Gene Therapy and Molecular Biology Vol 11, page 79
Gene Ther Mol Biol Vol 11, 79-92, 2007
Gene therapy trials for the treatment of high-grade
gliomas
Review Article
Adam M. Sonabend, Ilya V. Ulasov, Maciej S. Lesniak*
Division of Neurosurgery, The University of Chicago, Chicago, Illinois, USA
__________________________________________________________________________________
*Correspondence: Maciej S Lesniak MD, The University of Chicago, Division of Neurosurgery 5841 S. Maryland Avenue, MC 3026,
Chicago, IL 60637, USA; Tel: (773) 834-4757; Fax: (773) 834-2608; E-mail: [email protected]
Key words: Glioblastoma multiforme (GBM), brain tumor, glioma, clinical trial, gene therapy, virus
Abbreviations: adenoviral vectors with HSV-tk, (Ad-HSV-tk); billion infectious units, (BIU); central nervous system, (CNS);
Conditionally-replicative adenoviral vectors, (CRAd); coxsackie and adenovirus receptor, (CAR); ganciclovir, (GCV); glioblastoma
multiforme, (GBM); Herpes simplex virus type 1 thymidine kinase, (HSV-tk); herpes simplex virus, (HSV); neural stem cells, (NSCs);
Newcastle disease virus, (NDV); plaque forming units, (p.f.u.); retrovirus, (RV); retrovirus-mediated herpes simplex virus type 1
thymidine kinase gene therapy, (RV HSV-tk); viral producing cells, (VPC)
Received: 26 April 2007; Accepted: 7 May 2007; electronically published: June 2007
Summary
High-grade gliomas remain relatively resistant to current therapy. Local recurrence is a common feature and the
majority of patients progress despite conventional therapy. One modality-gene therapy-has shown a lot of promise
in early preclinical and clinical studies aimed at advancing the treatment of this disease. In this review, we provide
a comprehensive overview of clinical trials involving gene therapy in the field of neuro-oncology. The use of
different delivery vehicles, including liposomes, cells, and viruses, as well genes, especially cytokines and suicide
genes, are explored in detail. The unique features and advantages/disadvantages of the different vectors employed
are compared based on results of human studies. We discuss both the limitations and successes encountered in these
clinical trials, with an emphasis on the lessons learned and potential ways of improving current gene therapy
protocols.
A significant increase in survival of patients with
malignant brain tumors is a major goal of therapy and
thus, a wide variety of strategies are being explored. Some
of the experimental treatments are based on
immunotherapy, stem cell therapy, local chemotherapy
and radiotherapy (Liau et al, 1999; Lesniak et al, 2001; Yu
et al, 2001, 2004; Ehtesham et al, 2002; Ehtesham et al,
2005). In addition, gene therapy is becoming a promising
therapeutic alternative. Indeed, the fact that the majority of
brain tumors do not metastasize outside of the CNS may
allow for local delivery of vectors carrying therapeutic
genes (Immonen et al, 2004; Pulkkanen and Yla-Herttuala
2005).
In the last few decades, a considerable amount of
research dealing with gene therapy for glioma has been
conducted in vitro and in animal models. In the case of
human studies, the first clinical trials involving gene
therapy for gliomas were published in the 1990’s. These
pioneer studies consisted of retrovirus-mediated herpes
simplex virus type 1 thymidine kinase gene therapy (RV
HSV-tk) delivered by intra-tumoral injections of viral
producing cells (VPC) followed by systemic
I. Introduction
Gliomas are the most common form of primary
intracranial malignancy. Unfortunately, high-grade
gliomas like glioblastoma multiforme (GBM, WHO grade
IV) are the most frequently encountered and carry the
worst prognosis (Annegers et al, 1981; Louis et al, 2001).
The characteristic resistance to treatment shown by highgrade gliomas resides in their biological behavior and their
location within the central nervous system (CNS). As most
cancers, gliomas are subject to constant genotypic and
phenotypic alterations that can lead to treatment
resistance. Resistant cell populations get selected once a
therapy
is
administered.
In
addition,
most
chemotherapeutic agents cannot effectively reach all
tumor cells as the blood-brain-barrier limits the
penetration of these drugs to brain tumors (Lesniak and
Brem 2004). With respect to surgical treatment, the
complete resection of high-grade gliomas remains a
virtually impossible task since the nature of these tumors
is to infiltrate diffusely within surrounding brain
parenchyma (Ehtesham et al, 2005).
