Building A Better Virus Genetic Strategies To Increase Virulence In Tumor Cells

An alternative method for augmenting the oncolytic ability of an HSV-1 7134.5 mutant serendipitously emerged from further genetic analysis. To learn if other viral components were important in regulating PKR activation and eIF2a phosphorylation, a 7134.5 deletion mutant was sequentially passed in nonpermissive cells to select for isolates with restored capacity to replicate (40). These isolates had all sustained genetic rearrangements where the Us11 72 late promoter and most of the Us12 ORF, including the AUG initiation codon, were deleted (see Fig. 4). This extragenic or second-site suppressor mutation resulted in the IE expression of Us11, a dsRNA binding protein that inhibits PKR activation, and allows 7134.5 mutants to replicate efficiently in what were previously nonpermissive cells (40-43). The significant surprise was that although the suppressor mutant was capable of restored growth in cells that failed to support the replication of the 7134.5 parent virus, it essential remained as neuroattenuated as the parental 7134.5 mutant at the doses examined (up to 2 x 107 pfu). This established that it was possible to introduce additional mutations into the genome of an HSV-1 7134.5 mutant that dramatically improve its replicative ability in cancer cells without increasing neurovirulence in mice (55).

The attenuated neurovirulence profile of the suppressor mutant coupled with its dramatically improved replication properties made it an ideal oncolytic virus candidate. The antitumor activity of the suppressor virus was directly compared with the 734.5 mutant in three independent studies that utilized an animal model of different human cancers as well as a variety of viral genetic backgrounds (56-58). All of these studies demonstrated that incorporating the suppressor mutation into a 734.5 mutant virus resulted in

Fig. 4. Genetic structure of an attenuated, oncolytic HSV-1 y34.5 null virus with an additional growth enhancing mutation. The location of the y34.5 deletion (A) and the additional extragenic suppressor mutation (B) is shown on the viral genome. The unique long (UL) region and unique short (Us) region are shown as solid lines. Repetitive regions appear as open rectangles. This neuroattenuated suppressor variant contains two mutations: (1) both copies of the y34.5 gene have been replaced with sequences encoding P-glucuronidase (A); and (2) a 583 bp deletion (A) that spans the junction region where the viral Us segment joins the TRs component (B). The Us10, Us11, and Us12 open reading frames are shown. The segment of the Us12 open reading frame that is removed by the deletion is represented as a broken rectangle. The two RNA's that are synthesized appear as arrows above the open reading frames. Promoter elements that direct the synthesis of these RNAs appear as stars and each promoter is normally associated with either the Us10, Us11, or Us12 ORF (denoted by the number 10,11, or 12 at the lower right of each star). Note that the suppressor deletion removes the endogenous late Us11 promoter and a large segment of the Us12 ORF, including the ATG codon. This allows the transcript initiating from the immediate-early Us12 promoter in the TRs to direct the synthesis of the Us11 protein. Accumulation of Us11 at immediate-early times allows the suppressor mutant to sustain protein synthesis and thus replicate in nonpermissive cells that do not support the growth of y34.5 mutants. Furthermore, IE expression of Us11 also renders the virus resistant to interferon a, whereas simple A34.5 mutants remain exquisitely sensitive.

a dramatic improvement in the ability of the virus to inhibit tumor growth. A single injection of subcutaneous human prostate cancer tumors with 106 pfu of the suppressor virus reduced tumor volume by 50% or more in 60% of the treated animals, whereas animals treated with the y34.5 mutant were indistinguishable from mock treated animals (see Fig. 5) (56). Although inhibition of tumor growth was observed in animals treated with the y34.5 mutant at 10-fold higher doses of virus, equivalent doses of the suppressor virus still proved more effective. In addition, long-term responders were only seen in animals treated with the suppressor virus (56). A second study arrived at similar conclusions using an independently constructed virus (G47D) that contained a suppressor mutation in a G207 genetic background using a different tumor model. Following two treatments with 106 pfu of G47D, 66% of subcutaneous human gliomas implanted into athymic mice completely regressed and exhibited no signs of regrowth during a 3-mo follow-up period whereas only 25% of G207 treated gliomas responded accordingly. Moreover, G47D significantly prolonged the survival of tumor bearing animals (57). Likewise, a third study found that a 7134.5 mutant expressing Us11 as an

