Download Report

Scientific Update
Gene Therapy for Brain Tumors:
The Fundamentals
Herbert H. Engelhard, M.D., Ph.D., F.A.C.S.
Departments of Neurosurgery and Molecular Genetics, The University of Illinois at Chicago,
Chicago, Illinois
Engelhard HH. Gene therapy for brain tumors: The fundamentals.
Surg Neurol 2000;54:3–9.
BACKGROUND
Over the past two decades, significant advances have
been made in the fields of virology and molecular biology,
and in understanding the genetic alterations present in
brain tumors. The knowledge gained has been exploited
for use in gene therapy.
OBJECTIVE
The purpose of this article is to present an introduction
to the field of brain tumor gene therapy for the practicing
clinician.
RESULTS
A variety of gene therapy strategies have now been used
in the laboratory and in clinical trials for brain tumors.
They can be divided into five categories: 1) gene-directed
enzyme prodrug (“suicide gene”) therapy (GDEPT); 2)
gene therapy designed to boost the activity of the immune system against cancer cells; 3) oncolytic virus therapy; 4) transfer of potentially therapeutic genes—such as
tumor suppressor genes—into cancer cells; and 5) antisense therapy. GDEPT is the strategy that has been most
extensively studied.
CONCLUSIONS
To date, gene therapy has been found to be reasonably
safe and concerns related to adverse events such as
insertional mutagenesis have not been realized. Although
patients have not been cured, the development of this
therapy could still be considered to be at an early stage.
Current research is addressing factors that could be limiting the successful clinical application of gene therapy,
which remains an intriguing experimental option for patients with malignant brain tumors. © 2000 by Elsevier
Science Inc.
KEY WORDS
Antisense therapy, brain tumor treatment, DNA, glioblastoma multiforme, viral vectors.
Address reprint requests to: Dr. Herb Engelhard, Departments of Neurosurgery and Molecular Genetics, The University of Illinois at Chicago,
912 South Wood St., Chicago, IL 60612.
Received April 28, 2000; accepted May 5, 2000.
© 2000 by Elsevier Science Inc.
655 Avenue of the Americas, New York, NY 10010
xperimental use of gene therapy for brain tumors is currently a topic of great interest at
neurosurgical meetings and in the literature, and
tumor patients and their families often have questions about this type of treatment. The purpose of
this article is to present an introduction to the field
of gene therapy, and an organizational framework
for classifying the therapeutic strategies (involving
DNA, viruses and/or genes) that are being studied.
More than 8 years have now elapsed since the first
use of gene therapy for patients with malignant
brain tumors [20]. With the initiation of clinical
trials came great hope that the highly sophisticated
tools of molecular biology could be successfully
brought to bear against an otherwise intractable
disease [4]. Unfortunately, to date, the promise of
gene therapy has largely gone unfulfilled [30]. Yet
significant advances in the field continue to be
made [23], and new clinical protocols are being initiated every year (see http://clinicaltrials.gov/ct/gui).
“Gene therapy” can be defined as the transfer of
genetic material (usually DNA) into a patient’s cells
for therapeutic purposes [4]. When DNA is inserted
into a cell, the process is termed “transfection,”
with the inserted gene being called a “transgene.”
The transgene is transported to the nucleus where
it can become expressed as mRNA, then protein
(see Figure 1B). The transgene is actually a shortened version of the native gene— called “cDNA”—
which does not contain the portions that the cell
eliminates (by splicing or “post-transcriptional
modification”) to form the mRNA template. Once
foreign DNA is inside a cell, it can either integrate
into, or remain outside, the host cellular DNA. If it
remains outside, the transgene is termed “episomal.” If the transgene inserts itself into the host cellular DNA, there exists the theoretical possibility of
“insertional mutagenesis”—i.e., the production of a
mutation due to foreign DNA inserting into middle
of a gene, or changing the reading frame of the
“downstream” DNA. The possibility of creating such
E
0090-3019/00/$–see front matter
PII S0090-3019(00)00234-2
4
Surg Neurol
2000;54:3–9
Engelhard
A: Depiction of the usual processes of transcription, post-transcriptional modification, and translation, as would
occur in a tumor cell. In this scheme, the ribosomes would move from right to left along the mRNA template, to
produce the protein (translation). Cellular proteins produced would include normal proteins, as well as undesirable
“oncoproteins” causing the cell to be cancerous. B: Scheme for replacement gene therapy. While the usual processes
of transcription and translation are occurring (left side of figure), additional DNA is introduced into the cell by a viral
vector. This DNA moves into the nucleus and becomes expressed as mRNA, then a protein designed to be beneficial
to the host— e.g., a tumor suppressor protein. If the DNA does not integrate into the host genome, it is termed
“episomal.” C: Scheme for enzyme-directed pro-drug (“suicide gene”) therapy. The viral DNA causes an enzyme to be
produced. When a drug (such as ganciclovir) is subsequently given, it is converted into a toxic form by the enzyme.
