Gene Therapy (2000) 7, 867–874 2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00

www.nature.com/gt

ACQUIRED DISEASES RESEARCH ARTICLE

Conditionally replicating herpes simplex virus mutant,G207 for the treatment of malignant glioma: results ofa phase I trial

JM Markert1, MD Medlock2, SD Rabkin2, GY Gillespie1,3, T Todo2, WD Hunter2, CA Palmer4,F Feigenbaum2, C Tornatore5, F Tufaro6 and RL Martuza2

University of Alabama at Birmingham: 1Department of Surgery, Division of Neurosurgery; 3Department of Microbiology,4Department of Pathology, Division of Neuropathology; Georgetown University Medical Center: Departments of 2Neurosurgery and5Neurology; 6University of British Columbia Departments of Microbiology and Immunology, and Neurovir Therapeutics, Inc, SanDiego, CA, USA

G207 is a conditionally replicating derivative of herpes sim-plex virus (HSV) type-1 strain F engineered with deletionsof both g134.5 loci and a lacZ insertion disabling the UL39gene. We have demonstrated the efficacy of G207 in treatingmalignant glial tumors in athymic mice, as well as the safetyof intracerebral G207 inoculation in mice and in Aotus nan-cymai. We sought to determine the safety of G207 inocu-lation into cerebral malignant glial tumors in humans. Criteriafor inclusion into this dose-escalation study were the diag-nosis of histologically proven malignant glioma, Karnofskyscore >70, recurrence despite surgery and radiation ther-apy, and an enhancing lesion greater than 1 cm in diameter.

Keywords: HSV; glioblastoma; anaplastic astrocytoma; human; gene therapy

IntroductionMalignant gliomas are the most common primary malig-nant brain tumors, and are almost universally fataldespite aggressive therapies including surgery, radio-therapy and chemotherapy. Patients with glioblastomamultiforme (GBM), ie WHO grade IV astrocytoma, havea median survival of 12–18 months from initial diagnosisand 6–9 months after recurrence.1,2 Patients with anaplas-tic astrocytomas (AA), ie WHO grade III lesions, livelonger with a median survival of 36–40 months afterinitial diagnosis and 12–18 months after recurrence.3,4

Standard management of these tumors includes biopsyand/or tumor resection, followed by external beamradiotherapy, with treatment doses of approximately6000 cGy. Partial responses to chemotherapy are seen inapproximately 30% of tumors, but no significant changein mortality has been shown with this treatment.1 Due tothe lack of success with these standard treatments, recentefforts to improve survival have included various bio-logic therapies including monoclonal antibodies,immunotherapy and gene therapy.

Correspondence: MD Medlock, Georgetown University Neurosurgery,Pasquerilla One, 3800 Reservoir Road NW, Washington, DC 20007, USAReceived 8 February 2000; accepted 10 March 2000

Serial magnetic resonance images were obtained for volu-metric analysis. The trial commenced at a dose of 106

plaque forming units (p.f.u.) inoculated at a single enhancingsite and was completed when the 21st patient was inocu-lated with 3 × 109 p.f.u. at five sites. While adverse eventswere noted in some patients, no toxicity or serious adverseevents could unequivocally be ascribed to G207. No patientdeveloped HSV encephalitis. We found radiographic andneuropathologic evidence suggestive of anti-tumor activityand long-term presence of viral DNA in some cases. GeneTherapy (2000) 7, 867–874.

We have concentrated our efforts on the developmentof genetically engineered herpes simplex virus type 1(HSV-1) for the treatment of malignant glioma. HSV-1 isknown to be able to grow in neural tissue and we hypo-thesized and demonstrated that HSV-1 could growwithin and kill tumors derived from nervous systemcells.5 However, wild-type HSV-1 can cause a fatal,hemorrhagic, necrotizing encephalitis in humans. Ourstudies led to the development of G207, a conditionallyreplicating HSV-1. G207 contains mutations in both cop-ies of the virus’ diploid g134.5 gene, as well as a disablinglacZ insertion in UL39, the gene encoding the large sub-unit of the viral ribonucleotide reductase.6 As a result ofthese mutations, the intracerebral inoculation of G207into HSV-sensitive murine and simian primates is notpathogenic.6–8 These models mimic the most susceptiblehuman populations. Despite these mutations, G207retains anti-tumor efficacy in a wide variety of in vitroand in vivo gliomas6 and other tumor models.9–11 Thecombination of promising safety and efficacy profiles ledus to explore G207 as a therapeutic agent for malignantgliomas in a phase I, dose escalation study designedto determine the safety of stereotactic inoculation ofthis genetically engineered HSV-1 for the treatment ofrecurrent malignant glioma.