0 4 6 8 11 13 15 19 22 25 27 29 34 days after treatment

Fig. 5. Evaluation of y34.5 mutant derivatives that express Us11 at IE times as an antitumor agent in an animal model of human prostate cancer. Balb/c nu/nu mice (n = 5 for each treatment group) harboring established, subcutaneous PC3 tumors measuring approximately 50 mm3 received a single injection containing 2 x 106 pfu of either the y34.5 deletion mutant A34.5 (▲, dotted line) the suppressor mutant SUP (■), or a virus free lysate prepared from mock infected cells (♦). Tumors were measured every 2 d for 34 d and the average normalized values reflecting relative tumor size on each day were plotted. The initial tumor volume immediately prior to treatment was normalized to a relative size of 1.0. Error bars reflect the standard error of the mean. Reprinted with permission from ref. 56.

0 4 6 8 11 13 15 19 22 25 27 29 34 days after treatment

Fig. 5. Evaluation of y34.5 mutant derivatives that express Us11 at IE times as an antitumor agent in an animal model of human prostate cancer. Balb/c nu/nu mice (n = 5 for each treatment group) harboring established, subcutaneous PC3 tumors measuring approximately 50 mm3 received a single injection containing 2 x 106 pfu of either the y34.5 deletion mutant A34.5 (▲, dotted line) the suppressor mutant SUP (■), or a virus free lysate prepared from mock infected cells (♦). Tumors were measured every 2 d for 34 d and the average normalized values reflecting relative tumor size on each day were plotted. The initial tumor volume immediately prior to treatment was normalized to a relative size of 1.0. Error bars reflect the standard error of the mean. Reprinted with permission from ref. 56.

IE protein was more effective than a simple 7134.5 deletion mutant in inducing prolonged regression of several different human tumors implanted into athymic mice. Instead of using a laboratory HSV-1 strain, the latter study introduced a 7^4.5 deletion, either alone or in conjunction with a suppressor mutation allowing for IE Us11 expression, into a freshly isolated clinical HSV-1 strain (58). Finally, in addition to overcoming the block to protein synthesis seen in cells infected with a 7134.5 mutant derivative, the suppressor mutation, by allowing the production of Us11 as an IE protein, confers interferon resistance and allows a 7134.5 mutant virus to counteract this important arm of innate host defenses (45).

Other avenues to genetically modify 7134.5 mutant derivatives have met with limited success. In one approach, the 7134.5 gene was reintroduced into the viral genome such that it is expressed from a conditional promoter active only in dividing cancer cells. Although this virus demonstrated greater oncolytic activity and remains dramatically more attenuated than wild-type HSV-1, it also was more neurovirulent (LD50 2.7 x 107 pfu) than the parental 7134.5 mutant and serves to illustrate the potential drawbacks of viruses that carry wild-type alleles of the 7134.5 gene (59,60). The oncolytic potential of an engineered virus with a single copy of the 7134.5 gene and a mutation in a repetitive genome component thought to render it non-neuroinvasive has also been explored (see Fig. 6). This strain, R7020 and its clonal derivative NV1020, was originally developed as a vaccine candidate that expressed HSV-2 glycoproteins and while non-neuroinvasive, is substantially more neurovirulent than 7134.5 deficient viruses (61,62). Although R7020 and NV1020 replicate more effectively in tumors than a 7134.5 null mutant, they are not suitable for use in CNS tumors (63). A different strategy involved replacing the 7134.5 gene with a hybrid gene encoding a fusion protein where the amino terminus of the 7134.5 protein was joined to the C-terminal segment of the rodent GADD34 gene, myD116. Whereas this recombinant was sufficiently neuroattenuated, it was unable to enhance the survival of mice with syngeneic gliomas beyond that achieved with a 7134.5 mutant virus (21).

Finally 7134.5 mutant derivatives have been constructed that express ectopic transgenes, such as a soluble version of the immunostimulatory molecule B7-1 or the

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