Reporter Gene Technology

The road to clinical gene therapy trials is an arduous one but can be helped with the variety of reporter-gene technologies described in this chapter. As with any drug testing, there is a prerequisite period of preclinical testing, much of which takes place in animal models. As expected, there will be a strong inclination towards optical and radionuclide approaches, since these approaches are the most accessible and more rigorously tested among the imaging options. Furthermore, it is likely that a single investigator or a single group of investigators working on a specific gene therapy will most likely have to utilize a combination of modalities (e.g., optical and radionu-clide-based technologies) to efficiently test gene-therapy vectors and to facilitate their use in higher organisms. As testing progresses in higher species, efforts need to be made toward the radionuclide approaches, and substantial investment of time and money may be needed to develop PET-reporter probes. Eventually, human trials will employ PET, Gamma/ SPECT, and, in the future, MR-based methods. The decision and timing to use certain reporter systems over others is a relatively complex one, and we will attempt to simplify the decision-making process by providing the set of guidelines we use to make such decisions.

The first decision depends upon whether an investigator is attempting to study the biodistribution/pharmacokinetics of a gene-delivery vehicle or whether one is interested in monitoring gene expression. From an earlier discussion (see Section IV), the distribution of a vehicle can be directly imaged by directly labeling the vehicle with radioisotope, fluorescent, or MR-compatible markers. Distribution of injected vehicle can also be inferred from a reporter gene coupled to a constitu-

Before After

LIPO-HSV-1-Wc LIPO-HSV-1-flt infusion infusion

Figure 17 Human Brain Tumor HSV1-tk Suicide Gene Therapy Using Direct PET Imaging PET brain imaging of HSV1-f£ suicide gene therapy in a patient with [124I]FIAU as a reporter probe. A single, representative transverse image of a brain from a patient suffering from recurrent glioblastoma pre- and post-gene therapy treatment. Coregistration of [124I]FIAU-PET, MRI, [11C]methionine (MET)-PET, and FDG-PET before and after intratumorally infused liposome-gene complex containing HSV1-f£ (LIPO-HSV-1-f£). The white arrow marks the region within the tumor where specific [ 124I]FIAU retention was imaged after LIPO-HSV-1-f£ transduction (2nd row, 2nd column). The cross hairs in the right column indicate signs of necrosis after ganciclovir treatment (5 mg/kg twice a day over 14 days) that was started 4 days after vector application. The area of necrosis in the tumor as depicted by reduced methionine ([11C-MET]) uptake and decreased glucose [FDG] metabolism. See color insert for color version of this figure. (Images reproduced with permission from Ref. 107.)

Figure 17 Human Brain Tumor HSV1-tk Suicide Gene Therapy Using Direct PET Imaging PET brain imaging of HSV1-f£ suicide gene therapy in a patient with [124I]FIAU as a reporter probe. A single, representative transverse image of a brain from a patient suffering from recurrent glioblastoma pre- and post-gene therapy treatment. Coregistration of [124I]FIAU-PET, MRI, [11C]methionine (MET)-PET, and FDG-PET before and after intratumorally infused liposome-gene complex containing HSV1-f£ (LIPO-HSV-1-f£). The white arrow marks the region within the tumor where specific [ 124I]FIAU retention was imaged after LIPO-HSV-1-f£ transduction (2nd row, 2nd column). The cross hairs in the right column indicate signs of necrosis after ganciclovir treatment (5 mg/kg twice a day over 14 days) that was started 4 days after vector application. The area of necrosis in the tumor as depicted by reduced methionine ([11C-MET]) uptake and decreased glucose [FDG] metabolism. See color insert for color version of this figure. (Images reproduced with permission from Ref. 107.)

tive promoter. However, this method is less sensitive since the rate of gene transfer is always less than 100%, and often significantly lower.

