In Vivo Gene Delivery Methods

A wide range of methods are available for delivery of DNA or RNA into target cells. In vitro techniques such as direct microinjection and chemical (calcium phosphate) or physical (electroporation) transfection methods are not practical for in vivo application. This leaves “naked DNA” administration, liposomal gene delivery, and virally mediated gene transfer as the methods of choice for in vivo gene therapy application. Injection of “naked DNA” plasmids has been shown to result in intracranial gene expression.² However, with current technology, expression efficiency is very low and the practical value of this technique for brain tumor gene therapy remains to be seen. In contrast, liposomal gene transfer has been established as a promising therapeutic modality for brain tumors.³ Good transfer efficiency, safety, simple design, and their unique capability to carry, not only genetic material, but also proteins, chemicals, or physical particles into a cell make these artificial lipid bilayered vesicles useful and exciting therapeutic tools. However, viral-based vectors and therapeutic viruses are the absolute mainstay in the field of gene therapy.

Viral Therapies

Viruses have evolved as nature’s way to deliver genomic material into cells with unsurpassed efficiency, displaying an overwhelming repertoire of mechanisms. Since the early 1990s advances in the field of molecular biology have provided the tools necessary to manipulate viral genomes. The vast majority of viral vectors are composed of recombinant viruses, often engineered to exhibit attenuated phenotypes and harbor nonviral genes of interest. The search for and the design of appropriate vectors are directed by certain paradigms that depend on the clinical context. Simplified, in gene therapy applications for genetic or degenerative diseases the duration of the expression of the therapeutic gene is a foremost priority. In contrast, antitumor gene therapy generally seeks to destroy the target cell, making transduction efficiency much more important, and a short term gene expression appears to be sufficient. For tumor treatment, viruses have both been used as gene delivery vehicles (viral vectors) or as biological agents that attack and kill tumor cells directly (oncolytic viruses). Gene therapy strategies using adenoviral or retroviral vectors to deliver suicide genes to tumor cells have been the forerunners in the field and both have been used in clinical trials against human high-grade gliomas. In vivo transduction efficiency has been the main problem with retroviruses,⁴ which can deliver genes only to cells that are actively dividing.⁵ Insertional mutagenesis is a rare but feared side effect and can lead to the formation of secondary tumors resulting from oncogene activation or tumor-suppressor gene silencing due to the uncontrollable random insertion of the viral transgene into the human genome. Low transfection efficiency and lack of any therapeutic effect in phase III trials of RV-HSV-tk in newly diagnosed glioblastoma led to abandonment of retroviral strategies.⁶ First-generation adenoviruses (AdV) had been deprived of their replicative capabilities by deletion of variable portions (E1 or E3) of the viral genome.⁷ As these AdV vectors retain large portions of their viral genome, vectors and transduced cells are highly immunogenic once administered into the immunocompetent host. Consequently, therapeutic effects are short lived, though the enhanced immune response may noticeably contribute synergistically in the setting of anticancer therapy. Newer adenovirus strategies use either highly stripped down “gutless” AdV, requiring helper systems for production⁸ with minimized immune response and prolonged gene expression or conditional replication competent variants with inherent tumor selective affinity.⁹ An important aspect of antitumor gene therapy is the spatial distribution of the viral therapeutic. The therapeutic activity increases significantly from cell-limited gene expression over the so-called bystander effect of released therapeutic products to the active spread of replication-competent viruses. Replication-incompetent viral vectors have been proven ineffective in vivo, as only a small percentage of tumor cells can be infected under the most optimal circumstances with the delivered viral load.^10,11 Whether the viral therapeutic will replicate in the tissue is an important issue: Nonreplicating viruses are most commonly used to deliver toxic or therapeutic genes. Conditionally replicating viruses are based on replicating viruses but one or more of their viral genes are deleted to make them depend on cellular mechanisms, which are present only in tumor cells. Although this is a very attractive strategy, replicative health of most of these viruses is also impaired.^6,12 Replicating viruses are used to infect tumor tissues and viral progeny produced inside the target tissue infects surrounding cells to cause “local self-amplification.” A considerably small amount of virus can therefore be delivered to the target tissue to yield a much heavier therapeutic dose inside the tumor. Such a phenomenon does not exist in any other therapeutic modality. Newer, recombinant adenoviruses were engineered to be conditionally replicative in tumor cells or carried transgenes that are expressed only in specific cell types.¹³ Recombinant herpes viruses have also been commonly used in gene therapy protocols for brain tumors.⁶ Recombinant viral vectors based on vaccinia have also been used. In contrast to strategies that use viral vectors to transfer genes to the target cell, other researchers have experimented with oncolytic viruses, which preferentially replicate inside the tumor tissue and kill the tumor cells as a direct result of this infection.¹⁴ Other viruses including reovirus; Newcastle disease virus; and measles, vesicular stomatitis, and myxoma virus, have shown merit for infecting some types of brain tumor cells.¹⁵

In animal experiments viral infection can easily be monitored inside the target tissue or elsewhere using virally expressed fluorescent reporter genes. Fluorescent reporter genes can be engineered into most viruses, and when combined with tumor models that express other fluorescent proteins, temporal and spatial monitoring of both the oncolytic activity and/or side effects is greatly simplified.¹⁶

Immunotherapy/Immunogene Therapy Strategies

Cancer immunotherapy has been intensely studied in the field of malignant melanoma therapy, and the promise in that field among others led in October 2000 to the formation of the Immunotherapy Task Force for malignant brain tumors.¹⁷ As with gene therapy and oncolytic virotherapy, the focus of current immunotherapy studies of brain tumors is on tumors of neuroectodermal lineage, that is, glioblastoma and anaplastic astrocytoma. Though the transfer of this strategy to meningioma remains hypothetical at this point, a brief discussion of the current studies is merited.