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Sonabend et al: Gene therapy for glioma
administration of ganciclovir (GCV) (Oldfield et al, 1993;
Raffel et al, 1994; Kun et al, 1995). Since then, a growing
number of trials have gathered information regarding the
efficacy and safety of this emerging therapeutic approach
(Table 1, Figure 1). In this text, we will focus on the
clinical trials of gene therapy for the treatment of brain
tumors. The main vehicles and transgenes employed for
the purpose of gene therapy for gliomas will be explored
under this scope.
Table 1. Clinical adenovirus-mediated gene therapy studies for the treatment of malignant glioma. Abbreviations: Ad,
adenovirus; pfu, plaque-forming unit; Rv, retrovirus; Ab, antibody; vp, viral particle. Reproduced from Pulkkanen and YlaHerttuala 2005 with kind permission from Macmillan Publishers Ltd: [Molecular Therapy].
Figure 1. Median survival in clinical gene therapy trials for malignant glioma. Trials involving less than nine patients and/or trials where
full survival data is not available were excluded. Filled bars represent VPCs-mediated RV-HSV-tk gene therapy; open bars randomized
controlled group. (•) Adenovirus-mediated HSV-tk gene therapy; (o) ONYX-015 therapy; (!) adenovirus-mediated p53 gene therapy;
(") Herpes simplex type 1 mutant therapy Reproduced from Pulkkanen and Yla-Herttuala 2005 with kind permission from Macmillan
Publishers Ltd: [Molecular Therapy].
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Gene Therapy and Molecular Biology Vol 11, page 81
entrapped components into cells, compared to other types
of liposomes (Felgner et al, 1987; Felgner et al, 1989; Shi
and Pardridge 2000; Yoshida et al, 2004b). Moreover,
delivery of genes via DNA/liposome complexes to the
brain could be achieved by incorporating antibodies to the
transferrin receptor in order to facilitate passage across the
BBB (Shi and Pardridge 2000, Yoshida et al, 2004b).
Liposomes - as gene therapy vehicles for brain
tumors - have been tested in humans. A pilot clinical trial
to evaluate the safety and effectiveness of interferon #
gene therapy with a liposomal vector was performed in
five patients with recurrent malignant glioma
(glioblastoma multiforme or anaplastic astrocytoma)
(Yoshida et al, 2004a). Transgene expression and antitumor activity were detected in four patients. The general
and neurological condition of all patients was the same or
had improved 3 months after starting therapy, except for
one patient. Two patients showed a partial response (50%
tumor reduction) and two others had stable disease 10
weeks after beginning therapy. No direct toxicity
attributable to the liposomes was seen in the study.
A phase I/II clinical trial involving liposomal
delivery of HSV-tk followed by treatment with i.v. GCV
for 14 days has also been performed in patients with
recurrent glioblastoma (Jacobs et al, 2001). Transgene
expression was assessed by positron emission tomography
using a radiolabeled substrate for HSV-tk. In this study,
there was evidence of HSV-tk expression in one of the
five patients evaluated. Similarly, in a prospective phase
I/II clinical study, eight patients bearing recurrent
glioblastoma multiforme were treated with stereotactic
intra-tumoral convection-enhanced delivery of an HSV-tk
gene-bearing liposomal vector followed by systemic GCV
(Voges et al, 2003). Treatment was well tolerated without
major side effects. In two out of eight patients, a reduction
of tumor volume greater than 50% was noted.
Some authors argue that liposome-based systems are
less efficient for gene transfer than viral vectors. On the
other hand, the former have the hypothetical advantage of
being relatively safe (Schatzlein, 2001). Regardless of the
view, liposomes are not likely to surpass viral vectors as
tools for most of cancer gene therapy strategies
(Pulkkanen and Yla-Herttuala 2005). Due to the relative
safety that viral vectors have achieved in recent human
studies, the main reason for using liposomes seems to be
fading away in the context of the much greater transgene
expression achieved by viral vectors. Nevertheless, as long
as the lack of an effective therapy for gliomas remains, the
suitability of the best vector remains a matter of open
debate.
II. Strategies and approaches for gene
therapy of gliomas
Gene therapy consists of the delivery of a gene of
interest to tumor cells in order to control and when
possible, kill the growing tumor. The strategies chosen to
this end vary. In some cases, the idea is to reestablish a
tumor suppressor gene like p53 that is frequently disrupted
in a malignancy (Kim et al, 2001; Lang et al, 2003;
Geoerger et al, 2004). In other cases, the transgene is an
enzyme that elicits a toxic effect in the presence of a drug.
