Genetic Incorporation of Human Metallothionein into the Adenovirus Protein IX for Non-Invasive SPECT Imaging

As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus vectors is a promising strategy. However tracking the biodistribution of adenovirus vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do not give a full representation of the pharmacokinetics involved. Current non-invasive imaging strategies using reporter gene expression have been applied to analyze adenoviral vectors. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and human metallothionein. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional metallothionein (MT) as a component of their capsid surface. We demonstrate the feasibility of 99mTc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction. SPECT imaging of a mouse after administration of a 99mTc-radiolabeled virus showed clear localization of radioactivity to the liver. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. The ability we have developed to view real-time biodistribution in their physiological milieu represents a significant tool to study adenovirus biology in vivo.


Introduction
Barrier to gene therapy approaches for cancer As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus (Ad) vectors is a promising strategy. The existing approaches to gene therapy/ virotherapy of cancer can be divided into five broad categories: a) mutation compensation, b) molecular chemotherapy, c) genetic immunopotentiation, d) genetic modulation of resistance/sensitivity, and e) oncolytic therapy or virotherapy. Through 2009, Ad vectors were employed in a quarter of gene therapy clinical trials worldwide [1]. However tracking the biodistribution of Ad vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, nonquantitative, and do not give a full representation of the pharmacokinetics involved.

Current detection methods are inadequate for Ad vector systems
Several imaging studies have attempted to address this problem, including the use of positron emission tomography (PET) scanning to detect herpes simplex virus thymidine kinase (HSV-TK) as a reporter of oncolytic herpes simplex virus replication [2]. However, detection was restricted to infected cells expressing the reporter gene, which does not represent the physical distribution of the virus itself. Another group employed soluble hCEA and bhCG peptide markers as a way to monitor oncolytic measles virus therapy in mice, which correlated with therapeutic outcome but could not show viral localization [3]. Other conventional imaging systems for adenovirus based gene therapy have been designed to detect transgene expression of reporters such as green fluorescent protein (GFP) [4], somatostatin receptor type 2 (SSTR-2) [5,6], sodium iodide symporter [7], luciferase [8], and HSV-TK [9]. Despite their utility for assessing gene delivery and expression, these reporters by themselves are not suitable for monitoring physical biodistribution. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging.

Capsid protein IX is a potential location for labeling Ads
To overcome this limitation, we devised a novel approach to incorporate the human metallothionein (MT) protein as a fusion to the Ad minor capsid protein pIX (pIX). This would allow us to directly label Ads and determine if this approach can be used to monitor virus delivery in vivo by non-invasive imaging. Recent work by several groups has defined the C-terminus of pIX as a locus presenting incorporated ligands on the virus surface. Protein IX is a small polypeptide of 140 residues (14.7 kDa) that acts as a cement protein to stabilize hexon-hexon interaction and therefore the capsid structure itself [10]. Four trimers of pIX interact with a group of nine (GON) hexons in each facet of the icosahedron [11], resulting in 240 copies of the protein per virion [12]. In addition, pIX has been implicated as a transcriptional activator of several viral and cellular TATA-containing promoters, including adenoviral E1A, E4, and major late promoters [13]. Based on the understanding the pIX C-terminus is surface exposed [14,15], pIX has been exploited as a location to incorporate heterologous peptides (namely lysine octapeptide and polylysine) into its C-terminus for retargeting purposes [16]. A single chain variable fragment (scFv) against beta-galactosidase [17] and a single-chain Tcell receptor (scTCR) directed against the melanoma-associated antigen (MAGE) [18] were also successfully fused to pIX and assembled into virions. Based on these data, we also demonstrated that pIX would be a suitable location for incorporating reporter genes, such as firefly luciferase [19] and GFP [20,21]. However, these fusions require optical imaging techniques that are not clinically compatible and may be limited by depth of light penetration.