G207 clinical trialJM Markert et al

868

Gene Therapy

Results

Patient characteristicsTwenty-one patients were enrolled in the study betweenFebruary 1998 and May 1999 (Table 1). The mean age was54.1 years (range, 38–72). Fifteen men and six womenwere treated. Sixteen patients entered the study with adiagnosis of GBM. The mean age of GBM patients atdiagnosis was 56.1 years (range, 38–72). Patient No. 3originally was diagnosed with a GBM although a sub-sequent resection yielded tissue consistent with a gliosar-coma. This patient is included in the GBM group below.There were four patients with anaplastic astrocytoma andone with an anaplastic mixed glioma, hereafter groupedwith the AA patients. The mean age of AA patients atdiagnosis was 48 years (range, 46–54). Ten tumors werelocated primarily in the right hemisphere, 10 primarilyin the left hemisphere, and one was characterized asbilateral. Six tumors predominantly involved one frontallobe, five were parietal, three temporal, and one occipital;the remaining six had multilobar tumors. Seventeenpatients had undergone craniotomy for debulking andfour had undergone stereotactic biopsy without debulk-ing before inoculation. All patients had undergone pre-vious external beam radiotherapy with a minimum bio-logic effective dose of 5000 cGy. Ten patients hadundergone chemotherapy with one or more agents.

The date of original glioma diagnosis preceded G207treatment by a mean of 13.9 months (range, 2–65). InGBM patients, the diagnosis date preceded G207 treat-ment by a mean of 9.4 months (range, 4–15), while in AApatients, the diagnosis date preceded G207 treatment bya mean of 28.0 months (range, 2–65).

Table 1 Demographics

Patient Dose Age Pathology Location Time from Time fromNo. (p.f.u.) (years)/ diagnosis inoculation

Gender to to deathinoculation (last(months) follow-

up)(months)

1 1 × 106 54 m AA LP 65 62 1 × 106 46 m AA BF 20 133 1 × 106 54 m GBM LP 4 124 1 × 107 60 f GBM LF 12 105 1 × 107 46 f AA L T-P 2 (19)6 1 × 107 50 m GBM RT 8 67 3 × 107 62 m GBM R F-T 12 58 3 × 107 52 f GBM RF 9 (17)9 3 × 107 72 f GBM LF 12 5

10 1 × 108 60 m GBM RF 11 511 1 × 108 57 m GBM RT 11 312 1 × 108 63 f GBM LP 6 713 3 × 108 69 m GBM LT 10 214 3 × 108 48 f AA L F-P 33 115 3 × 108 38 m GBM RO 9 616 1 × 109 60 m GBM LP 6 1117 1 × 109 56 f GBM LF 5 (8)18 1 × 109 46 m AA R P-T 20 419 3 × 109 43 m GBM R F-T 12 (7)20 3 × 109 51 m GBM RF 15 521 3 × 109 50 m GBM R F-T 9 5

L, left; R, right; F, frontal; T, temporal; P, parietal; O, occipital.

Neurological statusThe mean mini-mental status examination (MMSE) scorepre-inoculation was 28.4 (s.d. 2.0). The mean MMSE scoreat day 4 was 27.4 (s.d. 5.1) and at 1 month was 27.4 (s.d.5.48). The mean MMSE score was 27.5 (s.d. 5.6) in the 12patients remaining in the study at 3 months.

The mean Karnofsky score pre-inoculation was 84.3(s.d. 12.5) and at 1 month was 83.8 (s.d. 13.6). The meanKarnofsky score was 81.7 (s.d. 18.5) in the 12 patientsremaining in the study at 3 months. An improvement inKarnofsky score was observed in six of 21 (29%) patientsat some time after inoculation.

Antibody status and conversionBefore inoculation, 14 of 19 patients were positive forHSV-1 antibody and five were negative. Two patients(Nos 14 and 19) did not have the presence or absence ofserum anti-HSV-1 antibodies determined before entryinto the protocol (Table 1). Patient No. 21, who wasinjected at the highest dose level, was the only HSV-1seronegative patient who became seropositive afterG207 treatment.

Patient No. 12 had a saliva culture positive for HSV-1at the 3 month follow-up visit. X-gal staining of this sam-ple was negative for lacZ expression. Southern blothybridization was carried out on this sample using threeenzymes and demonstrated that g 34.5 and ICP6 werepresent. Also, the cultured virus contained variousrestriction site polymorphisms that were not present inHSV-1, strain (F), the parent virus of G207 (NeurovirTherapeutics Inc). These findings indicate that the HSV-1 recovered from the saliva culture did not originate fromthe G207 inoculation. All other HSV cultures werenegative.