Herpes simplex virus type 1 thymidine kinase (HSV-tk) in
combination with ganciclovir is an illustrative example of
the latter principle; this system is one of the first and most
extensively studied gene therapy approaches in human
trials for gliomas (Klatzmann et al, 1998, Kun et al, 1995,
Oldfield et al, 1993, Prados et al, 2003, Raffel et al, 1994).
Other transgenes tested in the preclinical and phase 1
clinical settings are used to evoke an effective anti-tumor
immune response; these include interleukins and
interferons (Eck et al, 2001, Kunwar 2003, Okada et al,
2001, Okada et al, 2000, Yoshida et al, 2004a, Yoshida et
al, 2004b). Finally, antisense oligonucleotides have also
been explored as transgenes since these sequences can
shut down oncogenes that play a significant role in the
neoplastic phenotype (Zhang et al, 1998; Andrews et al,
2001; Ly et al, 2001; Matsuno and Nagashima 2004).
Although many of these modalities appear interesting and
highly sophisticated, only few turn out useful at the
bedside.
In order treat a brain tumor with gene therapy, a
transgene needs to be delivered into the tumor cells with
the aid of a vehicle. Vehicles vary; they include liposomes,
cells and viruses. In some cases, distinct vehicles are
combined and complemented by each other. For instance,
cells have been transfected to generate viral particles that
can target tumors. In this text, even though many
transgenes are explored, gene therapy systems for glioma
are discussed under the scope of the employed vectors.
III. Liposomal vectors
DNA injection has been shown to induce transgene
expression in target cells when introduced with the aid of a
carrier molecule. Liposomes are an illustrative example of
such principle. These vectors consist of artificially
produced lipid vesicles that can entrap drugs in either their
aqueous compartment or their lipid bi-layer (Yoshida et al,
2004a). In order to achieve transgene expression,
liposomes need to attach to the target cell surface, be
internalized, escape from endosomes, find a way to the
nucleus and finally, keep their DNA available for
transcription (Thomas and Klibanov, 2003).
Liposomes possess several characteristics that render
them suitable for clinical gene transfer trials: they are
simple and easy to prepare in large quantities; the prepared
liposomes can be sterilized; and they show no intrinsic
toxicity or tissue specificity (Yoshida et al, 2004a).
Molecular modifications can add special features to
liposomes in order to enhance their transgene delivery
capacity. For instance, liposomes bearing positively
charged molecules provide more efficient delivery of their
IV. Cells as vectors
Most recently, cells have been used as vehicles to
carry and produce viral vectors. Indeed, as discussed
elsewhere in this text, RV-based gene therapy trials
(Oldfield et al, 1993; Raffel et al, 1994; Kun et al, 1995;
Palu et al, 1999; Rainov, 2000; Colombo et al, 2005)
injected PA317 cells to produce viral particles. These VPC
derive from an embryonic mouse fibroblast cell line
(Lyons et al, 1995) and have a limited reach since they are
unable to migrate. In contrast, stem cells offer the capacity
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Sonabend et al: Gene therapy for glioma
to migrate towards tumor cells. In fact, neural stem cells
(NSCs) are a promising tool to target disseminated tumor
cells. NSCs have a tropism for infiltrating cancer cells, so
they can be used to deliver therapeutic agents directly to
tumor pockets that reside beyond the healthy appearing
surgical margin (Yip et al, 2003; Ehtesham et al, 2005).
Additionally, mesenchymal stem cells can also be used for
this purpose since these cells can migrate to encounter
malignant cells in the CNS (Nakamizo et al, 2005,
Nakamura et al, 2004).
In addition to fibroblasts and stem cells, genetically
modified glioma cells have been evaluated as gene therapy
vectors for brain tumors. In fact, a protocol for a phase I
study involving genetically modified autologous tumor
cells has been reported. Specifically, IL-4 and HSV-tk
were carried as transgenes (Okada et al, 2000). The same
group also announced a pilot study of vaccination with
irradiated autologous glioma and dendritic cells admixed
with IL-4 transduced fibroblasts to elicit an immune
response (Okada et al, 2001). The therapy was well
tolerated and there was no incidence of autoimmune
encephalitis in enrolled subjects (Okada personal
communication). From that trial, a case report has been
published (Okada et al, 2003). The therapy consisted in the
combination of fibroblasts transfected with IL-4 and
irradiated autologous glioma cells injected intradermally.
With respect to the immune response, the patient showed
infiltration of dendritic cells (CD1a+), CD4+ and CD8+ T
cells. In fact, the cells increased proportionally to the
amount of IL-4 produced at the each site. This patient
demonstrated partial clinical response. Treatment was well
tolerated and the patient survived for 10 months since the
initiation of the treatment.
