Herpesvirus vectors for therapy of brain tumors.
ABSTRACT Genetically modified, conditionally-replicating Herpes Simplex Virus Type 1 (HSV-1) vectors for the treatment of malignant glioma have provided encouraging results in the handful of Phase I and Phase II clinical trials conducted to date. In recent years, a number of new strategies have been developed to improve anti-tumor activity of these attenuated vectors, through either introduction of foreign gene inserts to enhance tumor killing through a variety of mechanisms, or through combination with existing treatment regimens, including radiation and/or chemotherapeutics. Another promising new approach has been the engineering of novel oncolytic HSV vectors that retain wildtype replication, but are targeted to tumor cells through a variety of mechanisms. This review summarizes the latest advances in herpesvirus-mediated oncolytic therapies from both preclinical results and clinical trials with oncolytic HSV vectors in patients, and their implication for design of future trials.
[show abstract] [hide abstract]
ABSTRACT: Novel approaches to treatment of malignant glioma, the most frequently occurring primary brain tumor, have included the use of a wide range of oncolytic viral vectors. These vectors, either naturally tumor-selective, or engineered as such, have shown promise in the handful of phase I and phase II clinical trials conducted in recent years. The strategies developed for each of the different viruses currently being studied and the history of their development are summarized here. In addition, the results of clinical trials in patients and their implication for future trials are also discussed.Neurotherapeutics 08/2009; 6(3):558-69. · 6.01 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Glioblastoma multiforme is one of the most common human brain tumors. The tumor is generally highly infiltrative, making it extremely difficult to treat by surgical resection or radiotherapy. This feature contributes to recurrence and a very poor prognosis. Few anticancer drugs have been shown to alter rapid tumor growth and none are ultimately effective. Oncolytic vectors have been employed as a treatment alternative based on the ability to tailor virus replication to tumor cells. The human neurotropic herpes simplex virus (HSV) is especially attractive for development of oncolytic vectors (oHSV) because this virus is highly infectious, replicates rapidly and can be readily modified to achieve vector attenuation in normal brain tissue. Tumor specificity can be achieved by deleting viral genes that are only required for virus replication in normal cells and permit mutant virus replication selectively in tumor cells. The anti-tumor activity of oHSV can be enhanced by arming the vector with genes that either activate chemotherapeutic drugs within the tumor tissue or promote anti-tumor immunity. In this review, we describe current designs of oHSV and the experience thus far with their potential utility for glioblastoma therapy. In addition, we discuss the impediments to vector effectiveness and describe our view of future developments in vector improvement.Expert Review of Neurotherapeutics 05/2009; 9(4):505-17.
[show abstract] [hide abstract]
ABSTRACT: Genetically engineered, conditionally replicating herpes simplex viruses type 1 (HSV-1) are promising therapeutic agents for cancer. They can replicate in situ, spread, and exhibit oncolytic activity via a direct cytocidal effect. In addition, oncolytic HSV-1 can transfer and express foreign genes in host cells. The phase I clinical study with G207, a double-mutated HSV-1, in recurrent malignant glioma patients has shown that oncolytic HSV-1 can be safely administered into human brains. The therapeutic benefits of oncolytic HSV-1 depend on the extent of both intratumoral viral replication and induction of host antitumor immune responses. We develop new-generation oncolytic HSV-1 by enhancing these properties while retaining the safety features. G47delta was created from G207 by introducing another genetic mutation. Compared with G207, G47delta showed 1) better stimulation of human antitumor immune cells, 2) better growth properties leading to higher virus yields and increased cytopathic effect in vitro, 3) better antitumor efficacy in both immuno-competent and -incompetent animals, and 4) preserved safety in the brain of HSV-1-sensitive mice. Preparation is under way for a clinical trial using G47delta in progressive glioblastoma patients. G47delta is also suited as a backbone vector for expressing foreign molecules. Using bacterial artificial chromosome and two DNA recombinases, we have created an "armed" oncolytic HSV-1 generation system that allows insertion of transgene(s) into the genome of G47delta in a rapid and accurate manner. We found that expression of immunostimulatory molecules can significantly enhance the antitumor efficacy of G47delta. Based on these advances, we anticipate that oncolytic virus therapy using oncolytic HSV-1 will soon be established as an important modality of cancer treatment.Frontiers in Bioscience 02/2008; 13:2060-4. · 3.52 Impact Factor
The Open Virology Journal, 2010, 4, 103-108 103
1874-3579/10 2010 Bentham Open
Herpesvirus Vectors for Therapy of Brain Tumors
Kevin A. Cassady and Jacqueline Nuss Parker*
Department of Pediatrics, Division of Infectious Diseases, University of Alabama at Birmingham, Birmingham, AL
Abstract: Genetically modified, conditionally-replicating Herpes Simplex Virus Type 1 (HSV-1) vectors for the
treatment of malignant glioma have provided encouraging results in the handful of Phase I and Phase II clinical trials
conducted to date. In recent years, a number of new strategies have been developed to improve anti-tumor activity of these
attenuated vectors, through either introduction of foreign gene inserts to enhance tumor killing through a variety of
mechanisms, or through combination with existing treatment regimens, including radiation and/or chemotherapeutics.
