(CANCER RESEARCH 55, 4752-4756. November 1, 1995]

Advances in Brief

Treatment of Human Malignant Meningiomas by G207, a Replication-competentMu11¡mutatedHerpes Simplex Virus I1

Takahito Yazaki,2 Herbert J. Manz, Samuel D. Rabkin, and Robert L. Martuza3

Georgetown Brain Tumor Center, Departments of Neurosurgery IT. Y.. S. D. /?.. R. L M.], Microbiology and Immunology IS. D. R.f, and Pathology IH. J. M.I, GeorgetownUniversity Medical Center. Washington. DC 20007

Abstract

We have demonstrated that replication-competent attenuated mutantsof herpes simplex virus type 1 ilISV-l i have therapeutic potential for

malignant gliomas. Moreover, a recently described multiple mutant HSV(termed C207) has properties which may allow human clinical trials. G207is able to replicate within and kill cells from three human malignantmeningiomas in cell culture. In nude mice harboring s.c. human malignantmeningioma <F5), G207 can inhibit growth in a dose-dependent fashion. In

nude mice harboring intracranial subdural human malignant meningioma (F5), one injection of G207 caused significantly decreased tumorgrowth and one apparent cure with neither neurological dysfunction norpathological changes in the surrounding brain. These results suggest thatG207 should be considered for therapeutic trials in the treatment ofmalignant meningioma refractory to currently available therapies.

Introduction

Meningiomas are the most common non-neuroepithelial-derived

brain tumors, constituting approximately 22% of all primary intracranial neoplasms (1). Although most benign convexity meningiomas aresurgically curable, approximately 3-11% of meningiomas are rapidlygrowing aggressive or malignant tumors (2-5). Therapeutic methods

for these meningiomas such as radiotherapy, chemotherapy, and/orhormonal chemotherapy are not very effective, and multiple recurrences requiring multiple surgeries are common (6-8). This led us to

examine a novel therapeutic approach using genetically engineeredviruses.

Previous studies demonstrated that attenuated, single-gene mutantsof HSV-14 were effective in killing malignant glioma cells (9-14) and

other nervous system tumors (15, 16). The herpes genes deleted oraltered in these studies included: (a) thymidine kinase (UL23; Refs. 9,12,13, and 16); (b) ribonucleotide reducíase(UL39; Refs. 11, 13, and17); and (c) y34.5 gene (RLI; Refs. 10 and 14). On the basis of ourprevious studies (9-11), we developed G207(18), a multigene mutantof HSV-1 with deletions at both y34.5 loci, which are required to

replicate in the brain or cause encephalitis (19), and a lacZ geneinsertion in the ICP6 gene which is required for efficient viral growthin nondividing cells but not in many dividing cells in tissue culture(20). These mutations make reversion to wild type highly unlikely andconfer important advantages that are essential in considering clinicaltrials, including: (a) acyclovir/ganciclovir sensitivities; (b) tempera-

Received 8/7/95; accepted 9/21/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This study was supported in part by NIH Grants CA60176 and NS32677.2 Present Address: Departments of Neurosurgery and Physiology, School of Medicine,

Keio University, Shinjuku-ku. Tokyo 160, Japan.1To whom requests for reprints should be addressed, at Department of Neurosurgery,

Georgetown University Medical Center, 3800 Reservoir Road, NW, Washington, DC20007.

4The abbreviations used are: HSV-1, herpes simplex virus type I; MOI, multiplicity

of infection: X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; pfu, plaque-forming unit.

ture sensitivity; and (c) markedly attenuated neuropathogenicity. Wedemonstrated that G207 has the potential to treat human malignantglioma U87 cells both in vitro and in vii-o (18). Malignant gliomas

were initially chosen as a therapeutic target for viral therapy becauseof their lack of systemic metastasis and their lack of response toconventional therapies. Malignant meningiomas have these sameproperties as well as several others which may make them ideal forvirus-based therapies: (a) location at the brain surface; (b) minimal

invasion into brain; and (c) multiple intercellular junctions. This studyexpands the range of human nervous system tumors that might betreated with the second generation HSV-1 mutant G207 to include

malignant meningiomas and demonstrates additional important features of this strategy, such as the relationship between effective doseand tumor size. The intracranial meningioma model described in thisarticle provides a useful system for testing therapeutic approaches tomalignant meningiomas.