This kills the cell, and adjacent tumor cells, even if they don’t have the enzyme (the “bystander effect”). D: Scheme for
antisense therapy. The antisense strand binds to the mRNA template, blocking the ribosome, stopping translation, and
activating cleavage by RNase H. The antisense DNA used is designed to be specific to the mRNA of an undesirable
protein, such as the c-myc oncoprotein.
1
Gene Therapy
1
Methods for Introducing DNA or Genes into Cells
Direct microinjection
Electroporation
Calcium phosphate transfection
Pneumatic delivery (the “gene gun”)
Cationic liposomes
Genetically-engineered viruses (adenovirus, herpes
simplex virus, adeno-associated virus, retroviruses)
Surg Neurol
2000;54:3–9
2
Categories: Gene Therapy for Cancer
WHAT ENTERS
PATIENT’S CELLS?
1) Gene-directed enzyme
prodrug therapy
2) Immuno-gene therapy
3) Oncolytic virus therapy
4) Therapeutic gene transfer
5) Antisense therapy
a mutation has been a source of fear in the use of
gene therapy. However, such a mutation could only
be passed to a patient’s children if it involved germ
line (as opposed to somatic) cells.
There are several different methods for introducing DNA or genes into cells (Table 1). In the beginning of gene therapy research, physical methods
such as direct microinjection, pneumatic delivery,
electroporation, and calcium phosphate transfection were used to get DNA into cultured cells (i.e., in
vitro). Later, cationic liposomes were used to transfer genes both in vitro and in vivo. Currently, the
most popular DNA delivery vehicles—“vectors”—
are modified viruses, which can also be used both
in cultured cells and in living organisms or patients.
Genetic material can be introduced into a patient
either through direct delivery to cells of the target
organ (in vivo technique), or by altering the genes
of cells which are initially outside the host, then
implanted into the affected area (ex vivo technique)
[4]. Glioblastoma was the first cancer to be treated
by gene therapy in humans using the in vivo technique. For brain tumor patients, the therapeutic
agent has usually been delivered by direct infiltration of brain (containing residual tumor cells) at the
time of tumor resection, or by means of stereotactic
implantations. Both of these approaches bypass the
blood-brain barrier and avoid systemic exposure to
the agent [1,4]. Although the majority of the basic
science research in gene therapy for brain tumors
has focused on gliomas, some studies have addressed the problems of brain metastases and leptomeningeal cancer [25,33].
Several different viruses have been used for gene
therapy, including herpes simplex virus, adenovirus, adeno-associated virus, and retroviruses. Retroviruses, such as the human immunodeficiency
virus (HIV), contain RNA that is reverse-transcribed
into DNA by reverse transcriptase, an enzyme encoded by the virus. For each type of virus, therapeutic genes and their regulatory elements are inserted into the viral DNA. The viruses are designed
to be unable to replicate (i.e., “replication defec-
5
Suicide gene or RNA
strand
Gene for antigen or
cytokine
Entire virus
Beneficial gene
Antisense DNA
tive”), by deliberately deleting essential viral genes
[4]. Such “engineered” viruses must be grown in
“helper” cell lines that replace the deleted functions
of the virus, and sometimes even have to be injected into the patient. Replication-defective viruses are used due to concerns that viral replication within the patient might lead to cellular
transformation and/or produce significant illness.