Another promising new approach has been the engineering of novel oncolytic HSV vectors that retain wildtype
replication, but are targeted to tumor cells through a variety of mechanisms. This review summarizes the latest advances
in herpesvirus-mediated oncolytic therapies from both preclinical results and clinical trials with oncolytic HSV vectors in
patients, and their implication for design of future trials.
Keywords: Oncolytic HSV therapy, brain tumor, G207, HSV1716, M032, chimeric HSV, C134, R5141, rQNestin34.5, phase I
trial, glioma, ?1(34.5).
a therapeutic challenge. For malignant glioma, the most
frequently occurring primary brain tumor, novel treatment
regimens have extended survival only by a few months.
Nearly three decades ago, advancements in molecular
biology, combined with
experimental therapies for a number of malignancies
including glioma, generated significant interest in the
application of genetically
replicating herpes simplex viruses (HSV-1) vectors for use
as anti-tumor agents. A number of recent reviews of the
history of oncolytic viral vectors, including those derived
from HSV-1, and their application for therapy of CNS
malignancies have summarized advances in oncolytic viral
therapy over the past several years [1-3]. The emphasis of
the current review is to provide an overview of the latest
approaches and technologies utilizing both attenuated,
conditionally replicating vectors, as well as HSV vectors that
replicate with wildtype efficiency but in a tumor-specific
fashion. These include:
Primary and metastatic tumors arising in the brain remain
research into viral-based
i) Introduction of foreign gene inserts that enhance
vector-mediated oncolysis through a variety of
ii) Use of chimeric HSV/human cytomegalovirus
(HCMV) vectors in which introduction of the HCMV
IRS1 or TRS1 genes into HSV backbone restores the
wildtype protein synthesis phenotype without
concomitant restoration of neurovirulence;
*Address correspondence to this author at the Department of Pediatrics,
Division of Infectious Diseases, University of Alabama at Birmingham,
Birmingham, AL 35294-0011, USA; Tel: 205-975-6549; Fax: 205-996-
7881; E-mail: JParker@peds.uab.edu
iii) Combining oncolytic HSV therapy with standard of
care therapies, including
iv) Development of wildtype, nonattenuated HSV vectors
that are retargeted to tumor-specific receptors;
v) Development of attenuated HSV vectors in which the
HSV-1 neurovirulence gene, gamma-1 34.5 (?134.5),
discussed in more detail below, is expressed from a
tumor-specific promoter (nestin 1); and
vi) Improved methods for delivery of oncolytic viral
vectors, including convection-enhanced delivery.
subsequent subsections, and summarized schematically in
Fig. (1). Finally, an update of latest clinical trial results, both
recently completed and pending, is provided at the end, and
summarized in Table 1.
