Repetitive monitoring of reporter gene expression in intact living animals is crucial for many applications, including cell trafficking, gene therapy studies, and transgenic models (Ray, P., et al. (2001) Semin. Nucl. Med. 31, 321-330). Noninvasive, real-time analysis of molecular events in intact living mammals is an active area of current research (See, e.g., Bremer, C. & Weissleder, R. (2001) Acad. Radiol. 8, 15-23). Several imaging technologies and new reporter genes are being studied for noninvasive imaging and quantitation of gene expression in living subjects. Some of the imaging modalities and established reporter genes include single photon emission computed tomography (SPECT) using Herpes Simplex Virus Type I thymidine kinase HSV1-tk, Somatostatin Type 2 receptor, and Sodium/Iodide Symporter as reporter genes. Positron emission tomography (PET) using HSV1-tk and Dopamine Type 2 Receptor as reporter genes, MRI with various reporter genes, and optical imaging approaches with fluorescence and bioluminescent reporter genes have also been studied. A detailed review of reporter gene approaches for use in living subjects can be found in Ray et al., supra.
Noninvasive imaging of reporter gene expression using various imaging modalities is playing an increasingly important role in defining molecular events in the field of cancer biology, cell biology, and gene therapy. It is important to be able to image reporter gene expression in living cells, animals, and humans using a single reporter construct. A single reporter gene would facilitate rapid translation of approaches developed in cells to preclinical models and clinical applications. To date, various methodologies exist that allow the imaging of reporter gene expression in living cells and animals noninvasively and repetitively. Confocal laser microscopy, two-photon laser microscopy, and several other techniques are available for real-time imaging of gene expression at the single cell level using fluorescence (Piston, D. W. Imaging living cells and tissues by two-photon excitation microscopy. Trends Cell Biol., 9: 66-69, 1999; Jakobs, S., et al., EGFP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy. FEBS Lett., 479: 131-135, 2000). For reporter gene imaging in living subjects PET, single photon emission computed tomography, magnetic resonance imaging, and optical imaging are well standardized and are being used extensively in small animal models (Ray, P., et al. Monitoring gene therapy with reporter gene imaging, Semin. Nucl. Med., 31: 312-320, 2001; Wu, J. C., et al. Optical imaging of cardiac reporter gene expression in living rats, Circulation, 105: 1631-1634, 2002; Bhaunik, S., and Gambhir, S. Optical imaging of renilla luciferase reporter gene expression in living mice, Proc. Natl. Acad. Sci. USA, 99: 377-382, 2002) and more recently with PET in humans (Yaghoubi, S. S., et al. PET imaging of FHBG in humans: a tracer for monitoring herpes simplex virus type 1 thymidine kinase suicide gene therapy, J. Nucl. Med., 41: 73P-74P, 2000; Jacobs, A., et al., Positron-emission tomography of vector-mediated gene expression in human gene therapy for gliomas, Lancet, 358: 727-729, 2001). These imaging techniques play important roles in defining critical pathways involved in tumorigenesis, metastasis, and evaluating the efficiency of gene therapy strategies (Vooijs, M., et al., Noninvasive imaging of spontaneous retinoblastoma pathway-dependent tumors in mice, Cancer Res., 62: 1862-1867, 2002; Gambhir, S. S. Molecular Imaging of cancer with positron emission tomography, Nat. Rev. Cancer, 2: 683-693, 2002, Yang, M., et al., Whole-body optical imaging of green fluorescent protein expressing tumors and metastases. Proc. Natl. Acad. Sci. USA, 97: 1206-1211, 2000). Each of these modalities has unique applications, advantages, and limitations that can be complementary to other modalities. A cell-based technique is not useful for whole body in vivo imaging studies, whereas techniques involved in imaging at the tissue or organism level do not have the resolution power to image gene expression at the cellular level. Among the whole body imaging modalities, the radionuclide-based techniques have high sensitivity, good spatial resolution, and are tomographic in nature but are somewhat limited by their higher cost and especially for PET, the need for a cyclotron for production of isotopes for most tracers. In contrast, optical imaging techniques (fluorescence and bioluminescence) represent a low cost and quick alternative for real-time analysis of gene expression in small animal models (Wu, supra; Yang, supra) but are limited by depth penetration and cannot be easily generalized to human applications.
To overcome the shortcomings of each modality, a multimodality approach should be very useful for detecting reporter gene expression. Combining two or more different technologies (e.g., PET with optical) through a unified vector would have the advantage of speed and ease of validating approaches in small animals that in turn can be translated to humans. Such a vector might be achieved by several different approaches. A single reporter gene can be investigated for a single substrate doubly labeled with different signatures such as a radioactive nuclide (suitable for radionuclide imaging) or a nonradioactive paramagnetic/bioluminescent/fluorescent molecule (suitable for magnetic resonance or optical imaging) and thus can be imaged by different imaging modalities. However, development of such substrates is often difficult because of the complex chemical nature of the biomolecules and limitations on required pharmacokinetics in vivo.
What is needed, therefore, is a single vector harboring two or more different reporter genes imaged by two or more different techniques (e.g., one radionuclide and one optical). Coexpression of multiple genes is generally achieved by using multiple promoters by insertion of an internal ribosomal entry site or by fusing the two (or more) genes into a single translational cassette (Ray, P., et al., supra, Semin. Nucl. Med., 31: 312-320). Our laboratory has successfully used tk (HSV1-sr39 thymidine kinase, an improved PET reporter gene over the wild-type HSV1-tk when using the guanosine analogues as tracer) and rl (renilla luciferase, a bioluminescence optical reporter gene) as separate imaging tools for studying the location, magnitude, and time variation of reporter gene expression in living subjects (Bhaumik, S., supra; Gambhir, S. S., et al., A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography, Proc. Natl. Acad. Sci. USA, 97: 2785-2790, 2000; Yu, Y., et al., Quantification of target gene expression by imaging reporter gene expression in living animals, Nat. Med., 6: 933-937, 2000).
The present inventors have now constructed and validated a novel tk and rl fusion protein imaged by microPET and bioluminescent optical CCD imaging modalities in tumor xenograft-bearing living mice. The present inventors have further constructed and validated triple fusion reporter constructs, including one combining a synthetic renilla luciferase (hrl), a red fluorescence protein (rfp or DsRed2) and a truncated version of sr39tk (ttk) that can be used to image in a single live cell using a fluorescence microscope and in living mice with both an optical cooled charged couple device (CCD) camera and microPET.