Non-invasive imaging using a pIX-MT fusion
MT is a ubiquitous, low molecular weight, metal-binding protein that participates in heavy metal metabolism and detoxification. Mammalian forms of MT bind seven metal ions in tetrahedral metal-thiolate clusters, including a commonly used medical isotope of technetium ( 99m Tc) useful for radioimaging by single photon emission computed tomography (SPECT) [22]. Several studies have shown that MT can be genetically engineered or conjugated with targeting proteins such as monoclonal antibodies [23,24,25] and streptavidin [26], and that 99m Tc can be bound in vitro to the complexes using a simple transchelation reaction.
In our current study, we sought to incorporate MT within pIX to determine if a fusion of this type could retain functionality in this context. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional MT as a component of their capsid surface. The alternative display of MT on the capsid may offer advantages with respect to direct functional applications of this gene product. Our innovative use of a structural fusion protein incorporating MT provides the non-invasive imaging advantages for detecting physical biodistribution and spread of Ad vectors after administration that is not possible employing a reporter gene. Further, the ability to noninvasively observe Ad function on a whole-body level, allows the possibility of detecting virus dissemination outside the tumor site(s) for monitoring clinical safety.

Cell culture
Human embryonic kidney epithelial (HEK293) cells were obtained from and cultured in the medium recommended by the American Type Culture Collection (Manassas, VA). The HEK293 cell line was grown in high glucose Dulbecco's Modified Eagle Medium (DMEM; Invitrogen; Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals; Lawrenceville, GA), 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. The cells were incubated at 37uC and 5% CO 2 under humidified conditions.

Animals
Female C57BL6 mice at 4-6 weeks of age were obtained from Charles River Laboratories (Wilmington, MA). All animals received humane care based on guidelines set by the American Veterinary Association. The experimental protocols involving live animals were reviewed and approved by the Institutional Animal Care and Use Committee of LSU Health Sciences Center at Shreveport (Protocol #P-08-017). For the SPECT imaging studies, mice were also pretreated with warfarin (5 mg/kg) dissolved in peanut oil and administered by subcutaneous injection at 3 days and 1 day prior to the Ad vector injections.

Recombinant Adenovirus Construction
A pIX-MT intermediate sequence in pUC57 was constructed by synthesizing a 932 bp DNA fragment (GenScript; Piscataway, NJ) corresponding to: amino acids 106-140 of the Ad serotype 5 (Ad5) pIX protein coding sequence, followed by a 15 amino acid linker (GGGGSGGGGSGGGGS), amino acids 1-61 of the human metallothionein 1A sequence, a 16 bp DNA linker sequence (TGAGCTAGCGACGTCA), and the 583 bp DNA sequence immediately upstream of the pIX gene (bp 4032-4614; Genbank accession AY339865). This synthetic DNA fragment was subcloned directly into the MfeI -BstXI sites of the AdenoVatorCMV5 shuttle vector (QBiogene; Carlsbad, CA). A turboGFP cDNA sequence (Axxora; San Diego, CA) was also subcloned into the CMV5 expression polylinker site of pAdenoVatorCMV5 for use as a reporter gene. The resulting pAdenoVatorCMV5-tGFP-pIX-MT shuttle vector was used to construct an adenovirus by homologous recombination with pAdEasy1 (containing the E1 and E3 deleted Ad5 backbone) in E. coli using methods previously described [27]. The resultant recombinant plasmid was linearized with Pac I and transfected into HEK293 cells to generate the Ad-tGFP-pIX-MT virus. Construction of the Ad-IX-EGFP and Ad-CMV-EGFP vectors used as controls has been described previously [20].

Virus propagation and purification
Viruses were propagated in HEK293 cells, which do not express wild-type pIX. Viruses were purified by double CsCl ultracentrifugation and dialyzed against Dulbecco's phosphate buffered saline containing 10% glycerol. Final aliquots of virus were analyzed for viral particle (v.p.) titer using absorbance at 260 nm and a conversion factor of 1.1610 12 viral particles per absorbance unit (v.p./OD260). Multiplicity of infection (m.o.i.) was determined using an Adeno-X Rapid Titer Kit (Clontech; Mountain View, CA) and represents the number of infectious units (i.f.u.) of virus. Viruses were stored at 280uC until use. The Ad-tGFP-pIX-MT virus titer used was 1.2610 11 v.p./mL and 1.4610 9 i.f.u./mL.

