Being dependent on the viability of the infected cells to produce an imaging signal, these strategies are not compatible with the cell-killing functions of oncolytic agents and are therefore inadequate for assessment of CRAd biodistribution, progeny production, and accumulation in tumors.Ī genetic technology of Ad capsid labeling involving fusing imaging motifs to the minor capsid protein IX (pIX) allows direct visualization of viral particles and overcomes the CRAd imaging limitation. 7Įxisting CRAd imaging strategies are based on detection of virus-encoded transgene expression as opposed to direct visualization of viral progeny particles.

Given that no single modality is capable of addressing all aspects of molecular imaging, construction of fusion reporters that combine the positive attributes of different modalities has recently been attempted for multimodality imaging applications. Imaging motifs such as mutated herpes simplex virus thymidine kinase (HSV-tk) 8 allow for use of common and clinically relevant imaging techniques such as computed tomography (CT), single-photon emission computed tomography (SPECT), or positron emission tomography (PET) using radioactive tracers, whereas monomeric red fluorescent protein (mRFP1) offers more economical optical fluorescence imaging options for in vitro and in vivo investigations. 4 – 7 Use of different types of reporter genes allowed exploiting a variety of signal detection techniques. We and others have reported adenoviral labeling systems based on incorporation of an imaging reporter into the E1A, E2B, and the fiber genes of Ad5. Various imaging strategies have been employed in the context of adenoviral (Ad) gene therapy vectors. 3 A flexible, dynamic, noninvasive CRAd imaging system remains clinically relevant and a promising prospect for future clinical evaluation of ovarian cancer virotherapy. 2 Although CRAd-based therapies have emerged as novel strategies for the treatment of ovarian cancer, clinical efficacy has been fleeting and information derived from clinical trials has yielded limited insight into factors limiting success. Conditionally replicative adenovirus (CRAd)-based virotherapy represents a promising option to treat malignancy through viral amplification, oncolysis, and lateralization-a mechanism that is distinct from other forms of cancer therapy. 1 It is evident that new therapeutic approaches are greatly needed for the treatment of women with this deadly disease. The 10-year relative survival rate for all women diagnosed with ovarian cancer is approximately 39%. OVARIAN CANCER remains the deadliest malignancy of the female reproductive tract, with over 14,600 deaths predicted in the United States for 2009. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro.

Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Replication and oncolysis were evaluated in ovarian cancer cells. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fluorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verified by molecular analyses.

A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for noninvasive dual-modality imaging of ovarian cancer virotherapy.

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