MRI tumor volumetricsMRI evaluations were performed using an eigenvaluealgorithm to minimize the introduction of interobservervariability or other bias.12 This algorithm measuredenhancing tumor volumes only, which was thought torepresent the viable portion of the tumors. A singlepatient had a pacemaker and underwent CT scans andvolumetric MRI data could not be performed. The meanenhancement volume was 39 cc before G207 inoculation,43 cc at 4 days, 55 cc at 1 month, 64 cc at 3 months, 26 ccat 6 months (Table 2). Six of 20 patients had a decreasein their enhancement volume between the preoperationscan and the 1 month post-inoculation MRI. Eight of 20patients had a decrease in their enhancement volumebetween the 4 day scan and the 1 month post-inoculationscan (Figure 1). Due to the variability in the time periodbetween the pre-inoculation scan and the inoculationdate, significant growth occurred in some tumors beforeinoculation. Therefore, the 4 day post-inoculation MRI is

Table 2 MRI volumetric data

MRI Number Mean (cc) Range (cc)

Pre-inoculation 20 39 3–904 day 20 43 4–1181 month 20 55 3–1543 month 13 64 2–2496 month 3 26 1–61

G207 clinical trialJM Markert et al

869

Figure 1 Individual patients’ MRI volumes are grouped by cohort. The x-axis is the number of days from injection. The y-axis is the growth ratio asdefined by the enhancing tissue volume, after inoculation divided by pre-inoculation. Volumes were calculated using an eigenvalue algorithm as describedin the text.

probably the best baseline for assessing any changes inenhancement that might be due to G207.

Patient No. 18 had a 25% decrease in enhancing massvolume between the 4 day (80 cc) and 1 month (61 cc)MRI. Patients 3 and 4 had prolonged decreases inenhancement volume at the site of inoculation lasting 5–9 months. Patient No. 3 developed growth of a satellitelesion approximately 2–3 cm from the initial inoculationsite. This lesion was treated with a second inoculation 11months after the first inoculation. The satellite lesion washistologically a gliosarcoma at autopsy. Patient No. 4 isdescribed in more detail below.

Progression and survivalAs of the time of submission four patients (AA 1, GBM3) remain alive a mean of 12.8 months (range, 7–19) fol-lowing inoculation. Mean time from inoculation to pro-gression was 3.5 months (range, 0–20; n = 21). Excludingsurviving patients, mean time from inoculation to deathwas 6.2 months (range, 1–13, n = 17). Mean survival fromdate of diagnosis for GBM patients was 15.9 months(range, 12–22, n = 13). Mean survival from date of diag-nosis for AA patients was 40.5 months (range, 24–71,n = 4). After G207 inoculation and subsequent pro-gression of tumor, four patients received chemotherapyand five patients underwent surgical debulking. PatientNo. 6 developed a liver metastasis of his GBM. No otherpatient developed a clinically or autopsy-detectedmetastasis.

Adverse eventsPatients 4, 12 and 14 underwent post-inoculation stereo-tactic biopsy to investigate clinical deterioration.

Patient 4’s tumor (1 × 107 p.f.u.) was histologically aGBM. Following G207 inoculation MRI showed a pro-gressive decrease in the enhancing mass with decreased

Gene Therapy

mass effect. There was no evidence of increased T2 signalto suggest inflammation (Figure 2). She was driving andliving independently with a Karnofsky score of 100. Ninemonths after inoculation she presented with an acute lossof consciousness. Stereotactic biopsy showed onlychanges consistent with necrosis. She remained comatosefor approximately 1 month before expiring. The autopsyrevealed pneumonia and subacute infarctions of bothmiddle and left anterior cerebral artery territories. Shehad no evidence of residual tumor or inflammation andHSV immunostaining was negative.

Patient 12’s tumor (1 × 108 p.f.u.) was histologically aGBM. She was stable following G207 inoculation, butapproximately 3 months later demonstrated a deteriorat-ing Karnofsky score, new focal deficits, and a progressivedecrease in mental status. This deterioration was notexplained by tumor progression on MRI. Stereotacticbiopsy revealed histologic evidence of tumor that wasadjacent to normal white matter. No evidence of HSVwas seen by immunostaining, and there was no evidenceof new inflammatory changes when compared with herprevious resection specimen. She continued to deterio-rate, and died 7.5 months after treatment. She had pre-viously participated in another phase I trial examininga radiation sensitizing agent. Her family refused furtherimaging and autopsy.

