The imaging paradigm we describe can be applied to any gene combination, and is based on the stable coexpression of two genes. Strict coexpression of two proteins in equimolar amounts can only be achieved by a fusion gene encoding both gene products [17
]. This approach, however, cannot be generalized as many fusion proteins may not yield functional activity or the fusion protein may not localize in the appropriate subcellular compartment. Fusion proteins may also induce immunogenic molecules, poorly suited for in vivo
studies. Coexpression of two distinct gene products is commonly achieved by using dual-promoter systems. However, when transgene expression is carefully examined in single cells rather than in cell populations in which total expression is averaged, coexpression is unreliable because of promoter interference or epigenetic transcriptional repression [18–20
The imaging paradigm we describe is not confined to one type of vector or transgene delivery system and could be extended to microinjected DNA and other viral vectors as well; this is because IRES-mediated coexpression is determined at the translational level. However, the efficacy of translation may depend on protein synthetic activity and cycling of the transduced cells. Indeed, cap-dependent translation is relatively inefficient during mitosis in mammalian cells because of the presence of underphosphorylated elF-4F [21
]. Eventually, it will be important to assess which IRES elements are reliable indicators of transgene coexpression in different tissues, taking into account the half-life of each encoded protein. These considerations will be important when using non-invasive imaging to assess organ (tissue) specificity, as well as level and duration of transgene expression.
In this report, we present as “proof of principle” the results of imaging studies with the HSV1-tk
gene positioned proximally in the IRES-based coexpression cassette (HSV1-tk
-IRES-geneX). Previously, we studied the range of HSV1-tk
expression levels when the HSV1-tk
gene was positioned distal to the IRES in combination with genes other than lacZ
). For example, when the HSV1-tk
gene was positioned distal to the IRES element and the gene encoding for the mutant low affinity nerve growth factor receptor (mLNGFR
) was positioned proximal (mLNGFR-IRES-HSV1-tk
), the average level of HSV1-tk
expression in a mixed population of transduced cells was two to three times lower [22
] compared to the expression levels observed in the current studies. Similar results were obtained with the HSV1-tk
(distal) and GM-CSF
(proximal) using the GMCSF-IRES-HSV1-tk
expression cassette [23
]. Nevertheless, the range of HSV1-tk
expression levels is more than adequate for non-invasive imaging in vivo
when the HSV1-tk
gene is positioned distal to the IRES. It is important to note that the expression of the IRES-linked genes was driven by the same promoter, retroviral LTR, in this and all of our previous studies. The level of expression of the both IRES-linked genes can be further increased by using promoters stronger than the retroviral LTR.
In most gene-therapy protocols, appropriate reporter substrates for direct imaging of transgene expression are not available, and their development and validation would require considerable time and effort. Furthermore, developing specific tracers for direct imaging would have to be repeated for each therapeutic transgene, and this is simply not feasible. An alternative approach is to use “indirect” imaging based on proportional coexpression of a reporter gene, such as HSV1-tk. This report demonstrates the feasibility of indirect reporter gene imaging to monitor the expression of therapeutic genes of interest by constructing appropriate IRES-based gene expression cassettes. For example, non-invasive imaging could facilitate the assessment of the intratumoral delivery of genes encoding different cytokines and costimulatory molecules (e.g., IL2, IL4, IL12, GM-CSF, INF-γ, B7, etc.), prodrug activating genes that can not be imaged directly (e.g., cytosine deaminase), tumor suppressor genes (e.g., p53). Non-invasive imaging could be applied to monitor the delivery and expression of angiogenic genes (e.g., VEGF) in cardiovascular gene therapy; genes that correct genetic deficiencies such as the Duschene muscular distrophy (dystrophin gene). Non-invasive imaging could also be applied to monitor gene expression and to track the genetically modified cells in immunotherapy (e.g., tumor vaccines), adoptive cell therapies (e.g., infusion of antigen-specific T-lymphocytes), and stem cell transfer (e.g., hematopoietic or muscle stem cells). Thus, the demonstration of proportional coexpression of a cis-linked transgene over a wide range of expression levels and the ability to image this range of coexpression non-invasively represents an important and essential step in development of the imaging paradigms discussed above.
In summary, we demonstrate that non-invasive imaging of a marker gene (HSV1-tk) can provide quantitative as well as topographical information related to the expression of a cis-linked transgene. This imaging paradigm could be applied to investigate the activity of specific promoter and enhancer elements in transgenes or genes targeted by homologous recombination. Furthermore, non-invasive imaging of HSV1-tk could be used to investigate and monitor a wide range of clinical gene therapy trials involving cis-linked transgenes; it could be readily applied to several ongoing and newly developing clinical gene therapy protocols by defining the location, magnitude and persistence of transgene expression over time.