A. Non-replicating viral vectors
Non-replicating vectors were initially considered
safer since the risk of uncontrolled infection leading to
neural and systemic toxicities is theoretically lower when
compared to their oncolytic counterparts. On the other
hand, transgene expression of non-replicating adenoviral
and RV vectors has been shown to be too deficient to
translate into significant clinical outcome.
Retroviruses have been proven safe for intracranial
injection and for a long time have been a popular vehicle
for gene therapy trials for glioblastoma multiforme.
Replication deficient RV in combination with VPC were
initially used for gene therapy trials. These cells were
injected into the tumor cavity and subsequently produced
and secreted the vectors (Oldfield et al, 1993; Raffel et al,
1994; Kun et al, 1995).
Interestingly, some studies have tested a bicistronic
RV-based system that carries interleukin-2 and HSV-tk in
patients with recurrent glioblastoma multiforme. A pilot
study with four patients showed varying transgene activity
in the tumors of the treated patients. (Palu et al, 1999). The
study was then extended to a larger population of patients
and evaluated safety, feasibility and biological activity of
treatment. This time a total of 12 patients received intratumoral injection of VPC followed by intravenous GCV.
Treatment was well tolerated with only minor, grade 1 and
2, adverse events. These included transient increase of
liver
transaminases
and
transient
leukocytosis.
Transduction of tumor cells was demonstrated in tumor
biopsies. A marked and persistent increase of intratumoral and plasma Th1 cytokine levels was demonstrated
after treatment. Results of this trial suggest that the
combined delivery of a suicide and a cytokine gene is safe,
it is capable of inducing transgene transduction, it can lead
to the activation of systemic cytokine cascade and there
are tumor responses in up to 50% of cases (Colombo et al,
2005).
In 2000, Rainov published one of the most
representative RV-based studies; the vector consisted of a
recombinant replication-deficient RV with HSV-tk
transgene as insert. The study consisted of a multicenter,
randomized, controlled phase III clinical trial (Rainov,
2000). RV vector particles were injected during surgery,
followed by systemic treatment with GCV. Unfortunately,
clinical outcomes including time to tumor progression,
progression-free median survival, median survival and 12month survival rates showed no significant differences
between treatment and control groups (Figure 2). This
study was significant because it was one of the few phase
III gene therapy trials performed in the setting of
malignant glioma which failed to reveal any efficacy.
Retroviruses have a series of disadvantges that make
them inefficient gene therapy vectors. For instance, RVs
exhibit a low transduction efficacy (Vile and Russell 1995;
Rainov and Ren 2003; Pulkkanen and Yla-Herttuala
2005). Moreover, RV can only integrate into the genomes
of replicating cells since they require the dissolution of the
nuclear membrane. This is important since gliomas also
contain non-cycling cells (Fueyo et al, 2000), implying
ineffective transgene expression when these vehicles are
used. In addition, the low transgene carrying capacity (8
V. Viral vehicles
Viral vehicles are naturally capable of transferring
genes into target cells. There are many kinds of viruses
that have been explored for the purpose of gene therapy
for brain tumors. These include herpes simplex virus
(HSV) (Markert et al, 2000; Rampling et al, 2000;
Papanastassiou et al, 2002; Kambara et al, 2005),
retrovirus (RV) (Oldfield et al, 1993; Raffel et al, 1994;
Rainov, 2000), measles virus (Phuong et al, 2003),
reovirus (Coffey et al, 1998; Yang et al, 2004), adenoassociated virus (Mizuno et al, 1998), Newcastle disease
virus (NDV) (Csatary and Bakacs 1999; Russell 2002),
Semliki Forest virus (Ren et al, 2003), vaccinia virus
(Gridley et al, 1998; Timiryasova et al, 1999; Chen et al,
2001), poliovirus (Gromeier et al, 2000; Jackson et al,
2001) and adenovirus (Miller et al, 1998; Dmitriev et al,
2000; Fueyo et al, 2000; Suzuki et al, 2001; Lamfers et al,
2002; van Beusechem et al, 2002; Chiocca et al, 2004).
Viral vectors can be divided into those that can replicate
and lyse tumor cells, also known as oncolytic vectors, and
those that do not replicate but can carry transgenes into
their targets, known as non-replicating vectors. In the case
of the latter kind, the therapeutic effect is solely achieved
due to the activity exerted by the transgene expressed in
target cells.
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kb approximately) and the risk of insertional mutagenesis
add to the list of disadvantages of these vectors.
