The paradigm for noninvasive imaging of therapeutic gene expression that we propose in this report relies on proportional coexpression of a “marker” gene and a “therapeutic” gene products. Usually, the coexpression of two gene products is achieved by using a dual-promoter approach. However, on an individual cell level, as opposed to cell populations in which total expression is averaged, proportional coexpression may be compromised because of promoter interference or transcriptional repression. The proportionality of IRES-mediated coexpression of two genes is determined at the translational level and depends on protein synthetic activity and cell cycle phase of the transduced cells [45
]. Strictly proportional (equimolar) coexpression of two genes under control of a single promoter can be achieved by using fusion gene approach.
To assess whether the fusion gene approach is applicable to the described imaging paradigm, we constructed the tkgfp
fusion gene consisting of the HSV-1-tk
marker gene (for noninvasibe imaging) and the gfp
gene, which served as model gene for different therapeutic transgenes. Transduction of different types of tumor cells with tkgfp
allowed the selection of transduced living cells by fluorescence microscopy and quantitation of tkgfp
expression with FACS analysis. Expression of tkgfp
induced sensitivity of the transduced tumor cells to GCV similar to that resulting from transduction of the cells with the same vector bearing the HSV-1-tk
gene alone. The levels of HSV-1-tk
gene expression in tkgfp
-transduced clones, as measured by the FIAU accumulation, were comparable to those observed by us previoulsy in RG2 and W256 tumor cells transduced with HSV1-1-tk
gene alone [22,23
] and were within the range that is adequate for noninvasive imaging with radioiodinated FIAU and gamma camera, single photon emission computed tomography (SPECT), or PET [22–24
]. Previously, we have shown that the FIAU/TdR accumulation ratio correlates highly with other independent measures of the HSV-1-tk
gene expression: namely, ganciclovir sensitivity (IC50
) and concentration of HSV-1-tk
Most importantly, we demonstrated that fusion gene approach provides a constant relationship between the expression of the HSV-1-tk and gfp genes over a wide range of expression levels. Thus, noninvasive images of HSV-1-tk expression should reflect distribution and level of expression of gfp or different therapeutic genes. Parametric images of therapeutic gene expression could be generated if the relationship between the levels of FIAU accumulation (HSV-1-tk expression) and functional measures of therapeutic gene expression in transduced tissues are known.
Limitations of the fusion gene-fusion protein approach is that is cannot be applied to proteins that may loose functionality as the result of fusion or will not be able to localize to the appropriate subcellular compartment. This approach cannot be applied to secretory proteins (e.g., cytokines, growth factors) because the HSV-1-tk marker enzyme should remain intracellular in order to catalyze the intracellular accumulation of radiolabeled FIAU. Potentially higher immunogenicity of fusion proteins may also limit their application in in vivo studies.
fusion gene can also be considered as therapeutic-marker gene construct, where tk
is used as therapeutic gene and gfp
as marker gene for in situ
detection or direct visualization. GFP is the most widely used marker protein for direct visualization of transgene expression in living cells and transparent tissues and organisms in vivo
]. Recently, Flotte et al. [53
] implemented a videoendoscopy technique for direct visualization of gfp
-transduced airway epithelium. Similar endoscopic technique for visualization of tkgfp
fusion gene expression could be useful for gene therapy directed to the gastrointestinal tract by using the HSV-1-tk
/GCV paradigm. Therefore, tkgfp
fusion gene could be used as a dual-marker gene for noninvasive imaging, direct endoscopic visualization, and in situ
detection with fluorescence microscopy.
From a vector design prospective, the tkgfp fusion gene has several positive features. A relatively small coding region of tk (1.2 kb) and gfp (750 bp) results in a tkgfp fusion gene that is less than 2 kb in size and allows for easy incorporation into virus vectors with small transgene capacities (e.g., adeno-associated virus vector). The transgene capacity of adeno-associated virus vectors is less than 5 kb, which makes cloning of two transgenes (therapeutic and selection gene) within the same vector difficult. The GFP-fluorescence is not dependent on substrates or cofactors, which enables direct detection of transgene expression in living cells. Therefore, the need for time-consuming immunocytochemistry and histochemistry for the detection of transduced cells can be avoided. This is particularly important because of the lack of a readily available antibody for HSV-1-tk. Furthermore, GFP-expressing cells can be rapidly isolated from a pool of transduced cells by FACS. This avoids time-consuming drug selection and allows for fractionation of cells with respect to the level of GFP expression and, in turn, the level of tk expression.