The Promise of Gene Therapy for Pancreatic Cancer

Abstract

Unlike for other digestive cancer entities, chemotherapy, radiotherapy, and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for patients with PDAC. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using nonviral therapeutic gene delivery, targeted transgene expression, or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC but may also significantly influence the field of gene-based molecular treatment of cancer.

Introduction: State of the Art

Pancreatic adenocarcinoma (PDAC) originates from the exocrine part of the pancreas. Despite the continued efforts of scientists and clinicians, PDAC is associated with a high death rate. Two-thirds of patients with PDAC die within 1 year of diagnosis, as the median survival hardly reaches 6 months for patients with advanced metastatic disease.¹ Pancreatic cancer cells maintain self-sufficiency in growth signals, are resistant to endogenous antiproliferative signals, evade apoptosis, have limitless replicative potential, and undergo tissue invasion and metastasis.^2,3 As a result, pancreatic cancer is usually resistant to conventional therapeutic approaches (chemotherapy, radiotherapy) and targeted biotherapies. Phase III clinical trials have shown that multidrug cocktails, such as FOLFIRINOX (folinic acid, fluorouracil, irinotecan, and oxaliplatin), can significantly improve the survival of patients with advanced pancreatic cancer.^4,5 In addition, new targeted therapies are emerging, such as nanoparticle albumin-bound (nab)-paclitaxel (Abraxane), a combination of albumin and paclitaxel that favors tumor cell targeting and enhances the clinical activity of the chemotherapy.^6,7 Last, promising innovative treatments based on patient genetics [poly(ADP-ribose) polymerase (PARP) inhibitor]⁸ or on targeting of the tumor microenvironment (hyaluronidase,⁹ sonic hedgehog inhibitors)¹⁰ are currently being evaluated in early-phase clinical trials. Nevertheless, the prognosis for patients with advanced pancreatic cancer remains poor and there is an urgent need for new therapeutic approaches such as gene therapy. In this review, we comment on the latest developments in PDAC gene therapy, including gene-based tumor cell sensitization to chemotherapy, tumor targeting, and the development of novel nonviral as well as oncolytic viral therapies within the French scientific community.

Gene Therapy to Treat Pdac: Advanced Clinical Trials

PDAC has been actively targeted by gene therapy approaches, and gene therapy products are currently in late clinical trials, alone or in combination with chemotherapeutic agents. TNFerade is the first gene therapy product that has advanced to a phase III clinical evaluation for pancreatic cancer.¹¹ However, a randomized, phase III, multicenter study demonstrated that TNFerade combined with standard-of-care was safe but failed to prolong survival in patients with locally advanced pancreatic cancer, as compared with standard-of-care alone.¹² Since then, REOLYSIN (wild-type reovirus serotype 3 Dearing) has been tested in phase II clinical trials in combination with gemcitabine and Abraxane in patients with metastatic pancreatic cancer. In addition, the first gene therapy product approved in China, Gendicine, a recombinant Ad–p53 gene therapy product for head and neck squamous cell carcinoma, was transferred for the management of patients with pancreatic cancer, but this is restricted to the Chinese market. Along the same line, Rexin-G, a retrovector bearing a cytocidal cyclin G1 construct,¹³ was approved in the Philippines and in the United States for the management of solid tumors, including pancreatic cancer.

Gene Therapy to Treat Pdac: Preclinical Studies and Early Clinical Trials in French Laboratories

Nonviral gene therapy of PDAC

Resistance to conventional drugs such as gemcitabine is responsible for the lack of efficacy of treatments for PDAC. Combining drugs such as gemcitabine with gene therapy agents capable of killing cancer cells by alternative pathways may bypass this resistance.