In general, viral vectors are advantageous because
they are: 1) selective for certain types of cells (e.g.,
dividing) and/or tissue (e.g., brain), 2) able to integrate DNA into the host genome well, and 3) relatively stable. The gene transfer efficiency of viruses
is generally higher than that of nonviral delivery
methods. Disadvantages of viruses can include: 1)
tissue toxicity, 2) generation of immunological
and/or inflammatory reactions, and 3) the limited
size of the gene that can be transferred [4,6]. Retroviruses, for instance, can integrate in stable fashion into the DNA of the patient’s tumor cells, but
they can only carry a small genetic “payload” and
have a high level of genetic variability.
The gene therapy strategies that have been used
against cancer can be divided into five basic categories: 1) gene-directed enzyme prodrug (“suicide
gene”) therapy (GDEPT); 2) gene therapy designed
to boost the activity of the immune system against
cancer cells; 3) oncolytic virus therapy; 4) transfer
of potentially therapeutic genes, such as tumor suppressor genes, into cancer cells; and 5) antisense
therapy [4]. The first three approaches are designed to destroy cancer cells. Gene transfer-based
immunotherapy—such as the use of gene therapy
to develop cancer vaccines— has also been called
“immunogene therapy.” In the last two approaches,
cancer cells may be destroyed, but the primary
objective of treatment is to alter the behavior (i.e.,
“phenotype”) of the target cells [4]. What enters the
patient’s cells in each case is given in Table 2.
6
Surg Neurol
2000;54:3–9
Gene Therapy for Tumor
Cell Destruction
The first gene therapy protocol for patients with
glioblastoma multiforme was initiated by Oldfield et
al. at the National Institutes of Health (NIH) in 1992
[20]. Pioneering preclinical studies demonstrating
the feasibility of the chosen approach (GDEPT) had
been performed by Martuza, Breakefield, and colleagues [5]. In the clinical trial, murine cells containing a retroviral vector coding for the herpes
simplex virus thymidine kinase (HSV-TK) gene were
administered intracranially; patients were then
treated with the antiviral drug ganciclovir. The thymidine kinase phosphorylates the ganciclovir, creating a toxic nucleotide analogue that blocks the
function of DNA polymerase. This leads to the death
of the target cell when it enters the DNA synthesis
phase of the cell cycle (see Figure 1C) [4]. This
approach to gene therapy (called GDEPT or “suicide gene therapy”) is still probably the most
widely recognized among neurosurgeons. The intent is to selectively put a gene into tumor cells,
thereby making them vulnerable to a drug that
would not normally affect them. Several other suicide gene prodrug combinations are currently under investigation [1,26].
The cellular “bystander effect” is a key component of “suicide” gene therapy. After the introduction of the lethal gene into a few of the tumor cells,
neighboring (uninfected) tumor cells are also killed,
due to uptake of activated drug through intercellular transfer and/or endocytosis. The bystander effect is very important, as not all of the tumor cells
are successfully transfected with the suicide gene.
The effect allows a large therapeutic result to occur,
despite the fact that the gene transfer process itself
is “inefficient,” i.e., only affects a small percentage of
cells [1,4,18]. An extensive review of GDEPT has
been published, which describes the vectors used,
enzyme/prodrug systems, and the bystander effect
in detail [19].
Results from several brain tumor trials using the
GDEPT approach have now been published [26].
Although the feasibility and safety of suicide gene
therapy for brain tumor patients have been established, the efficacy of the approach remains to be
clearly demonstrated. For instance, in the study by
Shands et al, median post-treatment survival time
was 8.6 months, and only 27% of the patients were
alive at 12 months [27]. In the study by Klatzman et
al, median survival was less than 7 months, with
25% of patients living longer than 12 months [14]. It
Engelhard
has now been reported that pediatric brain tumor
patients (with gliomas, ependymomas, and primitive neuroectodermal tumors) have also been safely
treated with retrovirus-mediated HSK-TK gene therapy [21]. Potential problems with this type of therapy are: 1) lack of transgene delivery to a sufficient
number of tumor cells, particularly those that are
deeply invasive, and 2) inability of ganciclovir
(which is water soluble) to penetrate the bloodbrain barrier (BBB) in regions of the brain where
the BBB is intact, but may still harbor competent
tumor cells. However, clinical trials of GDEPT are
still open for brain tumor patients; retrovirus or
recombinant adenovirus is being used to deliver the
HSV-TK gene (see http://clinicaltrials.gov/ct/gui).
