Conditionally Replicating Adenovirus Expressing TIMP2 Increases Survival in a Mouse Model of Disseminated Ovarian Cancer

Ovarian cancer remains difficult to treat mainly due to presentation of the disease at an advanced stage. Conditionally-replicating adenoviruses (CRAds) are promising anti-cancer agents that selectively kill the tumor cells. The present study evaluated the efficacy of a novel CRAd (Ad5/3-CXCR4-TIMP2) containing the CXCR4 promoter for selective viral replication in cancer cells together with TIMP2 as a therapeutic transgene, targeting the matrix metalloproteases (MMPs) in a murine orthotopic model of disseminated ovarian cancer. An orthotopic model of ovarian cancer was established in athymic nude mice by intraperitonal injection of the human ovarian cancer cell line, SKOV3-Luc, expressing luciferase. Upon confirmation of peritoneal dissemination of the cells by non-invasive imaging, mice were randomly divided into four treatment groups: PBS, Ad-ΔE1-TIMP2, Ad5/3-CXCR4, and Ad5/3-CXCR4-TIMP2. All mice were imaged weekly to monitor tumor growth and were sacrificed upon reaching any of the predefined endpoints, including high tumor burden and significant weight loss along with clinical evidence of pain and distress. Survival analysis was performed using the Log-rank test. The median survival for the PBS cohort was 33 days; for Ad-ΔE1-TIMP2, 39 days; for Ad5/3-CXCR4, 52.5 days; and for Ad5/3-CXCR4-TIMP2, 63 days. The TIMP2-armed CRAd delayed tumor growth and significantly increased survival when compared to the unarmed CRAd. This therapeutic effect was confirmed to be mediated through inhibition of MMP9. Results of the in vivo study support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of human patients with advanced ovarian cancer

Fatty acids: facts vs. fiction

During the last 100 years official dietary guidelines have recommended an increased consumption of fats derived from seeds while decreasing the consumption of traditional fats, especially saturated fats. These recommendations are being challenged by recent studies. Furthermore, the increased use of refining processes in fat production had deleterious health effects. Today, the number of high-quality studies on fatty acids is large enough to make useful recommendations on clinical application and everyday practice. Saturated fats have many beneficial functions and palmitic acid appears to be problematic only when it is synthesized due to excess fructose consumption. Trans fatty acids were shown to be harmful when they are manmade but beneficial when of natural origin. Conjugated linoleic acid has many benefits but the isomer mix that is available in supplement form differs from its natural origin and may better be avoided. The ω3 fatty acid linolenic acid has rather limited use as an anti-inflammatory agent - a fact that is frequently overlooked. On the other hand, the targeted use of long chain ω3 fatty acids based on blood analysis has great potential to supplement or even be an alternative to various pharmacological therapies. At the same time ω6 fatty acids like linoleic acid and arachidonic acid have important physiological functions and should not be avoided but their consumption needs to be balanced with long chain ω3 fatty acids. The quality and quantity of these fats together with appropriate antioxidative protection are critical for their positive health effects

Genetic Incorporation of a Herpes Simplex Virus Type 1 Thymidine Kinase and Firefly Luciferase Fusion into the Adenovirus Protein IX for Functional Display on the Virion

An advantage of the adenoviral vector is its molecular flexibility, which allows for vector tropism modifications for the purpose of cell targeting. In addition to targeting ligands, the capacity to incorporate heterologous peptides has allowed capsid incorporation of other functionalities. We have defined the minor capsid protein IX (pIX) as a locus capable of presenting incorporated ligands on the virion surface. Thus, we sought to exploit the possibility of incorporating functional proteins at pIX. In our current study, we sought to expand the potential utility of our capsid labeling strategy by developing simultaneous imaging capacity for dedicated small animal positron emission tomography and bioluminescence imaging on a single adenoviral vector. Therefore, we constructed an adenovirus that incorporates a fusion protein of herpes simplex virus type 1 thymidine kinase and firefly luciferase (Luc) (TK-Luc) into adenovirus capsid pIX. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional TK-Luc as a component of their capsid surface. Most importantly, Ad-pIX-TK-Luc retained dual enzymatic functions in vitro and in vivo. This dual-modality approach will allow dynamic or real-time imaging analysis of adenovirus-based interventions with maximized analytic flexibility and enhanced resolution potential

Survival analysis.

<p>Mice were monitored for survival. Survival data are plotted on a Kaplan-Meier curve (A). Treatment with Ad5/3-CXCR4-TIMP2 significantly enhanced survival when compared to treatment with Ad-ΔE1-TIMP2, Ad5/3-CXCR4, or PBS (p<0.05). (B), median survival in days for each treatment group.</p

TIMP-2 expression in excised epithelial ovarian tumor.

<p>At the time of sacrifice of mice due to tumor burden, tumors were collected from indicated treatment groups and examined for expression of TIMP2 by immunohistochemistry.</p

MMP-9 expression in excised epithelial ovarian tumor.

<p>At the time of sacrifice of mice due to tumor burden, tumors were collected from indicated treatment groups and examined for expression of MMP-9 by immunohistochemistry.</p

Genomic organization of armed CRAd and control viruses.

<p>Adwt300, is the wild- type Ad5 virus. Ad-ΔE1-TIMP2 is an E1-, E3-deleted replication-deficient Ad5 vector which expresses TIMP2 under the control of the CMV promoter in E1. Ad5/3-CXCR4 is an unarmed CRAd with a CXCR4 promoter in E1 to limit viral replication to the cancer cells in conjunction with a Ad5/3 modified fiber that contains the shaft and tail of Ad5 fiber and the Ad3 fiber knob. Ad5/3-CXCR4-TIMP2 is a CRAd with the CXCR4 promoter in E1, armed with TIMP2 in the E3B region and the aforementioned F5/3 modified fiber.</p

CD31 expression in excised epithelial ovarian tumor.

<p>Tumor conglomerates in the abdominal area were collected during sacrifice of the animals from indicated treatment groups and fixed. Five µM sections of paraffin blocks of tumor tissues were stained with human CD31 antibody to detect blood vasculature.</p