For investigators interested in monitoring transgene expression, a search for reporter probe(s) that may already exist for the transgene is mandatory. For example, for those who use the suicide gene, HSV1-tk, a number of reporter probes already exist, and direct-imaging protocols can be performed using radionuclide techniques. In the example of HSV1-tk, the decision to use [124I]FIAU over other probes such as [18F]FHBG is based on preliminary favorable evidence towards [124I]FIAU described earlier. However, the selection of the appropriate reporter probe is not trivial, and, as further tests are being performed, it is likely that the other reporter probes may prove to be just as or more efficient than FIAU, depending on specific cell-type, local pharmacoki-netics, mode of delivery of the transgene (viral vs. nonviral), etc. As we learn more about each reporter system, selection of specific transgene-reporter probe combinations will be disease-specific; it is likely that each reporter combination will have certain strengths and weaknesses depending on the disease model and target tissues. The investigator(s) will have to adjust accordingly. Future studies are bound to address these and other related issues.

If no reporter probe for the transgene exists, then indirect-imaging methods are needed. This requires the coupling of the therapeutic gene(s) with an optical, radionuclide-based or MR-based, reporter gene(s). As described earlier, a number of indirect methods are available, including the dual promoter approach, bidirectional approach, bicistronic approach, etc. The selection of 1 of these promoter configurations will largely depend upon the nature of the promoter of the therapeutic gene. If a robust, constitutive promoter drives the therapeutic gene, then the dual promoter, the bicistronic (IRES-mediated), or bidirectional approach may suffice. However, if a weak or tissue-specific promoter drives the therapeutic gene, then amplification strategies for either the reporter gene (and possibly the therapeutic gene) will need to be employed. Amplification strategies for tissue-specific promoters using the VP16 transactivating domain fused to the yeast GAL4 DNA-binding domain has been verified for use with reporter genes (108).

A variety of other factors also dictate the selection of reporter gene systems. For example, if a research group is intent on bringing a specific gene therapy to human application, it behooves them to use multimodality reporter-gene systems, i.e., use fused reporter genes such as HSV1-tk-Fluc (keeping in mind, HSV1-ik is not being used as a therapeutic gene in this case when the reporter probe is administered in nonphar-macological amounts). By coupling a therapeutic gene to a fused reporter gene, it will be possible to move quickly between the preclinical verification phase (optical imaging of Firefly luciferase) to the clinical phase (radionuclide-based imaging of HSV1-tk). Alternatively, if a research group strictly deals with small animals, then the use of optical reporter genes only may suffice. Along this line of reasoning, the use of large animals precludes the use of optical methods, and as a result, radionuclide-based or MR-based technologies become more important.

Imaging disease processes in the central nervous system (CNS) also limits the selection of reporter genes. Some of the radionuclide-based technologies, such as HSV1-ifc and its mutants, are limited since their reporter probes do not readily cross the blood-brain barrier (BBB). On the other hand, [18F]FESP, the reporter probe for D2R, easily crosses the blood-brain barrier and can be used to a certain extent in the CNS. Use of the D2R-[18F]FESP system in the CNS is limited in the striatum where endogenous dopamine receptors are present. Optical methods, both fluorescence and bioluminescence, can be utilized if specific spatial localization is not needed. The substrates of the bioluminescence methods, D-Luciferin and coelenterazine, easily cross the BBB, which facilitates the use of these reporter genes in the CNS.

By the same token, imaging in the lung, connective tissue, or cortical bone may limit the use of superparamagnetic-la-beled agents in MR imaging since it will be difficult to differentiate between the signal from the contrast agent and the signal of these anatomical structures, since they are identical in certain MR sequences.

Other important factors in the selection of reporter genes include the need for good spatial resolution (preference given to radionuclide and MRI-based imaging), repetitive imaging (preference given to bioluminescence and most radionuclide approaches), and image quantitation (preference given to ra-dionuclide, optical, and MRS-based methods). Cost, institutional infrastructure, requirement for support personnel, and the physical space required can also be significant factors that favor the optical and gamma camera methods and less so towards the PET and MR-based methods. Thus, a large number of factors have to be considered prior to the selection and implementation of reporter genes in living subjects. With careful planning, an optimal imaging strategy can be followed and be enormously helpful in the study of gene therapy and disease entities.

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