Of the gene therapy strategies used clinically to
treat cancer, immuno-modulatory trials have been
the most numerous. Although therapies designed to
elicit an immune response to tumors have been
sought for over a century with little success [1], the
advent of gene therapy “revolutionized” the field of
cancer immunotherapy [22]. Attempts at using gene
therapy to boost the immune system’s response
against cancer cells have often focused on activating cell-mediated immunity [4]. Tumor cells from an
experimental animal can be cultured in vitro, genetically modified to increase their tumorigenicity,
then irradiated and readministered subcutaneously
as a vaccine [22]. A variety of experimental approaches have been tested in cultured cells and
animal brain tumor models [4,26]. Past clinical trials for brain tumor immunogene therapy have centered on increasing production of cytokines such as
interleukin (IL)-2 and IL-4. MRI scans of some patients have shown tumor necrosis in response to
treatment [11,28]. New clinical trials to investigate
the use of local injection of allogeneic cytokineproducing cells, or dendritic cells (potent antigen
presenting cells) are being initiated.
In the oncolytic virus strategy, the viruses are
allowed to remain replication-competent. Viral replication within cancer cells results in lysis of the
cell, and the production of progeny virions, which
have the ability to infect and destroy adjacent cancer cells [4]. Herpes simplex virus 1 (HSV1) has
been a popular virus for oncolytic therapy. Although wild-type HSV1 is highly virulent and induces encephalitis in humans, research efforts have
produced a genetically-altered herpes virus with
low virulence for normal cells, but a retained ability
to target the glioma cells [15,17]. Strategies combining GDEPT and oncolysis are also being studied
[1].
Gene Therapy
Therapeutic Gene
Transfer and Antisense
Approaches
In the strategies described above, the ultimate goal
of the gene therapy—like chemotherapy or radiation therapy—is to kill tumor cells. Other approaches can be envisioned, however, in which the
transduced cells might continue to survive, but in
an altered form [4]. It would be ideal if gene therapy
could be used to “turn off” the genes causing the
cells to be cancerous (or replace lost or defective
genes), thereby restoring the normal control mechanisms limiting cellular proliferation and migration.
Such approaches have to be based on a precise
understanding of the molecular biology of brain
tumors. Fortunately, significant advances have
been made in this area: the initiation and progression of astrocytomas can now be related to: 1)
activation of cellular proto-oncogenes (including a
variety of growth factors and their receptors, intracellular messengers, cell cycle proteins and transcription factors), and/or 2) inactivating mutations
in tumor suppressor genes (such as those encoding
for the proteins p53, RB protein, p16INK4a, PTEN
protein, E2F-1, and p19ARF) [2,4,9,16,32].
The “replacement gene strategy” seeks to introduce a functional gene—such as a tumor suppressor gene—into the patient’s cells because the gene
is defective or absent (Figure 1B). Multicenter clinical trials to evaluate intratumoral injections of the
tumor suppressor gene protein p53 are currently
underway for patients with malignant glioma. p53
has been called the “guardian of the genome” because it coordinates the cell’s response to DNA
injury. When genetic damage occurs, p53 causes
growth arrest in the G1 phase of the cell cycle,
allowing time for DNA repair. If DNA injury exceeds
a critical repair threshold, p53 induces apoptosis
(“programmed cell death”) in order to stop the
perpetuation of potentially mutated cells [4]. p53
has been found to be functionally-inactivated in a
majority of malignant gliomas [16]; a comprehensive review of p53 and brain tumors has been published [12]. Although treatment with p53 has been
the prototype for this strategy, other gene therapies have been tested in experimental animals in
which the object is to express other beneficial proteins, such as one limiting angiogenesis [4,29,30].
Antisense-mediated gene inhibition has also been
considered a type of gene therapy. It is critical to
understand however, that in antisense therapy,
short segments of DNA—not a larger, functional
gene—are being introduced into the tumor cells.