Each of these therapies is discussed in more detail in
HERPES SIMPLEXVIRUS VECTORS
MUTATIONS OF ONCOLYTIC
mutants that lacked a functional thymidine kinase gene were
shown to be incapable of virus replication in both
nondividing cells and in the mammalian nervous system ;
subsequently, a tk deletion virus, named dlsptk, was shown
to be capable of establishing, but not reactivating from,
latency . This HSV mutant was shown in 1991 by
Martuza and colleagues to effectively kill glioma cell lines in
vitro and in subcutaneous tumors, and prolonged survival of
mice bearing intracranial tumors established from the same
glioma cell line . The tk gene deletion rendering this
mutant resistant to antiviral agents that target the viral
thymidine kinase prevented advancement of this virus to
clinical trials in patients. Nonetheless, these pioneering
studies demonstrated that HSV-1 derived vectors with
dlsptk mutants In the mid to late 1970s, an HSV-1
104 The Open Virology Journal, 2010, Volume 4 Cassady and Parker
attenuating mutations could be used for specific treatment of
rapidly growing tumors in the brain.
concept” studies from the dlsptk mutants emphasized the
need to develop viral vectors avirulent in normal brain but
capable of replication in actively dividing tumor populations.
The HSV-1 mutant hrR3 was constructed in which the
bacterial lacZ gene was introduced within the UL39 gene
encoding the viral ribonucleotide reductase (infected cell
protein 6, or ICP6) . The native thymidine kinase was
retained in this construct; as such this mutant retained
sensitivity to ganciclovir. Lack of a functional ICP6 protein
prevented the virus from replicating in nondividing cells.
However, in actively dividing cells, the virus was still able to
replicate, though progeny virus recovery was reduced as
compared to wildtype HSV. Additionally, anti-tumor activity
of rat brain tumors following treatment with hrR3 is
potentiated by ganciclovir treatment . The HSV-1 vector
G207 used in three Phase I clinical trials in the United States
also contains a lacZ insertion in the UL39 gene similar to
hrR3, in addition to deletions of both copies of the ?134.5
gene, described in the next section.
?-1 34.5 deleted HSV mutants. In 1990, Chou and
Roizman demonstrated that the neurovirulence function of
HSV-1 mapped to the diploid gene ?134.5, located in the
inverted repeat sequences flanking the Unique Long (UL)
segment of the viral genome . In later studies, the normal
Ribonucleotide reductase mutants. The “proof of
function of ICP34.5, the protein product encoded by the
?134.5 gene, was shown to preclude the shutoff of host
protein synthesis by recruitment of the host protein
phosphatase-1a, and subsequent dephosphorylation of the
eukaryotic translation initiation factor alpha (eIF-2?) .
Normally, following infection with wild type HSV-1,
production of double stranded RNA triggers an intracellular
stress response that causes the protein kinase R (PKR) to
phosphorylate eIF-2?, which mediates protein synthesis
shutoff. In normal non-mitotic cells, deletion or disruption of
both copies of the ?134.5 gene severely limit virus replication
due to the host PKR-mediated shutoff of host protein
synthesis. However replication of ?134.5-deleted HSV can
occur in tumor cells that possess complementing mutations.
Examples include ras overexpression , and alterations in
PKR and other cell signaling pathways . Of note,
deletion of the ?134.5 gene also impacts synthesis of the
complementary antisense DNA strands. To date, both
oncolytic HSV vectors tested in patient clinical trials are
based on the ?134.5-deletion platform (for recent reviews,
see [1, 2]).
(LATs), encoded on
oHSV VECTORS FOR DELIVERY OF FOREIGN
number of advantages, one being its capability for
introduction of large transgenes for combining oHSV
Oncolytic virus therapy using HSV-1 vectors have a
Fig. (1). Strategies to improve oHSV therapy of malignant glioma. Each of the different strategies discussed in detail in the text are shown
schematically. IRS= C134 chimeric HSV expressing IRS gene from HCMV; WT= retargeted viruses that have wildtype ICP34.5 but can
only infect cells with upregulated tumor-specific receptors; *?134.5= ICP34.5 expressed from tumor-specific promoters; ?= ?134.5 deleted
HSV Vectors for Brain Tumor Therapy The Open Virology Journal, 2010, Volume 4 105
therapy with expression of foreign genes that complement
the anti-tumor activity of the vector. There are currently a
number of different classes of transgenes being introduced
into oHSV vectors that are being evaluated as novel
therapies for glioma gene therapy. These include the
introduction of genes encoding pro-drug activating enzymes,
tumor suppressor genes, immune modulating genes, and
genes encoding proteins that inhibit tumor angiogenesis [13,
14] and references therein. A number of studies by our group
have demonstrated the antitumor efficacy of an IL-12
expressing oHSV, M002, in both syngeneic murine brain
tumor models and human glioma xenograft tumor models
. M002, like G207, is derived from the HSV-1 (F) strain
with deletions in both ?134.5 genes. The other notable
difference between M002 and G207 is that in M002, the
UL39 gene remains intact. A bicistronic expression cassette
encoding interleukin-12 p40 and p35 subunits from either
murine (M002) or human (M032) origin, and separated by an
internal ribosome entry sequence (IRES), were introduced
into both ?134.5 deleted sites. Production of a cGMP lot of
M032 for Phase I clinical trials through the NCI
Biopharmaceutical Development Program (BDP) has
recently been completed, and submission of an IND
application for approval by the United States Food and Drug
Administration (FDA) is planned for summer 2010.
