We live in a time of astounding technological advancement, which has enabled us to automate our lives, connect throughout the globe, and dig deep into molecular realm of our bodies. Sequencing the human genome has allowed us to unravel a wealth of information and our understanding of genetics and its impact in medicine continues to grow to the point that we now know where a certain gene is, what it expresses and how its absence or alteration manifests itself. The field of biomedical imaging has made significant advances as well - from extremely high-resolution anatomic imaging and functional imaging of physiologic and pathologic processes, to using novel modalities such as optical imaging to evaluate molecular features within the cellular environment. The latter has made it possible to image phenotypic markers of various genotypes that are implicated in human development, behavior and disease.
Reporter genes play a central role in genetic diagnostics, as they are responsible for encoding protein that can be rapidly and sensitively assayed as surrogate markers when fused to regulatory regions of genes of interest. They are used to assess whether a certain gene has been taken up or expressed in the cell. In that capacity, they are used to study promoter/enhancer elements that affect expression, and mRNA sequences that control message stability and regulate translation efficiency, and provides means to determine the location, magnitude, and persistence of reporter gene expression. All of the above and more is made possible through the development of highly advanced molecular imaging techniques, such as optical imaging. RGs are increasingly being used for imaging-based evaluation and monitoring of cell therapy, stem cell therapy, & immune therapy.
Reporter Gene Imaging involves non-invasive, repetitive and quantitative analysis of reporter gene expression. It follows the following basic steps: RG transcription -> enzyme/receptor production -> entrapment of imaging reporter probe -> imaging signal (which can be a radioisotope/photochemical reaction/magnetic resonance metal cation based on the imaging modality used). Non-RG based imaging techniques that are currently available, depend on direct probes that bind to their targets and are retained in tissues based on direct afﬁnity between the target molecule and the reporter probe, such as radiolabeled ligands and radiolabeled anti-/minibodies e.g: dopamine D2 receptor antagonist 3-(2-[18F]ﬂuoroethyl)spiperone (FESP), or on indirect probes that sequester in tissues as a result of the biological activity of the target molecule, e.g. 2-deoxy-2-[18F]ﬂuoro-D-deoxyglucose (FDG) that gets phosphorylated and trapped in cells.
RG-based in-vivo molecular imaging provides non-invasive and highly specific means to detecting several molecular processes such as gene expression or protein-protein interactions, and follow trafficking and targeting of cells. This enables us to optimize drug and gene therapy, assess disease progression at a molecular level and allow rapid, reproducible, and quantitative monitoring of time-dependent influences on gene products.
Optical imaging is one such molecular imaging technique that detects visible light that is generated in living cells using two methods:
Fluorescence uses photon-emitting proteins (such as green ﬂuorescent protein, GFP) to label and isolate cells by ﬂow cytometry, to track microscopically the subcellular distribution of various molecules, and monitor the production of a gene product.
Bioluminescence depends on enzymatic reaction between a luciferase enzyme (firefly, renilla) and its substrate (luciferin) to produce visible light that is detected and quantified using highly sensitive cooled charge coupled device (CCD) cameras.
There have been significant advances in MRI and ultrasonography-based imaging of subcellular processes. Acoustically analyzable nanoparticles have been used to detect speciﬁc receptors, e.g. antibodies to tissue factor coupled to the perﬂuorocarbon nanoparticles imaged by ultrasound.
Nuclear medicine, on the other hand, utilizes isotopes that emit high energy particles like gamma rays and positrons to label probes, which readily penetrate tissue and be detected outside of the body. Gamma- or positron emitting isotopes can be incorporated into molecular probes such as receptor-speciﬁc ligands and enzyme-speciﬁc substrates, or retained in tissues. Various PET reporter probes (PRP) have been used to image the expression of their respective imaging reporter genes (IRGs). These IRGs may encode enzymes that phosphorylate specific PRPs that cause intracellular entrapment, receptors that can be bound by specific PRP, or cell membrane transporters that facilitate flow and accumulation of specific PRPs into cells.
RGI SPECT imaging of pulmonary micrometastasis (figure) -PEG-3 promoter linked to an RG and the plasmid DNA hooked up to a cationic gene delivery vehicle, which was then injected into a mouse melanoma metastasismodel and imaged with SPECT to visualize RG expression in tumor micrometastasis. Courtesy: Cell Picture Show, Cell Press.
PRPs may be viral-based or human-gene based. HSV1 thymidine kinase (HSV1-tk) and its mutant derivatives are the most extensively studied and applied viral-based IRGs. Of note, immunogenicity of viral-based IRGs can be detrimental to adoptively transferred therapeutics cells expressing them, shortening the duration of PET IRG expression, or eradicating the therapeutic cell expressing the PET IRG. On the other hand, human gene-based PRPs may accumulate in cells that express the endogenous gene, or the imaging gene may function like the endogenous gene, perturbing the cells in which it is expressed. Human thymidine kinase 2 (hmtk2) is one such human gene product, which is ubiquitously expressed in mitochondria of mammalian cells, inaccessible to uracil nucleoside analogs, and has a non-immunogenic truncated version produced in cytoplasm without nonspecific accumulation of its PRPs.