Surg Neurol
2000;54:3–9
3
7
Target Genes for Antisense cDNA Transfection
Basic fibroblast growth factor (bFGF)
Protein kinase C, isotype ␣ (PKC␣)
Insulin-like growth factor 1 (IGF-1)
Urokinase-type plasminogen activator receptor (UPAr)
Transforming growth factor-␤ (TGF-␤)
Vascular endothelial growth factor (VEGF)
Undesirable genes in the tumor cells are being “targeted;” therefore, this strategy could be called
“gene targeted therapy.” Viral vectors may or may
not be used. The term “antisense” refers to the fact
that the therapeutic strands are complementary to
the coding (i.e., “sense”) genetic sequence of the
target gene. Antisense constructs hybridize (i.e.,
bind) in an antiparallel orientation to the mRNA
template, through Watson-Crick base pairing [8].
Two main antisense strategies have been employed: 1) transfection of cells with antisense cDNA,
and 2) treatment of cells with antisense oligodeoxynucleotides (ODNs). The former strategy has
been successfully used against glioma cells in vitro
and in animal models with the gene targets listed in
Table 3 [8,13].
Antisense ODNs are easy to synthesize, and are
readily taken up by cells by pinocytosis and/or
receptor-mediated endocytosis. After binding to
the target mRNA template, formation of a DNA: RNA
“heteroduplex” produces gene inactivation either
through steric blocking of the ribosome complex,
or by triggering mRNA cleavage by RNase H [8]
(Figure 1D). Direct ODN infusion into the brains of
animals has shown extensive penetration and minimal toxicity [10,31]. In animal models, transcription of a variety of genes has been successfully
blocked within the brain, using antisense ODN infusions [8]. Clinical trials with ODNs are now proceeding for a variety of cancers.
Conclusions
Impressive advances have been made in the fields
of basic virology and molecular biology over the
past two decades. The knowledge gained has been
exploited for use in gene therapy. Although gene
therapy has not yet produced a cure for brain cancer, several strategies have been shown to be feasible and reasonably safe for brain tumor patients.
Some patients treated with GDEPT lived more than
4 years [24]. Factors currently limiting further success might include: 1) lack of delivery of the therapeutic agent to deeply-invasive tumor cells, 2) lack
of adequate target (i.e., tumor) cell specificity, 3)
8
Surg Neurol
2000;54:3–9
potential pathologic (and/or immune) response of
the brain to the virus (or therapeutic molecule), 4)
low transfection efficiency and/or transient persistence of the gene, and 5) lack of identification of the
appropriate antineoplastic strategy and/or target
gene(s) [4,7,30].
At the present time, there is simply no way to add
or replace a gene throughout widespread areas of
the CNS [34]. Viruses (in particular) are very large
compared to conventional drugs and may have limited penetration into the brain. This may explain
why it has been possible to “cure” animals with
brain tumors using gene therapy, but not patients.
Animal brain tumors are much smaller than human
tumors, and not as deeply invasive into normal
brain. It is also clear that in contrast to the hereditary gene disorders, malignant brain tumors are
polygenetic—and thus far more complex—in terms
of their pathogenesis [26]. Different glioblastoma
patients express different sets of genes which culminate in the malignant phenotype. Genetic variability may even exist within different parts of one
patient’s tumor. Therefore, it is likely that the first
successes with gene therapy will probably be seen
in the treatment of other diseases.
Yet gene therapy continues to offer the potential
for providing treatments that are more precise,
more effective, and less toxic, than conventional
therapy [24]. It is likely that the development of
gene therapy is still in an early stage. Much ongoing
effort is being directed at developing improved vectors and increasing transduction efficiency [3]. A
new, more precise stereotactic surgery technique
has recently been reported, which should improve
saturation of intraparenchymal tumors with the
gene therapy vector [23]. Combination therapies
are being studied— either combinations of gene
therapies, or gene therapy used synergistically with
conventional techniques such as chemo- and/or radiation therapy [4,30]. Neurosurgeons (and others)
still envision using molecular biology to help cure
malignant brain tumors; because of this, the clinical
and basic science research will certainly continue.
The author thanks Mr. Kanti Bansal and Ms. Jill Hohbein for
assistance in performing background research for this article, and Dr. Kern Guppy for his thoughtful review. Dr. Engelhard’s laboratory is supported by a generous gift from the
Valerie Landis Research Endowment Fund of the University
of Illinois at Chicago.
Engelhard
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
REFERENCES
1. Alavi JB, Eck SL. Gene therapy for malignant gliomas.
Hemat Oncol Clin N Am 1998;12:617–29.
