Adenoviral vector-mediated gene therapy for gliomas: coming of age

Abstract

Introduction: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and it carries a dismal prognosis. Adenoviral vector (Ad)-mediated gene transfer is being developed as a promising therapeutic strategy for GBM. Preclinical studies have demonstrated safety and efficacy of adenovirus administration into the brain and tumor mass in rodents and into the non-human primates' brain. Importantly, Ads have been safely administered within the tumor resection cavity in humans.

Areas covered: This review gives background on GBM and Ads; we describe gene therapy strategies for GBM and discuss the value of combination approaches. Finally, we discuss the results of the human clinical trials for GBM that have used Ads.

Expert opinion: The transduction characteristics of Ads, and their safety profile, added to their capacity to achieve high levels of transgene expression have made them powerful vectors for the treatment of GBM. Recent gene therapy successes in the treatment of retinal diseases and systemic brain metabolic diseases encourage the development of gene therapy for malignant glioma. Exciting clinical trials are currently recruiting patients; although, it is the large randomized Phase III controlled clinical trials that will provide the final decision on the success of gene therapy for the treatment of GBM.

Keywords: Fms-like tyrosine kinase 3 ligand; HSV1-TK; dendritic cells; glioblastoma multiforme; high-capacity adenovirus; immunotherapy.

Figure 1. Mechanism of action and bystander effect of adenovirus vectors encoding HSV1-thymidine kinase

Herpes Simplex Virus Type 1-thymidine kinase (TK) phosphorylates the non-toxic prodrug ganciclovir (GCV) to GCV-monophosphate, which after conversion into a triphosphorylated GCV by cellular kinases is incorporated into the duplicating DNA chain during the S phase of the cell cycle, leading to apoptotic cell death of proliferating cells. While GCV can freely diffuse through cells, triphosphate-GCV is a highly charged molecule and accesses neighboring cells through gap junctions, inducing apoptotic cell death in non-transduced proliferating cells surrounding the infected cell.

Figure 2. Mechanism of action and bystander effect of vectors encoding mIL-13-PE

Ad.mIL-4.TRE.mIL13 vector encodes for chimeric toxin mIL-13-PE (mutated human interleukin-13), which targets and kills glioma cells expressing IL-13Rα2, absent in non-tumor cells. Upon binding to IL-13Rα2, PE toxin (Pseudomonas endotoxin) is internalized and inhibits elongating factor 2 (EF2), blocking protein synthesis and leading to tumor cell death. The release of mIL-13-PE from infected cells provides a strong bystander effect. As an extra safety feature, the adenovirus vector encodes for mIL-4 that binds and blocks physiological IL-13/IL-4R, present in normal brain cells.

Figure 3. Therapeutic targets to enhance antitumor immunity using Ad vectors

FLT3L drives the generation of myeloid and lymphoid derived DC populations. CD40L enhances the expression of co-stimulatory ligands such as CD86/CD80 on DCs and also stimulates the release of cytokines including IL-12, IL-18 and RANTES/CCL5. IL-12 promotes the development of CD4+ TH1 cells and the release of IFN-γ and IL-2 by CD8+ T cells and NK cells, thus increasing cytotoxicity against tumor cells. IFN-γ and IL-2 also play a role in the generation of memory T cells. Additionally, IFN-γ enhances MHC I expression on tumor cells and facilitates their recognition by CD8+ T cells. APC: antigen presenting cell, Ad: adenovirus, DC: dendritic cell, TCR: T cell receptor; Antigen.

Figure 4. Mechanisms of tumor-induced immune suppression

Glioma cells inhibit T cell function through cytokines that act directly on T cells, like: TGFβ, IL-10, IL-6, which inhibit T cell production of IL-2, IL-4, IFN-γ, expression of IL2R and CD247, signaling downstream of the T cell receptor and activation of CTLs by T helper cells. This results in inhibition of T cell activation and proliferation. Through enhanced production of reactive oxygen species by PGE2, glioblastomas also directly induce T cell apoptosis. IL-2, IL-4, IFN-γ can be used to stimulate T cell function and aid anti-glioma therapies. Glioma cells can also inhibit T cell function indirectly by impairing differentiation, maturation, activation and function of antigen presenting cells (APC), reducing the expression of co-stimulatory molecules (CD80/CD86), MHC-I and MHC-II, decreasing the production of immunostimulatory cytokines TNFα, IL-2, IL-4, IL-12. Therapies to increase production of CD80/86 and immunostimulatory molecules show promise as adjuvant therapies for glioma. In addition, glioma cells produce cytokines (VEGF, PDGF, LIF, GDNF, IL-6, IL-10, CCL2) which alter hematopoietic lineages to promote differentiation towards immunosuppressive phenotypes and increase the number of Myeloid Derived Suppressor Cells (MDSCs) and regulatory T cells (Tregs). These are recruited to the tumor microenvironment and circulate back to lymphoid organs to suppress anti-tumor immune responses further. Abbreviations used: Arg1, arginase 1; CCL2, Chemokine ligand 2 (also known as monocyte chemotactic protein -1 MCP-1); EGF, epidermal growth factor; GDNF, glial derived neurotrophic factor; GM-CSF, granulocyte macrophage colony stimulating factor; HO-1 heme oxygenase 1; iNOS, inducible nitric oxide synthase (also known as NOS2); LIF, leukemia inhibitory factor; PGE2, Prostaglandin E2; PDGF, platelet-derived growth factor; TGFβ, Transforming growth factor beta; TNFα, Tumor necrosis factor alpha, VEGF, vascular endothelial growth factor.

Figure 5. Efficacy of the combined conditional cytotoxic, immune stimulatory gene therapy strategy (Ad-TK and Ad-Flt3L) in murine models of GBM

A. Expression of TK was assessed by immunostaining in CNS-1 rat GBM tumors in vivo and in vitro. Cell death was evaluated by flow cytometric analysis. The efficacy of Ad-TK+GCV was evaluated in mice and rats bearing syngeneic CNS-1 and GL26 tumors, respectively. B, Ad-mediated expression of Flt3L was assessed by immunostaining in J3T canine GBM cells. Rats bearing intracranial GBM were treated with Ad-Flt3L, control vector encoding b-Gal or saline. Infiltration of DCs in the brain tumor was assessed 5 days after treatment. The efficacy of Ad-Flt3L was evaluated in mice and rats bearing syngeneic CNS-1 and GL26 tumors, respectively. C, Rats were implanted with F98, 9L or CNS-1 tumors in the brain and treated with Ad-TK+Ad-Flt3L Microphotographs show the neuropathology (Nissl) of moribund saline-treated rats and of Ad-TK+Ad-Flt3L-treated long-term survivors. Graphs show Kaplan Meier survival curves of tumor-bearing rats (*p<0.05 vs saline, Long rank test).