Fluorescence Microscopy
Epifluorescence microscopy was performed using an inverted Eclipse TE300 microscope (Nikon Instruments, Melville, NY) equipped with a CoolSNAP fx monochrome CCD camera (Roper Scientific -Princeton Instruments; Trenton, NJ). Images were acquired with a 20X objective and processed with IPLab imaging software (Scanalytics, Inc.; Fairfax, VA).

Assessment of Replication of Virus
Increase in the copy number of the E4 gene was used as a surrogate to determine virus propagation. At 1, 2, 3, and 4 days post-infection of HEK293 cells, aliquots of culture media (300 mL) were removed and frozen at 280uC until use. Total DNA was purified using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA). E4 gene copy number was determined using specific E4 primers and TaqMan probes for real-time polymerase chain reaction (PCR) as previously described [28] with TaqMan fast reagents (Applied Biosystems, Framingham, MA) and measured against a standard curve made from known amounts of adenovirus genome.

Thermostability assay
Fifty thousand HEK293 cells were subcultured overnight into 24-well tissue culture plates with 1 ml DMEM medium containing 10% FBS. Prior to infection, the viruses were incubated for 0, 15, 45, 60, and 90 min time periods at 45uC, and subsequently, the heat-treated viruses were used to infect the HEK293 cells. At 24 h post infection, the plates were analyzed for relative fluorescence intensity of GFP using a Fluoroskan Ascent Microplate Fluorometer (Thermo Fisher Scientific; Waltham, MA).

Radiolabeling of virus and gel chromatography
For radiolabeling Ad virions, 99m Tc-pertechnetate was reduced in the presence of glucoheptonate using a commercially available kit (DRAXIS Specialty Pharmaceuticals Inc.; Kirkland, Quebec, Canada) for 15 min at room temperature and added directly to aliquots of Ad at a ratio of 10:1. Incubation with Ads proceeded for 60 min at 37uC. Purification of free 99m Tc from labeled adenovirus virions was performed by gel filtration using Sephacryl S-200 HR (GE Healthcare; Piscataway, NJ). Sephacryl resin was loaded into a 20 mL disposable column (Bio-Rad) and pre equilibrated with phosphate buffered saline (PBS) containing 5% glycerol. One mL aliquots of radiolabeled virus were applied to the columns, and 10 drop fractions were collected using an automated fraction collector. The viral particle (v.p.) titer of each fraction was determined by measuring the absorbance at 260 nm in a spectrophotometer, using a conversion factor of 1.1610 12 v.p./ OD260 [29]. The radioactivity was measured with a dose calibrator (Capintec, Inc.; Ramsey, NJ).

Metal competition assay
Aliquots of the radiolabeled Ad-tGFP-pIX-MT were incubated in the absence or presence of non-radioactive competitor metal ions. After addition of competitor metals using 100X stock solutions, the aliquots were incubated at room temperature for 15 min. The adenovirusbound and free 99m Tc were separated by gel chromatography using micro spin columns (Bio-Rad; Hercules, CA), and quantified using a NaI (Tl) c-counter (PerkinElmer Inc.; Waltham, MA).

HPLC analysis of 99m Tc radiolabel stability
Aliquots of the radiolabeled Ad-tGFP-pIX-MT were incubated for 30 min at 27uC in PBS containing 5% glycerol, with the absence presence of 50% mouse serum (Sigma-Aldrich; St. Louis, MO). Aliquot were also incubated for 30 min with 50% mouse serum at increasing temperatures (37,42,45,50, and 55uC). Afterwards, adenovirus particles were analyzed by HPLC as previously described [30] on a Dionex DX500 Chromatography System (Dionex Corp; Sunnyvale, CA) using a strong anion exchange Bio-Monolith QA HPLC column (Agilent technologies; Santa Clara, CA). The HPLC system was equipped with in-line UV (260 nM) and Nal(Tl) gamma scintillation detectors interfaced to a multichannel analyzer. Software analysis was performed for the UV detector using Chromeleon 6.80 (Dionex) and was performed for the gamma scintillation detector using PeakSimple 3.85 (SRI Instruments, Torrance, CA).

