Noninvasive Imaging of Lentiviral-Mediated Reporter Gene Expression in Living Mice

Transcription

1 Noninvasive Imaging of Lentiviral-Mediated Reporter Gene Expression in Living Mice Abhijit De, 1,2 Xiaoman Zhou Lewis, 2 and Sanjiv Sam Gambhir 1,2,3,4, * 1 The Crump Institute for Molecular Imaging, 2 Department of Molecular & Medical Pharmacology, 3 UCLA Jonsson Comprehensive Cancer Center, and 4 Department of Biomathematics, University of California at Los Angeles School of Medicine, Los Angeles, California , USA *To whom correspondence and reprint requests should be addressed at the Crump Institute for Molecular Imaging, Department of Molecular & Medical Pharmacology, UCLA School of Medicine, B3-399A BRI, 700 Westwood Plaza, Los Angeles, CA Fax: (310) sgambhir@mednet.ucla.edu. Lentiviral-mediated gene delivery holds significant promise for sustained gene expression within living systems. Vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 1-based lentiviral vectors can be used to introduce transgenes in a broad spectrum of dividing as well as nondividing cells. In the current study, we construct a lentiviral vector carrying two reporter genes separated by an internal ribosomal entry site and utilize that virus in delivering both genes into neuroblastoma cells in cell culture and into cells implanted in living mice. We utilize two reporter genes, a mutant herpes simplex virus type 1 (HSV1) sr39tk as a reporter gene compatible with positron emission tomography (PET) and a bioluminescent optical reporter gene, firefly luciferase (Fluc), to image expression in living mice by an optical chargecoupled device (CCD) camera. By using this lentivirus, neuroblastoma (N2a) cells are stably transfected and a high correlation (R ) between expressions of the two reporter genes in cell culture is established. Imaging of both reporter genes using micropet and optical CCD camera in living mice is feasible, with the optical approach being more sensitive, and a high correlation (R ) between gene expressions is again observed in lentiviral-infected N2a tumor xenografts. Indirect imaging of HSV1-sr39tk suicide gene therapy utilizing Fluc is also feasible and can be detected with increased sensitivity by using the optical CCD. These preliminary results validate the use of lentiviral vectors carrying reporter genes for multimodality imaging of gene expression and should have many applications, including imaging of xenografts, metastasis, and cell trafficking as well as noninvasive monitoring of lentiviral-mediated gene delivery and expression. Key Words: lentivirus, reporter gene, firefly luciferase, herpes simplex virus type-1, thymidine kinase, molecular imaging, micropet, tumor xenograft, suicide gene therapy INTRODUCTION Retroviral-based gene transfer provides effective means for the delivery, integration, and expression of transgenes in mammalian cells in culture as well as in vivo [1]. In contrast to oncogenic retroviruses, lentiviruses have the capability to deliver genetic materials into cells irrespective of their state of division [2]. Further, by introducing a relatively strong internal promoter such as cytomegalovirus (CMV), lentiviruses can be made highly useful [3,4]. In some cases, efforts have been made to introduce a tissuespecific promoter to get tissue-specific expression of the gene of interest [5,6]. In other cases, attempts have been made to include genetic regulators within the vector system to achieve more control of target gene expression [7]. With all of these different modifications, lentiviruses are likely to play a greater role in gene therapy applications. As safe and high-titer lentiviral vectors continue to be developed, efficient delivery of transgenes into target cells, irrespective of their dividing state, will likely become more frequent, and the application of these vectors in different gene therapy trials including cancer trials should grow [8 11]. Imaging gene expression entails determining the location(s) of those cells expressing a particular gene of interest as well as monitoring the magnitude and time variation of gene expression. In a therapeutic setting involving animal models, the expression of more than one gene may be necessary because the therapeutic protein may consist of multiple subunits or several proteins may be required. Further, in addition to the functional transgene the expression of a reporter gene from a multigene vector /03 $

