A novel bicistronic high-capacity gutless adenovirus vector that drives constitutive expression of herpes simplex virus type 1 thymidine kinase and tet-inducible expression of Flt3L for glioma therapeutics

Abstract

Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.

FIG. 1.

Structure and in vitro characterization of therapeutic bicistronic HC-Ad. (A) Structure and transcriptional regulation of HC-Ad-TK/TetOn-Flt3L. (B) CNS1 GBM cells and rat astrocytes in primary culture were infected with HC-Ad-TK/TetOn-Flt3L with or without the inducer doxycycline (Dox). Flt3L expression was assessed by immunofluorescence and ELISA. *, P < 0.05 versus mock and infected controls, by one-way ANOVA followed by Tukey's test. (C) CNS1 GBM cells and astrocytes in primary culture were infected with the bicistronic HC-Ad and incubated with or without the prodrug GCV. TK expression was assessed by immunofluorescence, and cell death was determined by flow-cytometric analysis of annexin V/propidium iodide-stained cells as shown in representative dot plots. *, P < 0.05 versus mock and infected controls, by one-way ANOVA followed by Tukey's test.

FIG. 2.

Efficacy and Flt3L expression after intratumoral administration of therapeutic bicistronic HC-Ad. (A) HC-Ad-TK/TetOn-Flt3L (16/group) or saline (14/group) was delivered intratumorally into 6-day intracranial CNS1 tumors. (B) For preimmunization studies, animals were preimmunized with either saline or Ad-0. Two weeks later, animals were implanted with CNS1 tumor cells and then treated 6 days later with an intratumoral injection of either saline or HC-Ad (8 to 10/group). (C) Kaplan-Meier survival curves of naïve tumor-bearing rats treated with bicistronic therapeutic HC-Ad. *, P < 0.05 versus saline (log-rank test). (D) Kaplan-Meier survival curves of preimmunized animals treated with bicistronic therapeutic HC-Ad. *, P < 0.05 versus saline (log-rank test). (E) Levels of Flt3L protein in brain hemispheres ipsilateral (ips) and contralateral (cont) to the bicistronic HC-Ad injection site were assessed by ELISA at 5 and 30 days after treatment (5/group). *, P < 0.05 versus the contralateral value (Student's t test). (F) DNA was isolated from both brain hemispheres (5/group), and Flt3L transgene copies were quantified by qPCR at 5 and 30 days after delivery of bicistronic HC-Ad. *, P < 0.05 versus the contralateral value (Student's t test).

FIG. 3.

Biodistribution of bicistronic HC-Ad vector genomes in tumor-bearing animals. HC-Ad vector genomes were quantified in the tissues indicated at 5 days (A), 30 days (B), 6 months (C), and 1 year (D) after intratumoral injection of bicistronic HC-Ad into naïve, tumor-bearing rats. The solid line indicates the detection limit.

FIG. 4.

Neuropathological analysis of brains at 5 days, 30 days, 6 months, and 1 year after intratumoral administration of bicistronic HC-Ad to naïve, tumor-bearing Lewis rats. Images of TK and Flt3L immunoreactivity are higher-magnification images from the brain hemisphere ipsilateral to the HC-Ad injection. TH, tyrosine hydroxylase; MBP, myelin basic protein; MHCII, major histocompatibility complex II.

FIG. 5.

Behavioral assessment of long-term survivors 1 year after treatment with bicistronic HC-Ad. Behavioral assessment was performed before and after amphetamine treatment in long-term survivors 1 year after intratumoral administration of bicistronic HC-Ad into naïve, tumor-bearing rats. Naïve, age-matched rats were used as controls. (A) Rotational behavior. (B) Right-forelimb-use asymmetry. (C) Spontaneous motor behavior. (D) Rearing behavior.