Patient 14’s tumor (3 × 108 p.f.u.) was histologically anAA. She developed an acute change in mental status anddysphasia within 24 h of inoculation, but developedneither fever nor an abnormal elevation in white bloodcell count. Repeat MRI scan performed 1 day after inocu-lation demonstrated progression of her enhancing mass,but no evidence of edema or hemorrhage. A minimalamount of hemorrhage was seen in the region of theneedle track as well as increased enhancement on an MRIdone 8 days after inoculation, but no change in the

G207 clinical trialJM Markert et al

870

Gene Therapy

Figure 2 Patient No. 4 MRIs showing regression. Patient No. 4 T1-weighted gadolinium enhanced MRI images. Volumes pre-injected: 21.5 cc; 1 month23.4 cc; 6 months 21.1 cc; 9 months 17.1 cc.

amount of edema. The increase in enhancement (from50 cc to 117 cc) appeared to occur primarily in the pre-viously nonenhancing portion of the tumor and not inthe surrounding brain parenchyma. A stereotactic biopsywas performed 14 days after inoculation to verify that noencephalitis had developed. Three biopsy specimenswere taken; one in the region of G207 inoculation, as wellas 1 and 2 cm distant. These demonstrated viable tumorwith increased cellularity compared with her pre-treat-ment biopsy, consistent with tumor progression. Therewas no evidence of inflammatory changes or other find-ings to suggest encephalitis. Immunostaining of all threespecimens for HSV was negative, as was viral culture forHSV. PCR was negative for lacZ sequence and positivefor HSV-1 sequences pol and gB. Inadequate sampleremained for retesting this result. The patient improvedwith dexamethasone administration and became ambu-latory but remained confused and somewhat dysphasic.She died 37 days after G207 inoculation, but no autopsywas obtained.

Patients 6 and 19 had temporal lobe masses injectedand both required extensions of their post-inoculationhospital stays. Patient 6 had mild increased weakness

and tumor progression seen on MRI scan. His increasedweakness responded promptly to increased dexame-thasone administration. Patient 19 developed a slowedaffect within 12 h of inoculation and a CT scan at 24 hdemonstrated punctate hemorrhages associated with thefive inoculation sites. He responded to increased dexame-thasone and regained his pre-operative level of functionwithin 2 days.

Tissue analysisBiopsy specimens from six patients were analyzed afterinoculation. Three specimens were from the post-inocu-lation diagnostic biopsies described above. Specimensfrom four patients (Nos 1, 3, 7 and 8) were from re-resec-tions performed 60, 157, 56 and 97 (mean 93) days afterinoculation, respectively.

Patients 3 and 8 had specimens that were positive forboth HSV-1 and lacZ sequence by PCR, indicating thatG207 DNA was present in the specimen. These two posi-tive specimens were from resections done 157 and 56days after inoculation at dose levels of 1 × 106 and3 × 107 p.f.u.

The brains of five patients (Nos 2, 3, 4, 11 and 20) were

G207 clinical trialJM Markert et al

871available for analysis at autopsy (four were formalinfixed, the fifth was frozen at −80°C). No evidence ofencephalitis, white matter toxicity, or inflammatorychanges was present. HSV-1 immunostaining was nega-tive. Patient No. 2 had a bifrontal, ‘butterfly’ glioma. His-tological examination of three of these five brainsrevealed that their tumors remained localized to the leftinternal capsule and thalamus (No. 3), right temporallobe (No. 11), and right frontal lobe (No. 20). PatientNo. 20 was diagnosed at the age of 50 and was injected14 months after his original diagnosis of a GBM. Pro-gression of disease led to an autopsy 5 months afterinoculation. His right frontal tumor showed the necrosisand pseudopallisading typical of a GBM. There was noevidence of infiltration into or across the corpus callosumin four of these five patients. Patient No. 4 died due to astroke 9 months after inoculation and had no evidence ofGBM on serial sections of the brain.

No specimen from any patients had evidence ofencephalitis on routine hematoxylin and eosin staining,and no HSV-1 antigen was detected by immunostaining.

DiscussionWe report the results of the first North American humantrial of a herpes simplex virus specifically engineered forthe treatment of intracerebral malignancy.13 We haveshown that doses up to 3 × 109 p.f.u. of this conditionallyreplicating virus can be inoculated safely into braintumors without the development of encephalitis. Nopatients developed MRI, laboratory or pathologic evi-dence of encephalitis. We are still following our four sur-viving patients and have not documented any evidenceof long-term toxicity from this treatment. While somepatients developed complications frequently seen withmalignant glioma, including death, none of these compli-cations or deaths could be unequivocally ascribed toG207.

However, it is not always possible to know the causeof adverse events in this group of patients. Two patientsfrom our study merit specific discussion. Patient No. 6developed a liver metastasis, which was first identifiedafter treatment with G207. While rare, extraneural met-astases are seen with GBM in 0.1–0.5% of cases, usuallyin the lung, lymph nodes, bone and liver.14,15 While thepossibility that this was related to G207 therapy cannotbe excluded, no other metastatic lesions were found inour trial.