2 doi: /s (03) can aid in the experimental assessment of gene transfer efficiency and in the optimization of the gene transfer process. For a majority of therapeutic genes, direct monitoring of their expression in living subjects is difficult and therefore an indispensable tool to check the expression of the delivered gene is by imaging expression of a correlated reporter gene indirectly [12,13]. There are several ways to link the activity of two genes, including a fusion gene, a bicistronic approach with an internal ribosomal entry site (IRES), as well as several other approaches [14]. The ability to induce expression of multiple genes from a lentiviral vector may extend important benefits in targeting various diseases. To develop multigene lentiviral vectors, several groups have tested strategies based on inclusion of an IRES [12] or insertion of internal promoters [15] or splicing-based lentiviral vectors [16]. With the IRES-based approach for linking two or more genes, a translational cis-acting element, the encephalomyocarditis virus (EMCV) IRES sequence, is most often used to allow translation of two open reading frames from a single mrna. A variety of imaging techniques based on reporter gene approaches using both positron emission tomography (PET) [17 20] and optical bioluminescence approaches [21,22] are potentially useful. These approaches utilize FIG. 1. Diagrammatic representation showing an IRES-based bicistronic lentivector used to link the activity of two reporter genes. The first gene (HSV1-sr39tk) is linked to the second gene (Fluc) by an EMCV IRES sequence. When this combination of genes is packaged within a lentivector for cell targeting, the virus delivers this DNA cassette to the host cell nucleus followed by a reverse transcription reaction. The two genes are integrated within the host genome irrespective of the cell division state and start producing messenger RNA as the host genome replicates. These mrna molecules are translated to proteins within the host cytoplasm. By using FHBG as a PET reporter probe one can quantitatively image the HSV1-sr39TK activity within living subjects. The same is also true for the Fluc gene, in that by administration of D-Luciferin, a substrate for FLUC, light production can be imaged. 682

3 reporter genes that encode reporter proteins which can be detected in a living animal through injection of specific substrates [reviewed in 14]. The power of these techniques is that they can noninvasively image the location(s), magnitude, and time variation of reporter gene expression in intact living subjects. Well-known methods harnessing the power of reporter gene technology, which are noninvasive, quantitative, and applicable to animals and human, are radioisotope-based techniques such as PET and single photon emission tomography (SPECT). Using PET and SPECT it is possible to image reporter gene expression in living subjects both repeatedly and noninvasively [23]. Reporter genes with optical signatures (e.g., fluorescence and bioluminescence) are rapidly growing as a low-cost alternative for real-time analysis of gene expression in small animal models. Among this category, bioluminescent reporter proteins, which can produce light by utilizing appropriate substrates, are gaining popularity. Recently, several technical advances in developing highly sensitive detection devices have led to the use of cooled charged-coupled device (CCD) cameras capable of imaging very low levels of visible light emitted from internal body organs of rodents in biological research [24]. A mouse can be placed in a light-tight dark box and projection images of CCD-imaged bioluminescence can be superimposed on photographic images of the mouse to image quantitatively and repetitively the bioluminescent signal from a given location. We have previously validated the reproducibility and quantitative capability of this approach [21]. The goals of this study were to: (1) develop a lentivirus carrying both PET and bioluminescence optical reporter genes driven by a robust CMV promoter, (2) demonstrate that the lentivirus is capable of leading to significant levels of expression of both reporter genes in various cell lines, (3) demonstrate correlated expression of both reporter genes in both cell culture and in living subjects, and (4) demonstrate the utility of the vector in noninvasively monitoring suicide