2. Alemany R, Gomez-Manzano C, Balague C, Yung WKA,
18.
Curiel DT, Kyritsis AP, Fueyo J. Gene therapy for
gliomas: molecular targets, adenoviral vectors, and
oncolytic adenoviruses. Exp Cell Res 1999;252:1–12.
Amado RG, Chen ISY. Lentiviral vectors: The promise
of gene therapy within reach? Science 1999;285:
674 – 6.
Bansal K, Engelhard HH. Gene therapy of brain tumors. Current Oncology Reports 2000, in press.
Carter BS, Zervas NT, Chiocca EA. Neurogenetic surgery: current limitations and the promise of gene- and
virus-based therapies. Clin Neurosurg 1999;45:226 –
46.
Chen J, Bezdek T, Chang J, Kherzai AQ, Willingham T,
Azzara M, Nisen PD. A glial-specific, adenovirus vector for brain tumor gene therapy. Cancer Res 1998;
58:3504 –7.
Dewey RA, Morrissey G, Cowsil CM, Stone D, Bolognani F, Dodd NJ, Southgate TD, Klatzmann D, Lassman
H, Castro MG, Lowenstein PR. Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy:
implications for clinical trials. Nat Med 1999;5:1256 –
63.
Engelhard HH. Antisense oligodeoxynucleotide technology: potential use for the treatment of malignant
brain tumors. Cancer Control 1998;5:163–70.
Engelhard HH, Butler AB, Bauer KD. Quantification of
the c-myc oncoprotein in human glioblastoma cells
and tumor tissue. J Neurosurg 1989;71:224 –32.
Engelhard HH, Narang C, Homer R, Duncan H. Urokinase antisense oligodeoxynucleotides as a novel
agent for malignant glioma: In vitro and in vivo studies
of uptake, effects and toxicity. Biochem Biophys Res
Com 1996;227:400 –5.
Fueyo J, Gomez-Manzano C, Yung WKA, Kyritsis A.
Targeting in gene therapy for gliomas. Arch Neurol
1999;56:445– 8.
Fulci G, Ishii N, Van Meir EG. P53 and brain tumors:
from gene mutations to gene therapy. Brain Pathol
1998;8:599 – 613.
Hall WA, Flores EP, Low WC. Antisense oligonucleotides for central nervous system tumors. Neurosurgery 1996;38:376 – 82.
Klatzmann D, Valery CA, Bensimon G, Marro B, Boyer
O, Mokhtari K, Diquet B, Saltzmann JL, Philippon J. A
phase I/II study of herpes simplex virus type I thymidine kinase “suicide” gene therapy for recurrent glioblastoma. Hum Gene Ther 1998;9:2595–2604.
Kramm CM, Chase M, Herrlinger U, Jacobs A, Pechan
PA, Rainov NG, Sena-Esteves M, Aghi M, Barnett FH,
Chiocca EA, Breakefield XO. Therapeutic efficiency
and safety of a second-generation replicationconditional HSV1 vector for brain tumor gene therapy. Hum Gene Ther 1997;3:1354 – 61.
Lang FF, Yung WKA, Sawaya R, Tofilon PJ.
Adenovirus-mediated p53 gene therapy for human
gliomas. Neurosurgery 1999;45:1093–1104.
Mineta T, Rabkin SD, Yazaki T, Hunter WD, Martuza
RL. Attenuated multi-mutated herpes simplex virus-1
for the treatment of malignant gliomas. Nat Med 1995;
9:938 – 43.
Namba H, Tagawa M, Iwadate Y, Kimura M, Sueyoshi
K, Sakiyama S. Bystander effect-mediated therapy of
Gene Therapy
19.
20.
21.
22.
23.
24.
25.
26.
27.
Surg Neurol
2000;54:3–9
experimental brain tumor by genetically engineered
tumor cells. Hum Gene Ther 1998;9:3– 4.
Niculescu-Duvaz I, Spooner R, Marais R, Springer
CJ. Gene-directed enzyme prodrug therapy. Bioconjugate Chem 1998;9:4 –22.
Oldfield EH, Ram Z, Culver KW, Blaese RM, DeVroom
HL, Anderson WF. Gene therapy for the treatment of
brain tumors using intra-tumoral transduction with
the thymidine kinase gene and intravenous ganciclovir. Hum Gene Ther 1993;4:39 – 69.