Besides RV, adenoviral vectors constitute a popular
option for gene therapy of brain tumors. In fact, these
vehicles are currently being used in approximately one
quarter of all gene therapy trials (McConnell and
Imperiale, 2004). These vectors are rendered replication
deficient by deletion of early regions of viral genomes. In
contrast to RV, non-replicating adenoviruses offer a
considerable deal of advantages. Adenoviral vectors
transduce both dividing and quiescent cells (both kind
found in gliomas) (Pulkkanen and Yla-Herttuala 2005;
Sonabend et al, 2006), are efficient with regard to
transgene expression and exhibit a safety record as shown
in human trials (Sandmair et al, 2000; Chiocca et al, 2004;
Lichtenstein and Wold 2004). In addition, adenoviruses
can be engineered to restrict transduction (Fueyo et al,
2003, van Beusechem et al, 2002) and transgene
expression to the desired target cell population (Parr et al,
1997; Shinoura et al, 2000; Vandier et al, 2000; Kambara
et al, 2005). These features are important in order to
decrease toxicity.
In the year 2000, Sandmair et al., published the first
clinical trial involving adenoviral vectors (Sandmair et al,
2000). The study compared retroviral vs. adenoviral
delivery of HSV-tk. It included 21 patients with primary
(n=8) and recurrent (n=13) gliomas that were treated with
intra-operative viral injection followed by i.v. GCV as
adjuvant therapy. As control, one group of patients
received Ad-LacZ (no GCV in these patients). Follow-up
included MRI scans to assess disease status (Figure 3). No
serious secondary effects were reported. Surprisingly,
median survival time of the group treated with Ad-HSV-tk
(15 months) was significantly longer than that of RVHSV-tk (7.4 months) and Ad-LacZ (8.3 months) (p<
0.012) (Figure 4) (Sandmair et al, 2000).
Figure 2. Kaplan-Mayer survival graphs of patients with RV HSV-tk and GCV treatment vs. standard treatment from a phase III clinical
trial with patients with GBM (Rainov, 2000). (A) Graph showing time to death (overall survival time). (B) Graph showing time to death
(overall survival time) for all patients in whom GBM was confirmed by central pathology review. Differences between groups are not
significant. Reproduced from Rainov, 2000 with kind permission from Human Gene Therapy.
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Sonabend et al: Gene therapy for glioma
Figure 3. VPC-mediated RV-HSV-tk gene therapy was compared to Ad-HSV-tk gene therapy (Sandmair et al, 2000). MRI follow-up 3
months after treatment. (A-C) MRI of patient 10 shows a right temporal GBM, (A) before, (B) day 1 and (C) 3 months after VPC
mediated RV-HSV-tk treatment. Shown is a subtotal surgical resection and fast regrowth of the tumor despite the gene therapy. (D-F)
MRI of patient 19 shows a left recurring frontal anaplastic astrocytoma before (D), 1 day (E) and 3 months (F) after the operation,
radiation and adenovirus-mediated gene therapy. Shown is a total tumor resection and no signs of tumor regrowth 3 months after the
treatment. (J-L) MRI of patient 21 shows a left recurring frontoparietal GBM before (J), 1 day (K) and 3 months (L) after reoperation
and adenovirus mediated gene therapy. Shown is a subtotal tumor resection and no signs of tumor regrowth 3 months after treatment.
Reproduced from Sandmair et al, 2000 with kind permission from Human Gene Therapy.
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Figure 4. Kaplan-Mayer survival graph of patients after Ad or VPC-RV-mediated or HSV-tk gene therapy for GBM. The difference in
outcome between the patients with adenovirus-mediated gene therapy (n=7) and VPC-RV-mediated gene therapy (n=7) was statistically
significant (p < 0.012, Fisher exact test). Patients who received #-galactosidase marker gene (n=7) served as controls. X, Means survival
time for each group. Reproduced from Sandmair et al, 2000 with kind permission from Human Gene Therapy.
These results encouraged Immonen and colleagues to
continue the evaluation of Ad-HSV-tk in a phase IIb
randomised, controlled trial (Immonen et al, 2004). Thirtysix patients with operable primary or recurrent malignant
glioma were randomized to receive AdvHSV-tk injection
(n=17) followed by i.v. GCV or standard care consisting
of radical excision (additional radiotherapy in patients
with primary tumors) (n=19). The primary end-point was
survival as defined by death or surgery for recurrence.