Materials and Methods

Viruses and Cells. G207 was constructed as described (18) and containsdeletions of both copies of the y34.5 gene as well as a lacZ insertion into theICP6 gene. Stocks of G207 were grown in African green monkey kidney(Vero) cell cultures, and virus titration was performed as described previously(9). One human malignant meningioma cell line (F5) was kindly provided byAnil G. Menon (University of Cincinnati, Cincinnati, OH; Ref. 21), and theother two human malignant meningioma cell lines (GPSM4, GPSM5) werekindly provided by Gabriel Pulido-Cejudo (Health Canada, Ottawa, Ontario,

Canada). Original tumors of these cell lines were histologically diagnosed asmalignant meningiomas. These cells were cultured in RPMI lf>4() mediumsupplemented with 10% inactivated PCS and antibiotics. F5 cells were alsomaintained by monthly passaging in the s.c. space of nude mice. Histologicaldiagnosis, immunohistochemical reactivity against S-100 and vimentin, and a

karyotypic finding of a deletion of chromosome 22 of s.c. F5 tumors were thesame as in the original tumor (5).

Cell Culture Cytotoxicity. Subconfluent monolayers of meningioma cellsin 25-cm2 tissue culture flasks were infected at a MOI of 0.1 pfu/cell while

controls were mock infected with virus buffer (150 ml NaCl, 20 mM Tris,pH 7.5). Viable cell numbers were determined by trypan blue exclusion ondays 1-6.

Animal Studies. Six-week-old female athymic BALB/c nude mice (nu/nu)

purchased from the National Cancer Institute (Bethesda, MD), each weighing12-20 g, were kept in groups of five or less, housed in sterile cages, and had

free access to autoclaved food and water. All animal procedures were approvedby the Georgetown University Animal Care and Use Committee. For surgicalprocedures, each mouse was anesthetized with an i.p. injection of a 0.25-0.30

ml solution consisting of 84% bacteriostatic saline, 10% sodium pentobarbital(1 mg/kg; Abbott Laboratories, Chicago, IL), and 6% ethyl alcohol.

s.c. Tumor Therapy and X-gal Staining. F5 tumors were removed asep-tically from the flanks of host mice, minced into 1-mm pieces, and transplanted

into additional mice for study. Mice harboring s.c. tumors (approximately 6mm in diameter) were randomly divided into two groups (n = 8/group) andtreated intraneoplastically with either 5 X IO7 pfu of G207 virus suspended in

50 fil virus buffer and DMEM (1:1) or with 50 /xl mock-infected extract,prepared from mock-infected cells using the same procedures as those used forvirus inoculum. Mice harboring s.c. tumors (approximately 10 mm in diame-

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MULTIMUTATED HSV THERAPY FOR MENINGIOMAS

ter) were randomly divided into three groups (n = 8/group). Group 1 wastreated intraneoplastically with 5 X IO6 pfu of G207 in 50 ¿ilvirus buffer andDMEM (1:1), group 2 was treated intraneoplastically with 5 X IO7 pfu of

G207 in 50 ^1 virus buffer and DMEM (1:1), and group 3 was treated withmock-infected extract (50 ¿xl)alone. The tumor diameter was determined by

external caliper measurements. Tumor growth ratio was determined by(/ X H' X /])/(/ X w X A)day(),where / is length, w is width, and h is height.

Statistical differences in growth ratio were assessed by using an unpaired i test.For pathological studies, one mouse in group 1, and three mice in groups 2

and 3 were sacrificed on day 24. Animals were perfused with 2% paraform-aldchyde-5 HIM [ethylenebis(oxyethylenenilrilo)] tetraacetic acid-2 mM magnesium chloride in 0.1 M 1,4-piperazinediethanesulfonic acid buffer (pH 7.3).The s.c. tumors were removed and stained by X-gal as described previously

(11). After being frozen on dry ice, tumors were sectioned on a freezingmicrotome. The sections (20 /im) were mounted onto gelatin-coated glassslides, stained with X-gal again overnight, and counterstained with Carmalum.

