Evaluation of E1A double mutant oncolytic adenovectors in anti-glioma gene therapy

Abstract

Malignant glioma, in particular glioblastoma multiforme (GBM), represents one of the most devastating cancers currently known and existing treatment regimens do little to change patient prognosis. Conditionally replicating adenoviral vectors (CRAds) represent attractive experimental anti-cancer agents with potential for clinical application. However, early protein products of the wild type adenovirus backbone--such as E1A--limit CRAds' replicative specificity. In this study, we evaluated the oncolytic potency and specificity of CRAds in which p300/CPB and/or pRb binding capacities of E1A were ablated to reduce non-specific replicative cytolysis. In vitro cytopathic assays, quantitative PCR analysis, Western blot, and flow cytometry studies demonstrate the superior anti-glioma efficacy of a double-mutated CRAd, Ad2/24CMV, which harbors mutations that reduce E1A binding to p300/CPB and pRb. When compared to its single-mutated and wild type counterparts, Ad2/24CMV demonstrated attenuated replication and cytotoxicity in representative normal human brain while displaying enhanced replicative cytotoxicity in malignant glioma. These results have implications for the development of double-mutated CRAd vectors for enhanced GBM therapy.

Fig. 1

Schematic representation of deletional mutations applied to CRAds. Blue and red boxes indicate regions of E1A gene which encode high-affinity binding domains for the host proteins p300 and pRB, respectively. An X placed over the arrow pointing towards p300 or pRb boxes illustrates the ablation of these regions from a particular construct. ITR; inverted terminal repeat at left of adenovirus genome. Black arrow in Ad24CMV and Ad2/24CMV diagram represents the presence of CMV promoter.

Fig. 2

Analysis of induced cytopathic effect by conditionally replicating adenoviruses. Oncolytic potential of mutant vectors was evaluated by crystal violet staining in U87MG, U373MG, GL261, A172, Kings, and N.10 cells. Cells were infected with the panel of viruses at the indicated doses (vp/cell). After 10 days of culture, adherent cells were stained with crystal violet.

Fig. 3

Effect of E1A mutations on adenoviral transcription in glioma. A: The mouse glioma cell line GL261 and human glioma cell lines were infected with AdWT, Ad24, Ad24CMV, and Ad2/24CMV vectors at 10 vp/cell. Twenty-four hours after infection, transcripts were quantified by real-time PCR using primers recognizing adenovirus E1A. E1A transcript copy number for each mutant virus is represented as fold E1A copy/ng RNA over AdWT. B: The same analysis as for (A) was performed in two GBM tissue samples, T25 and T26.

Fig. 4

Analysis of viral replication in glioma. A: Human glioma cells U87MG, U373MG, A172, N.10, Kings, mouse glioma cells GL261, and (B) primary GBM tissue T25 and T26 were infected with AdWT, Ad24, Ad24CMV, and Ad2/24CMV viruses at a dose of 1,000 (A) and 500 (B) vp/cell. After 4 hr adsorption, cells were rinsed with PBS and allowed to continue incubation with growth media. Twenty-four and 48 hr after infection with each virus, replication activity was quantified by measuring the amount of E1A DNA copies/ng DNA by qPCR. Bar graphs represent fold change of E1A DNA copies/ng DNA over AdWT.

Fig. 5

Specificity of protein production and replication mediated by E1A-mutant CRAds. A: Human glioma U373MG or normal human astrocytes (NHA) cells were infected with AdWT, Ad24, AdCMV24, or Ad2/24CMV vectors at 1,000 vp/cell. After 4 hr adsorption, virus containing media was aspirated and replaced with fresh media supplemented with 10% FBS. Cell lysates were examined 24 hr after viral infection for E1A protein expression by Western blot. Replications assays were also conducted for NHA (B) and normal human brain (D). CRAd-induced toxicity in NHA was analyzed by crystal violet staining (C). E1A detection was achieved using a polyclonal antibody against Ad5 E1A proteins. Mock- and AdWT-infected cells were used as comparative controls and actin antibodies were used for quality control. Data represent results from two independent experiments. E1A DNA copies were quantified and bar graphs represent copies of E1A DNA/ng DNA. “*” shows P-value <0.05.