Positron emission tomography reporter genes and reporter probes: gene and cell therapy applications

Abstract

Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.

Keywords: Genetically Modified Therapeutic Cells; Imaging Therapeutic Transgenes or Cells; PET Imaging Reporter Genes; Positron Emission Tomography; Therapeutic Transgenes.

Fig 1

Mechanism of positron emission tomography (PET) imaging. PET is a molecular imaging modality useful for pre-clinical and clinical imaging of molecular events in living subjects. PET imaging involves a positron emitting radioisotope labeled imaging probe and instrumentation that can detect in a coincidence manner the radiation emitted when positrons from the imaging probe collide with nearby electrons. In this manner, PET cameras acquire data about the precise location of imaging probes inside the body of a living subject and the amount of imaging probe accumulated at every site at any given time. PET imaging probes usually accumulate at a site through binding to receptor molecules, interaction with an enzyme, or a cellular transport mechanism.

Fig 2

Traditional classification of PET IRG/PRP systems based on their mechanism of action. A) The reporter gene encodes an enzyme that catalyzes chemical transformation of the reporter probe, thereby the reporter probes gets trapped within cells expressing the reporter gene. B) The reporter gene encodes a protein receptor that can be specifically bound by a radiolabeled ligand reporter probe. C) The reporter gene encodes a protein transporter that transports the radionuclide reporter probe into the cells expressing the reporter gene. Illustrations reprinted from review article by Penuelas et al.

Fig 2

Traditional classification of PET IRG/PRP systems based on their mechanism of action. A) The reporter gene encodes an enzyme that catalyzes chemical transformation of the reporter probe, thereby the reporter probes gets trapped within cells expressing the reporter gene. B) The reporter gene encodes a protein receptor that can be specifically bound by a radiolabeled ligand reporter probe. C) The reporter gene encodes a protein transporter that transports the radionuclide reporter probe into the cells expressing the reporter gene. Illustrations reprinted from review article by Penuelas et al.

Fig 2

Traditional classification of PET IRG/PRP systems based on their mechanism of action. A) The reporter gene encodes an enzyme that catalyzes chemical transformation of the reporter probe, thereby the reporter probes gets trapped within cells expressing the reporter gene. B) The reporter gene encodes a protein receptor that can be specifically bound by a radiolabeled ligand reporter probe. C) The reporter gene encodes a protein transporter that transports the radionuclide reporter probe into the cells expressing the reporter gene. Illustrations reprinted from review article by Penuelas et al.

Fig 3

The mechanism and consequences of PRG immunogenicity. The PRG is transcribed and translated in genetically engineered therapeutic cells; peptides derived from the PRG are displayed on the cell surface in the context of MHC class I molecules. If these peptides have never been encountered by the host immune system (i.e., they are 'foreign'), they are detected by CD8+ T cells, which then kill the therapeutic cells, leading to treatment failure. In contrast, if the PRG is sufficiently similar or identical to a gene normally expressed by the host ('self' rather than 'foreign') it is less likely that the therapeutic cells will be detected as 'foreign' and eliminated.

Fig 4

Direct imaging of a therapeutic transgene's products with PET. Specific PET probes may be designed and developed to directly image the messenger ribonucleic acids (mRNA) transcribed from a therapeutic transgene (TG) or the protein translated from its mRNA. Examples of potential mRNA PET imaging probes are radiolabeled antisense oligo-nucleotides (RASONs) or modifications of RASONs that are complementary to a specific region of an mRNA. There are also PET imaging probes that specifically detect a certain protein by binding to it or even detect the protein's function. Examples are specific detection of D₂R with [¹⁸F]FESP and specific detection of HSV1-sr39TK enzyme activity with [¹⁸F]FHBG. However, developing a new specific PET probe to image the expression of any TG requires significant expenditure of resources and may not be practical.

Fig 5

Example of direct gene therapy monitoring with PET. Direct imaging of HSV1-sr39tk/GCV suicide gene therapy progress in C6 glioma xenografted immunodeficient mice. Four C6sr39 tumor xenografts were implanted subcutaneously on 4 sites of the nude mouse shown. All 4 tumors highly accumulated [¹⁸F]FHBG and [¹⁸F]FDG prior to starting GCV treatment (week 0). The mouse was administered daily IP injections of GCV (100 mg/kg) for two weeks, during which time period the tumors regressed (3 of them visually eradicated) and [¹⁸F]FHBG and [¹⁸F]FDG accumulation declined to background levels. The mouse was monitored up to three weeks after halting GCV treatment. The tumors re-grew, but only accumulated [¹⁸F]FHBG at background levels, despite robust ability to accumulate [¹⁸F]FDG. Reprinted from research article by Yaghoubi et al.

Fig 6

Five genetic constructs enabling linkage of the expression of a therapeutic transgene to a PET imaging reporter transgene for indirect monitoring of the pharmacokinetics of the therapeutic transgene. Depending on the therapeutic transgene and the PET imaging reporter transgene used these constructs should allow expression of both transgenes in a correlated manner in a population of cells. Reprinted from figure 9.5 of a book chapter by Yaghoubi et al.