SPECT/CT scanning of Ad-infected mice
Two mice were injected via the tail vein with approximately 1 mCi of the radiolabeled Ad-tGFP-pIX-MT, and image acquisition was started at 60 min afterwards. Similarly, one mouse was injected with approximately 1 mCi of 99m Tc-glucoheptonate and one mouse was injected with approximately 1 mCi of 99m Tcpertechnetate. The animals were anesthetized using isoflurane and fixed in a prone position on the bed and center of rotation relative to the gantry of a dedicated small animal trimodality PET/SPECT/ CT system (Gamma Medica-Ideas, Northridge, CA). After 60 min, 3D SPECT imaging was performed in a step-and-shoot manner using the following acquisition parameters: 64 projections, 30 seconds/projection (35-minute image acquisition), with a 140 keV photopeak 610% window. SPECT 3D reconstruction was carried out using Amira software (Gamma Medica-Ideas). Immediately after SPECT imaging was performed, CT images were acquired using the same coordinates as SPECT with 256 projections and 102461024 projection matrix size and a voltage of 60 kV; reconstructions were performed using filtered back-projections. Reconstructed data was saved as DICOM files and were imported for further analysis to AMIDE (version 0.9.2; Andy Loening) freeware. Volumes of interest (VOIs) from each image were manually defined and image analyses were performed; the AMIDE ROI statistics tool was used to obtain the mean voxel values for the VOI. The percent activity for each VOI was determined as (VOI mean voxel value6VOI voxel number)/(total image mean voxel value6total image voxel number)610.

Statistical analysis
All data are expressed as means 6 SE. In vitro experiments were performed in triplicate. Statistical analysis was carried out using Student's t-test, or for multiple comparisons, two-way ANOVA using GraphPad Prism version 5.0 software. Statistical significance was set at P,0.05.

Constructing an adenovirus vector containing a pIX-MT fusion
We initiated this work to construct an adenovirus by using an E. coli recombination system with a shuttle vector and the adenovirus backbone plasmid. As a labeling system, we incorporated the MT coding sequence as a C-terminus fusion to the pIX protein. We used the E. coli recombination system to generate a recombinant replication deficient Ad containing an E1A expression cassette with tGFP sequence under control of the CMV promoter. The resulting recombination inserted shuttle vector sequences contain-ing the expression cassette and pIX-MT fusion gene into the pAdEasy1 adenovirus backbone replacing the E1A and E1B sequences. (Fig. 1A). The identity of the recombinant genome was validated by restriction enzyme digestion and PCR analysis and was confirmed by DNA sequencing (data not shown). The resulting pAd5.CMV5-tGFP-pIX-MT plasmid vector was used to rescue an Ad virion by linearization with Pac I and transfection into HEK293 cells. After transfection, positive Ad plaques were detected by fluorescence microscopy (Fig. 1B). Propagated Ad-tGFP-pIX-MT virus was purified by double CsCl ultracentrifugation. After virus purification, a recombinant pIX-MT fusion protein was clearly detected as a 21 kD band by western blot analysis of the purified virus using an antimetallothionein antibody (Fig. 1C). This band corresponds to the expected molecular weight of the pIX-MT, and was absent in purified Ad-CMV-EGFP virus. As a control, both the Ad-tGFP-pIX-MT and the Ad-CMV-EGFP virus showed 60 kD bands by western blot analysis using an anti-Ad fiber protein. These results indicate that correct introduction of the MT protein into the viral capsid of an adenovirus by fusing it with the minor capsid protein IX allows the rescue of an infectious virion.