can escape ??134.5 first generation oncolytic HSV treatment
[16, 17]. One hypothesis is that these mutants are unable to
maintain prolonged replication in the tumor, and therefore their
ability to spread throughout the tumor mass is compromised by
the innate antiviral host responses. To counteract these
responses impeding ??134.5 HSV replication in infected tumor
cells, the IRS1 gene from a distantly related herpesvirus, Human
Cytomegalovirus (HCMV), was introduced into a ??134.5 HSV
background. IRS-1 has been previously demonstrated to
selectively restore late viral protein synthesis . Additionally,
Preclinical and clinical evidence indicates that tumor cells
since the HCMV gene was evolutionarily distant enough from
HSV-1 genes, it was hypothesized that IRS-1 lacked the
neurovirulence function of ?134.5 and thus could restore late
viral protein synthesis without restoring neurovirulence. Two
chimeric HSVs were engineered; C130, a ??134.5 HSV
engineered to express the HCMV TRS1 gene and C134, a
??134.5 HSV engineered to express HCMV IRS1. Results
indicated that insertion of the HCMV TRS1 or TRS1 gene into
a ??134.5 HSV resulted in a virus with restored late viral protein
synthesis and improved replication in malignant glioma cells
both in vitro and in vivo . This improved replication
translated to a better anti-tumor response as well, when directly
compared to tumors treated with parent ??134.5 HSV only.
Importantly, the HSV/HCMV chimeric viruses were also
demonstrated to be safe following direct intracranial inoculation
in mice, but with significantly different LD50 values. While the
LD50 value measured for C134, which expressed IRS1, was
identical to that of its parent ??134.5 HSV, the LD50 value of
C130, encoding the TRS1 gene, was increased by nearly 100-
fold as compared to C134. Despite the increased virulence of
C130, both HSV/HCMV chimeric viruses have safety profiles
similar to oHSV (HSV1716 and G207) that have been
successfully used in Phase I clinical trials [20, 21]. Due to its
excellent anti-tumor efficacy and minimal neurotoxicity, C134
has been advanced for clinical development. An application was
submitted, and received approval, for production of C134 using
current Good Manufacturing Practices (cGMP) through the
National Cancer Institute’s Rapid Access to Intervention
Development (RAID) Program for the translation of novel
experimental therapeutics into Phase I trials in patients.
