Enhancement of Vaccinia Virus Based Oncolysis with Histone Deacetylase Inhibitors

Histone deacetylase inhibitors (HDI) dampen cellular innate immune response by decreasing interferon production and have been shown to increase the growth of vesicular stomatitis virus and HSV. As attenuated tumour-selective oncolytic vaccinia viruses (VV) are already undergoing clinical evaluation, the goal of this study is to determine whether HDI can also enhance the potency of these poxviruses in infection-resistant cancer cell lines. Multiple HDIs were tested and Trichostatin A (TSA) was found to potently enhance the spread and replication of a tumour selective vaccinia virus in several infection-resistant cancer cell lines. TSA significantly decreased the number of lung metastases in a syngeneic B16F10LacZ lung metastasis model yet did not increase the replication of vaccinia in normal tissues. The combination of TSA and VV increased survival of mice harbouring human HCT116 colon tumour xenografts as compared to mice treated with either agent alone. We conclude that TSA can selectively and effectively enhance the replication and spread of oncolytic vaccinia virus in cancer cells

Adenovirus-Mediated Expression of the p14 Fusion-Associated Small Transmembrane Protein Promotes Cancer Cell Fusion and Apoptosis In Vitro but Does Not Provide Therapeutic Efficacy in a Xenograft Mouse Model of Cancer.

Adenoviruses (Ads) are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1)-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo

FAST protein expression in A549 cells decreases cellular metabolic activity, promotes apoptosis and concomitant loss of membrane integrity.

<p><b>A)</b> A549 cells were infected at an MOI of 10 with AdEmpty or AdFAST. Cellular metabolic activity was assessed every 24 h until 96 hpi using an MTS assay. The average of three independent experiments (n = 3) done in triplicate is plotted with the standard error of the mean. Values were normalized to mock infected cells at 24 hpi. *p<0.05 compared to mock infected cells at their corresponding timepoints. **p<0.05 comparing AdFAST to AdEmpty infected cells. <b>B)</b> A549 cells were infected with AdEmpty or AdFAST at an MOI of 1, 10, 50 or 100. An MTS assay was conducted 72 hpi. The average of three independent experiments (n = 3) done in triplicate ± the standard error of the mean is shown with normalization to mock infected cells. *p<0.05 compared to mock infected cells. **p<0.05 comparing AdFAST to AdEmpty treated cells. <b>C)</b> A549 cells were treated with AdEmpty or AdFAST at an MOI of 10, or left uninfected, and crude protein extracts prepared 24 hr later and examined for total or cleaved caspase 3 by immunoblot. As positive controls, A549 cells were treated with 100 μM etoposide for 24 h, or 1 μM staurosporine for 6 h. Alpha-tubulin served as a loading control. <b>D)</b> A549 cells were infected with AdEmpty, AdFAST or VSVΔ51 using a range of MOI. Relative cell membrane integrity was measured based on the lactate dehydrogenase levels in the supernatant 72 hpi. The average of two independent experiments (n = 2) is shown with each experiment done in triplicate. Error bars denote the standard error of the mean. *p<0.05 compared to cells only. **p<0.05 when comparing AdFAST to AdEmpty infected cells.</p

FAST protein expression induces extensive cell fusion in 293 cells.

<p><b>A)</b> 293 cells were infected with AdEmpty or AdFAST at an MOI of 1 and stained with Giemsa stain 18 h later. Images were captured using bright field microscopy (20x objective). A region of fused cells for the AdFAST-treated cells is outlined with a dotted line, and several nuclei within the syncytium are indicated with asterisks (*) <b>B)</b> Fusion index of 293 cells infected with AdEmpty or AdFAST. The fusion index for two fields of view were determined, and the average with the standard deviation is depicted in the graph. <b>C)</b> 293 cells were infected with AdRFP or AdFAST/RFP at MOI 10 and observed using fluorescence microscopy at 48 h post infection. All images were taken using a 20x objective. <b>D)</b> 293 cells were infected at a MOI of 10 with AdEmpty or AdFAST and relative metabolic activity was determined using an MTS assay over a 96 h interval every 24 h. Experiments were completed in triplicate and the average of three independent experiments is shown (n = 3). Values were normalized to mock infected cells at 24 hpi. Error bars denote the standard error of the mean. *p<0.05 compared to mock infected cells. **p<0.05 when comparing AdFAST to AdEmpty infected cells.</p

Ad-mediated FAST protein expression from an E1-deleted vector does not affect virus growth or yield.

<p><b>A)</b> All viruses are early region 1 (E1) and early region 3 (E3) deleted. AdFAST-HA has a single HA tag linked to the C terminus through a glycine linker. ITR denotes inverted terminal repeats and Ψ is the packaging signal. RFP is the red fluorescent protein and CMV represents the cytomegalovirus enhancer/promoter. <b>B)</b> 293 and A549 cells were infected with the control virus AdEmpty or AdFAST-HA at an MOI of 10. Whole cell lysates were collected 48 hpi. Samples were probed for the HA tag, Ad5 fibre and alpha-tubulin for a loading control. The top band denoted by * in the A549 samples is non-specific. <b>C)</b> 293 cells were infected with AdEmpty or AdFAST at an MOI of 1 and whole cell lysates were collected at 6, 18, 24 and 30 hpi. Samples were probed for Ad5 fibre and alpha-tubulin was used as a loading control. <b>D)</b> Supernatants collected from 293 cells infected with AdEmpty or AdFAST at an MOI of 1 were used to conduct plaque forming assays to determine the number of viral progeny at various times post-infection.</p

AdFAST does not induce fusion or promote survival in immunodeficient CD-1 mice with subcutaneous A549 tumors.

<p><b>A)</b> CD-1 nude mice harbouring subcutaneous A549 tumors were intratumorally injected with PBS, 5x10⁸ pfu AdEmpty or AdFAST. Five days post injection, tumors were excised, fixed, sectioned and subjected to hematoxylin and eosin staining. Results are representative of 2–3 mice (10x objective). <b>B)</b> CD-1 mice with subcutaneous A549 tumors intratumorally injected with PBS, 5x10⁸ pfu AdEmpty or AdFAST were measured at day 20 post injection. The line in each column represents the average of the associated treatment group. <b>C)</b> A Kaplan-Meier survival curve shows the percentage survival of CD-1 mice with subcutaneous A549 tumors intratumorally injected with PBS, AdEmpty or AdFAST over time. Each treatment group consisted of 5 mice.</p

Identification of Genetically Modified Maraba Virus as an Oncolytic Rhabdovirus

To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations