Figure 16 Indirect PET Imaging Using a Bidirectional Transcriptional Approach (A) Target (therapeutic) gene expression can be measured indirectly by imaging reporter-gene expression if expression of the 2 genes is 'linked.' Both genes can be simultaneously expressed from 2 minimal cytomegalovirus (CMV) promoters that are regulated by a single bi directional tetracycline-responsive element (TRE). The rTetR-VP16 fusion protein is produced constitutively from a CMV promoter. When the rTetR-VP16 fusion protein binds to doxycycline, this complex binds to the TRE regulatory sequence and substantially enhances expression from the 2 minimal CMV promoters. The target gene A in 1 coding region and a reporter gene (for example, a reporter kinase such as HSV1-sr39f£) in the alternative coding region are transcribed simultaneously into 2 mRNA molecules. Translation of the 2 mRNA molecules yields two distinct proteins in amounts that are directly correlated with each other. (B, see pg. 474) Quantitative imaging of the location(s) and magnitude of PET reporter-gene expression by trapping of a PET tracer inside the cell (for example, by phosphorylation of [18F]FHBG by the HSV1-sr39TK reporter protein) provides an indirect measure of target-gene expression. Sequential microPET (positron-emission tomography) imaging studies of a nude mouse carrying 4 tumors. Four tumor cell lines—2 positive controls (constitutive reporter-gene expression), 1 negative control, and 1 inducible line (reporter-gene expression induced by doxycycline)—were injected subcutaneously into 4 separate sites in a single mouse. When tumors reached a size of at least 5 mm, the mouse was imaged with 9-(4-[18F] fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG). Doxycycline was then added to the water supply for 7 days. The mouse was then scanned again with [18F]FHBG. Doxycycline was removed from the water supply for the next 7 days, and the mouse was again scanned with [18F]FHBG. The locations of the 4 tumors and the mouse outline are shown by the dotted regions of interest. All images are 1-2 mm coronal sections through the 4 tumors. The % ID/g (% injected dose per gram tissue) scale for [18F]FHBG is shown on the right. The negative control tumors show no gene expression, and the positive control tumors show increased expression over the time course. The tumor on the top right, with inducible gene expression, initially does not accumulate [18F]FHBG, then at 7 days after addition of doxycycline, induction of reporter-gene expression traps [18F]FHBG. Seven days after withdrawal of doxycycline, there is decreased induction and minimal trapping of [18F]FHBG. The [18F]FHBG image signal correlates well with target-gene expression (not shown). See color insert for color version of this figure. (Images reproduced with permission from Ref. 23.)

Figure 16 Continued.

sis occurs in the region where [124I]FIAU uptake was previously shown, suggesting successful suicide gene transfer to a portion of the tumor. Those patients who could not accumulate detectable levels of [124I]FIAU in their infused tumors were found to have a low mitotic index in their tumors.

With this small yet extremely exciting study, we are able gain a significant amount of knowledge about human gene therapy using in vivo imaging methods. For example, the investigators were able to identify and localize successful gene transfer in 1 patient 68 h after tumor infusion. Also, in the patient where [124I]FIAU uptake was seen, only a fraction of the tumor was sequestering [124I]FIAU. This suggests that the rest of the tumor was either not transduced or that the transduction was too weak to detect. Additional follow-up scans of the patient would help to determine the outcome of the heterogeneously treated tumor as well as the importance of reporter-probe uptake. In the patients that did not demonstrate [124I]FIAU uptake, the failure to see [124I]FIAU reporterprobe accumulation may be due to sheer lack of transducible cells or failure of gene transfer. Future studies of these and other issues are certain to be extremely helpful in understanding the effectiveness of gene therapy.

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