Gene Therapy for Mesothelioma
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- Vachani, A., Moon, E. & Albelda, S.M. Curr. Treat. Options in Oncol. (2011) 12: 173. doi:10.1007/s11864-011-0153-5
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Mesothelioma represents an especially good target for gene therapy since few effective therapies exist, the disease remained relatively localized until late in its course, the tumor can be accessed relatively easily through the chest wall, and the thin layer of mesothelial cells offers a large surface area for efficient, rapid, and diffuse gene transfer. Gene therapy trials in mesothelioma have shown safety, and some limited evidence of efficacy. We present a review of clinical trials that have been performed in mesothelioma and describe several new approaches currently being pursued.
Approximately 20 years ago, advances in molecular genetics and gene transfer technology made the development of “gene therapy” (the modification of the genetic makeup of cells for therapeutic purposes) a possibility for medicine. The disorders originally proposed as targets for gene therapy were inherited, recessive disorders in which transfer of a normal copy of a single defective gene could potentially prevent or alter the course of a disease. Clear, but slow progress has been made in the areas of genetic diseases such as blindness  and inherited immune deficiencies .
It soon became apparent that the range of target diseases could be extended to acquired diseases, such as cancer, and now many early phase clinical trials have now been conducted in various malignancies. Although these trials have demonstrated good safety results, there has been relatively limited efficacy.
Cancer gene therapy strategies used in mesothelioma
Suicide Gene Therapy
transduction of tumor cells with a gene encoding for a specific enzyme that induces sensitivity to an otherwise benign agent
HSV thymidine kinase
Cytokine Gene Therapy
Expression of immuno-stimulatory cytokines from tumor cells will activate the immune system in situ resulting in more effective anti-tumor immune
IL-2, Interferons (α,β, or γ)
Gene-Modified T-cells for Adoptive Transfer
modify T-cells with tumor antigen specificity and with enhanced properties
Mesothelin specific T-cells
Vectors used in gene therapy
Gene delivery efficiency is an important requirement for successful gene therapy. To this end various viral and non-viral vectors have been engineered including replicating and non-replicating viruses, bacteria, and liposomes . Each varies in regards to the targeted cell type, DNA carrying capacity, in-vivo gene transfer efficiency, and inflammatory response induced. Though no one vector is suitable for all diseases, one can tailor the vector to the specific disease of interest.
The most widely used vector is the recombinant, replication incompetent adenovirus. Several characteristics of the adenoviral vector make it attractive for gene therapy for cancer (but not genetic diseases). It is: i) able to transfect various target cell types in vivo, even when the cells are non-dividing, ii) transduce with high efficiency rates, and iii) achieve high level, but transient gene transfection [4, 5].
Adenoviral-based delivery is accompanied by significant local and systemic inflammation—an early innate component involving a cytokine surge and a later acquired immune response involving neutralizing antibodies and cytotoxic lymphocytes.
Importantly, the safety record of adenoviral vectors in humans has been excellent.
The principal advantages of this vector derive from its availability to accomplish efficient gene transfer in vitro in a broad range of targeted cells, with the capacity to achieve integration into the host genome and long term expression. However, retroviral vectors can achieve gene transfer only to dividing cells. They are labile in vivo since complement and other blood components inactivate the virion.
To circumvent the inability of retroviruses to infect non-dividing cells, vector systems based on the lentivirus genus of retroviruses, which includes human immunodeficiency virus (HIV), have been developed [6, 7]. Because these viruses are more complex than other retroviruses, and because of obvious safety concerns, development has been slow and cautious. Like retroviruses, they must be used ex vivo.
The adeno-associated virus (AAV) [8, 9] is a defective parvovirus with a single strand DNA genome and a naked protein coat. AAV has not been associated with any known human disease state, suggesting a significant safety margin for this vector. In vivo transduction may be possible. Long term expression can be seen.
Vaccinia/fowl pox vectors
Vaccinia is a double-stranded DNA virus whose entire life cycle takes place within the cytoplasm of infected cells. Due to its role in the eradication of smallpox, it has been used extensively in humans and is very safe.
Vaccinia is being explored as a vector for delivery of cancer therapeutic genes, as a carrier for tumor antigens and/or immunostimulatory molecules to develop cancer vaccines, and as a replication-selective, tumor-specific oncolytic virus [10, 11].
The related Fowl Pox vectors have been used primarily as cancer vaccines.