A second patient, No. 14, developed mental statuschanges and dysphasia which could have been due tosurgical trauma, tumor edema, or viral toxicity. However,fever was not present. MRI showed an increase inenhancement, which appeared to correspond to tumor-ous regions that had not enhanced before G207 inocu-lation. No increase in the amount of hyperintense T2 sig-nal was appreciated after inoculation. Although biopsyspecimens taken from three sites did not show encepha-litis or HSV antigen, and cultures were negative for HSVfrom all three sites, stereotactic biopsies have limitations.Unfortunately, no autopsy was obtained, so a definitivedetermination of the reason for her decline cannot bemade. While the possibility of G207-related toxicity can-not be excluded, six patients were treated at higher doselevels (including one AA patient) without any similarevents.

Gene Therapy

The HSV-1 antibody status of our patients mirroredthat of the general population.16 Of the five patients nega-tive for HSV-1 antibody before inoculation, patientNo. 21 injected with 3 × 109 p.f.u., seroconverted. Thissuggests that despite the known immunosuppressiveeffects of malignant glioma and chronic dexamethasonetreatment, an immune response to HSV-1 can bemounted by at least some fraction of malignant gliomapatients after treatment at the highest dose levels of G207.

Neuropathologic evaluation was performed on fiveautopsied brains after G207 treatment. One GBM patientwho had been treated previously with surgery and radi-ation had no evidence of residual or recurrent glioma atautopsy. The tumors of three other patients remainedlocalized to a single geographic region. This finding isvery atypical in malignant gliomas, which by microscopicexamination almost always extend (often by single cellsseen scattered through sections or in subpial locations)by infiltration into the underlying white matter, generallycrossing into the contralateral hemisphere via thecorpus callosum.

Malignant glioma was chosen for study due to its lackof response to traditional therapeutics and near uniformlethality. Ninety percent of gliomas recur locally, within2 cm of their resection margin, and systemic metastasesare rare. As a result, these brain tumors are excellent tar-gets for intervention using conditionally replicatingviruses such as HSV-1 which replicate and kill tumorcells, while sparing normal nervous tissue.

Currently, many centers are investigating a variety ofvectors for use in treating malignant gliomas. The vectorsmost widely studied to date have been retrovirus andadenovirus. Retroviruses are difficult to produce at hightiters, require packaging cell lines, have significant limitsto genetic insert size, have a potential for insertionalmutagenesis and do not transduce non-dividing cells,including quiescent tumor cells.17 Adenoviruses do notrequire a producer cell line and can generate relativelyhigh levels of foreign gene product, but this is usuallydue to transient gene expression, and may be associatedwith an overwhelming inflammatory response. Further-more, adenoviruses are promiscuous in their host cellrange, and are also limited in the size of genetic insertthat may be utilized. Most adenovirus vectors now inclinical trials are non-replicating.17 Recently, a replicatingadenovirus (Onyx-0115) has been tested in head and neckcancers and has entered testing in human glioma therapy.

HSV-1 is a DNA virus with a well-studied genome.Approximately 90% of the adult population has acquiredantibodies due to previous exposure to the virus.18 Whileneurovirulent in its wild-type form, a variety ofmutations can be introduced which abrogate this toxicity.The neurovirulence gene, g134.5, is present in two copiesin the wild-type virus. Both copies can be deleted whileallowing the virus to maintain its anti-tumor effects.19

Additionally, HSV-1 requires the enzyme ribonucleotidereductase for replication. Viruses defective in ribonucleo-tide reductase can still replicate in rapidly dividing cellsby presumably using the cellular ribonucleotidereductase provided in trans. Such viruses cannot, how-ever, replicate and lyse post-mitotic cells such as neuronsand most other cells making up the vast majority of nor-mal adult brain.17,20

A major and unique advantage of HSV-1 as a vectorfor treating gliomas and other malignancies is the avail-

G207 clinical trialJM Markert et al

872

Gene Therapy

ability of effective anti-viral medications already in clini-cal use (eg acyclovir, ganciclovir), which could be utilizedif excessive toxicity resulted from treatment with the vec-tor. G207 contains deletions in both copies of the virus’diploid g134.5 gene and a lacZ insertion disabling thelarge subunit of the viral ribonucleotide reductase.6 As aresult of the ribonucleotide reductase mutation, G207 ishypersensitive to these antiviral compounds when com-pared with wild-type HSV-1.6 G207 has been studied ina variety of rodent and primate models designed todetermine both its efficacy as an antiglioma agent as wellas its safety.6–8 Antitumor effects and increased survivalhave been seen in immunocompromised mice withhuman xenografts, as well as in other murine tumormodels in immunocompetent animals. It is evident thatthe oncolytic effect of the virus accounts for at least aportion of its anti-tumor effects; other studies suggestthat an anti-tumor immune response can also be inducedby the virus in animal models.10,11,21

