Dynamic reporter gene imaging in translational research

ED03-01 Molecular-genetic studies of disease and our understanding of the multiple and converging pathways that are involved in disease development (e.g., oncogenesis and tumor progression) have expanded rapidly over the past decade. The era of molecular medicine has begun and the benefits to individual patients are widely expected to be realized in the near future. For example, the formerly unresponsive and rapidly fatal gastrointestinal stromal tumors (GIST) and chronic myelogenous leukemia (CML) have shown remarkable responses to imatinib mesylate (Gleevac) treatment, a drug that targets several receptor tyrosine kinases (cKit and Bcr-Abl, respectively) that are mutated or constitutively over-expressed. Molecular-genetic imaging in living organisms has also experienced exceptional growth over the past 10 years, and can be defined as “the macroscopic visualization of cellular processes in space and time at the molecular level of function”. It has its roots in molecular and cell biology as well as in imaging technology, chemistry and radiochemistry. Three imaging strategies: based on “direct” and ”indirect” assessments of molecular-genetic processes, as well as “bio-marker” or “surrogate” imaging have been combined with three imaging technologies: radionuclide-, magnetic resonance- and optical-based imaging systems. The “direct” molecular imaging motif builds on established relationships between chemistry/radiochemistry and imaging. Bioconjugate chemistry linking specific binding motifs and bioactive molecules to paramagnetic particles for MR imaging or to radionuclides for PET and gamma camera imaging. This interactive relationship has existed for many years and continues to expand through the development of new relationships and focused interactions between molecular/cellular biologists, chemists, radiochemists, imagers and clinicians. The next generation of direct molecular imaging probes will come from better interactions between pharmaceutical companies, academia and hospitals. Such interactions are now being pursued with the objective to develop and evaluate new compounds for imaging; compounds that target specific molecules (e.g., DNA, mRNA, proteins) or activated enzyme systems in specific signal transduction pathways. However, a constraint limiting direct imaging strategies is the necessity to develop a specific probe for each molecular target, and then to validate the sensitivity, specificity and safety of each probe for specific applications prior to their introduction into the clinic. Examples of how dynamic imaging of direct-acting probes can contribute to drug development and patient care will be presented. Biomarker or surrogate imaging that reflects endogenous molecular/genetic processes is particularly attractive for expansion and translation into clinical studies in the near-term. This is because existing radiopharmaceuticals and imaging paradigms may be useful for monitoring down-stream changes of specific molecular/genetic pathways in diseases such as cancer (e.g., FDG PET). Biomarker imaging is very likely to be less specific and more limited with respect to the number of molecular-genetic processes that can be imaged. Nevertheless, it benefits from the use of radiopharmaceuticals that have already been developed and are currently being used in human subjects. This application strategy is most dramatically illustrated by the use of [18F]FDG PET to image the response, recurrence and progression of particular tumors (e.g., Gleevac treatment of GIST). The translation and application of biomarker imaging paradigms into patient studies, using clinically-approved radiopharmaceuticals or contrast agents, will be far easier than either the direct imaging or reporter transgene imaging paradigms. Reporter gene imaging studies will be more limited in patients compared to that in animals, due to the necessity of transducing the target tissue or cells with specific reporter constructs, or the production of transgenic animals bearing the reporter constructs. Ideal vectors for targeting specific organs or tissue (tumors) do not exist at this time, although this is a very active area of human gene therapy research. Each new vector requires extensive and time-consuming safety testing prior to regulatory approval for human administration. Nevertheless, reporter gene imaging, particularly the genetic labeling of cells with reporter constructs, has several advantages. There are now three well-defined human genes (hNIS, hNET and hSSTR2) with complimentary, clinically approved, radiopharmaceuticals for PET or gamma camera imaging in patients. These complimentary pairs (gene + probe) are excellent candidates for future reporter gene imaging in patients. Importantly, these human genes are less likely to be immunogenic compared to the reporter genes currently used in animals (e.g., viral thymidine kinases, luciferases, fluorescent proteins). It should also be noted that a single reporter gene - reporter probe pair can be used in different reporter constructs to image many different biological and molecular-genetic processes. Once a complimentary reporter-pair (gene + probe) has been approved for human studies, the major regulatory focus will shift to the particular backbone and regulatory sequence of the reporter construct and to the vector used to target reporter transduction to specific cells or tissue, both ex vivo and in vivo. The major factor limiting translation of reporter gene imaging studies to patients is the “transduction requirement”; target tissue or adoptively administered cells must be transduced (usually with viral vectors to achieve high transduction efficiency) with reporter constructs for reporter gene imaging studies. At least two different reporter constructs will be required in most future applications of reporter gene imaging. One will be a “constitutive” reporter that will be used to identify the site, extent and duration of vector delivery and tissue transduction or for identifying the distribution/trafficking, homing/targeting and persistence of adoptively administered cells (the “normalizing” or denominator term). The second one will be an “inducible” reporter that is sensitive to endogenous transcription factors, signaling pathways or protein-protein interactions that monitor the biological activity and function of the transduced cells (the “sensor” or numerator term). The initial application of such double-reporter systems in patients will most likely be performed as part of a gene therapy protocol or an adoptive therapy protocol where the patients own cells are harvested (e.g., lymphocytes, T cells or bone marrow-derived progenitor cells), transduced with the reporter systems and expanded ex-vivo, and then adoptively re-administered to the patient. For example, adoptive T cell therapy could provide a venue for imaging T cell trafficking, targeting, activation, proliferation and persistence. These issues could be addressed in a quantitative manner by repetitive PET imaging of the double-reporter system described above in the same subject over time. We remain optimistic; the tools and resources largely exist and it is only a matter of time before these imaging paradigms will become more generally available. Once in place, Cancer Clinical Trials and Personalized Medicine will be able to benefit from the noninvasive imaging paradigms desctibed above; similar to the benefits of sequential FDG PET scans performed today in order to monitor GIST tumor response and recurrence. The ability to visualize transcriptional and post-transcriptional regulation of endogenous target gene expression, as well as specific intracellular protein-protein interactions in patients will provide the opportunity for new experimental venues in patients. They include the potential to image the malignant phenotype (e.g., signal pathway activity) of an individual patient’s tumor at a molecular level and to monitor changes in the phenotype over time. The potential to image a drug’s effect on a specific target molecule ro signal transduction pathway in an individual patient’s tumor provides the opportunity for monitoring treatment response at the molecular level. Because this paradigm requires the use of “diagnostic” reporter-gene transduction-vectors that target specific organs or tissue (tumors), this requirement will initially limit the translation and application of reporter gene technology to patients. However, direct and surrogate molecular imaging may begin to fill this gap over the next decade.