The INSERM U1037 group in Toulouse used this principle in the first in-human clinical trial, based on the use of nonviral vectors to transfer anticancer genes that sensitize PDAC cells to gemcitabine chemotherapy, namely somatostatin receptor type 2 (SSTR2), deoxycytidine kinase (DCK), and uridine monophosphate kinase (UMK).¹⁴ We have previously demonstrated that expressing SSTR2, a G protein-coupled receptor, in PDAC experimental tumors strongly inhibits cell proliferation, tumor progression, and tumor angiogenesis, and induces cancer cell apoptosis,¹⁵ through activation of the caspase-3 and PARP pathways.¹⁶ In addition, SSTR2 synergizes with tumor necrosis factor (TNF)-α receptors to enhance cancer cell death by apoptosis.¹⁷ In greater detail, SSTR2 was initially found to inhibit cell proliferation by stimulating tyrosine phosphatase activity.¹⁸ Interestingly, SSTR2-expressing cells produced somatostatin-like immunoreactivity that corresponded mainly to somatostatin 14, indicating the induction of a negative autocrine loop. Accordingly, the SSTR2–somatostatin axis was demonstrated to induce a local and a distant antitumoral bystander effect through the inhibition of vascular endothelial cell growth factor (VEGF) production and the activation of SSTR3 on tumor blood vessels to control angiogenesis,¹⁵ and of SSTR1 on tumor cells to control the progression of distant tumors,¹⁹ respectively. DCK and UMK are two key kinases involved in gemcitabine metabolism, and we demonstrated that expression of DCK::UMK fusion genes chemosensitizes cancer cells to this drug.²⁰ This trial (TherGAP) included 22 patients with advanced PDAC who were not eligible for surgery. Two ultrasound endoscopy-guided intratumoral injections of the gene therapy product, 1 month apart, combined with chemotherapy infusion, were safe and well tolerated, without serious side effects. Interestingly, a population of patients with locally advanced tumors and high levels of plasma α₂-macroglobulin at diagnosis benefited from this treatment, with two of these patients surviving for 2 years after gene therapy. This early-phase clinical trial demonstrated that intratumoral gene delivery is safe and feasible in patients with pancreatic cancer who cannot undergo surgery. Efficacy will be further evaluated in a forthcoming phase II clinical trial that should start in 2016 in several French centers.

The success of gene therapy protocols relies strongly on the identification of gene delivery vectors that are safe and easy to manufacture, and with significant DNA delivery efficacy. Polyethylenimine (PEI), a cationic polymer, was chosen for the TherGAP clinical trial as it complexes therapeutic DNA to prevent degradation, and permits intracellular delivery. Substantial progress has been made in the design and development of nonviral vectors that could be used to target primary tumors or distant metastases.

A more efficient version, L-PEI, with lower cytotoxicity, has been developed from the “parental” PEI. The modifications include substitution of the amino groups of PEI either with acetate, butanoate, hexanoate, or alkylcarboxylate groups.^21

–24 Although these amino group substitutions reduced the number of amino groups on PEI and thereby its cytotoxicity, they did not improve transfection efficacy. One of the remarkable properties of PEI is its high buffering capacity due to the great density of secondary and ternary amino groups capable of inducing a proton sponge effect in endosomes, allowing plasmid DNA delivery into the cytosol. The CNRS group in Orléans has demonstrated that histidine can be used advantageously to destabilize membranes to increase the buffering capacities of the resulting polymer,²⁵ to enhance transfection efficiency, and to reduce cytotoxicity.²⁶ The absence of cell division strongly limits nuclear delivery of plasmid DNA in quiescent cells. The same group has identified that sequences recognized by the NF-κB transcription factor can improve the nuclear import of plasmid.^27,28 In addition, inserting E3 early proteins of adenoviruses can mediate active migration of large plasmid DNA molecules along the microtubules toward the nucleus when linked to the plasmid DNA.²⁹ Thus, equipping plasmid DNA with such devices may contribute to the development of “artificial” viruses challenging viral vector gene transfer efficacy.

The high propensity of PDAC to develop metastasis is a key factor in the low 5-year survival rate observed, even in patients with early-stage disease who are candidates for surgery. The liver is the most common anatomical site of PDAC metastasis, but the lungs can also be affected. This is particularly true among long-term survivors (>5 years) after resection of the pancreas for adenocarcinoma, for whom the most common site for disease recurrence is the lung.^30,31 Beyond PDAC, the lung is an organ often affected by metastasis of numerous types of cancers. Therefore, the development of vectors capable of transfecting the lungs should provide new tools in the management of metastatic diseases. In this context, a screen based on imaging transgene expression and aiming at the discovery of new nonviral gene delivery vectors that are effective in vivo was performed in the TIRO (Transporters in Imaging and Radiotherapy in Oncology) laboratory in Nice. The results showed that the tetrafunctional block copolymer 704 was capable of transfecting the lungs. Tetrafunctional block copolymers have a tetrafunctional structure consisting of four poly(ethylene oxide)/poly(propylene oxide) blocks centered on an ethylenediamine moiety. They form small complexes with DNA, as observed by electron microscopy.³² This formulation was shown to result in higher levels of reporter gene expression than the GL67A formulation that was used in a clinical trial in patients with cystic fibrosis.³³ The inflammatory response associated with this gene transfer was lower than that induced by the GL67A formulation, and the 704 formulation was amenable to repeated administrations.³⁴ As this formulation essentially targets type I and type II pneumocytes,³⁴ its application in oncology would involve therapeutic, diffusible transgenes, ectopically expressed by lung epithelial cells and exerting a selective effect on implanted metastatic cells. The proof of principle of this strategy was demonstrated on lung metastasis of colorectal cancer and osteosarcoma.³⁵