As an alternative to the viral vectors, a variety of non-viral vectors have also been developed for in vivo and in vitro gene delivery. Several general strategies have been developed to achieve this end, including liposomes, polymers, and molecular conjugates [12, 13]. For the most part, these strategies appear to be less efficient than the various viral vectors described above and they do not result in prolonged transgene expression.
Antisense therapy relies on inhibition of gene expression, accomplished with a targeted oligonucleotide delivered either intravenously or intratumorally leading to diminished transcription of the complementary mRNA. The oligonucleotide is usually modified to enhance stability. More recently, siRNA has been used in preclinical models, but has not yet moved to clinical trials.
Clinical investigations in malignant pleural mesothelioma
Suicide gene therapy
Suicide gene therapy involves transduction of tumor cells with a gene encoding for a specific enzyme that induces sensitivity to an otherwise benign agent. In essence, a “prodrug” is transformed into a toxic metabolite by the enzyme introduced into the cells with subsequent accumulation leading to tumor cell death or “suicide” .
An advantage of suicide gene therapy is the induction of a “bystander effect”—the killing of neighboring cells not transduced with the vector. A commonly studied suicide gene is the herpes simplex virus-1 thymidine kinase (HSVtk) gene which makes transduced cells sensitive to the nucleoside analog gancyclovir (GCV). GCV is metabolized poorly by mammalian cells and thus it is usually non-toxic. However, after conversion to GCV-monophosphate by HSVtk, it is metabolized rapidly by endogenous kinases to GCV-triphosphate which acts as a potent inhibitor of DNA polymerase and competes with normal mammalian nucleosides for DNA replication [14, 15].
Sterman and colleagues initiated a series of Phase 1 clinical trials of adenovirus (Ad.HSVtk/GCV) gene therapy in advanced MPM patients to assess toxicity, gene transfer efficiency, and immune response induction [16, 17]. Subsequent to a single intrapleural administration of Ad.HSVtk vector, GCV was given intraveneously twice daily for 2 weeks.
Dose-related intratumoral HSVtk gene transfer was demonstrated in 23 of 30 patients with those treated at a dose of equal to or greater than 3.2 × 1011 particle forming units (pfu) having evidence of HSVtk protein expression at tumor surfaces and up to 30–50 cell layers deep by immunohistochemical assessment.
Overall, the therapy was well-tolerated with minimal side effects and dose-limiting toxicity was not reached. Anti-tumor antibodies and anti-adenoviral immune responses, including high titers of anti-adenoviral neutralizing antibody and proliferative T-cell responses were generated in both serum and pleural fluid.
A number of clinical responses (i.e. survival of more than 3 years) were seen at the higher dose levels with two patients showing long periods of survival (one alive for seven years and one still alive after 10 years) . One of the two surviving patients had demonstrable reduction of tumor metabolic activity as assessed by serial 18-fluorodeoxyglucose positron emission tomography (18FDG PET) scans over several months. This relatively long response period was likely due to induction of a secondary immune bystander effect of the Ad.HSVtk/GCV instillation.
Schwarzenberger and colleagues conducted a Phase 1 trial using irradiated ovarian carcinoma cells retrovirally-transfected with HSV (PA1-STK cells) that were instilled intrapleurally followed by GCV for 7 days. Minimal side effects were seen and Tc radiolabeled PA1-STK cells demonstrated preferential adhesion to the tumor lining the chest wall. There were also some post-treatment increases in the percentage of CD8 + T lymphocytes in the pleural fluid. However, no significant clinical responses were seen .
Cytokine gene therapy
The rationale for cytokine gene therapy is that high level expression of immunostimulatory cytokines (such as interleukin-2 [IL-2], IL-12, tumor necrosis factor [TNF], granulocyte-mononcyte colony stimulating factor [GM-CSF], or interferons [α,β, or γ]) from tumor cells will activate the immune system in situ resulting in a more effective anti-tumor immune response without having to target specific antigens. The advantages of gene therapy over systemic administration of these agents includes lower toxicity, higher local concentrations, much longer persistence of the cytokine, and advantages relating to cytokine secretion by the tumor cell itself.
