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Vesicular acetylcholine transporter plays a crucial role in the cholinergic system, and its alterations is implicated in several neurodegenerative disorders. We recently developed a PET imaging tracer [18F]VAT to target VAChT in vivo with high affinity and selectivity. Here we report in vitro characterization of [3H]VAT, a tritiated counterpart of [18F]VAT. Using human VAChT-rich cell membrane extracts, a saturated binding curve was obtained for [3H]VAT with Kd = 6.5 nM and Bmax = 22.89 pmol/mg protein. In the [3H]VAT competition-binding assay with a panel of CNS ligands, binding inhibition of [3H]VAT was observed using VAChT ligands, the Ki values ranged from 5.41 to 33.3 nM. No inhibition was detected using a panel of other CNS ligands. In vitro [3H]VAT autoradiography of rat brain sections showed strong signals in the striatum, moderate to high signals in vermis, thalamus, cortex, and hippocampus, and weak signals in cerebellum. Strong [3H]VAT ARG signals were also observed from striatal sections of normal nonhuman primates and human brains. Competitive ARG study with human striatal sections demonstrated strong ARG signals of [3H]VAT in caudate and putamen were blocked significantly by either VAChT ligand TZ659 or (−)-vesamicol, but not by the σ1 receptor ligand Yun-122. ARG study also indicated that signal in the striatal sections from PSP human brains was lower than normal human brains. These data provide solid evidence supporting [18F]VAT as a suitable PET radiotracer for quantitative assessment of VAChT levels in vivo.

Nine new compounds targeting on transient receptor potential vanilloid-4 (TRPV4) were synthesized and their biological activities toward TRPV4 were determined using freshly isolated mouse skin macrophages through live cell Ca(2+) imaging assay. Three compounds 4b, 4c, and 4i exhibit higher percentages of activating TRPV4 in vitro as 48.1%, 59.3% and 33.5%, comparable with 56.4 % activation response of a reported TRPV4 agonist GSK1016790A (3). The compound 4i was chosen for (11)C-radiosynthesis using its phenol precursor 4g to react with [(11)C]methyl iodide. The radiosynthesis was achieved with good radiochemical yield (16 ± 5%), high chemical and radiochemical purity (> 95%), and high molar activity (16-21 GBq/μmol, decay corrected to the end of bombardment, EOB n ≥ 4). Furthermore, the initial ex vivo biodistribution study showed that [(11)C]4i had relative higher uptakes the kidney, liver and small intestine compared to other tissues and quick washout from the animal body.

A novel caspase-3 substrate-based probe [(18)F]-CP18 was evaluated as an in vivo positron emission tomography (PET) imaging agent for monitoring apoptosis in tumors.Uptake of [(18)F]-CP18 in cell assays and tumors was measured. Caspase-3/7 activities in cell lysates and tumor homogenates were determined. Autoradiography,Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and cleaved caspase-3 immunostaining were performed on adjacent tumor sections to identify areas of apoptosis.The in vitro cell assays showed caspase-3-dependent uptake of [(18)F]-CP18 in tumor cells when treated with an apoptosis inducer. The in vivo microPET imaging signal of [(18)F]-CP18 in xenograft tumors correlated with the ex vivo caspase-3/7 activities in these tumors. Furthermore, tumor autoradiographies of [(18)F]-CP18 in tumor sections matched adjacent sections stained by TUNEL and caspase-3 immunohistochemistry (IHC).[(18)F]-CP18 demonstrated high affinity and selectivity for activated caspase-3 both in vitro and in vivo, and the results support [(18)F]-CP18 as a promising new PET imaging agent for apoptosis.