Secondary end-points were all-cause mortality and tumor
progression as determined by MRI. Findings were also
compared with historical controls (n = 36) from the same
unit over 2 years preceding the study. Ad-HSV-tk
treatment produced a clinically and statistically significant
increase in mean survival from 39.0 +/- 19.7 to 70.6 +/52.9 weeks (P = 0.0095). The median survival time
increased from 37.7 to 62.4 weeks (Figure 5). The
treatment was well tolerated (Immonen et al, 2004).
Nevertheless, as in the case of RV, replication
deficient adenoviruses are not very efficient in their
capacity for transgene expression. Indeed, a phase I
clinical trial of p53 gene therapy was performed using a
replication-defective adenoviral vector with wild type p53
(Ad-p53, INGN 201) against malignant brain tumors
(Lang et al, 2003). The vector was stereotactically injected
intratumorally via an implanted catheter. Treated tumor
specimens were obtained and analyzed afterwards. In all
patients, exogenous p53 protein was detected within the
nuclei of astrocytic tumor cells and transgene expression
induced apoptosis of targeted cells. However, with the use
of this replication-defective vector, transgene expression
was limited to within 5 mm of the injection site.
B. Replicating viral vectors
Many scientists favor replication competent vectors
over replication defective counterparts. It is thought that
since oncolytic viruses exhibit higher replication,
infectivity and transgene expression, these vectors could
offer a significant advantage. The Ad-p53 cited INGN 201
trial, among other comparative studies suggest this
observation (Ichikawa and Chiocca 2001; Lang et al,
2003). Nevertheless, these theoretical advantages remain
to be proven by efficacy endpoints such as patient
survival.
In the oncolytic virus category, the vectors that have
made it to the bedside are based on HSV, the adenovirus
and New Castle virus. In the case of HSV oncolytic
vectors, a few have been tested in phase I clinical trials
where their safety has been proven. Even though only
modest results have thus far been obtained with these
viruses, their effectiveness remains to be tested in future
trials (Markert et al, 2000; Rampling et al, 2000;
Papanastassiou et al, 2002).
G207 is one of the HSV-based oncolytic vectors.
This virus has deletions of both $(1)34.5 loci and a lacZ
insertion disabling the UL39 gene. With these deletions,
HSV-1 no longer produces hemorrhagic, necrotizing
encephalitis characteristic of wild-type HSV-1 infection in
human CNS (Shah et al, 2003). This vector was tested in a
dose-escalation phase I study for patients with malignant
glioma. The study included 21 patients with recurrent
malignant glioma. Dosage varied from 1x106 plaque
forming units (p.f.u.) inoculated at a single site to 3x109
p.f.u. at five sites. No serious adverse effects were
attributed to this treatment and no patient developed HSV
encephalitis. Radiographic and neuropathologic evidence
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Sonabend et al: Gene therapy for glioma
suggestive of anti-tumor activity and long-term presence
of viral DNA was documented in 8/20 cases (Markert et
al, 2000). There are current plant to continue the
evaluation of this vector in Phase Ib and Phase II clinical
trials and recruitment for these studies has begun (Shah et
al, 2003).
HSV 1716, an oncolytic vector based on wild type
HSV-1 17 strain, contains a single deletion in ICP34.5.
This vector was also tested in a phase I clinical trial
(Rampling et al, 2000). A total of nine patients with
relapsed malignant glioma received intra-tumoral
injections of doses up to 1x105 p.f.u. There were no cases
of encephalitis and no adverse clinical symptoms. Of nine
patients treated, four remained alive and neurologically
stable at 14 to 24 months after the vector’s administration
(Rampling et al, 2000). In addition, the same group
conducted another phase I study with HSV 1716 in twelve
patients with recurrent malignant glioma (same dose of
1x105 p.f.u.) (Papanastassiou et al, 2002). In the latter
study, the authors documented viral replication in tumors
resected four to nine days posterior to injection date. HSV
DNA was assessed by PCR at the sites of inoculation in
ten patients and at distal tumor sites in four. Interestingly,
viral replication took place in both HSV-seropositive and seronegative patients (Papanastassiou et al, 2002).
Conditionally-replicative adenoviral vectors (CRAd)
have been tested in clinical trials as well. CRAd vector
dl1520, so called ONYX 015 is a pioneer in this category.
Its replication selectivity is explained as follows: E1B 55K
is an early expression adenoviral protein that binds and
inhibits p53, consequently prolonging host cell life and
favoring viral progeny (Bischoff et al, 1996; Heise et al,
1997; Ramachandra et al, 2001). ONYX 015 has a
deletion in the viral genomic region coding E1B 55K.