Fig 7

[¹⁸F]FESP and [¹⁸F]FHBG coronal images of (A) a Swiss Webster and (B) a Nude mouse at three different time points after co-injecting equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D₂R intravenously. (A) 2.0 X 10⁹ and (B) 0.1 X 10⁹ pfu of both Ad-CMV-D₂R and Ad-CMV-HSV1-sr39tk were co-injected into the tail vein of a Swiss Webster mouse and a Nude mouse, respectively. The mice were then scanned for [¹⁸F]FESP and [¹⁸F]FHBG activity. For the [¹⁸F]FESP images times 1, 2, and 3 correspond to 3, 10, and 24 days after the injection of the adenoviruses, respectively. For the [¹⁸F]FHBG images times 1, 2, and 3 correspond to 5, 12, and 26 days after the injection of the adenoviruses, respectively. The images are scaled for injected dose. These mice were representatives of a larger data set. Reprinted from research article by Yaghoubi et al.

Fig 7

[¹⁸F]FESP and [¹⁸F]FHBG coronal images of (A) a Swiss Webster and (B) a Nude mouse at three different time points after co-injecting equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D₂R intravenously. (A) 2.0 X 10⁹ and (B) 0.1 X 10⁹ pfu of both Ad-CMV-D₂R and Ad-CMV-HSV1-sr39tk were co-injected into the tail vein of a Swiss Webster mouse and a Nude mouse, respectively. The mice were then scanned for [¹⁸F]FESP and [¹⁸F]FHBG activity. For the [¹⁸F]FESP images times 1, 2, and 3 correspond to 3, 10, and 24 days after the injection of the adenoviruses, respectively. For the [¹⁸F]FHBG images times 1, 2, and 3 correspond to 5, 12, and 26 days after the injection of the adenoviruses, respectively. The images are scaled for injected dose. These mice were representatives of a larger data set. Reprinted from research article by Yaghoubi et al.

Fig 8

PET IRG based imaging of the kinetics of a TC population. TCs are genetically engineered to stably express the PET IRG ex vivo. Following confirmation of PET IRG expression TCs are injected into the living subject. At any desired time point after TC administration the specific PRP is injected into the living subject and the subject is scanned for the biodistribution of the PRP. Quantitative PRP biodistribution analysis then allows studying the kinetics of the TCs in the subject.

Fig 9

(A). Description of the procedures involved in the preparation of genetically engineered CTLs and their infusion into the recurrent glioma tumor resection site. (B). MRI and PET over MRI superimposed brain images of a patient recieving genetically engineered cytolytic T cells expressing HSV1-tk for imaging and safety purposes. Images were acquired approximately two hours after [¹⁸F]FHBG injection. The patient had a surgically resected tumor (1) in the right parietal lobe and a new non-resected tumor in the center (2), near corpus callosum of his brain. The infused cells had localized at the site of tumor 1 and also trafficked to tumor 2. [¹⁸F]FHBG activity is higher than the brain background at both sites. Background [¹⁸F]FHBG activity is low within the Central Nervous System due to its inability to cross the blood brain barrier. Background activity is relatively higher in all other tissues. Activity can also be observed in the meninges. The tumor 1/meninges and tumor 2/meninges [¹⁸F]FHBG activity ratio in this patient was 1.75 and 1.57, respectively. Whereas the average resected tumor site/meninges and intact tumor site to meninges [¹⁸F]FHBG activity ratio in control patients was 0.86 and 0.44, respectively. Figure 9A is a reprint of figure 13.9 from a book chapter by Penuelas et al. and Figure 9B is a reprint of figure 1 from a case study report by Yaghoubi et al.

Fig 9

(A). Description of the procedures involved in the preparation of genetically engineered CTLs and their infusion into the recurrent glioma tumor resection site. (B). MRI and PET over MRI superimposed brain images of a patient recieving genetically engineered cytolytic T cells expressing HSV1-tk for imaging and safety purposes. Images were acquired approximately two hours after [¹⁸F]FHBG injection. The patient had a surgically resected tumor (1) in the right parietal lobe and a new non-resected tumor in the center (2), near corpus callosum of his brain. The infused cells had localized at the site of tumor 1 and also trafficked to tumor 2. [¹⁸F]FHBG activity is higher than the brain background at both sites. Background [¹⁸F]FHBG activity is low within the Central Nervous System due to its inability to cross the blood brain barrier. Background activity is relatively higher in all other tissues. Activity can also be observed in the meninges. The tumor 1/meninges and tumor 2/meninges [¹⁸F]FHBG activity ratio in this patient was 1.75 and 1.57, respectively. Whereas the average resected tumor site/meninges and intact tumor site to meninges [¹⁸F]FHBG activity ratio in control patients was 0.86 and 0.44, respectively. Figure 9A is a reprint of figure 13.9 from a book chapter by Penuelas et al. and Figure 9B is a reprint of figure 1 from a case study report by Yaghoubi et al.