We identified and validated [18F]-CP18, a DEVD (the caspase 3 substrate recognition motif) containing substrate-based compound as an imaging tracer for caspase-3 activity in apoptotic cells. CP18 was radiolabeled with fluorine-18 using click chemistry. The affinity and selectivity of CP18 for caspase-3 were evaluated in vitro. The biodistribution and metabolism pattern of [18F]-CP18 were assessed in vivo. [18F]-CP18 positron emission tomography (PET) scans were performed in a dexamethasone-induced thymic apoptosis mouse model. After imaging, the mice were sacrificed, and individual organs were collected, measured in a gamma counter, and tested for caspase-3 activity. In vitro enzymatic caspase-3 assay demonstrated specific cleavage of CP18. In vivo, [18F]-CP18 is predominantly cleared through the kidneys and urine, and is rapidly eliminated from the bloodstream. There was a sixfold increase in caspase activity and a fourfold increase of [18F]-CP18 retention in the dexamethasone-induced thymus of treated versus control mice. We report the use [18F]-CP18 as a PET tracer for imaging apoptosis. Our data support further development of this tracer for clinical PET applications.

Current gene therapy protocols often suffer from an inability to monitor the site, level and persistence of gene expression following somatic DNA delivery. Herpes simplex virus 1 thymidine kinase (HSV1-tk) is currently under intensive investigation as a reporter gene for in vivo imaging of reporter gene expression. The presence of the HSV1-tk reporter gene is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled thymidine analogues or acycloguanosine HSV1-TK substrates and subsequent detection, by positron emission tomography, of trapped, phosphorylated product. To improve the efficacy of the HSV1-tk PET reporter gene system, both alternative substrates and mutations in the HSV1-tk gene have been described. We used a replication defective adenovirus to deliver the HSV1-sr39tk mutant enzyme and the wild-type HSV1-tk enzyme to mice. HSV1-sr39TK demonstrates greater sensitivity than wild-type HSV1-TK enzyme in vivo, using 9-[(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine as probe, following adenovirus-mediated hepatic expression in mice. Using this adenoviral delivery system, the location, magnitude and duration of HSV1-sr39tk PET reporter gene expression could be non-invasively, quantitatively and repetitively monitored for over 3 months by microPET.

Gene therapy protocols are hampered by the inability to monitor the location, magnitude, and duration of ectopic gene expression following DNA delivery. Consequently, it is difficult to establish quantitative correlations and/or causal relationships between therapeutic gene expression and phenotypic responses in treated individuals. One approach to monitor “therapeutic gene” expression indirectly is to incorporate reporter genes that can be imaged in vivo into bicistronic transcription units, along with the therapeutic genes. Expression of the dopamine D2 receptor (D2R) and herpes simplex virus thymidine kinase (HSV1-TK) can both be monitored, in vivo , by positron-emission tomography (PET). We created ad.DTm, an adenovirus containing a cytomegalovirus (CMV) early promoter–driven transcription unit, in which the D2R gene is placed proximal to an encephalomyocarditis virus internal ribosomal entry site (IRES) and a modified HSV1-tk gene is placed distal to the IRES. Following intravenous ad.DTm injection into mice, correlated hepatic D2R and HSV1-sr39tk PET reporter gene expression was demonstrated. Repeated microPET scanning quantitated both D2R-dependent sequestration of a positron-emitting ligand and HSV1-TK-dependent sequestration of a positron-emitting product. It is possible, in living mice, to investigate noninvasively and to measure quantitatively and repeatedly correlated expression of two coding regions from a bicistronic transcription unit over a 3-month period following adenovirus delivery.

Biodistribution, magnitude and duration of a therapeutic transgene's expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRG's 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 gene's expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D(2)R), 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-D(2)R express both reporter genes with good correlation (r(2) = 0.93). Similarly, a high correlation (r(2) = 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 D(2)R expression with 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl) guanine ([(18)F]FHBG) and 3-(2-[(18)F]fluoroethyl)spiperone ([(18)F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.