gene therapy in living subjects. We used a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET reporter gene and firefly luciferase (Fluc) as a bioluminescent reporter gene. A bicistronic lentivirus vector (CS-CMVsr39tk-I-Fluc) carrying HSV1-sr39tk in the first cistron and Fluc in the second cistron separated by an EMCV IRES was constructed (Fig. 1). We achieved the goals of this study using both cell culture and imaging studies performed with micropet and optical CCD imaging in living mice. RESULTS N2a Cells Infected with the CS-CMVsr39tk-I-Fluc Lentivirus Show Stable and Correlated Expression of both the HSV1-sr39tk and the Fluc Reporter Genes We constructed a bicistronic lentiviral vector with HSV1- sr39tk in the first cistron and Fluc in the second cistron FIG. 2. HSV1-sr39TK and FLUC enzyme activity in lentiviral (CS-CMVsr39tk-I- Fluc)-infected N2a cells. The TK activity is expressed as percentage conversion of 8-3 H-PCV to its phosphorylated form per microgram protein per minute. The FLUC activity is expressed as RLU per microgram protein per second. Error bars represent SEM for triplicate measurements. (A) Relative HSV1-sr39TK enzyme activity in lentiviral CS-CMVsr39tk-I-Fluc-transduced N2a cells compared to parental N2a cells as a negative control. (B) Relative FLUC enzyme activity in lentiviral CS-CMVsr39tk-I-Fluc-transduced N2a cells compared with parental N2a cells as a negative control. (C) Correlation between HSV1-sr39TK (y axis) and FLUC (x axis) enzyme activity in lentiviral CS-CMVsr39tk-I-Fluctransduced N2a cells as determined by assaying different numbers of infected cells. The correlation coefficient is R separated by an EMCV IRES (Fig. 1). To demonstrate that the constructed lentivirus was capable of infecting cells, leading to significant expression of both reporter genes, 683

4 doi: /s (03) FIG. 3. Optical CCD and micropet imaging of a mouse implanted with N2a cells stably expressing HSV1-sr39tk and Fluc reporter genes. (A) Optical CCD image for Fluc expression on day 9, micropet FDG scan to check tumor viability on day 10, and micropet FHBG scan on day 11 for HSV1-sr39tk gene expression. Tumors were grown by injecting N2a cells transduced in culture by lentivirus (CS-CMVsr39tk-I-Fluc) (R) and control tumors an equal number of parental N2a cells (L). B, brain; GI, gastrointestinal tract; L, left tumor; R, right tumor. (B) Graph showing correlation of HSV1-sr39tk and Fluc gene expression in tumor xenografts. The correlation was established based on repeated scanning of four mice on days 8, 10, and 12 with both the micropet and the optical CCD camera. Individual mean FHBG %ID/g values versus maximum photons/s/cm 2 /sr values calculated from the regions of interest (ROI) on the respective scanned images of the tumor-bearing mice are plotted. Each of the 12 data points represents ROI values from bioluminescence (x axis) and micropet (y axis) images of the same mouse on the same day. The correlation coefficient is R we infected N2a cells with CS-CMVsr39tk-I-Fluc and repetitively assayed for expression of both reporter genes by utilizing cell extracts and FLUC (Fluc refers to the gene and FLUC to the enzyme) and HSV1-sr39TK (tk refers to the gene and TK to the enzyme) enzyme assays over a period of 4 months (see Table 1). TK assay of the virustransduced N2a cells showed approximately 2-fold higher activity than that of untransduced N2a cells (Fig. 2A). FLUC assay with the protein extracts from the same cells showed approximately 1000-fold higher signal than the control cells (Fig. 2B). Further, we also determined the correlation of the two reporter genes in the virus-infected N2a cells by assaying different numbers and batches of cells. We observed a high correlation (R ) between expression of HSV1-sr39tk and Fluc genes in the N2a cells (Fig. 2C). Similar results were seen in 293T, RG2, and C6 cell lines with stable and correlated expression (data not shown). Lentivirus (CS-CMVsr39tk-I-Fluc)-infected N2a cells stably expressing both reporter genes were used for subsequent studies. 684