The application of imaging genetics includes clinical translational avenues such as gene and cell therapy. Radionuclide-based IRGs have thus far been the only IRGs to provide whole-body images of transgene expression in humans. The first clinical trial of reporter gene based cell imaging in patients with glioma involving therapeutic autologous cytolytic T cells (CTLs) that stably expressed HSV1-tk injected into the resection cavity over a period of 5 weeks. Following infusion of all the cells a whole-body [18F]FHBG PET scan demonstrated above-background [18F]FHBG signal at the site of cytolytic T-cell infusions and revealed trafficking of these cells to a remote recurrent tumor in the patient's corpus callosum.
Additionally, molecular imaging techniques provide ways to study whole-body kinetics of therapeutic cells (TCs), allowing for monitoring presence, location, quantity, proliferation, survival and status of TCs in animals or patients at any desired time-point following their administration. They also provide means to study the proliferation and persistence of genetically engineered therapeutic cells.
Recently, there has been a lot of interest in using genetic imaging techniques to help understand neurological disease processes such as Alzheimer's disease, schizophrenia, bipolar disorder as well as various cancers such as pancreatic, prostate and breast cancer. This has led to an active discussion on the ethical issues involved with tests that detect the endophenotypes in at-risk individuals in order to guide early intervention. This situation raises various clinical, social and legal concerns regarding meaningful interpretation of the results and disposition of the tested individuals.
One case in point is the use of fMRI and PET to detect early cognitive decline in Alzheimer’s disease. Other such tests have been introduced that contribute as jigsaw pieces to the puzzle of predictive medicine. In some cases, such as BRCA1/2 testing, identifying patients at a high risk for developing breast cancer is useful as it allows for definitive early intervention and is subsequently related to significant positive outcomes. While there is some merit to acquiring this information through molecular tests and imaging in Alzheimer's disease where some scope of early intervention is being advocated, it has been a challenge to apply this approach with other diseases. Recent studies focusing on imaging altered expression of COMT gene affecting prefrontal dopamine levels have shown to have an association with development of schizophrenia. Functional and anatomic imaging identifying these phenotypic changes may predict an early-stage or preclinical schizophrenia, but reporting such information without subsequent management options creates an ethical dilemma for the care provider. Without the availability of a definite therapeutic option in early or preclinical schizophrenia, such information may not only be unuseful in terms of management but also predisposes the patient to significant personal and social challenges.
The complex nature of biochemical and functional profile of the brain precludes definite validity of these tests which are primarily based on inferring endophenotypic changes related to chemical alteration in the brain. Furthermore, this new information poses to question the currently established definitions of personal, social and medical identity of individuals, which is a unique sociological dilemma. The psychosocial implications of these results in terms of the profound anxiety and discomfort that it brings to the patient and family are immense. Having such a diagnosis or prognosis may have a profoundly negative impact on subjects, such as increasing the risk of depression and suicide in an otherwiseasymptomatic or at least subclinical person. Also important to note is that early intervention through psychotropic medication in such individuals is unsafe and likely without any benefit. Complex issues such as these related to key personal, social, legal and ethical aspects of imaging genetics have spurred a lively debate within the field of genethics.
Current evidence suggests that these tests lack specificity, and a whole array incidental findings and the challenges associated with their clinical reporting further confound the situation. As clinical application of imaging genetics involves gleaning useful information from the individual’s genome studies and brain scans, significant false-positives are encountered. This may be in part due to a positive result implicating a gene of unclear signficance, or when a positive gene is associated with a condition mimicking the disorder. While the diagnostic accuracy and reliability continues to improve for imaging genetics tests for various oncologic and neurologic disorders, the legal, social and medical consequences of a false positive result, no matter how infrequent, may have insurmountable negative effects.
Given the multifactorial complexity of underlying pathophysiology that underlies any disease, the presence of a genetic or even a clinical trait associated with any medical condition does not cause illicit the disorder itself, and safe and ethical clinical application of such tests remains dubious at this time. The use of these studies also bring to question important issues of privacy and autonomy. The conventional definition of legal competence as it relates to these individuals may also be brought into question, which is exceptionally difficult as the implicated disorder has no manifestation at that time.
We, as a scientific community continue to strive for innovation in diagnostics and excellence in clinical prowess, but we also need to remain insightful of inadvertent and potentially devastating implications of such tests on various aspects of human life. This creates an opportunity for us as to apply robust empirical methods to ensure clinical usefulness of such tests, and finding ways to couple it with effective therapeutic options for early intervention as warranted by the information they provide, leading to positive outcomes. These steps when executed meticulously and judiciously, harness the true potential of the exciting field of imaging genetics, leading us into the era of ethically-sound, outcomes-oriented, image-guided precision medicine.