Such deletion restricts the replication of this CRAd to
neoplastic cells with defective p53 pathway (Bischoff et
al, 1996; Heise et al, 1997; Hall et al, 1998; Habib et al,
2002). Nevertheless, more recently it has been suggested
that E1B 55kd deleted ONYX 015 replicates in neoplastic
cells due to aberrations in cancer cell nuclear mRNA
export rather than by p53 alteration (O'Shea et al, 2004;
O'Shea et al, 2005; Parato et al, 2005).
Figure 5. Kaplan-Mayer survival graphs for patients treated with Ad-HSV-tk gene therapy and randomized controls bearing malignant
gliomas. (A) All patients. (B) Patients with GBM. Log-rank regression analysis was performed. Reproduced from Immonen et al, 20045
with kind permission from Macmillan Publishers Ltd: [Molecular Therapy].
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A phase I clinical trial of ONYX 015 injection into
peri-tumoral regions of 24 patients with recurrent
malignant gliomas was published (Chiocca et al, 2004).
Treatment consisted of intra-cerebral injections of ONYX015. Patients were assigned into cohort groups to test
different doses (n=6 per cohort). Doses varied from 1x107
p.f.u. to 1x1010 p.f.u. into a total of 10 sites within the
resection cavity. None of the 24 patients experienced
serious adverse events related to ONYX-015. The median
time to progression after treatment with ONYX-015 was
46 days (range 13 to 452 + days). The median survival
time was 6.2 months (range 1.3 to 28.0 + months). This
trial proved that injection of up to 1x1010 p.f.u. of ONYX
015 into brain surrounding a resected malignant glioma is
safe in humans.
In addition to those discussed in this text, several
human trials involving adenoviral vectors for glioma have
been published to date (Trask et al, 2000; Eck et al, 2001;
Germano et al, 2003; Smitt et al, 2003) (Table 1).
Although conclusions regarding therapeutic effectiveness
vary, in the general, these adenoviral vectors appear safe
for intracranial injection.
In the case of NDV, the virus was used for a pediatric
patient bearing glioblastoma multiforme. The vector was
administrated intravenously on a daily basis; neurologic
improvement and tumor regression were reported (Csatary
and Bakacs 1999). In 2006, a phase I/II trial of intravenous
NDV oncolytic virus in recurrent glioblastoma multiforme
was released (Freeman et al, 2006). The study included
patients diagnosed with recurrent GBM based on imaging
studies. NDV-HUJ, the oncolytic HUJ strain of Newcastle
disease virus was administrated. The first part of the study
utilized an accelerated intrapatient dose-escalation
protocol with one-cycle dosage steps of 0.1, 0.32, 0.93, 5.9
and 11 billion infectious units (BIU) of NDV-HUJ (1 BIU
= 1X109 EID (50) 50% egg infectious dose) followed by
three cycles of 55 BIU. The virus was administered by
intravenous infusion over 15 min. In the second part,
patients received three cycles of 11 BIU. All patients
without progressive disease were maintained with two
doses of 11 BIU i.v. weekly. Eleven of the 14 enrolled
patients (11-58 years, Karnofsky performance scale 5090%) received treatment. Toxicity was minimal (fever was
seen in 5 patients). Maximum tolerated dose was not
achieved. Anti-NDV hemagglutinin antibodies appeared
within 5-29 days. NDV-HUJ was recovered from blood,
saliva and urine samples and one tumor biopsy. One
patient achieved a complete response. Intravenous NDVHUJ was found to be well tolerated. The authors
concluded that the high tolerability of NDV warrants
future studies to asses the clinical anti-tumoral effect of
this therapy.
modifications in their genomes and their structural
proteins.
Among the CRAd that can potentially be tested in
humans, Ad5-!24 is an interesting example. This
adenoviral vector carries a 24-bp deletion in the E1 viral
genoma. This deletion impairs the vector’s capacity to
interfere with Rb protein. Nevertheless, Ad5-!24
replicates in and destroys cancer cells with deficient Rb
(Fueyo et al, 2000), a pathway that is commonly altered in
gliomas (Hamel et al, 1993; Ueki et al, 1996). Ad5-!24
has been tested in vivo in human glioma xenografts in
nude mice. A single dose of this vector induced 66.3 %
inhibition and multiple injections an 83.8 % inhibition of
tumor growth in this model. On the other hand, normal
fibroblast or cancer cells with restored Rb activity were
resistant to this virus (Fueyo et al, 2000).