Intracranial Tumor Therapy. We established an intracranial tumormodel to simulate treatment of meningioma patients. Tumors were generatedby transplanting F5 tumor fragments 1 mm in diameter into the right frontalsuhdural space. Mice harboring tumors were randomly divided into two groups(n = 9/group). Fifteen days later, 5 x IO6 pfu of G207 in 5 ß\virus buffer and

DMEM (1:1) or mock extract (5 fil) were inoculated intraneoplastically, andanimals were followed up until death. Because of limitations in virus titer andthe larger volume that would be necessary and which could cause excessiveleakage and damage to the brain, we could not test the 5 x IO7 pfu/ml dose in

the subdural tumor model. For studies of tumor size, animals were sacrificed,perfused with 2% paraformaldehyde-5 mM [ethylenebis(oxyethyleneni-trilo)]tetraacetic acid-2 mM magnesium chloride in 0.1 M 1,4-piperazinedi

ethanesulfonic acid buffer (pH 7.3), and brains were removed with skull bone.Brains were placed in 10% formalin and then decalcified for 6 h in décalcification solution (8% hydrochloric acid, concentrated, 10% formic acid indistilled water). Microtome sections (5 firn) were mounted on glass slides andstained with hematoxylin and eosin.

Results

In Vitro Cytopathic Efficacy. To determine whether G207 couldreplicate in and destroy malignant meningioma cells, human malignant meningioma cell lines F5, GPSM4, and GPSM5 were infectedwith G207 at a MOI of 0.1. All three cell lines were efficientlydestroyed by G207 within 6 days (Fig. la), the cytopathic effectappeared on day 1 postinfection, and >99% cytotoxicity was evidenton day 6.

In Vivo Treatment of s.c. Tumors. The effect of G207 infectionon s.c. tumor xenografts was examined. F5 tumor fragments weretransplanted into BALB/c (nu/nu) mice. Growing tumors approximately 6 mm in diameter were evident within 2 weeks and were 10mm in diameter within 3 weeks, at which time each tumor was treated

with one injection of G207 or mock extract. For 6-mm tumor frag

ments, there was limited tumor growth and no significant difference intumor diameter between control and 5 X IO7 pfu of 0207-treated

tumors for the first 7 days after injection of G207. From day 10onward the tumor size between the two groups diverged (Fig. \b).When the experiment was terminated on day 28 because of the largetumor burden in the control animals, the mean tumor growth ratio wassignificantly different between G207-treated and control groups

(P < 0.001; unpaired t test; Fig. \b). Next we tested whether largertumors could also be successfully treated. When tumors were allowedto grow to 10 mm in diameter before treatment, tumor growth wasrapid from the time of treatment, and G207 was effective in adose-dependent manner (Fig. lc). On day 24 when the experiment

was terminated due to tumor burden in the controls, the mean tumorgrowth ratio was significantly inhibited (P < 0.005 with 5 X IO7 pfu,P < 0.01 with 5 X 10" pfu; unpaired / test) in G207-treated tumors

when compared to control tumors treated with mock extract. Therewas also a significant difference between the 5 X 10ft pfu of G207-treated tumors and the 5 X IO7 pfu of G207-treated tumors on day 24

(P < 0.05; unpaired t test; Fig. lc). Tumors larger than 10 mm couldnot be tested in this model system due to the large tumor burden thatwould be necessary in the control group.

To assess the spread of replication-competent virus in F5 tumors invivo, animals in the 10-mm treatment group were sacrificed on day 24after treatment. These tumors were fixed and stained with X-gal toexamine the extent of ß-galactosidase expression. Mock-infected tumors showed no ß-galactosidaseexpression (data not shown). Tumorstreated at a low dose (5 X IO6 pfu; Fig. 2, a and b) showed lacZ

expression in the area adjacent to the needle tract. In contrast, tumorstreated at doses one log higher (5 X IO7 pfu; Fig. 2, c and d) showed

lacZ expression that was more diffuse with islands of lacZ-positive

cells throughout the tumor and extending to the capsule. However, inboth cases X-gal-positive cells were seen at a distance from infected

foci, indicative of viral infection spreading a large distance from thesite of injection. Within positive foci, necrotic areas were evident.