5 FIG. 4. Optical CCD images of tumor xenografts showing Fluc gene expression over time. Tumors were implanted sc with lentiviral (CS-CMVsr39tk- I-Fluc)-infected N2a cells on the right shoulder (R) and same number of parental N2a cells on the left shoulder (L) on day 0. The mouse was repeatedly scanned with the CCD on days 0, 4, 7, 11, and 14 as the tumors grew. The highest expression was observed on day 11, after which internal necrosis within the tumors likely resulted in lowered intensity of FLUC signal on day 14. All images shown are the visible light image superimposed on the optical CCD bioluminescence images. FIG. 5. Monitoring of HSV1-sr39tk suicide gene therapy indirectly by Fluc reporter gene expression in a tumor xenograft model. In a set of six mice, control tumors (L) with N2a cells infected with lentivirus CS-CMVFluc and experimental tumors (R) with N2a cells infected with lentivirus CS-CMVsr39tk-I-Fluc were implanted with cells. Tumors were allowed to grow for 6 days before GCV treatment was started (day 0). GCV was administered daily at a dose of 25 mg/mouse/day. Follow-up scans with D-Luciferin on days 3 and 7 of GCV treatment showed significant decrease in the FLUC signal at the site of the experimental tumor (R), whereas the control tumor (L) continued to grow with increasing light signal. 685

6 doi: /s (03) TABLE 1: Data showing sr39tk and FLUC enzyme activity over a period of 4 months in lentiviral (CS-CMVV-sr39tk-I- Fluc)-infected N2a cells Month sr39tk activity a FLUC activity b Means SEM. a % Activity/ g protein/min. b RLU/ g protein/s. N2a Cells Stably Expressing HSV1-sr39tk/Fluc Implanted into Living Mice Show Significant and Correlated Signals with both the MicroPET and the Optical CCD Cameras To check for reporter gene expression in living subjects, we performed imaging studies in four living mice by subcutaneously (sc) injecting control N2a cells at one site and virus (CS-CMVsr39tk-I-Fluc)-infected stably expressing N2a cells at the second site. We allowed the tumors to grow until they reached a palpable size of 5 6 mmin diameter. On day 9, scanning for Fluc expression with the CCD camera showed average maximum (max) value of (mean SEM) photons/s/cm 2 /steridian (sr), which is significantly higher (P 0.005) compared to the control tumor value of photons/s/ cm 2 /sr. For the same set of mice, micropet imaging with [2-18 F]fluoro-2-deoxyglucose (FDG) on day 10 verified that both experimental and control tumors have similar FDG uptake levels, and %ID/g (% injected dose per gram of tissue), respectively, supporting similar cell viability of both tumors. MicroPET scanning with 9-([4-18 F]fluoro-3-hydroxymethylbutyl)guanine (FHBG) showed an average of %ID/g in the experimental tumor, statistically significantly (P 0.05) above the control tumor, which has an average value of %ID/g (Fig. 3A). MicroPET imaging at earlier time points, when tumors size was 5 mm in diameter, did not show any significant signal from experimental tumors compared to background and control tumors. Further, repeated scanning of living mice with both the CCD camera and FHBG micropet on the same day on days 8, 10, and 12 of tumor growth showed a high correlation (R ) between HSV1-sr39tk and Fluc expression (Fig. 3B). These results demonstrate that the level of expression of both reporter genes is sufficiently high to be detected in living subjects and, as seen in cell culture, the expression of the two reporter genes remains highly correlated. Serial CCD Imaging of Living Mice Implanted with Control and Stably Expressing HSV1-sr39tk/Fluc N2a Cells Demonstrates Significant and Increasing Signal as Tumors Grow We implanted another set of four mice with experimental (infected with CS-CMVsr39tk-I-Fluc in cell culture) and control N2a cells on the right and left shoulder, respectively. The first CCD scanning performed 2 h after cell implantation showed average max signal of photons/s/cm 2 /sr in the right shoulder, which is significantly higher (P 0.005) than the left shoulder value of (Fig. 4). On day 4, the Fluc signal intensity was further reduced, with a max value of photons/s/cm 2 /sr, which is likely consistent with the death of some of the implanted cells at the tumor site. Repetitive CCD imaging on days 7 and 11 showed an increase in signal with values increasing to as high as and photons/s/cm 2 /sr, respectively. Further scanning on day 14 showed a decreased mean maximum value of photons/s/cm 2 /sr. These results also reflect that tumors grown with N2a cells remain viable and maintain reporter gene expression for a period of 2 weeks. Also, the optical CCD camera is sensitive enough to detect bioluminescence emitted by 500,000 or even lower numbers (which may be the case on day 4) of