Targeting gene expression to maximize efficacy and safety

Despite careful intratumoral administration, therapeutic DNA was recovered in the blood of patients in the TherGAP trial, suggesting systemic leakage of the transgene product in the vasculature or release by necrotic tumor cells.¹⁴ Considering the potentially toxic nature of the DCK::UMK transgene, important side effects or adverse effects might be anticipated. This is also true for the other strategies described in this review. Naldini's group, in 2006,³⁶ took advantage of the differential expression of microRNA (miRNA) in different tissues to restrict transgene expression to target tissues. This breakthrough, known as the “detargeting approach,” is significant as it allows immune clearance of the transgene to be overcome. The group in Orléans³⁷ and others^38,39 have developed an alternative approach called the “programming approach”; it differs from the detargeting approach in that the endogenous RNA interference (RNAi) machinery is not used to suppress expression of the transgene in nontarget cells but, rather, to induce its expression in target cells. The overall strategy relies on the use of an miRNA cassette (miR T) positioned in the 3′ untranslated region (UTR) of a transcriptional repressor. When the miRNA of interest is present, it binds to the miR T cassette and induces the degradation of the repressor mRNA in targeted cells. In the absence of repressor protein, RNA polymerase II binds to the promoter to transcribe expression of the transgene. The CNRS UPR4301 group in Orléans provided experimental evidence that this programming approach is as robust, specific, and tight as the detargeting approach.³⁷ In the same line of research, Pichard and colleagues³⁸ from INSERM U948 in Nantes demonstrated that this strategy was transposable to engineered lentiviral vectors, in vitro and in vivo. In the context of PDAC, tumor cells express an opposite pattern of miRNA as compared with normal, surrounding, healthy cells.^40,41 miRNA-21, miRNA-155, miRNA-210, and miRNA-10a/b are the most upregulated miRNAs in human PDAC tissues and are indicative of poor survival, poor response to treatment, and development of metastases. On the other hand, miRNA-34a, let-7, miRNA-96, miRNA-148a, miRNA-217, and miRNA-375 are often downregulated in PDAC.⁴² Such a differential expression pattern has been used to control expression of therapeutic genes in PDAC, using one of the genetic switches described previously.^43,44 Altogether, these series of data generated from various laboratories indicate that this programming approach might be valuable, first to program expression of the transgene in target cells and second to prevent expression of the cytotoxic transgene in normal healthy cells.

Oncolytic virotherapy for PDAC

Pancreatic carcinogenesis is associated with a series of changes that provide a selective growth advantage to the cancer cells. However, these very changes (among them, lack of interferon response, elevated metabolic activity, and disengagement of cell cycle control) render cancer cells highly sensitive to viral infection. As a consequence, selectively replicating viruses (oncolytic viruses) represent a promising new therapeutic strategy. These viruses can be naturally “cancer selective” for their replication or may require genetic modifications. A clinical trial watch report has summarized the use of oncolytic virus for cancer therapy.⁴⁵

In the case of PDAC, several viruses have been explored for their ability to replicate and to inhibit tumor growth in experimental models.^46
–48 Preclinical studies have demonstrated that conditionally replicative adenoviruses (CRAds) can be exploited for PDAC therapy, when combined with gemcitabine.⁴⁹ Pioneering studies demonstrated that intratumorally injected ONYX-15 combined with gemcitabine was well tolerated in a phase I/II clinical trial for patients with PDAC.⁵⁰ Current refinements in CRAds as a result of a German–French collaboration comprise modifications of the hexon to improve viral replication in both tumor and stromal cells,⁵¹ as PDAC is characterized by a substantial stromal reaction that impedes the intratumoral dissemination of therapeutic drugs.

