Optical Imaging

Today, the Genelux family of viral and bacterial constructs expresses a variety of proteins that can be used for optical imaging, including luciferases and fluorescent proteins. These proteins allow for the detection of tumors and metastases located either on or near body surfaces, easily-accessible parts of the body (allowing bronchoscopy, endoscopy, laparoscopy etc.), or which can be exposed surgically. Optical imaging could thus be used for the detection and diagnosis of tumors and metastases in patients with melanoma or other surface tumors. Optical imaging markers are also potential tools to monitor the therapeutic effect of our oncolytic viruses. Further, tumors and metastases infected by our viral constructs can be detected intra-operatively by surgeons with relatively simple equipment, allowing them to verify successful removal of all malignant tissues to obtain clean margins.

GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria. It is a naturally autofluorescent protein which exhibits bright green fluorescence when exposed to blue light. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression.

 


 

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Ruc-GFP Fusion Construct

Genelux scientists have incorporated Ruc-GFP, a luminescent fusion protein, into the Genelux platform technology (GL-ONC1) to enable non-invasive cancer diagnosis, staging and monitoring. This particular protein is a combination of two proteins: one bioluminescent derived from glowing sea pansies, and one fluorescent protein from jellyfish.

 

Visualization of implanted glioma tumors colonized by vaccinia virus carrying Ruc-GFP construct under a stereofluorescent microscope.

 

When injected into the bloodstream of a mouse, the GL-ONC1 virus “colonizes” or finds and enters a tumor and begins to replicate. As it makes copies of itself, it also makes copies of the GFP protein, allowing scientists to visualize tumors with simple optical imaging equipment.

 

 

The pictures (above) show the effect of GL-ONC1 when administered to female nude mice. The breast cancer became infected with the intravenously (i.v.) delivered vaccinia virus, which can be detected by optical imaging as green fluorescence in the presence of blue excitation light.

The boundary of the tumors is clearly visible, allowing distinction between tumorous tissues and non-tumorous tissues. One clinical application is fluorescent marking of tumor margins by the engineered virus, assisting surgeons during tumor removal procedures. Over time, the breast tumors are eradicated by the virus, and the green fluorescence signal also disappears. The same green fluorescence signal can also be used to mark tumors in live animals (see below) after being injected with GL-ONC1. Once marked, the antitumor therapeutic outcome can be monitored noninvasively in real time.

 

Visualization of cancerous tumor in live mouse

 

Preclinical Breast Cancer Study

In a pivotal preclinical study published by Genelux scientists in Cancer Research (October 2007), human breast cancer tumors colonized by GL-ONC1 exhibited growth, inhibition, regression through complete eradication in 130 days in 95% of mice tested, and results were monitored utilizing GFP light emission. Below are images of a tumor 14 days, 28 days and 56 days after the virus has been injected into the bloodstream of a mouse against a control mouse whose tumor is untreated.

  • 14 days after virus injection vs. Control: Virus enables tumor illumination
  • 28 days after virus injection vs. Control: Significant reduction in tumor size over control/less visible light
  • 56 days after virus injection vs. Control: Nearly complete regression/no light; virus is eliminated completely from body

 

Light emission (GFP) was used to monitor breast tumor growth/regression

 

Ongoing human clinical trials involving GL-ONC1 may similarly utilize this optical imaging capability to provide early visual evidence of therapeutic effect/efficacy (surface or near-surface tumor colonization followed by shrinkage or elimination) in cancer patients. (Such findings would be considered secondary outcomes in a Phase I study focused on Safety and Dose Escalation, and would likely be confirmed in subsequent tumor biopsies).

Genelux scientists are continuing to test a variety of fluorescent proteins, because true deep-tissue imaging in whole mammals has been constrained by limitations below certain tissue “depths.” (Light emission/detection is hindered by absorbance of light by hemoglobin and the autofluorescence of the body tissue).

 


 
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TurboFP635

Among the fluorescent proteins that emit light efficiently in the “optical window” (low absorbance of light by hemoglobin and low autofluorescence of body tissue) is the emerging Genelux technology TurboFP635 (scientific name “katushka”). TurboFP635 is derived from the sea anemone Entacmaea quadricolor and shows efficient light emission in this optical window.

When expressed by the oncolytic vaccinia virus, the detection of tumors (using fluorescent optical imaging techniques) improves significantly as can be seen in Figure 1. While GFP-imaging alone allowed detection of superficial parts of the tumor, presence of TurboFP635 resulted in delineation of a much higher proportion of the tumor tissue with much less background caused by autofluorescence. Therefore, expression of alternative fluorescent proteins like TurboFP635 allows detection of tumor cells with higher specificity (less autofluorescence “noise”) and sensitivity (less light absorption due to hemoglobin).

 

TurboFP635

 

Figure 1: A mouse bearing two tumors (black and white image on the left, tumors encircled with yellow dotted line) was injected with a virus encoding GFP and TurboFP635. Both proteins are made in the same concentration, but the TurboFP635 signal is much stronger. On the colored images the mouse skin is blue, GFP green and TurboFP635 is red. When detected on the same spot (e.g. small spot on the tail), the expression of both proteins results in the yellow color (GFP+TurboFP635 image).