. The risk of reactivation was felt to be low based on a study showing that, after adult rats were infected with wild-type HSV-1 and latency was established, intracerebral inoculation of hrR3 failed to cause HSV-1 reactivation (8). There is, however, a risk of viral mutations that restore a wild-type pheno-type based on a study showing that, when a strain of HSV-1 with a deletion of the y34.5 gene is growing, a suppressor mutation in two viral genes can occur, enabling the virus to acquire the wild-type HSV-1 phenotype of sustained late protein synthesis (9).

Concern about these risks led to the design of vectors with dual mutations (group 3). One example of a double mutant HSV-1 vector is 3616UB, generated by introducing a mutation in uracil DNA glycosylase into the y34.5 mutant R3616 (10). The enhanced safety of the double-mutant 3616UB compared with the single-mutant R3616 was demonstrated in a study in which cultured neurons could still occasionally sustain the replication of R3616, whereas no neurons supported the replication of 3616UB. The most thoroughly studied combination is mutations in hrR3 and y34.5, a combination found in two vectors, G207 and MGH-1, with the former having completed phase I safety trials. At an multiplicity of infection of 1, cultured rodent gliosarcoma cells are completely killed within 4 d of infection with single mutant hrR3, whereas the double mutants R3616 and MGH-1 result in approx 50% killing (11). Thus, the enhanced safety of double-mutant vectors may compromise their oncolytic potential.

The greater oncolytic effect of a single mutant can be combined with the reduced virulence of a double mutant by deleting ribonucleotide reductase and keeping the y34.5 gene under transcriptional control of the cell cycle-regulated B-myb promoter, creating a novel oncolytic virus, Myb34.5, that is as oncolytic as a ribonucleotide reductase single mutant and whose safety is currently being investigated (12).

Oncolytic HSV can be further modified by adding anticancer genes, thus combining viral-based with gene-based therapies. One logical selection would be to use ganciclovir, because the HSV oncolytic viruses already express thymi-dine kinase. However, combining the HSV-TK/GCV strategy with herpes oncolysis can produce complex results because GCV inhibits the propagation of herpes vectors. In gliomas, GCV-mediated oncolysis of tumor cells expressing HSV-TK exceeds GCV-mediated inhibition of hrR3 propagation (50), but in cell lines derived from colorectal carcinomas, GCV's antiviral effect predominates, and GCV promotes the survival of hrR3-infected cells in culture (51). This distinction may be attributable to differences in these tumors' levels of gap junctions. When using HSV-TK-positive herpes vectors, after allowing sufficient time for intratumoral vector spread, GCV could be administered as a safety-ensuring agent that blocks vector spread beyond the tumor. If the tumor happens to express high levels of gap junctions, an additional GCV-mediated oncolysis will occur.

Replacement of the large subunit of HSV-1 ribonucleotide reductase with the CYP2B1 gene has led to the generation of an HSV-1 vector, rRp450, that can kill tumor cells through three modes—viral oncolysis and rendering infected cells sensitive to CPA and GCV. Subcutaneous tumors established from glioma cell lines in immunodeficient mice only regress when treated with rRp450, CPA, and GCV (67).

Oncolytic HSV-1 vectors are being employed in three clinical trials, all designed to treat cancer and two designed to treat brain tumors, as of September 2001 (3).


This single-stranded RNA virus commonly infects and replicates in anterior horn motoneurons, producing devastating paralytic illnesses in humans. However, deletion of one region of the viral genome and substitution with a sequence from human rhinovirus significantly attenuates this virus, so that it cannot grow in neurons, yet still infects/grows in and kills glioma cells. In another application, poliovirus was disabled so that it would infect tumor cells/grow in them and kill them, but the produced viral progeny was then unable to infect additional cells further.

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