In Vivo Treatment of Intracranial Tumors. We next studied theeffect of G207 infection on right frontal subdural F5 tumors that weretreated with G207 (5 X IO6 pfu) or mock extract 15 days after tumor

implantation. All animals treated with mock extract died by day 31. Astatistically significant increase in survival was seen in G207-treatedanimals compared with controls (P < 0.01; Wilcoxon test and Cox-

Mantel test; Fig. 3). Brains were removed from three animals at thetime of death in the control group and from three survivors in theG207-treated group who were sacrificed at day 65. Sections stained

234

Days poslmfecbon

I" I 1 1 1 1 1 1

3 6 9 12 15 18 21 24 27 30Days after treatment

5 10 15Days after treatment

Fig. 1. Effect of G207 on human malignant meningiomas in vitro and in vivo. at cytopathic efficacy of G207 in vitro. Three human malignant meningioma cell cultures were infectedwith G207 al a MOI of 0.1. The data plotted are the mean of triplicate wells, b and r, s.c. tumor growth of F5 malignant meningioma cells in BALB/c (nu/nu) mice. Mice harborings.c. F5 tumors [approximately 6 mm (b) and 10 mm (c) in diameter] were treated with G207 or mock-infected extract (n = 8/group). The mean tumor growth rate was significantlyinhibited in G207-treated tumors compared to control tumors treated with mock extract. For rapidly growing 10-mm tumors, two different viral doses were tested (c).

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MULTIMUTATED HSV THERAPY FOR MENINGIOMAS

Fig. 2. Pathological examination of G2()7-treated s.c. malignant meníngioma tumors after staining with X-gal. On day 24 postinfection, animals from 10-mm tumor fragment-implanted mice were sacrificed and tumors fixed, s.c. meningioma tumors were removed and slained wilh X-gal solution. Tumors treated at a low dose (5 x 10* più;a, and h) showedlacZ expression in the area adjacent to the needle tract. In contrast, tumors treated at doses one log higher (5 X IO7 pfu; r and d) showed lacZ expression that was more diffuse with

islands of lacZ-positive cells throughout the tumor and extending to the capsule. Bars, 4.5 mm in a and c, 1.1 mm in b and d.

1UUt90-80-70-50-40-30-20-10-Ltri-oU3*-9i^-.-

^-f

0 —¿�Mock^

0G207A

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

Days after implantation

Fig. 3. Extended survival of mice wilh inlracranial (subdural) malignant meningiomaF5 tumors treated with G207. BALB/c (nu/nu) mice with suhdural tumors (15 dayspostimplantation) received either 5 X H)*1pfu G207 or mock extract (n —¿�9/group).

G207-treated animals had a prolonged median survival and three were alive at day 65(P < 0.01 with Wilcoxon or Cox-Mantel test).

with hematoxylin and eosin revealed large tumors in all three controlanimal brains (Fig. 4, a-c). Malignant meningioma F5 cells invadedinto the skull and s.c. space. The tumors had neovascularization andcaused brain compression. Two G207-treated survivors had smalltumors in the subdural space (Fig. 4, d and e) and one had no tumor(Fig. 4/). G207-treated tumors had larger and lighter cell bodies,fewer numbers of cells, and larger numbers of necrotic cells comparedto control tumors. No inflammation, hemorrhage, necrosis, or otherpathological effects were seen in the surrounding brain tissue.

Discussion

We previously demonstrated that HSV-thymidine kinase-defi-cient mutants, y34.5-deleted mutants, and HSV-ribonucleotidereductase-deficient mutants have therapeutic potential for centralnervous system tumors (9-11, 15, 16). However, each of theseviruses has limitations that could preclude use in humans. Therefore, to provide adequate safeguards for possible clinical trials, wedeveloped a multiple gene mutant of HSV-1, G207, with deletionsat both y34.5 loci and a /acZgene insertion in the ICP6 gene (18).G207 was avirulent upon cultured primary rat neurons and astro-cytes and also upon intracerebral inoculation of mice and HSV-

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MULTIMUTATED HSV THERAPY FOR MENINGIOMAS

Fig. 4. Pathological evaluation of malignant meningioma F5 tumors implanted subdurally. Mock-injected (a-c) BALB/c (nu/nu) mice were fixed at Ihe time of death. G207-treated(d-f) BALB/c (nu/nu) mice with implanted tumors were sacrificed, perfused with fixative, and their brains with skull bone were removed. They were fixed, decalcified, and stainedwith H & E. All mock-injected animals (a-c) showed large tumors. 0207 treatment was associated with decreased tumor size (d and e) or no detectable tumor (/), and caused nopathological change in the underlying brain (d-f). n = 8/group. Bar, 2.4 mm.

sensitive nonhuman primates (18). G207 inhibited s.c. glioblas-

toma U87 tumor growth in vivo and prolonged survival of micewith intracerebral tumors. G207 provides many advantages forclinical therapy including: (a) multiple large mutations make reversion to the wild type unlikely; (h) retained sensitivity to gan-

ciclovir or acyclovir; (c) temperature sensitivity which wouldcompromise viral replication in the presence of encephalitis andfever; and (d) the lacZ gene provides a sensitive means to trackviral replication both in the tumor and outside the tumor.

