Mohammad Namavari

Quantification of target gene expression by imaging reporter gene expression in living animals

Quantification of target gene expression by imaging reporter gene expression in living animals

Regioselective radiofluorodestannylation with [18F]F2 and [18F]CH3COOF: A high yield synthesis of 6-[18F]fluoro-l-dopa

A protected 6-trimethylstannyl dopa derivative 6 has been synthesized for the first time as a precursor for the preparation of 6-[18F]fluoro-L-dopa. The tin derivative 6 readily reacted with electrophilic radiofluorinating agents such as [18F]F2 and [18F]AcOF. The [18F]fluoro intermediate 7 was easily hydrolyzed with HBr and the product 6-[18F]fluoro-L-dopa was isolated after HPLC purification in a maximum radiochemical yield of 25%, ready for human use. The various intermediates, the stannyl precursor 6 and the final product (after 18F decay) were all fully characterized by 1H, 13C, 19F and 119Sn NMR as well as high resolution mass spectroscopy.

Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors.

Biodistribution, magnitude and duration of a therapeutic transgenes expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRGs expression by a PET reporter probe (PRP) in living animals. We validate the simple approach of co-administering two distinct but otherwise identical adenoviruses, one expressing a therapeutic transgene and the other expressing the PRG, to track the therapeutic genes expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D2R), each regulated by the same cytomegalovirus (CMV) promoter, have been inserted into separate adenoviral vectors (Ad). We demonstrate that cells co-infected with equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D2R express both reporter genes with good correlation (r2 = 0.93). Similarly, a high correlation (r2 = 0.97) was observed between the expression of both PRGs in the livers of mice co-infected via tail-vein injection with equivalent titers of these two adenoviruses. Finally, microPET imaging of HSV1-sr39tk and D2R expression with 9-(4-[18F]fluoro-3-hydroxymethylbutyl) guanine ([18F]FHBG) and 3-(2-[18F]fluoroethyl)spiperone ([18F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.

Seeing is believing: non-invasive, quantitative and repetitive imaging of reporter gene expression in living animals, using positron emission tomography.

The ability to monitor reporter gene expression in living animals and in patients will permit longitudinal examinations both of somatically transferred DNA in experimental animals and patients and of transgenic constructs expressed in experimental animals. If investigators can non‐invasively monitor the organ and tissue specificity, the magnitude and the duration of gene expression from somatically transferred DNA and from transgenes, conceptually new experimental paradigms will be possible. If clinicians can non‐invasively monitor the location, extent and duration of somatically transferred genes, they will be better able to determine the correlations between expression of therapeutic genes and clinical outcomes. We have developed two reporter gene systems for in vivo reporter gene imaging in which the protein products of the reporter genes sequester positron‐emitting reporter probes. The “PET reporter gene” dependent sequestration of the “PET reporter probes” is subsequently measured in living animals by Positron Emission Tomography (PET). We describe here the principles of PET reporter gene/PET reporter probe in vivo imaging, the development of two imaging systems, and the validation of their ability to non‐invasively, quantitatively and repetitively image reporter gene expression in murine viral gene transfer and transgenic models. J. Neurosci. Res. 59:699–705, 2000

Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules.

Human epidermal growth factor receptor type 2 (HER2) is a well-established tumor biomarker that is overexpressed in a wide variety of cancers and that serves as a molecular target for therapeutic intervention. HER2 also serves as a prognostic indicator of patient survival and as a predictive marker of the response to antineoplastic therapy. The development of 18F-labeled biomolecules for PET imaging of HER2 (HER2 PET) is very important because it may provide a powerful tool for the early detection of HER2-positive tumor recurrence and for the monitoring of HER2-based tumor treatment. Methods: In this study, anti-HER2 monomeric and dimeric protein scaffold molecules [ZHER2:477 and (ZHER2:477)2, respectively] were radiofluorinated at a reasonable radiochemical yield (13%–18%) by use of site-specific oxime chemistry. The resulting radiofluorinated protein scaffold molecules were then evaluated as potential molecular probes for small-animal HER2 PET by use of a SKOV3 tumor–bearing mouse model. Results: The 4-18F-fluorobenzaldehyde conjugated aminooxy-protein scaffolds [18F-N-(4-fluorobenzylidene)oxime (FBO)-ZHER2:477 and 18F-FBO-(ZHER2:477)2] both displayed specific HER2-binding ability in vitro. Biodistribution and small-animal PET imaging studies further revealed that 18F-FBO-ZHER2:477 showed rapid and high SKOV3 tumor accumulation and quick clearance from normal tissues, whereas 18F-FBO-(ZHER2:477)2 showed poor in vivo performance (low tumor uptake and tumor-to-normal tissue ratios). The specificity of 18F-FBO-ZHER2:477 for SKOV3 tumors was confirmed by its lower uptake on pretreatment of tumor-bearing mice with the HER2-targeting agents ZHER2 and trastuzumab. Moreover, small-animal PET imaging studies revealed that 18F-FBO-ZHER2:477 produced higher-quality tumor imaging than 18F-FBO-(ZHER2:477)2. 18F-FBO-ZHER2:477 could clearly identify HER2-positive tumors with good contrast. Conclusion: Overall, these data demonstrate that 18F-FBO-ZHER2:477 is a promising PET probe for imaging HER2 expression in living mice. It has a high potential for translation to clinical applications. The radiofluorination method developed can also be used as a general strategy for the site-specific labeling of other proteins with 18F. The protein scaffold molecules used here are attractive for the further development of PET probes for other molecular targets.