stably expressing N2a cells located near the surface of a nude mouse. Ganciclovir-Mediated Destruction of N2a Tumor Cells Implanted in Mice and Stably Expressing HSV1- sr39tk and Fluc Genes Can Be Imaged by Monitoring FLUC Signal in the CCD Camera We implanted six mice with CS-CMV-Fluc-infected N2a cells (as control) and CS-CMVsr39tk-I-Fluc-infected N2a cells on the left and right shoulders, respectively. After 6 days of tumor growth, before the start of ganciclovir (GCV) treatment (day 0), optical CCD imaging showed an average max value of photons/s/cm 2 /sr in the experimental tumors. After 3 and 7 days of GCV treatment, the FLUC signal decreased to and photons/s/cm 2 /sr, respectively, in the experimental tumors (Fig. 5). The control N2a tumors with only Fluc expression continued to grow over the treatment days with max signals of , , and photons/s/cm 2 /sr on days 0, 3, and 7, respectively. Lower FLUC signal in the control tumors is likely due to a lower number of transduced N2a cells or lower levels of expression per cell. This statement can be supported by the observed low 16.4 relative light units (RLU)/ g protein/s value in the assayed control cell lysate over the experimental cell lysate value of RLU/ g protein/s at the time of cell implantation. The slight decrease in the signal on day 7 (which is day 14 of tumor growth in total) is likely due to the onset of internal tumor necrosis within the relatively large xenografts. Further, micropet imaging with FDG verified viability of the tumors, which had not responded to GCV, as well as the growth of control tumors not expressing HSV1-sr39tk (data not shown). FDG images were useful to detect the remaining mass of the experimental tumor after 7 days of GCV treatment. 686

7 Direct Injection of Lentivirus into N2a Tumor Xenograft in Living Mice Demonstrates Detectable Optical CCD and FHBG MicroPET Signals That Are Significantly Less Than Those Observed in Cells Infected in Cell Culture and Then Implanted into Mice We implanted four mice sc with N2a cells in both shoulder regions, and when tumors reached 5 mm diameter, we injected the right tumor on each mouse with the CS-CMVsr39tk-I-Fluc lentivirus of titer 138 RLU/ g protein/s/ml. We then serially imaged the mice with D-Luciferin by optical CCD camera on days 2, 4, and 7 and with FHBG by micropet on days 1 and 8. We implanted four additional mice with N2a cells stably expressing (by the same lentivirus infection) both reporter genes on the right shoulder and with control N2a cells on the left shoulder. These mice were scanned with the optical CCD camera on days 0 (2 h after cell implantation), 4, and 7 and FHBG micropet on days 2 and 8. Scanning with optical CCD camera of the mice that received direct virus injection into the tumors showed average max values at the injected tumor site on days 2, 4, and 7 of , , and photons/s/cm 2 /sr, respectively, which is significantly higher than the control tumor value of photons/s/cm 2 /sr (Fig. 6, left). Simultaneous CCD scans of mice in which tumors were implanted with the same number of ex vivo virus-infected N2a cells showed average max values of , , and photons/s/cm 2 /sr on days 0, 4, and 7, respectively (Fig. 6, right). FHBG micropet scanning of the mice with direct virus injection at the tumor site on days 1 and 8 showed %ID/g FHBG of and , respectively (Fig. 7), whereas mice implanted with virus-infected cells showed FHBG %ID/g of only on day 8. FHBG micropet on day 2 after cell implantation could not detect any significant signal at the experimental tumor site, as the tumor size at that point was below the scanner s spatial resolution limit. No significant FLUC or HSV1-sr39TK signal was observed elsewhere within the mouse. These results reflect that if vesicular stomatitis virus G protein (VSVG)-pseudotyped virus is introduced at an early stage of tumor growth, it can deliver the reporter genes successfully into the target cells but with lesser efficiency ( 10-fold lower) than using cells infected in cell culture and then implanted into mice. Further, by increasing the virus titer or injected volume one can achieve a better signal (data not shown). DISCUSSION We developed a lentiviral vector in which both a PET reporter gene (HSV1-sr39tk) and a bioluminescent reporter gene (Fluc) are linked in a bicistronic vector using an EMCV IRES. We utilized the lentivirus to demonstrate stable and