We have now extended the use of G207 to the treatment ofmalignant meningiomas. In cell culture, spreading infection andmonolayer destruction occurred in all three human malignant meningiomas tested at a MOI of 0.1. Intraneoplastic inoculation of G207virus into s.c. xenografts of malignant meningioma F5 cells in nudemice resulted in a significant decrease of tumor growth rate in vivo.Using F5 cells, we were able to establish an intracranial subduralmeningioma model which had histológica! characteristics similar tohuman meningiomas. Intraneoplastic inoculation of G207 virus intosubdural xenografts of F5 in nude mice induced significantly prolonged survival, with three animals still alive on day 65 compared to100% lethality in the control group at day 31. One G207-treated

animal had no detectable tumor on histológica! examination. G207produced no detectable change in the underlying normal brain. Theseresults demonstrate that G207 has the potential to destroy malignantmeningioma in situ. Moreover, our studies with two viral doses in

larger (10 mm diameter) s.c. tumors suggest that for large, rapidlygrowing tumors, a larger dose of virus may be necessary. Since G207is an attenuated replication-competent virus, its growth is slower thanwild-type HSV. We therefore postulate that virus growth and tumor

growth are in competition and that the virus dose and spacing ofinoculations may need to be adjusted relative to the size of the tumorand its rate of growth. Additional studies in various tumor types ofdifferent sizes and growth rates are necessary.

Acknowledgments

We thank Dr. Anil G. Menon for providing F5 cells, Dr. Gabriel Pulido-

Cejudo for providing GPSM4 and GPSM5 cells, and Dr. William D. Hunter,Dr. Toshihiro Mincta, Dr. Shin-Ichi Miyatake, Kent C. New, Anu Iyer, Dale

Gibson, Dr. Joseph T. Newsome, and the Georgetown University ResearchResource Facility staff for their technical assistance.

References

1. Rachlin, J. R., and Rosenblum, M. L. Etiology and biology of meningiomas. In:O. Al-Mefty (ed.), Meningiomas, pp. 27-36. New York: Raven Press, 1991.

2. Cho, K. G., Hoshino, T., Nagashima, T., Murovic, J. A., and Wilson, C. B. Predictionof tumor doubling time in recurrent meningiomas. Cell kinetics studies with bro-modeoxyuridine labeling. J. Neurosurg., 65: 790-794, 1986.

3. Alvarez, F., Roda, J. M., P'erez Romero, M., Morales, C., Sarmiento, M. A., andBi'azquez, M. G. Malignant and atypical meningiomas; a reappraisal of clinical,

histológica!, and computed tomographic features. Neurosurgery, 20: 688-694, 1987.

4. LeMay, D. R., Bucci, M. N., and Farhat, S. M. Malignant transformation of recurrentmeningioma with pulmonary metastasis. Surg. Neurol., 31: 365-368, 1985.

4755

on June 26, 2020. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MULTIMUTATED HSV THERAPY FOR MENINGIOMAS

5. Mirimanoff, R. O.. Dosoretz, D. E., Linggood, R. M., Ojemann, R. G., and Martuza,R. L. Meningioma: analysis of recurrence and progression following neurosurgicalresection. J. Neurosurg., 62: 18-24, 1985.

6. Markwalder, T. M., Gerber, H. A„Waelti, E., Schaffner, T., and Markwalder, R. V.Hormonotherapy of meningiomas with medroxyprogesterone acetate. Immunohisto-

chemical demonstration of the effect of medroxyprogesterone acetate on growthfractions of mcningioma cells using the monoclonal antibody Ki-67. Surg. Neurol.,JO: 97-101, 1988.

7. Thomas, H. G., Dolman, C. L., and Berry, K. Malignant meningioma: clinical andpathological features. J. Neurosurg., 55: 929-934, 1981.