Analysis of viral DNA replication
To determine DNA replication properties of Ad-tGFP-pIX-MT, the following experiment was performed. Ad E4 copy numbers were analyzed after cells were infected with virus. In brief, 10 i.f.u./cell of Ad-tGFP-pIX-MT, Ad-CMV-EGFP, or Ad-IX-EGFP was used to infect the Human embryonic kidney epithelial (HEK293) cell line. Aliquots of medium were collected on 1, 2, 3, and 4 days post-infection. Total DNA was extracted from the medium (which was used to incubate infected cells) and analyzed for Ad5 viral E4 DNA copy number (Fig, 2). Medium from uninfected cells was also obtained at each time point to serve as a base line for viral replication. At 1 day post-infection, the E4 copy number for Ad tGFP-pIX-MT and Ad-IX-EGFP were observed to be approximately 52.6 and 7.5 copies per 100 mL aliquot of medium, while the E4 copy number value for Ad-CMV-EGFP was 12. By 4 days post-infection, Ad5 E4 copy number for each of the adenoviral constructs was dramatically increased to approximately 41,200, 75,900, and 81,700 copies per 100 mL aliquots of media for Ad-tGFP-pIX-MT, Ad-IX-EGFP, and Ad-CMV-EGFP, respectively. Taken together, these data indicate that the adenovirus constructs with modified pIX genes (tGFP-pIX-MT and Ad-IX-EGFP) showed similar replication patterns in 293 cells, and this replication is comparable to an adenovirus construct with a wild-type pIX gene (Ad-CMV-EGFP).

Thermostability of Ad-tGFP-pIX-MT virions
Modification of pIX may destabilize the capsid structure; this possibility was assessed by comparing the thermostability of Ad-tGFP-pIX-MT to a control Ad-CMV-EGFP virus containing the wild-type pIX and to an Ad-IX-EGFP virus incorporating the enhanced GFP (EGFP) protein as a fusion to pIX. Virus samples were incubated at 45uC for various times and then quantified in terms of infectious titer (Fig. 3). Beginning at 15 min of incubation, the infectious titer of all viruses decreased in an exponential fashion; the infectious titer for Ad-CMV-EGFP and Ad-tGFP-pIX-MT were 70.8% and 40.4% of initial titers, respectively, while the infectious titer for Ad-IX-EGFP was only 4.8% if the initial titer. Viral titers continued to decrease between 15 and 90 min of incubation, with the time for 50% remaining titer of Ad- CMV-EGFP at 26.7 min, Ad-tGFP-pIX-MT at 11.9 min, and Ad-IX-EGFP at 3.2 min. These results indicate that the MT addition to pIX did negatively affect the thermostability of Ad-tGFP-pIX-MT following exposure of the virus to high temperature compared to a virus containing wild-type pIX. However, when comparing the thermostability curves, this effect was significantly less than that of incorporating a pIX-EGFP fusion (two-way ANOVA, P,0.001).

Radiolabeling of Ad-tGFP-pIX-MT with 99m Tc
We tested whether Ad-tGFP-pIX-MT could be radiolabeled by 99m Tc binding in vitro. In this representative experiment, 99m Tcpertechnetate was reduced in the presence of glucoheptonate for 15 min at room temperature and added directly to the adenovirus solution. Incubation with Ad proceeded for 60 min at 37uC. Purification of free 99m Tc from labeled adenovirus virions was performed by gel filtration using a Sephacryl S-200 column. As shown in Fig. 4, the specific activity of the peak at fraction 30 corresponding to the highest adenovirus infectious activity was approximately 0.13 mBq/v.p. for the Ad-tGFP-pIX-MT. Importantly, an adenovirus construct Ad-CMV-EGFP containing the wild-type pIX showed lower specific activity at fraction 30 (0.006 mBq/v.p.), possibly from nonspecific 99m Tc binding to free sulfhydryl groups [31] on the Ad capsid surface. These results demonstrate the feasibility of 99m Tc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction.