Analysis of a 1.3-kilobase segment of 5′-flanking DNA from the barbiturate-inducible P450BM-1 gene (CYP106) of Bacillus megaterium revealed two open reading frames. One, BM1P1, encodes 98 amino acids and is located 267 base pairs upstream from the sequence encoding cytochrome Pf450BM-1 but in the opposite orientation. The second, BM1P2 (88 amino acids), is 892 base pairs upstream from the P450BM-1 coding sequence and in the same coding strand. The expression of BM1P1 and BM1P2 was strongly stimulated in cells grown in the presence of pentobarbital, and the BM1P1 gene product exerted positive control on expression of P450BM-1. When a 177-base pair fragment encompassing the overlapping promoter regions of the P450BM-1 and BM1P1 genes was used as a probe in DNA binding assays, the BM1P1 and BM1P2 gene products and Bm3R1 (the repressor protein regulating the barbiturate-mediated expression of P450BM-3) could bind individually, but the addition of BM1P1 or BM1P2 to a binding mixture containing Bm3R1 completely prevented the appearance of a Bm3R1 binding band. When a 208-base pair fragment containing a Barbie box sequence and located upstream of the 177-base pair fragment was used as a probe, only a Bm3R1 binding band was detected. Although neither BM1P1 and BM1P2 appeared to bind to this 208-base pair fragment, their presence strongly inhibited the binding of Bm3R1 to the same probe. The evidence suggests that BM1P1 and BM1P2 may, in part, act as positive regulatory proteins involved in the expression of the P450BM-1 gene by interfering with the binding of the repressor protein, Bm3R1, to the regulatory regions of P450BM-1.

In a previous publication (He, J.-S., and Fulco, A. J.(1991) J. Biol. Chem. 266, 7864-7869), we reported that a 15-17-base pair DNA sequence (designated a Barbie box element) in the 5′-regulatory regions of cytochrome P450BM−1and P450BM−3 genes from Bacillus megaterium was recognized by a barbiturate-regulated protein. It is now recognized that essentially all eukaryotic and prokaryotic genes whose 5′-flanking regions are known and that encode barbiturate-inducible proteins contain the Barbie box element. A 4-base pair sequence (AAAG) is found in the same relative position in all Barbie box elements. In B. megaterium, mutation of the Barbie box located in the P450BM−1 gene leads to the constitutive synthesis of cytochrome P450BM−1 and a 10-fold increase of expression of Bm1P1, a small gene located upstream of the P450BM−1 gene, that encodes a putative regulatory protein. Mutation of the P450BM−3 Barbie box significantly increased the expression of both P450BM−3 and Bm3P1 (another small gene located upstream of the P450BM−3 gene that encodes a second putative regulatory protein) in response to pentobarbital induction but left the basal levels unaffected. In gel mobility shift assays, Bm3R1, a repressor of the P450BM−3 gene, was found to specifically interact with the Barbie box sequences of the B. megaterium P450 genes. Mutated Barbie boxes showed a decreased binding affinity for Bm3R1 compared to their wild type (unmutated) counterparts. Barbie box sequences were also shown to specifically interact with putative positive regulatory factors of B. megaterium cells. These putative positive factors were induced by pentobarbital and were also present at high levels during late stationary phase of B. megaterium cell cultures grown in the absence of barbiturates. The mutated Barbie box sequences had greater binding affinity for these positive factors than did unmutated Barbie box sequences. DNase I footprinting analysis of the 5′-flanking region of the P450BM−1 gene revealed that these positive factors protected a segment of DNA covering a portion of the Barbie box sequence and a small flanking region. Similar footprinting experiments with the 5′-flanking region of the P450BM−3 gene failed, however, to unambiguously reveal protected sequences in the Barbie box region. The evidence suggests that the positive factors and Bm3R1 compete with each other for binding to the Barbie box region, especially in the 5′-flanking region of the P450BM−1 gene, and for putative roles in the regulation of transcription from the B. megaterium P450 genes. This inference was further supported by evidence derived from a nested-deletion analysis in and around the Barbie box of the P450BM−1 regulatory region that showed that a repressor binding site and a positive factor binding site overlap at the Barbie box sequence. In toto, these experimental results indicate that, in B. megaterium at least, the Barbie box sequences are important cis-acting elements for coordinately regulating the barbiturate-mediated expression of the P450BM−1 and P450BM−3 genes and the genes encoding their positive regulatory factors.