In one study, the INSERM U1037 team in Toulouse demonstrated that engineered herpes simplex type 1 viruses (HSV-1) are efficient in blocking experimental tumor growth, used alone or in combination with gemcitabine.⁵² HSV-1-based viruses infect various tumor cell types, do not integrate into the genome of infected cells, and are safe in patients; in addition, there are several anti-HSV-1-specific drugs available in case of adverse events. Also, cell killing of entire cancer cell populations can be achieved rapidly with a relatively low dose of virus. The most widely used strategy to restrict HSV-1 replication to cancer cells is to delete genes that are important for viral replication as well as the evasion of innate immune responses. These include the IFN-related protein kinase R (PKR) antiviral response.⁵³ ICP6 encodes the large subunit of viral ribonucleotide reductase (RR), an enzyme involved in de novo synthesis of deoxynucleotides.⁵⁴ In the absence of viral RR, viral replication depends on host cell RR activity, which has been reported to be higher in cancer cells.⁵⁵ Consequently, HSV-1 mutants with deletions in the ICP6 gene preferentially replicate in actively dividing cells such as malignant cells. Viral double-stranded RNAs activate various cellular antiviral responses, including PKR, which phosphorylates and inactivates the protein initiation factor eIF-2α, thus inhibiting protein synthesis and host cell growth. Amongst other functions that have evolved to overcome the innate cellular responses against infection, viral γ₁34.5 interacts with cellular protein phosphatase-1α, resulting in dephosphorylation of eIF-2α, thus inhibiting the action of PKR.^48,53 This allows the reinitiation of protein translation and robust viral replication. Deleting γ₁34.5 may restrict the viral cytopathic effect and replication to malignant cells, in which eIF-2α phosphorylation is deregulated as a result of the activation of the K-ras pathway.⁵⁶ First-generation oncolytic HSV-1 viruses, such as OncoVex and HF10, which have reached clinical applications, were ablated for the γ₁34.5 viral protein and resulted in reduced therapeutic efficacy.⁴⁸ In the work performed by the INSERM U1037 group in Toulouse, the HSV-1-based virus Myb34.5, in which expression of the γ₁34.5 viral protein is controlled by a tumor-specific B-Myb promoter, efficiently replicates and kills human PDAC cells. When injected in orthotopic human tumors developed in nude mice, Myb34.5 successfully replicates, strongly inhibits tumor progression, and blocks metastatic dissemination. The Myb34.5 antitumor effect is potentiated by gemcitabine administration, both in vitro and in vivo. Interestingly, PDAC tumors may offer an exquisite “niche” for Myb34.5 replication as they express high levels of cellular ribonucleotide reductase activity, while nucleotide influx is strongly impaired. This work stems from the use of second-generation, HSV-1-based virus in early-phase clinical trials in patients with advanced PDAC.

Parvovirus may also be of great interest for PDAC therapy. The oncolytic parvovirus H-1 (H-1PV) belongs to the Parvoviridae family and is composed of a small, nonenveloped, icosahedral capsid containing a linear, single-stranded DNA of about 5 kb in size. The mechanism underlying the natural selective replication as well as the specific toxicity of the virus for rat and human cancer cells is complex and multifactorial. If H-1PV binds and infects normal and transformed permissive cells equivalently, it is thought that the molecular abnormalities encountered in cancer cells favor nuclear envelope breakdown, viral gene expression, viral DNA amplification, capsid assembly, virion maturation, and cytopathic effect.^57,58 Variable levels of constitutive activation of the specific metabolic/signaling pathways leading to these cellular events may explain the difference in potency of H-1PV, as permissive, semipermissive, and a few resistant cancer cell types have been described.⁵⁷