Metal competition assay
The heavy metals that can be bound by MT include Ag, Au, Bi, Cd, Co, Cu, Fe, Hg, Ni, Pb, Sn, Tc, and Zn, [32], as well as Re [33]. To determine the specificity of 99m Tc binding, we performed a metal competition assay using increasing concentrations of CdCl 2 , CoCl 2 , CuCl 2 , or ZnCl 2 . As shown in Fig. 5, the bound radioactivity was competed with increasing concentrations of metal, with half maximal concentrations required for displacement of 99m Tc. Importantly, these results correlate with the relative order of in vitro binding affinities to MT determined to be Cu . Cd . Zn . Co [34]. In addition, Cu was capable of displacing 99m Tc from the radiolabeled Ad-tGFP-pIX-MT between 0.1 and 1 mM. However, this concentration of Cu required for displacement is much higher than physiological levels of Cu determined in serum of normal mice, at approximately 400 ng/mL (0.006 mM) [35].

Stability of 99m Tc radiolabel on the Ad-tGFP-pIX-MT virions
Serum stability of the radiolabel on Ad-tGFP-pIX-MT was investigated by incubating virions for 30 min in 50% normal mouse serum (Fig. 6). In the absence of serum, we could detect no loss of radioactivity at room temperature, 27uC or 37uC after incubation for 30 min (data no shown). However, compared with radiolabeled virus incubated in the absence of serum, approximately 80% of the radioactivity was still bound in 50% serum after incubation at 27,   37, and 42uC. This loss of approximately 20% radioactivity at these temperatures may represent loss from low affinity binding sites to 99m Tc. When the virus was incubated at higher temperatures, the remaining bound radioactivity was decreased to 46% radioactivity at 45uC, 40% radioactivity at 50uC, and 19% radioactivity at 55uC. This result is similar to the results in Fig. 3 showing inactivation of virus infectivity at 45uC, as well as previous data demonstrating virus inactivation at temperatures between 48 and 54uC [36].

SPECT analysis of adenovirus administration in vivo
After radiolabeling the Ad-tGFP-pIX-MT, we determined whether SPECT imaging could be used to monitor Ad biodistribution and uptake in vivo. In the experiment shown in Fig. 7, a female C57BL6 mouse was injected intravenously with 99m Tc-labeled Ad-tGFP-pIX-MT. One hour after administration, the animal was imaged; clear localization of activity to the liver (4568%) was seen (Fig. 7C), and bladder activity (3263%) was also detected. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. As a control, in vivo imaging of a mouse in Fig. 7A injected with 99m Tc-pertechnetate alone showed no liver signal, with localization to stomach (1463%) and bladder (1662%). Likewise, in vivo imaging of a control mouse in Fig. 7B injected with 99m Tc-glucoheptonate alone also showed no liver activity, with activity localization to kidneys (2268%).
Liver tropism of administered Ad5 vectors results in rapid sequestration. Key components of this process have recently been elucidated, involving Ad capsid interaction with multiple vitamin K-dependent coagulation factors [37]. Specifically, coagulation factor 6 (FX) binds to the capsid hexon protein and mediates the majority of the Ad5 liver tropism, presumably via heparin sulfate proteoglycan-mediated pathways [38,39,40]. Pretreatment with the drug warfarin, which inhibits vitamin K-dependent coagulation factor production, can deplete circulating FX levels and decrease the liver tropism of i.v. administered Ad5. As shown in Fig.7D, warfarin pretreated mice exhibited a decrease in liver activity (1468%) compared with untreated mice (Fig. 7C), as well as a diffuse peritoneal imaging pattern. Together, these data illustrate that the pIX-MT fusion retains its metal binding capacity at the pIX locale and can function in an in vivo context, thus making non-invasive imaging analysis feasible.