Purpose To demonstrate the efficacy of repeated, non-invasive optical imaging of reporter gene expression in monitoring the ability of bi-specific recombinant molecules (i) to “transductionally untarget” adenovirus from Coxsackie and Adenovirus Receptor (CAR)-dependent infection of normal tissue and (ii) to “transductionally retarget” infection to specific target cells. Procedures sCAR-EGF is a recombinant, bi-specific molecule containing the soluble portion of CAR fused to Epidermal Growth Factor. The sCAR moiety binds to the virus and blocks CAR-dependent adenovirus infection. The EGF moity binds to cellular EGF receptors. We used non-invasive optical imaging of firefly luciferase to repeatedly monitor, in living animals, the ability of sCAR-EGF (i) to “transductionally untarget” systemically administered Ad.CMVfLuc, an adenovirus that constitutively expresses luciferase, from normal tissues and (ii) to “transductionally redirect” adenovirus infection in mice to xenograft tumors that express elevated epidermal growth factor (EGF) receptor levels. Results Systemic injection of sCAR-EGF “coated” adenovirus expressing firefly luciferase from the CMV early promoter, reduces expression of the reporter gene in the liver and facilitates expression of the reporter gene in tumor xenografts expressing high levels of the EGF-receptor. Conclusion Both liver “untargeting” and tumor “retargeting” of adenovirus by recombinant sCAR-EGF can be imaged non-invasively using a luciferase reporter gene.

Purpose To demonstrate that non-invasive bioluminescent imaging can monitor restricted expression from a nonreplicating adenovirus in which the cyclooxygenase-2 (COX-2) promoter drives firefly luciferase. Procedures Adenovirus in which the COX-2 promoter drives the firefly luciferase imaging gene was injected intratumorally into xenografts that express relatively low and relatively high levels of COX-2. Adenovirus that expresses Renilla Luciferase from the cytomegalovirus early promoter was co-injected, to normalize for injection, leakage, vascularization, etc. COX-2 restricted firefly luciferase and global Renilla Luciferase activities were measured by optical imaging techniques both in vivo and in isolated tissues. Results Dramatic reduction in hepatic luciferase expression after intravenous viral injection can be imaged non-invasively in living animals. Following intratumoral injection, luciferase levels in tumor xenografts that express differing endogenous COX-2 levels reflect the luciferase levels observed when these cells are infected in cell culture. Essentially no luciferase expression is observed in liver following intratumoral injection. Conclusion Both tissue restricted expression and transcriptional redirection to tumors expressing COX-2 can be imaged non-invasively following injection of Adenovirus expressing firefly luciferase from the COX-2 promoter.

Background— We have previously validated the use of micro-positron emission tomography (microPET) for monitoring the expression of a single PET reporter gene in rat myocardium. We now report the use of a bicistronic adenoviral vector (Ad-CMV-D2R80a-IRES-HSV1-sr39tk) for linking the expression of 2 PET reporter genes, a mutant rat dopamine type 2 receptor (D2R80a) and a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk), with the aid of an internal ribosomal entry site (IRES). Methods and Results— Rat H9c2 cardiomyoblasts transduced with increasing titers of Ad-CMV-D2R80a-IRES-HSV1-sr39tk (0 to 2.5×10 8 pfu) were assayed 48 hours later for reporter protein activities, which were found to correlate well with viral titer ( r 2 =0.96, P r 2 =0.98, P r 2 =0.97; P 9 pfu of Ad-CMV-D2R80a-IRES-HSV1-sr39tk and saline, respectively. Forty-eight hours later and weekly thereafter, rats were assessed for D2R80a-dependent myocardial accumulation of 3-(2-[ 18 F]fluoroethyl)spiperone ([ 18 F]-FESP) and HSV1-sr39tk–dependent sequestration of 9-(4-[ 18 F]fluoro-3-hydroxymethylbutyl)guanine ([ 18 F]-FHBG) using microPET. Longitudinal [ 18 F]-FESP and [ 18 F]-FHBG imaging of experimental rats revealed a good correlation between the cardiac expressions of the 2 PET reporter genes ( r 2 =0.73; P Conclusions— The IRES-based bicistronic adenoviral vector can potentially be used in conjunction with PET for indirect imaging of therapeutic gene expression by replacing 1 of the 2 PET reporter genes with a therapeutic gene of choice.