H-1PV efficacy and safety were tested in a rat orthotopic model of PDAC, involving either rat cells in a syngeneic rat⁵⁹ or human carcinoma cells in an immuno-incompetent model.^59,60 Intratumoral administration of H-1PV after inoculation of rat PDAC cells in the pancreas led to a rapid burst of viral gene expression in the tumor and the surrounding pancreatic tissue. H-1PV transcripts were also detected in lymphoid organs. From about 10 days after virus administration, viral transcripts started to disappear from normal pancreas, but they persisted at the tumor site. This selectivity was obtained without any significant toxicity.⁶¹ Other experiments, involving subcutaneous human PDAC tumors in immuno-incompetent mice, nonpermissive to H-1PV, led to similar conclusions.^59
–61 The efficacy of H-1PV was also tested in combination therapies, in the context of PDAC. Considering that H-1PV induces cancer cell killing by a different mechanism (one that is cathepsin dependent), oncolytic H-1PV could potentially kill cells resistant to gemcitabine. In combination studies, gemcitabine was administered first, followed by treatment 2 weeks later with H-1PV.⁵⁹ The results showed enhanced efficacy when compared with either treatment used as a monotherapy. Of note, the therapeutic improvements were obtained without any additional toxicity, measured by clinically relevant parameters in bone marrow, liver, and kidney function.⁵⁹ In addition, the improved oncolytic activity of H-1PV- and gene-directed enzyme prodrug therapy when using the yCD/5-FC (yeast cytosine deaminase/5-fluorocytosine) combination resulted in the drastic inhibition of tumor cell spreading and a subsequent increase in long-term survival.⁶⁰ In addition to its action as an oncolytic virus, H-1PV has also shown promise as an immunotherapeutic agent. From a basic point of view, it is thought that the stimulation of the immune response is a result of the primary oncolysis of tumor cells, which, in turn, increases tumor antigen presentation, activates dendritic cells, and potentiates the release of proinflammatory cytokines.⁶² To amplify this immunogenic action in the context of PDAC, a concomitant injection of H-1PV and IFN-γ was shown to increase the response of splenocytes against the tumors and to reduce the titer of H-1PV-neutralizing antibodies, resulting in increased survival in rats.⁶³ Another approach involved the production of CpG-enriched H-1PV. Systemic administration of the virus led to early natural killer (NK) and T cell infiltration of the tumors, elevated IFN in serum and spleen, and prolonged survival in PDAC rats.⁶³ Altogether, the preclinical data obtained in glioma or PDAC models (see previously), as well as data obtained in tumor-free rats or animals bearing breast, cervical, or gastric carcinomas, or Burkitt lymphomas, reviewed by Angelova and colleagues,⁵⁷ demonstrate the significant anticancer activity of H-1PV, associated with a general lack of toxicity.

Considering the clinical application of H-1PV in patients, a French study, in which the skin metastases of 12 patients with melanoma, breast adenocarcinoma, lung large cell carcinoma, pancreatic carcinoma, or kidney leiomyosarcoma were injected with escalating doses of H-1PV, reported only mild signs of toxicity. The presence of viral DNA and proteins was detected in the injected tumor lesions, as well as at distant tumor sites, suggesting a systemic spread of the virus. In light of the lack of significant toxicity encountered in this early clinical trial, an oncolytic H-1PV trial (ParvOryx01) proposing escalating doses of H-1PV was initiated in patients with malignant brain tumors,⁶⁴ after injection into the primary site, or intravenous administration, and was found to be safe. Altogether, the clinical trials reported so far involving administration of H-1PV in patients with cancer have demonstrated that this virus is safe and devoid of clinically relevant side effects. In addition, the ParvOryx01 trial, which has just been completed and is under current clinical assessment, provided intriguing evidence of viral intracellular replication, induction of viral and cellular cytotoxic effectors, and activation of specific T cell responses,⁶⁵ which could be translated in the future to the therapy of pancreatic cancer.

Concluding Remarks

Over the past 40 years, a multidisciplinary approach combining surgery, chemotherapy, and radiation has led to a dramatic improvement in survival for patients affected by malignant diseases. However, this benefit has not yet been achieved in patients with PDAC. Unacceptable toxicities of the standard treatments, and resistance, are at the core of this lack of progress. As discussed herein, anticancer gene delivery and targeting, and new oncolytic viruses, have proved to be efficient in experimental models, and have advanced, or will soon advance, to clinical trials in France.

Footnotes

Acknowledgments

G.V. acknowledges funding from the Plan Cancer 2009–2013. P.C. acknowledges funding from the Région Midi-Pyrénées.

Author Disclosure

No competing financial interests exist.

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