Robinson and colleagues conducted the first clinical trial of intratumoral cytokine gene delivery in MPM patients using a recombinant partially replication-restricted vaccinia virus (VV) that expressed the human IL-2 gene . Serial VV-IL-2 vector injections over a period of 12 weeks into chest wall lesions of six patients with advanced MPM resulted in minimal toxicity with no demonstrable evidence of vector spread to patient contacts. Though no significant regression of tumor was seen, modest intratumoral T-cell infiltration was detected on post-treatment biopsy specimens. As measured by reverse transcriptase polymerase chain reaction (rtPCR), VV-IL-2 mRNA was detected in biopsy specimens for up to 6 days post-injection (though declined to low levels by day 8) despite the generation of significant levels of anti-VV neutralizing antibodies .
Vero cells, which are immortalized monkey fibroblasts capable of expressing human proteins, have also been studied as a cytokine delivering vector in humans. Fourteen patients received four courses of injections of Vero cells expressing IL-2. The treatment was well tolerated with no significant adverse effects. Levels of circulating IL-2 were detected in half of the patients with one patient demonstrating transient tumor regression and one with disease stabilization for 4 months. To our knowledge, this approach is not being pursued further .
A Phase 1 Ad.Interferon-β (Ad.IFN-β) dose escalation trial in MPM and metastatic pleural malignancies demonstrated successful gene transfer in 7 of the 10 patients by measurement of pleural fluid IFN-β mRNA or protein [21, 22]. Anti-tumor immune responses, including humoral responses to known tumor antigens (e.g. SV40 Virus Tag, mesothelin) and unknown tumor antigens were elicited in 7 of 10 patients. Four patients demonstrated meaningful clinical responses defined as disease stability and/or partial regression on 18FDG-PET and CT imaging 2 months after vector administration.
In light of the encouraging results, a second study was performed with the aim of augmenting these immunologic and clinical response [23•]. Based on preclinical studies showing enhanced effects after two doses , this trial involved two administrations of Ad.IFN-β (levels ranging from 3 × 1011 to 3 × 1012 viral particles) via an indwelling pleural catheter separated by 1 to 2 weeks was conducted in seventeen patients (10 with MPM and 7 with malignant pleural effusions). Again, overall treatment was well tolerated and anti-tumor humoral immune responses similar to that seen in the initial trials were induced. Several patients had meaningful clinical responses (mixed and/or partial responses) as determined by pre- and post-vector delivery PET/CT scans. However, high anti-adenoviral neutralizing antibodies titers were detected, even with a dose interval as short as 7 days, inhibiting effective gene transfer of the second dose.
Based on preclinical studies showing synergy between Ad.IFN and systemic chemotherapy, our group at Penn has initiated a trial of Ad.IFN-α in combination with chemotherapy for MPM patients.
One especially promising new area in gene therapy is the use of lentiviral or retroviral vectors to transduce T cells with modified T-cell receptors engineered to attack specific tumor antigens . This can be done by introduction of totally artificial chimeric T-cell antigen receptors (CARs) that use single chain antibody fragments to define antigen specificity and intracellular fragments of both the T-cell receptor and accessory molecules (such as CD28 or 4-1BB) to enhance activation .
This approach has shown success in patients with melanoma [27, 28] and our group and others have strong preclinical data to support using T cells targeted to attack mesothelin expressing tumor [29, 30].
A clinical trial with mesothelin-targeted T cells at the University of Pennsylvania will begin in early 2011.
Summary and future directions
In the past two decades, much experimentation using gene therapy has been done pre-clinically, and some clinical trials for MPM have been performed. In general, these trials have shown safety, but only intermittent efficacy.
In vivo gene transfer has been clearly achievable, but with the vectors currently available, it has been very difficult to transduce more than a small percentage of tumor cells, and this is usually only accomplished by local injection. This limitation has thus doomed the approaches that do not have strong bystander effects (i.e. oncogene inactivation or replacement of tumor suppressor genes).
Currently, most of the efforts in MPM gene therapy are focused on immunogene therapy—delivery of inflammatory cytokines or gene-modified T cells. Future trials will need to incorporate these novel approaches in combination with standard of care therapies (e.g. chemotherapy, pleurectomy).
At this point in time, gene therapy for mesothelioma remains experimental and limited to a few referral centers. However, the practicing clinician can participate in moving this approach forward by not taking a nihilistic approach to MPM, but by discussing the option of participating in clinical trials with his patients. The most important criteria for participation would be a good performance status and a willingness to participate in a clinical trial.
No potential conflicts of interest relevant to this article were reported.