Positron-Emission Tomography Reporter Gene Expression Imaging in Rat Myocardium

Background—This study examines the quantitative accuracy, detection sensitivity, and time course of imaging the expression of a mutant herpes simplex type-1 virus thymidine kinase (HSV1-sr39tk) PET reporter gene in rat myocardium by using the PET reporter probe 9-(4-[18F]-Fluoro-3-Hydroxymethylbutyl)-Guanine ([18F]-FHBG) and a small-animal PET (microPET). Methods and Results—In 40 rats, adenovirus expressing HSV1-sr39tk driven by a cytomegalovirus promoter (Ad-CMV-HSV1-sr39tk, 1×106 to 1×109 pfu) was injected through a thoracotomy directly into the left ventricular myocardium. After 3 days, myocardial perfusion was imaged with [13N]-ammonia for delineating the left ventricular myocardium, followed by imaging the expression of the reporter gene with intravenous [18F]-FHBG. The total myocardial [18F]-FHBG accumulation was quantified in percent of injected dose (%ID). Immunohistochemistry and autoradiography demonstrated HSV1-sr39tk enzyme (HSV1-sr39TK) and accumulation of [18F]-FHBG in the inoculated myocardium in 3 rats each. In 24 rats with various viral titers, the %ID was correlated with ex vivo well counting (r2=0.981, P <0.0001) and myocardial HSV1-sr39TK activity by tissue enzyme activity assay (r2=0.790, P <0.0001). Myocardial [18F]-FHBG accumulation was identified at viral titers down to 1×107 pfu. In 6 rats serially imaged up to day 17, myocardial [18F]-FHBG accumulation on microPET peaked on days 3 to 5 and was no longer identified on days 10 to 17. Conclusions—HSV1-sr39tk reporter gene expression can be monitored with [18F]-FHBG and microPET in rat myocardium quantitatively and serially with high detection sensitivity. Cardiac PET reporter gene imaging offers the potential of monitoring the expression of therapeutic genes in cardiac gene therapy.

Quantitative imaging of gene induction in living animals

Methods to repeatedly, non-invasively, and quantitatively image gene expression in living animals are rapidly emerging and should fundamentally change studies of gene expression in vivo. We previously developed assays utilizing positron emission tomography (PET) to image reporter gene expression. In this paper we: (1) describe a new bi-directional, tetracycline-inducible system that can be used to pharmacologically induce target gene expression and to quantitatively image induced expression by using a PET reporter gene; (2) demonstrate the potential of this system in transient and stable cell transfection assays; and (3) demonstrate the ability to repetitively and quantitatively image tetracycline and tetracycline analog induction of gene expression in living animals. We utilize the dopamine type-2 receptor (D2R) and the mutant herpes-simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter genes to validate this system. We utilize microPET technology to show that quantitative tomographic imaging of gene induction is possible. We find a high correlation (r2 = 0.98) between ‘target’ and reporter gene expression. This work establishes a new technique for imaging time-dependent variation of gene expression both from vectors with inducible promoters and in transgenic animals in which pharmacologic induction of gene expression must be monitored. These techniques may be applied both in gene therapy and for the study of gene expression in transgenic animals.

Molecular Imaging of EGFR Kinase Activity in Tumors with 124I-Labeled Small Molecular Tracer and Positron Emission Tomography

Positron emission tomography (PET) with epidermal growth factor receptor (EGFR) kinase-specific radiolabeled tracers could provide the means for noninvasive and repetitive imaging of heterogeneity of EGFR expression and signaling activity in tumors in individual patients before and during therapy with EGFR signaling inhibitors. We developed the synthesis and 124I-radiolabeling of the (E)-But-2-enedioic acid [4-(3-[124I]iodoanilino)-quinazolin-6-yl]-amide-(3-morpholin-4-yl-propyl)-amide (morpholino-[124I]-IPQA), which selectively, irreversibly, and covalently binds the adenosine-triphosphate-binding site to the activated (phosphorylated) EGFR kinase, but not to the inactive EGFR kinase. The latter was demonstrated using in silico modeling with crystal structures of the wild type and different gain-of-function mutants of EGFR kinases. Also, this was demonstrated by selective radiolabeling of the EGFR kinase domain with morpholino-[131I]-IPQA in A431 human epidermoid carcinoma cells and Western blot autoradiography. In vitro radiotracer accumulation and washout studies demonstrated a rapid accumulation and progressive retention postwashout of morpholino-[131I]-IPQA in A431 epidermoid carcinoma and in U87 human glioma cells genetically modified to express the EGFRvIII mutant receptor, but not in the wild-type U87MG glioma cells under serum-starved conditions. Using morpholino-[124I]-IPQA, we obtained noninvasive PET images of EGFR activity in A431 subcutaneous tumor xenografts, but not in subcutaneous tumor xenografts grown from K562 human chronic myeloid leukemia cells in immunocompromised rats and mice. Based on these observations, we suggest that PET imaging with morpholino-[124I]-IPQA should allow for identification of tumors with high EGFR kinase signaling activity, including brain tumors expressing EGFRvIII mutants and nonsmall-cell lung cancer expressing gain-of-function EGFR kinase mutants. Because of significant hepatobiliary clearance and intestinal reuptake of the morpholino-[124I]-IPQA, additional [124I]-IPQA derivatives with improved water solubility may be required to optimize the pharmacokinetics of this class of molecular imaging agents.