Besides detection of infectious or foreign organisms the immune system has the capability to recognize and even eliminate cancerous cells. For that response to be effective, certain preconditions need to be met. Tumor-associated antigens (TAAs) need to be expressed and the tumor environment needs to be “immune-compatible,” as certain tumors can generate a local and systemic state of immune suppression. Evidence suggests both membrane and intracellular elements can serve as TAAs.¹⁸ However; despite its remarkably high selectivity, the natural immune response falls short in terms of efficiency to control advanced, sizable cancers. The limit of a patient’s immune response seems to be in the range of less than a million tumor cells, which is a negligible fraction compared to the billions of cells of an average sized brain tumor.¹⁹ In that regard, immunotherapy can be defined as strategies aiming to amplify the natural immune response to cancerous cells. Both arms of the immune response, the initiating cascade and the executing effector phase, can be targeted.

Earlier attempts of immunotherapy included the use of tumor lysates and microbe preparations such as nonspecific immune activators with little success.²⁰ With increasing knowledge of mechanisms underlying antigen presentation and immune modulation, the direction shifted toward highly specific stimulation of the antitumor response. The main strategies comprise the use of cytokines,²¹ active vaccination with tumor antigen-pulsed dendritic cells,²² and adoptive immunotherapy using cytotoxic T cells raised and expanded ex vivo.²³ Among others, cytokines interleuking-2 (IL-2), IL-4, IL-12, IL-18, interferon (IFN), granulocyte-macrophage colony-stimulating factor (GM-CSF), and B7-2 have been used with promising results alone, in combinations, or as adjuvants to cell-based immunotherapy.²⁴ Applications range from direct peripheral infusion and convection-enhanced delivery to in vivo cytokine gene therapy and ex vivo gene transfer to antitumor dendritic or cytotoxic T cells. Active antitumor vaccination involves the collection of blood mononuclear cells and the in vitro isolation and priming of dendritic cells. Considered the most potent antigen presenting cells, dendritic cells pulsed with tumor cells or tumor lysate can effectively prime T-helper cells once reinjected into the patient’s body, leading to an efficient mounting of an antitumor response by cytotoxic T lymphocytes. In contrast, adoptive immunotransfer focuses on ex vivo expansion and reinjection of tumor-specific effector cells. Several cell types have been described with potential for adoptive immunotherapy: lymphokine-activated killer cells, tumor infiltrating lymphocytes, cytotoxic T lymphocytes, and others.

What Has Been Achieved So Far?

Herpes simplex virus and adenovirus are among the first recombinant virus species used for gene therapy applications. Both viruses have been extensively used both in preclinical and clinical studies against various types of tumors and have shown promise against meningioma. Similarly, poliovirus²⁵ and herpesvirus²⁶ replicons have been found to effectively transduce meningioma cells. Other viruses such as reovirus,²⁷ myxoma virus,²⁸ and retroviruses²⁶ have been reported to be ineffective against meningioma cultures, despite their potential against other kinds of brain tumor.

Herpes simplex virus and adenovirus have been the most common recombinant virus species used for gene therapy attempts. Both viruses have been extensively used both in preclinical and clinical studies against various types of tumors. Herpes simplex was the first oncolytic virus tested against meningiomas.²⁹ Herpes simplex virus is a large, enveloped virus that belongs to the alpha-herpesvirus family and has a 152-kb double-stranded linear DNA genome. The wild-type virus has 80 densely packed genes, approximately 50% of which are necessary for viral replication. Almost 30 kilobases can be replaced with transgenes without affecting viral replication. The virus can infect both replicating and nonreplicating cells. Lytic infection of herpesviruses has three phases (α, β, and γ) and genes taking part in these three phases are responsible for regulating transcription, promoting DNA synthesis and creating a favorable environment for protein synthesis, respectively. Earlier genes in the genome have a suppressor role on late genes. Recombinant HSVs have been designed by making changes in or deleting α-genes (e.g., G47Δ, NV1020), β-genes (DLSPTK, G207), or γ-genes (R3616, G207). Herpes simplex is a large virus with a large transgene capacity, is stable, infects many cell types, and has animal models available for toxicity. Absence of proinflammatory effects and bone marrow toxicity, the availability of antiherpetic chemotherapy, and the absence of genomic integration are advantages of the HSV for gene therapy. In contrast, the large size of the genome, presence of preexisting HSV immunity in humans, and risk of encephalitis from primary or latent infection are the basic concerns associated with HSV. G207, which is the only HSV that was used in experimental studies against meningioma, had been tested in a phase 1 clinical trial against human glioblastoma.^30–32 Magnetic resonance imaging (MRI) responses and few long-term survivors have been reported in these glioblastoma trials, with no chronic toxicity observed in survivors.