Safety of G207 has been demonstrated by intracerebralinoculation in naive animals including sensitive mousestrains (BALB/c, A/J strain) and highly susceptible pri-mates (Aotus nancymai). No evidence of encephalitis,either clinically or histologically, has been seen in theseanimals with G207 doses up to 109 p.f.u. In contrast,intracerebral inoculation of wild-type virus results inlethality in both animal models at doses of 103 p.f.u.Long-term evaluation of Aotus has shown no clinical evi-dence of G207-related toxicity for as long as 3 years afterinoculation. Further Aotus studies confirmed that G207was not detectable in other organs via direct culture orat autopsy.7

Our phase I study necessarily has some limitations.First, to minimize surgically-induced morbidity whichcould confound the interpretation of results, a biopsy wasnot performed before treatment. As a result, some or allof the enhancing mass in some patients could be radi-ation necrosis rather than viable tumor. We elected notto perform a pre-inoculation biopsy or resection becausepost-surgical MRI changes may have masked the MRIchanges of subclinical viral infection. We were initiallyconcerned that pre-inoculation surgery would havecaused gliosis, inflammation and vascular repair, provid-ing proliferating cells that could support productiveinfection of non-neoplastic tissue.

Second, we were not able to prove in vivo replicationof the virus due to ethical concerns over inoculationimmediately preceding operation for resection. All of ourresection specimens were obtained following disease pro-gression, and thus were not obtained within a time-frameduring which we would expect to see unequivocalevidence of G207 replication.

Third, to minimize potential surgical complicationsthat might have interfered with evaluation of G207safety, single site inoculations of G207 in minimal vol-umes (0.1–0.3 cc) were performed, except at the highestdose level (0.2 cc in each of five sites) where volumerequirements necessitated multiple site inoculations.Limited viral distribution within the tumor may haveinterfered with efficacy.

Our study suggests that G207 can be safely inoculatedinto human brain tumors at doses up to 3 × 109 p.f.u.Further studies with this agent in the treatment of humanglioma are warranted.

Table 3 Dose escalation schedule

No. patients Dose (p.f.u.) Volume per locus No. loci(cc)

3 106 0.1 13 107 0.1 13 3 × 107 0.1 13 108 0.1 13 3 × 108 0.1 13 109 0.3 13 3 × 109 0.2 5

Materials and methods

Trial designA phase I dose escalation design was employed using amodified halfılog incremental scheme (Table 3). An initialdosage level of 106 p.f.u. or active HSV-1 particles, waschosen based upon preclinical efficacy studies in miceand safety data obtained in both mice and primate stud-ies.7,8 At each dose level, patients were entered in cohortsof three, with a 10-day waiting period before inoculationof the next patient within each cohort. After accrual toeach cohort was completed, we waited 28 days to observefor potential acute toxicities before proceeding with doseescalation. These waiting periods allowed assessment ofpossible acute toxicity before proceeding with the nextinoculation. This protocol and its amendments wereapproved by the Institutional Review Boards of bothGeorgetown University Medical Center and the Univer-sity of Alabama at Birmingham, and reviewed by theRecombinant DNA Advisory Committee of the NationalInstitutes of Health and the Food and Drug Adminis-tration.22

Inclusion and exclusion criteriaPatients included in the trial had MRI or CT evidence ofrecurrent or progressive malignant glioma despite stan-dard therapy (Table 4). Standard therapy was defined as

Table 4 Enrollment criteria

I Biopsy-proven glioblastoma multiforme, anaplasticastrocytoma or gliosarcoma.

I One centimeter of enhancing tissue that would not requiretransgression of the brainstem, basal ganglia or ventricleduring a stereotactic inoculation.

I Failed radiation therapy (>5000 cGy) more than 4 weeksbefore inoculation.

I Failed surgery more than 4 weeks before inoculation.I No chemotherapy within 6 weeks of inoculation.I Karnofsky score greater than or equal to 70.I Willing to practice birth control.I No pregnant or lactating females.I No history of encephalitis, multiple sclerosis or other CNS

infection.I HIV seronegative.I Tumor growth on MRI following the last treatment

undertaken.I Normal hematologic, hepatic and renal function.I No prior participation in a viral therapy protocol.I No active aphthous ulcers.I No change in steroid dose within 2 weeks of the inoculation.I No current treatment with any medication active against HSV

(eg acyclovir).I 18 years of age and able to give informed consent.

G207 clinical trialJM Markert et al

873surgery and/or biopsy followed by external beam radio-therapy (>5000 cGy biologic effective dose). Somepatients received chemotherapy before enrollment, butno chemotherapy could be given within 6 weeks of treat-ment with G207 inoculation to prevent confounding oftrial results. Additionally, patients could not havereceived debulking surgery within 4 weeks of inoculationto minimize the possibility of G207 viral infection of cellsactive in the normal reparative response. All patients hadhistopathological confirmation of their diagnosis bycentral review.