8. Inoue, H., Tamura, M., and Koizumi, H. Clinical pathology of malignant meningiomas. Acta Neurochir., 73: 179-191, 1984.

9. Martuza, R. L., Malick, A., Market, J. M., Ruffner, K. L., and Coen. D. M.Experimental therapy of human glioma by means of genetically engineered virusmutant. Science (Washington DC), 252: 854-856, 1991.

10. Market, J. M.. Malick, A., Coen, D. M., and Martuza, R. L. Reduction and eliminationof encephalitis in an experimental glioma therapy model with attenuated herpessimplex mutants that retain susceptibility to acyclovir. Neurosurgery, 35: 597-603,1993.

11. Mineta, T., Rabkin, S. D., and Martuza, R. L. Treatment of malignant gliomas usingganciclovir-hypersensitive, ribonucleotide reductase-deficient herpes simplex viralmutant. Cancer Res., 54: 3963-3966, 1994.

12. Jia, W. W., McDermont, M., Goldie, J., Cyander, M., Tan, J., and Tufaro, F. Selectivedestruction of gliomas in immunocompetent rats by thymidine kinase-defectiveherpes simplex virus type I. J. Nati. Cancer Insl., 86: 1209-1215, 1994.

13. Boviatsis, E. J., Scharf, J. M., Chase, M., Harrington, K., Kowall, N. W., Breakefield,X. O., and Chiocca, E. A. Antitumor activity and reporter gene transfer into rat brainneoplasms inoculated with herpes simplex virus vectors defective in thymidine kinase

or ribonucleotide reducíase.Gene Therapy, /: 323-331, 1994.

14. Chambers, R., Yancey Gillespie, G., Soroceanu, L., Andreansky, S., Chatterjee, S.,Chou, J., Roizman, B., and Whitley, R. J. Comparison of genetically engineeredherpes simplex viruses for the treatment of human malignant glioma. Proc. Nati.Acad. Sci. USA, 92: 1411-1415, 1995.

15. Market, J. M., Coen, D. M.. Malick, A., Mineta, T., and Martuza, R. L. Expandedspectrum of viral therapy in the treatment of nervous system tumors. J. Neurosurg.,77: 590-594, 1992.

16. Kaplitt, M. G., Tjuvajev, J. G., Leib, D. A., Berk, J., Pettigrew, K. D., Posner, J. B.,Pfaff, D. W., Rabkin, S. D., and Blasberg, R. G. Mutant herpes simplex virus inducedregression of tumors growing in immunocompetent rats. J. Neuro-Oncol., 19: 137-147, 1994.

17. Boviatsis, E. J., Park, J. S., Sena-Esteves, M., Kramm, C. M., Chase, M., Efird, J. T.,Wei, M. X., Breakefield, X. O., and Chiocca, E. A. Long-term survival of rats

harboring brain neoplasms treated with ganciclovir and a herpes simplex virus vectorthat retains an intact thymidine kinase gene. Cancer Res.. 54: 5745-5751. 1994.

18. Mineta, T., Rabkin, S. D., Yazaki, T., Hunier. W. D., and Martuza, R. L., Attenuatedmulti-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nat.Med., /: 938-943, 1995.

19. Chou, J., Kern, E. R., Whitley, R. J., and Roizman, B. Mapping of herpes simplexvirus-1 neurovirulence to y34.5, a gene nonessential for growth in culture. Science(Washington DC), 250: 1262-1266, 1990.

20. Goldstein, D. J., and Weller, S. K. Herpes simplex virus type 1-induced ribonucleotide reducíaseactivity is dispensable for virus growth and DNA synthesis: Isolationand characterization of an ICP6 lacZ insertion mutant. J. Virol., 62: 196-205, 1988.

21. Yazaki, T., Takamiya, Y., Costello, P. C., Mineta, T., Menon, A. G., Rabkin, S. D.,and Martuza, R. L. Inhibition of angiogenesis and growth of human non-malignantand malignant meningiomas by TNP-470. J. Neuro-Oncol., 23: 23-29, 1995.

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1995;55:4752-4756. Cancer Res   Takahito Yazaki, Herbert J. Manz, Samuel D. Rabkin, et al.   Replication-competent Multimutated Herpes Simplex Virus 1Treatment of Human Malignant Meningiomas by G207, a

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