In spite of these advances, a major limitation of
currently available adenoviral vectors is the poor infection
of tumor cells. The initial transduction process of
adenoviruses requires the knob domain of fiber protein to
bind the coxsackie and adenovirus receptor (CAR). Even
though CAR is widely present in most tissues, it is poorly
expressed in gliomas (Bergelson et al, 1997; Tomko et al,
1997; Miller et al, 1998; Asaoka et al, 2000; Grill et al,
2001; Lamfers et al, 2002). Our group has previously
examined whether changes in adenoviral tropism can
enhance gene transfer in the context of malignant glioma
(Zheng et al, 2007). For this purpose, we assessed several
receptors that are over-expressed on tumor cells and tested
a series of adenoviral vectors that recognize these
receptors and carry luciferase trangene: Ad5-RGD which
binds %v#3/%v#5; Ad5/3 which contains adenovirus
serotype 3 knob and binds to CD46; Ad5-Sigma which
incorporates the reovirus sigma knob and binds to
junctional adhesion molecule-1; and Ad5-pk7 which
contains the polylysine motif and binds heparan sulfate
proteoglycans. We also investigated the Ad5-CAV1
vector, which contains the knob of canine adenovirus type
1, a virus previously shown to infect glioma via an
unknown mechanism. To evaluate the efficiency of viral
transduction associated with all these structural
modifications, expression of luciferase transgene both in
vitro and in vivo was studied on glioma cell lines. Our
results indicate that all five modified vectors attained
higher mean luciferase activity vs. control. Among them,
Ad5-CAV1 and Ad5-pk7 attained the highest transduction
efficiency independent of different tumor lines or infection
time. Most importantly, Ad5-pk7 achieved 1000-fold
increased transgene expression in human glioma
xenografts in vivo (Zheng et al, 2007).
Ad5-!24RGD, a new adenoviral vector with a viral
fiber modification, was obtained by insertion of an ArgGly-Asp (RGD) motif into the fiber knob of vector !24
(Suzuki et al, 2001). Such fiber modification enhances
viral infection of the target cells by interaction of the
inserted motif with %v integrins abundantly expressed in
glioma (Lamfers et al, 2002). Ad5-&24RGD exhibited
higher oncolytic activity than Ad5-!24. This vector was
tested in vivo in nude mice with s.c. human malignant
glioma IGRG121 xenografts. Intratumoral injection
resulted in complete tumor regression in 9 of 10 mice and
C. Future perspectives
Since the use of adenoviruses in human trials for
gliomas has led to promising results in clinical outcomes,
a considerable variety of CRAd are being developed and
tested to further investigate their use. These novel vectors
are constructed to preferentially target glioma cells and
remain relatively harmless to the surrounding tissue. To
this end, adenoviral vectors have been subject to
87
Sonabend et al: Gene therapy for glioma
long-term survival in all treated mice compared to
controls. The oncolytic activity of this vector was shown
to be enhanced by irradiation such that the same
therapeutic effect was achieved when a 10-fold lower viral
dose was applied (Lamfers et al, 2002).
In addition to the modification of viral structural
proteins, transcriptional targeting has been explored as a
strategy to achieve specificity of gene therapy in gliomas.
To this end, we have explored various promoters based on
their activity in these tumors relative to normal tissues
(Ulasov et al, 2007a,b). Some of these promoters could be
incorporated into the genome of viral vectors or elsewhere
in other gene therapy systems to restrict the expression of
transgenes into cells that present high activity of the
promoter. For instance, we have evaluated midkine,
survivin and CXCR4 promoters in the context of their
tumor specificity for gliomas (Ulasov et al, 2007b).
Among these, the survivin promoter demonstrated the
highest level of mRNA expression in primary tumor
samples and cell lines. Transcriptional targeting was
confirmed by infection of glioma cells with an adenovirus
expression vector containing a survivin-driven luciferase
reporter gene. Of the tested promoters, minimal level of
survivin activity was detected in normal human liver and
brain (Ulasov et al, 2007b).
with retrovirus VPCs HSV-tk system, showed that no
prolongation of survival was achieved.
In the proximate future, questions related to clinical
efficacy will be answered since some of the vectors
recently tested in phase I clinical trials will be subject to
clinical outcome assessment in phase II and phase III
studies that are on the way. As the first generations of
gene therapy constructs are been evaluated, new and more
sophisticated systems are approaching the patient bedside.
For all these reasons, clinicians that treat patients with
malignant brain tumors should follow the course of gene
therapy for this devastating disease.
Acknowledgments
This work was supported by a grant from the
National Institute of Neurological Disorders and Stroke
(NINDS), K08 NS046430 (ML).
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