Patients underwent screening with a history, physicalexamination, chemistry profile, complete blood count,urinalysis, HIV serology, HSV-1 and HSV-2 antibody tit-ers, HSV-1 cultures of saliva and blood, serum beta-HCG,electrocardiogram, chest radiograph, and volumetricmagnetic resonance imaging (MRI). Screening volumetricMRI included axial imaging: fast spin echo, FLAIR, andT1 (pre- and post-gadolinium). T1-weighted images inthe coronal and sagittal planes were also included. Theimage matrix was 256 × 192 and the field of view was200 mm. Scan thickness was 10 mm with interscan spac-ing of 2 mm. Laboratory studies were performed byCovance Laboratories (Indianapolis, IN, USA) unlessotherwise specified.

The date of progression was defined as the time atwhich there was clinical progression, increased steroiddose dependence or any increase in enhancement onimaging. Toxicities were rated using the classificationdeveloped by the National Institutes of Health.23

Virus handling and operative procedureProduction of G207 following current Good Manufactur-ing Practices (cGMP) was performed under contract atBioReliance Corporation (formerly Magenta, Rockville,MD, USA), using a process developed at NeuroVir Thera-peutics Inc (Vancouver, BC, Canada). The upstream pro-cess was roller bottle based and utilized a freeze–thawstep to release virus from cells. Major purification andconcentration steps in the downstream process were achi-eved using size exclusion chromatography and ultracen-trifugation, respectively. This process yielded approxi-mately 3 × 1010 p.f.u. final product per 100 roller bottlerun with a specific activity of >5 × 108 p.f.u./mg. Thevirus was then stored in 1.0 ml cryovials containing0.12 ml of G207 suspended in the storage buffer D-PBS/10% glycerin at −60°C. Immediately before surgery,the cryovial with virus was removed from storage andthawed in a bath at 37°C, then centrifuged for 10 s. Analiquot was diluted with sterile saline for injection (USP)to the concentration appropriate for each dose cohort,then transported to the operating room on ice. All hand-ling of the virus and materials potentially contaminatedwith virus was conducted in accordance with BiosafetyLevel 2 precautions.

On the morning of surgery, the patients underwentCosman-Roberts-Wells stereotactic head frame appli-cation under local anesthesia, followed by a contrast-enhanced CT scan and target localization. All targetswere chosen by the neurosurgeon/investigator at eachstudy site, with the goal of injecting virus in the enhanc-ing (probably actively growing) regions and avoidinginoculation of central non-enhancing (probably necrotic)regions of tumor.

The patients were then taken to the operating room

Gene Therapy

where the virus was stereotactically injected. Needleswith stylets (0.7 mm diameter, 220 mm length, Radionics,Burlington, MA, USA) were used for each inoculation.The volume of each needle, as well as the volume of thedead space present during each inoculation, was calcu-lated pre-operatively to allow for precise dosing. Targetpoints were calculated in a standard fashion. A slow,deliberate administration schedule was adopted to avoidreflux of virus into the needle and along the needle track.The needle was passed to the target, the stylet wasremoved, and the virus was injected slowly over a 2-mininterval. Following inoculation, the syringe and needlewere left in place for 2 min to allow interstitial diffusionof the inoculum. The stylet was then reinserted over 2min. Finally, the needle was withdrawn over 2 min.Patients in the final cohort underwent five inoculationsaccording to the same procedure. These five loci wereselected by the surgeon to include the superior andinferior poles, as well as three additional equatoriallocations of enhancement. No significant egress of theviral preparation was noted along the needle tract dur-ing surgery.

The patients were observed in the hospital for 4 days,and a MRI scan was obtained before discharge. Encepha-litis was considered if a fever greater than 38°C waspresent for greater than 48 h, deterioration in neurologi-cal status occurred, or there was progressive hemorrhageand/or swelling inside or around the inoculated enhanc-ing tissue. When deemed appropriate by the surgeon,stereotactic biopsies were performed, and examined forhistologic and immunohistochemical evidence of HSVencephalitis. Polymerase chain reaction (PCR) was per-formed to look for evidence of HSV and lacZ stainingwhen appropriate. Empiric intravenous acyclovir admin-istration was planned for patients whose clinical patternwas suspicious for HSV encephalitis.

Patients were evaluated with history, physical examin-ation, HSV antibody titer, saliva and blood HSV cultures,as well as MRI pre-operatively and at 4 days, 1 month,3 months, 6 months and 1 year following inoculation.Neurological examination included mini-mental statustesting, Karnofsky grading, and neurological assessment.Patients had sera and saliva cultured at each follow-upvisit for evidence of HSV shedding. Follow-up MRI’swere classified as complete response, partial response,stable disease or progressive disease. Patients whoshowed signs of clinical or MRI disease progression weredeclared a treatment failure and cleared to pursueadditional therapy. Even after disease progression,patients were included in clinical, MRI and autopsyfollow-up whenever possible.