Imaging progress of herpes simplex virus type 1 thymidine kinase suicide gene therapy in living subjects with positron emission tomography

Molecular imaging of a suicide transgenes expression will aid the development of efficient and precise targeting strategies, and imaging for cancer cell viability may assess therapeutic efficacy. We used the PET reporter probe, 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)guanine ([18F]FHBG) to monitor the expression of a mutant Herpes Simplex Virus 1 thymidine kinase (HSV1-sr39tk) in C6 glioma tumors implanted subcutaneously in nude mice that were repetitively being treated with the pro-drug Ganciclovir (GCV). [18F]-Fluorodeoxyglucose ([18F]FDG), a metabolic tracer, was used to assess tumor cell viability and therapeutic efficacy. C6 glioma tumors stably expressing the HSV1-sr39tk gene (C6sr39) accumulated [18F]FHBG prior to GCV treatment. Significant declines in C6sr39 tumor volumes and [18F]FHBG and [18F]FDG accumulation were observed following 2 weeks of GCV treatment. However, 3 weeks after halting GCV treatment, the tumors re-grew and [18F]FDG accumulation increased significantly; in contrast, tumor [18F]FHBG concentrations remained at background levels. Therefore, [18F]FHBG can be used to detect tumors expressing HSV1-sr39tk, susceptible to regression in response to GCV exposure, and the effectiveness of GCV therapy in eradicating HSV1-sr39tk-expressing cells can be monitored by [18F]FHBG scanning. [18F]FHBG and [18F]FDG imaging data indicate that exposure of C6sr39 tumors to GCV causes the elimination of [18F]FHBG-accumulating C6sr39 cells and selects for re-growth of tumors unable to accumulate [18F]FHBG.

Pharmacokinetic Assessment of the Uptake of 16β-18F-Fluoro-5α-Dihydrotestosterone (FDHT) in Prostate Tumors as Measured by PET

The aim of this study was to develop a clinically applicable noninvasive method to quantify changes in androgen receptor (AR) levels based on 18F-16β-fluoro-5α-dihydrotestosterone (18F-FDHT) PET in prostate cancer patients undergoing therapy. Methods: Thirteen patients underwent dynamic 18F-FDHT PET over a selected tumor. Concurrent venous blood samples were acquired for blood metabolite analysis. A second cohort of 25 patients injected with 18F-FDHT underwent dynamic PET of the heart. These data were used to generate a population-based input function, essential for pharmacokinetic modeling. Linear compartmental pharmacokinetic models of increasing complexity were tested on the tumor tissue data. Four suitable models were applied and compared using the Bayesian information criterion (BIC). Model 1 consisted of an instantaneously equilibrating space, followed by a unidirectional trap. Models 2a and 2b contained a reversible space between the instantaneously equilibrating space and the trap, into which metabolites were excluded (2a) or allowed (2b). Model 3 built on model 2b with the addition of a second reversible space preceding the unidirectional trap and from which metabolites were excluded. Results: The half-life of the 18F-FDHT in blood was between 6 and 7 min. As a consequence, the uptake of 18F-FDHT in prostate cancer lesions reached a plateau within 20 min as the blood-borne activity was consumed. Radiolabeled metabolites were shown not to bind to ARs in in vitro studies with CWR22 cells. Model 1 produced reasonable and robust fits for all datasets and was judged best by the BIC for 16 of 26 tumor scans. Models 2a, 2b, and 3 were judged best in 7, 2, and 1 cases, respectively. Conclusion: Our study explores the clinical potential of using 18F-FDHT PET to estimate free AR concentration. This process involved the estimation of a net uptake parameter such as the ktrap of model 1 that could serve as a surrogate measure of AR expression in metastatic prostate cancer. Our initial studies suggest that a simple body mass–normalized standardized uptake value correlates reasonably well to model-based ktrap estimates, which we surmise may be proportional to AR expression. Validation studies to test this hypothesis are underway.