STANDARD OF CARE THERAPIES IN COMBINATION
WITH oHSV THERAPY
paradigms for patients with newly diagnosed glioma was
made following the report that combination of ionizing
radiation with temozolomide (TMZ) administration resulted
in significantly improved survival benefit vs treatment with
either therapy alone . In this study, the median two year
The most recent shift in standard of care treatment
Past, Present and Pending oHSV Trials in Patients with Malignant Glioma
Virus Parent Strain Tumor-Targeting Mutation(s) Trial Description Trial Status References
Phase 1 dose escalation, safety closed 
Phase 1b, single dose, assess
intratumoral virus replication
Phase Ib, safety of resection
HSV1716 17 Both ?134.5 gene copies deleted
Phase III ongoing nexxusscotland.com website
Phase 1 dose escalation, safety closed 
Phase Ib, two dose regimen, one
intratumor, one into resection
Both ?134.5 gene copies deleted,
lacZ gene inserted into UL39 gene
Phase Ib closed Markert et al., submitted
Both ?134.5 gene copies deleted
Phase I, safety and dose
Both ?134.5 gene copies deleted,
HCMV gene IRS1 introduced
within UL3/UL4 intergenic region
Phase I safety and dose
106 The Open Virology Journal, 2010, Volume 4 Cassady and Parker
survival of patients treated with the combination therapy was
over 26% vs 10% two year survival of patients that received
radiation therapy only. It is not unreasonable to predict that
combining oncolytic HSV therapies with standard of care
therapies like ionizing radiation and chemotherapeutic drugs
would also result in improved survival benefit vs either
therapy alone. A number of reports support this, and are
colleagues demonstrated that administration of high doses of
ionizing radiation (IR) 24 and 48 hours following direct,
intratumoral inoculation of R3616, a ??1345 HSV, lysed
glioma tumor explants much more efficiently than treatment
with either therapy alone . More recently, the underlying
mechanism mediating enhanced viral replication was linked
to upregulation of late viral promoters by the host p38
protein . These results are discussed in more detail
??134.5HSV and ionizing radiation. In 1998, Advani and
drugs. Likewise, combination of TMZ treatment with oHSV
vectors was also shown to improve lysis of tumor cells
resistant to destruction by either agent alone . Aghi and
colleagues discovered that the DNA repair mechanisms
elicited following treatment with TMZ enhance intratumoral
replication of oHSV and kill tumor cell lines normally
resistant to either therapy alone. These data support reports
of combination radiation or chemotherapy with oncolytic
viral therapy in other malignancies outside the brain [27, 28].
oHSV therapy in combination with chemotherapeutic
TUMOR-SPECIFIC RETARGETING OF oHSV
the ??134.5 HSV platform is their inability to maintain
efficient replication in CNS tumors. One solution is to
engineer viruses in which the ?134.5 gene is only expressed
in tumor cells, but not in adjacent normal tissue. Two of the
strategies developed to enable tumor-specific ICP34.5
expression are i) to modify virus glycoproteins to enter cells
via a tumor-specific receptor, while preventing interaction
with normal cell surface receptors, and ii) to express ICP34.5
under the control of a tumor-specific promoter. In a series of
elegant experiments, Zhou and Roizman and colleagues were
able to re-target a wildtype HSV vector to the IL-13Ralpha2
receptor, which is expressed on the surface of many
glioblastoma cell lines, as well as on anaplastic astrocytomas
[29, 30]. HSV mutant R5111 contains both copies of 34.5
intact, but the known heparan binding sites on glycoproteinB
(gB) and gC were mutated. R5111 also contains a chimeric
gD that was constructed to express IL-13. R5111 was able to
infect cells following interaction of the chimeric gD/IL13
with the IL13Ralpha2 on the tumor cell lines tested .
Mutations were then introduced into R5111 to construct a
virus (R5141) in which binding to either HveA or nectin-1,
its native receptor has been completely abolished .
Current challenges for receptor re-targeted viruses like
R5141 include development of an appropriate and FDA-
approved cell line for production of highly purified, high
titer virus stocks for preclinical safety and efficacy testing in
mice, and for Phase I clinical trials in patients.
As mentioned earlier, one of the primary limitations of
employs the use of tumor-specific promoters driving
expression of the ICP34.5 protein. One example is the
The second strategy for targeted expression of ICP34.5
promoter for nestin-1, an intermediate filament protein
shown to be upregulated in a high percentage of human
glioma cell lines, but not normal human astrocytes. Studies
by Kambara and colleagues demonstrated that rQNestin34.5,
an HSV engineered to express ICP34.5 from the nestin-1
promoter, was able to improve survival of mice bearing
intracranial tumors as compared to treatment with the parent
virus (lacking both ?134.5 gene copies and containing a
disrupted UL39 gene) only . In vivo neurotoxicity studies
showed that rQNestin34.5 was not more neurovirulent than
its ?134.5-deleted parent.
Another example of a tumor-specific promoter driving
ICP34.5 expression is the mutant HSV KeM34.5. KeM34.5
was derived from G207, but the lacZ gene within the UL39
gene has been replaced with the Musashi-1 promoter driving
ICP34.5 expression . Musashi 1 is a neural RNA-binding
protein whose expression is upregulated in glioma cell lines.
