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Scientic 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;54:39. BACKGROUND

Over the past two decades, signicant advances have been made in the elds 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 eld 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 ve 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 genessuch as tumor suppressor genesinto 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 eld 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 rst 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 unfullled [30]. Yet signicant advances in the eld continue to be made [23], and new clinical protocols are being initiated every year (see http://clinicaltrials.gov/ct/gui). Gene therapy can be dened as the transfer of genetic material (usually DNA) into a patients 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 modication) 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 mutagenesisi.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

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A: Depiction of the usual processes of transcription, post-transcriptional modication, 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 gure), 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 benecial 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 dont 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 specic to the mRNA of an undesirable protein, such as the c-myc oncoprotein.

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Methods for Introducing DNA or Genes into Cells

Categories: Gene Therapy for Cancer

Direct microinjection Electroporation Calcium phosphate transfection Pneumatic delivery (the gene gun) Cationic liposomes Genetically-engineered viruses (adenovirus, herpes simplex virus, adeno-associated virus, retroviruses)

WHAT ENTERS PATIENTS CELLS?


1) Gene-directed enzyme prodrug therapy 2) Immuno-gene therapy 3) Oncolytic virus therapy 4) Therapeutic gene transfer 5) Antisense therapy Suicide gene or RNA strand Gene for antigen or cytokine Entire virus Benecial gene Antisense DNA

a mutation has been a source of fear in the use of gene therapy. However, such a mutation could only be passed to a patients 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 vehiclesvectors are modied 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 rst 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 inltration 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 immunodeciency 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-

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 signicant 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 efciency 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 inammatory 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 patients 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 ve 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 rst three approaches are designed to destroy cancer cells. Gene transfer-based immunotherapysuch 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 patients cells in each case is given in Table 2.

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Gene Therapy for Tumor Cell Destruction


The rst gene therapy protocol for patients with glioblastoma multiforme was initiated by Oldeld 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, Breakeeld, 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 inefcient, 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 efcacy 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

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 sufcient 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 eld of cancer immunotherapy [22]. Attempts at using gene therapy to boost the immune systems 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 modied 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].

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Therapeutic Gene Transfer and Antisense Approaches


In the strategies described above, the ultimate goal of the gene therapylike chemotherapy or radiation therapyis 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, signicant 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 genesuch as a tumor suppressor geneinto the patients 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 cells 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 benecial 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 DNAnot a larger, functional geneare being introduced into the tumor cells.

Target Genes for Antisense cDNA Transfection

Basic broblast 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 elds 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 specicity, 3)

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potential pathologic (and/or immune) response of the brain to the virus (or therapeutic molecule), 4) low transfection efciency and/or transient persistence of the gene, and 5) lack of identication 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 polygeneticand thus far more complexin 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 patients tumor. Therefore, it is likely that the rst 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 efciency [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. Engelhards laboratory is supported by a generous gift from the Valerie Landis Research Endowment Fund of the University of Illinois at Chicago.

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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.
Catherine D. DeAngelis, M.D., MPH The Plight of Academic Health Centers JAMA 2000;283:2438 9

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