Note added in proofSince acceptance of our manuscript, we have been noti-fied of the death of patients 17 and 19, 9 and 10 monthsfollowing injection.

AcknowledgementsWe dedicate this paper to the memory of James MacDow-ell ‘Mac’ Markert, III. We wish to thank Thomas Mikkel-son, MD for performing the volumetric MRI measure-ments; John Gnann, MD for serving as study monitor;Jeff Ostrove, PhD and Paul Johnson, PhD for coordinat-ing virus preparation and performing X-gal staining and

G207 clinical trialJM Markert et al

874

Gene Therapy

Southern blot hybridization. Ann Lowe, MD and SherylOsborne, RN, Jolene Lewis, RN and Joy Dritschillo, RNfor assistance with protocol preparation; and Fred Lake-man, PhD for PCR.

References1 Walker MD et al. Randomized comparisons of radiotherapy and

nitrosoureas for the treatment of malignant glioma after surgery.New Engl J Med 1980; 303: 1323–1329.

2 Subach BR et al Morbidity and survival after 1,3-bis(2-chloroethyl)-1-nitrosourea wafer implantation for recurrentglioblastoma: a retrospective case-matched cohort series. Neuro-surgery 1999; 45: 17–22; discussion 22–23.

3 Prados MD et al. Highly anaplastic astrocytoma: a review of 357patients treated between 1977 and 1989. Int J Radiat Oncol BiolPhys 1992; 23: 3–8.

4 Chamberlain MC, Kormanik P. Salvage chemotherapy withtaxol for recurrent anaplastic astrocytomas. J Neurooncol 1999;43: 71–78.

5 Martuza RL et al. Experimental therapy of human glioma bymeans of a genetically engineered virus mutant. Science 1991;252: 854–856.

6 Mineta T et al. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nature Med 1995; 1:938–943.

7 Hunter WD et al. Attenuated, replication-competent herpes sim-plex virus type 1 mutant G207: safety evaluation of intracerebralinjection in nonhuman primates. J Virol 1999; 73: 6319–6326.

8 Sundaresan P et al. Attenuated, replication competent herpessimplex virus type I mutant G207: safety evaluation in mice. JVirol 2000; 74: 3832–3841.

9 Yazaki T et al. Treatment of human malignant meningiomas byG207, a replication-competent multimutated herpes simplexvirus 1. Cancer Res 1995; 55: 4752–4756.

10 Toda M et al. In situ cancer vaccination: an IL-12 defectivevector/replication-competent herpes simplex virus combination

induces local and systemic antitumor activity. J Immunol 1998;160: 4457–4464.

11 Todo T et al. Systemic antitumor immunity in experimentalbrain tumor therapy using a multimutated, replication-com-petent herpes simplex virus. Hum Gene Ther 1999; 10: 2741–2755.

12 Peck DJ et al. Cerebral tumor volume calculations using plani-metric and eigenimage analysis (see comments). Med Phys 1996;23: 2035–2042.

13 Rampling R et al. Therapeutic replication-competent herpesvirus (letter; comment). Nature Med 1998; 4: 133.

14 Choucair AK et al. Development of multiple lesions duringradiation therapy and chemotherapy in patients with gliomas.J Neurosurg 1986; 65: 654–658.

15 Pasquier B et al. Extraneural metastases of astrocytomas andglioblastomas: clinicopathological study of two cases andreview of literature. Cancer 1980; 45: 112–125.

16 Hashido M et al. Changes in prevalence of herpes simplex virustype 1 and 2 antibodies from 1973 to 1993 in the rural districtsof Japan. Microbiol Immunol 1999; 43: 177–180.

17 Cobbs C, Markert J. Gene therapy of glioma: a review. Perspec-tives Neurol Surg 1999; 10: 1–20.

18 Whitley RJ, Herpes simplex viruses. In: Fields BN, Knipe DM,Howley PM (eds). Field Virology. Lippincott-Raven Publishers:New York, 1996, pp 2297–2342.

19 Markert JM et al. Reduction and elimination of encephalitis inan experimental glioma therapy model with attenuated herpessimplex mutants that retain susceptibility to acyclovir. Neurosur-gery 1993; 32: 597–603.

20 Mineta T et al. CNS tumor therapy by attenuated herpes simplexviruses. Gene Therapy 1994; 1(Suppl. 1): S78.

21 Toda M et al. Herpes simplex virus as an in situ cancer vaccinefor the induction of specific anti-tumor immunity. Hum GeneTher 1999; 10: 385–393.

22 Andreansky S et al. Treatment of intracranial gliomas inimmunocompetent mice using herpes simplex viruses thatexpress murine interleukins. Gene Therapy 1998; 5: 121–130.

23 Common Toxicity Criteria. National Institutes of Health, NationalCancer Institute: Bethesda, 1984.