Treatment of human glioma tumors established intracranially
in mice resulted in significantly improved survival of tumor
bearing mice, and this effect was dose-dependent.
specific receptors allowing specific delivery of wildtype
virus, or through tumor-specific
controlling ICP34.5, are promising strategies for glioma
therapy. It remains to be seen whether they will be safe for
administration in humans.
Thus retargeting of oHSV vectors, either through tumor-
CONVECTION-ENHANCED DELIVERY (CED) OF
efficient delivery of drugs or therapeutics, including oHSV,
into the tumor site. The blood brain barrier (BBB) can
significantly impede delivery of novel therapeutic drugs and
agents to tumors inaccessible to stereotactic injection of
oHSV. Even when the tumor mass is accessible, migrating
tumor cells distal from the primary mass are often left
untouched, due to inadequate distribution at tumor
inoculation . One recently described approach to
improve distribution of oHSV vectors into the tumor mass is
the use of convection enhanced delivery (CED). In CED,
multiple catheters are placed stereotactically within the
tumor mass and surrounding the tumor/resection area.
Hadjipanayis et al. demonstrated the CED of a replication
defective HSV vector, d106, significantly improved survival
of tumor-bearing mice when the therapy was combined with
IR or TMZ . However there was no significant
difference in median survival rates of mice treated with d106
+ CED alone (without IR or TMZ). Nevertheless, CED using
any of the oHSV vectors described earlier in this review is a
promising approach to ensure adequate and widespread
distribution in the tumor.
Another obstacle in the treatment of malignant glioma is
ADVANCING ONCOLYTIC HSV VECTORS INTO
PATIENTS WITH MALIGNANT GLIOMA: STATUS
OF CLINICAL TRIALS
vectors have been published to date [17, 20, 21, 37, 38].
These trials were initiated more than a decade ago with the
first-generation oHSV vectors HSV1716 (United Kingdom)
and G207 (United States). Both HSV1716 (derived from
wildtype strain 17) and G207 (derived from wildtype strain
A total of 5 clinical trials in patients utilizing oHSV
HSV Vectors for Brain Tumor Therapy The Open Virology Journal, 2010, Volume 4 107
“F”) have one or both of the attenuating mutations (?134.5
deletion and UL39 disruption), respectively, and described
earlier in this review. The details of these trials have been
adequately summarized elsewhere [1, 39].
ionizing radiation was administered following G207 in a
group of 9 patients (Markert JM, personal communication)
and report of the results are pending final analysis. Finally,
according to their website, [http://www.nexxusscotland.com/
Crusade Laboratories in Glasgow, Scotland has initiated a
Europe-wide Phase III clinical trial using HSV1716.
A Phase Ib study has just been completed in which
initial Phase I safety studies in patients. cGMP production of
M032, mentioned earlier, is complete and, pending final
sterility testing, submission of an IND application to the
FDA is planned for mid to late 2010. The chimeric
HSV/HCMV, C134, is also being submitted for cGMP
production through the NCI BDP. Production date has not
yet been set, but protocols for growing up high titer virus
production lots have already been developed and the process
is anticipated to proceed quickly. Table 1 summarizes the
past and present oHSV clinical trials in patients, as well as
second generation viruses poised to enter the clinical trial
arena in the next 12-18 months.
At least two other promising oHSV vectors are close to
malignant glioma continues to evolve with the advent of new
therapeutics, and discovery of new tumor-specific targets to
be engineered into oHSV vectors. Examples of novel targets
include markers of glioma progenitor cells (GPCs), as well
as tumor-specific delivery and IR-mediated or expression of
proteins involved in signal transduction pathways. Gene
expression profile analyses of G207-treated tumor specimens
from the G207 Phase Ib study  should provide new
insight as to correlates of responders to oHSV therapy vs
nonresponders. Regardless, inasmuch as the oHSV trials in
patients to date have overwhelmingly demonstrated safety of
direct intratumoral inoculation and surrounding adjacent
tissue, advancement of 2nd and 3rd generation viruses into
Phase I clinical trials, alone and in combination with current
standard of care therapies, is eagerly anticipated.
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