Packer RJ, Raffel C, Villablanca JG, Tonn JC, Burdach
SE, Burger K, LaFond D, McComb JG, Cogen PH,
Vezina G, Kapcala LP. Treatment of progressive or
recurrent pediatric malignant supratentorial brain tumors with herpes simplex virus thymidine kinase
gene vector-producer cells followed by intravenous
ganciclorvir administration. J Neurosurg 2000;92:
249 –54.
Parney IF, Hao C, Petruk KC. Glioma immunology and
immunotherapy. Neurosurgery 2000;46:778 –92.
Qureshi NH, Bankiewicz KS, Loius DN, Hochberg FH,
Chiocca EA, Harsh IV GR: Multicolumn infusion of
gene therapy cells into human brain tumors: technical report. Neurosurgery 2000;46:663–9.
Ram Z, Culver KW, Oshiro EM, Viola JJ, DeVroom HL,
Otto E, Long Z, Chiang Y, McGarrity GJ, Muul LM, Katz
D, Blaese RM, Oldfield EH. Therapy of malignant brain
tumors by intratumoral implantation of retroviral
vector-producing cells. Nat Med 1997;3:1354 – 61.
Ram Z, Walbridge S, Oshiro EM, Viola JJ, Chiang Y,
Mueller SN, Blaese RM, Oldfield EH. Intrathecal gene
therapy for malignant leptomeningeal neoplasia. Cancer Res 1994;54:2141–5.
Sasaki M, Plate KH. Gene therapy of malignant glioma:
Recent advances in experimental and clinical studies.
Annals Oncol 1998;9:1155– 66.
Shand N, Weber F, Mariani L, Bernstein M, Gianella-
28.
29.
30.
31.
32.
33.
34.
9
Borradori A, Long Z, Sorensen AG, Barbier N. A phase
1-2 clinical trial of gene therapy for recurrent glioblastoma multiforme by tumor transduction with the herpes simplex thymidine kinase gene followed by ganciclovir. Hum Gene Ther 1999;10:2325–35.
Sobol RE, Fakhrai H, Shawler D, Gjerset R, Dorigo O,
Carson C, Khaleghi T, Koziol J, Shiftan TA, Royston I.
Interleukin-2 gene therapy in a patient with glioblastoma. Gene Ther 1995;2:164 –7.
Tanaka T, Manome Y, Wen P, Kufe DW, Fine HA. Viral
vector-mediated transduction of a modified platelet
factor 4 cDNA inhibits angiogenesis and tumor
growth. Nat Med 1997;3:437– 42.
Weyerbrock A, Oldfield EH. Gene transfer technologies for malignant gliomas. Current Opinion Oncol
1999;11:168 –73.
Whitesell L, Geselowitz D, Chavany C, Fahmy B, Walbridge S, Alger JR, Neckers LM. Stability, clearance,
and disposition of intraventricularly administered oligodeoxynucleotides: implications for therapeutic application within the central nervous system. Proc Natl
Acad Sci USA 1993;90:4665–9.
Wick W, Furnari FB, Naumann U, Cavenee WK, Weller
M. PTEN gene transfer in human malignant glioma:
sensitization to irradiation and CD95L-induced apoptosis. Oncogene 1999;18:3936 – 43.
Yu JS, Burwick JA, Dranoff G, Breakefield XO. Gene
therapy for metastatic brain tumors by vaccination
with granulocyte-macrophage colony-stimulating
factor-transduced tumor cells. Hum Gene Ther 1997;
8:1065–72.
Zlokovic BV, Apuzzo ML. Cellular and molecular neurosurgery: pathways from concept to reality-part I:
target disorders and concept approaches to gene
therapy of the central nervous system. Neurosurgery
1997;40:789 – 803.
third sign of rebellion is the refusal of certain departments,
faculty members, or both to accept patients with managed
care health plans. This has now spread to private practitioners who
are tired of providing bargain medicine at bargain prices. Some
individuals worry that this could lead to a 2-class system of care,
which only makes me wonder what they believe we have now.
A
—Catherine D. DeAngelis, M.D., MPH
“The Plight of Academic Health Centers”
JAMA 2000;283:2438 –9