We are developing assays for noninvasive, quantitative imaging of reporter genes with positron emission tomography (PET), for application both in animal models and in human gene therapy. We report here a method to improve the detection of lower levels of PET reporter gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET reporter gene. The HSV1-sr39tk mutant was identified from a library of site-directed mutants. Accumulation (net uptake) of the radioactively labeled substrates [8- 3 H]penciclovir ([8- 3 H]PCV), and 8-[ 18 F]fluoropenciclovir (FPCV) in C6 rat glioma cells expressing HSV1-sr39tk is increased by a factor of ≈2.0 when compared with C6 cells expressing wild-type HSV1-tk. The increased imaging sensitivity of HSV1-sr39tk when FPCV is used is also demonstrated in vivo both with tumor cells stably transfected with either HSV1-tk or HSV1-sr39tk, and after hepatic delivery of HSV1-tk or HSV1-sr39tk by using adenoviral vectors. The use of HSV1-sr39tk as a PET reporter gene and FPCV as a PET reporter probe results in significantly enhanced sensitivity for imaging reporter gene expression in vivo .

We previously showed [Q. Liang, A.J. Fulco, J. Biol. Chem., 270 (1995) 18606–18614) that the binding of Bm3R1 repressor to Barbie box elements and operator sites in the 5′-flanking regions of the P450BM-3 and P450BM-1 (CYP102 and CYP106) genes in Bacillus megaterium was a critical factor in their regulation at the level of transcription. We now describe experiments that delineate specific roles for Bm3R1 in the barbiturate-mediated induction of these genes. We directly demonstrate the interaction of Bm3R1 with Barbie box and operator sequences and show that high in vivo levels of Bm3R1 prevent putative positive factors from binding to Barbie box elements, strongly inhibit the expression of the P450 genes, prolong the lag phase of growth in Bacillus megaterium cultures and increase the sensitivity of the cells to the growth-inhibitory effects of barbiturates. Finally, our data suggest that there may be two forms of Bm3R1, either of which can interact with OIII, the bicistronic operator sequence.

We previously reported (Liang, Q., He, J.-S., and Fulco, A.J. (1995) J. Biol. Chem. 270, 4438-4450) that Bm3R1, a repressor regulating the expression of P450BM-3 in Bacillus megaterium, could bind to Barbie box sequences in the 5'-flanking regions of barbiturate-inducible genes. We've now shown that pentobarbital does not inhibit in vitro binding of Bm3R1 to the P450BM-3 and P450BM-1 Barbie boxes (BB3 and BB1), although the palindromic operator sequence (OIII) of P450BM-3 did have a strong competitive effect on such binding. G39E-Bm3R1, a mutant of Bm3R1, did not bind to either Barbie box. In the presence of Bm3R1, portions of the regulatory regions of P450BM-3 and P450BM-1 were protected from DNase I digestion. These included 11 of the 15 base pairs of BB3 plus 7 base pairs 3' to BB3, BB1 plus 16 base pairs 3' to BB1, and, in the 5'-flanking region of P450BM-1, segments covering most of two palindromic sequences (OII and OIII) of 24 and 52 base pairs. These DNase I-protected regions (including OIII) showed considerable sequence identity, especially in a conserved poly(A) motif. Barbiturates did not inhibit binding of Bm3R1 to OI. OII in vitro while G39E-Bm3R1 did not bind. The regulatory effects of Bm3R1 on P450BM-1 and P450BM-3 were also evaluated in vivo using heterologous chloramphenicol acetyltransferase constructs and Western blotting. In the G39E mutant strain, both P450BM-1 and P450BM-3 were constitutively expressed, and the regulatory proteins Bm1P1 and Bm3P1, although still pentobarbital-inducible, had significantly higher basal levels of synthesis. In toto, our results show that Bm3R1 represses both P450BM-1 and P450BM-3 expression and that it may effect this by coordinate binding to operator and Barbie box sequences to produce looping of the P450BM-1 and P450BM-3 regulatory regions through protein-protein interaction.