Discussion
We have shown herein that our novel pIX-MT labeling strategy for adenovirus yields rescuable virions. The pIX protein is a structural component of the Ad that stabilizes hexon-hexon interactions; there are 240 pIX molecules per virion. With a capacity of 7 metal ions bound per pIX-MT fusion protein, as many as 1680 metal ions can bind per virion. The highly conserved metallothionein structure potentially offers the additional advantage of lower immunogenicity, by ''shielding'' the Ad from preexisting humoral immunity [41]. Direct western blot analysis of purified Ad-tGFP-pIX-MT produced a protein band that corresponded to a pIX-MT fusion. This data indicates that the pIX-MT fusion was successfully incorporated into the Ad pIX locale. Most importantly, Ad-tGFP-pIX-MT demonstrated specific 99m Tc binding in vitro when incubated with 99m Tc-glucoheptonate. This study also demonstrated our ability to create a virus that encompasses a protein motif useful for SPECT imaging. The majority of previous imaging strategies have focused on incorporating reporter gene using a single imaging modality whereas; our unique virus incorporates the potential for multiple imaging modalities.
Knowledge of biodistribution is crucial in assessing the efficacy of Ad mediated therapies. Clinical trials completed so far have had to rely on conventional histology of biopsy specimens and analysis of body fluids to detect virus; the majority of biodistribution studies have involved PCR-based techniques. For example, after intravenous administration at the highest dose of a replication deficient Ad5CMV-p53, the presence of a unique 89 bp amplicon specific to Ad5CMV-p53 DNA was detectable by Q-PCR in the plasma of 4 of 5 patients at day 14 and 2 of 3 assessable patients at day 28 [42]. In addition, the presence of Ad5CMV-p53 DNA was detected within biopsies of tumor metastasis distant from the intravenous administration site in 6 of 7 assessable patients. However, these results only provide data on the presence of a nucleotide sequence and not on the presence of viral vector  particles. In addition, these results highlight the shortcomings of current vector detection methods: the need to acquire multiple biopsies using an invasive procedure that is prone to sampling error and is concomitantly impractical for repeated monitoring of the entire tumor.
Radiotracer imaging technologies that can measure the distribution of radiolabeled tracers in vivo are now widely available and have a wide range of research and clinical applications. Two classes of radiotracer imaging systems exist: those designed to imaging SPECT radionuclides such as 99m Tc and those designed to image PET radionuclides such as 64 Cu. Thus, besides SPECT imaging using 99m Tc, the pIX-MT could also be used for non-invasive PET imaging, using 64 Cu binding. SPECT and PET techniques are able to image as low as 10 -10 to 10 -12 M of radiolabeled moieties. Each imaging modality has advantages and disadvantages, and thus has specific applications. In general, PET has higher spatial resolution and sensitivity, and is easier to quantify than SPECT. However, SPECT radiotracers are cheaper and much more widely available. Likewise, next-generation SPECT systems have dramatically increased sensitivity. In addition to evaluation of biodistribution by initially in vitro 99m Tc radiolabeled replication deficient Ads, we expect virions containing the pIXmetallothionein fusion proteins can also be potentially labeled using 188 Re [33]. This method could offer the advantage of dual-functional SPECT imaging and radionuclide therapy [43,44,45].
In total, as hypothesized these data clearly establish the functionality of pIX-MT when incorporated into the adenovirus capsid at the pIX locale. The ability to view real-time molecular biodistribution events in their physiological milieu represents a significant tool to study adenovirus biology in vivo. Although in vitro studies can serve as a simplified controlled model to study Ad behavior in tumor cells, their simplicity strips them of the complexities of a three-dimensional tumor environment that has profound effects on Ad performance. Ultimately, the highest yield of valuable information about Ads will come from in vivo preclinical studies and application of Ad vectors in patients.
It has become clear that cell-specific transductional retargeting of Ad vectors is of fundamental importance with respect to deriving their full benefit in the context of the gene therapies [46,47,48]. These transductional retargeting strategies, whereby Ad viruses are designed to selectively infect target cells, have the potential for reducing deleterious side effects and increasing the therapeutic index of these agents. The pIX-MT labeling system demonstrated herein, offers the potential for noninvasive dynamic imaging of Ad biodistribution that can be easily used by existing nuclear medicine imaging modalities. Importantly, this tool can be used for preclinical development of transductional retargeted Ads, and it would be practical in the clinical setting to monitor the therapeutic application of these agents.