correlated expression of these two reporter genes in cell culture utilizing several cell lines. Next, we demonstrated the ability to image with both optical CCD and micropet the expression of cells stably transfected by lentiviral infection in cell culture as well as the ability to use the lentivirus to stably transfect cells growing as a tumor in living mice. We also demonstrated the ability to monitor the response of HSV1-sr39tk/GCV suicide gene therapy with Fluc optical CCD imaging. The strategy of linking the expression of two different genes with an IRES has been previously used successfully in the construction of adenoviral [26], oncoretroviral [27], and lentiviral [12,16] vectors. This is, however, the first study that we know of to demonstrate the utility of a bicistronic lentiviral vector specifically used for multimodality imaging, including monitoring suicide gene therapy. The N2a cells infected in cell culture with the lentivirus reflect a heterogeneous population of cells, some of which likely express more or less of both the reporter genes. We attempted to sort the cell populations using a bioluminescence-activated cell sorting technique [28], but due to relatively low levels of light production (in the presence of D-Luciferin), compared to fluorescence-based cell sorting [29], we were unable to sort the cells to achieve a more homogeneous population. In future studies it will be helpful to develop lentiviral vectors that also include a fluorescent reporter gene, e.g., green fluorescent protein (GFP) or red fluorescent protein, to obtain a cell population with known transduction efficiency. This might also allow imaging using fluorescence optical techniques adapted for imaging of living subjects [30]. Although a high correlation in HSV1-sr39tk and Fluc expression is observed with the heterogeneous population of N2a cells, it will be important in future studies to show correlation across several clonal populations once such populations can be selected using a fluorescent reporter. Both micropet and optical CCD imaging techniques used in our study demonstrate the ability to image the lentiviral-mediated stably transfected N2a cells in living mice using xenograft models. The levels of reporter gene expression are clearly sufficient as the signals detectable are well above control N2a cells and other tissues. The FLUC signal is clearly much higher than HSV1-sr39tk and far fewer numbers of cells can be detected using the optical CCD approach compared to the micropet approach at the depth of location studied. The HSV1-sr39tk reporter gene was placed in the proximal cistron because of previously reported attenuation of expression of the gene placed in the distal cistron [13], and the more sensitive reporter gene (Fluc) was therefore placed in the distal position. Further, the overall sensitivities of the two systems are dependent on several factors. In the optical bioluminescent system, the signal can be affected by tissue depth, concentration of reporter protein, dose of reporter probe, and regional hemoglobin concentration (which absorbs emitted bioluminescence). In the PET-based approach, the [ 18 F]FHBG signal is less dependent on tissue depth and is not altered by regional hemoglobin levels. 687

8 doi: /s (03) FIG. 6. Optical CCD imaging of Fluc gene expression over 7 days in a living mouse carrying xenografts. On the left, tumors were implanted sc with parental N2a cells on both shoulders and allowed to grow to a palpable size of 5 mm diameter before lentivirus (CS-CMVsr39tk-I-Fluc) was injected directly into the right tumor (R) only once. The animals were scanned for Fluc expression on days 2, 4, and 7 after virus injection. On the right, tumors were implanted sc with parental N2a cells on the left shoulder (L) and with lentiviral (CS-CMVsr39tk-I-Fluc)-infected N2a cells on the right shoulder (R). These animals were scanned on days 0 (2 h after cell implantation), 4, and 7. FIG. 7. FHBG micropet images of transverse sections of a mouse on days 1 and 8 with HSV1-sr39tk gene expression in a tumor xenograft. N2a parental cells were implanted sc on both shoulders and allowed to grow to a palpable size of 5 mm diameter before lentivirus (CS-CMVsr39tk-I-Fluc) was injected directly into the right tumor (R) only once. L indicates left tumor. Further, the distinct advantage in using the micropet is that it provides depth-independent, tomographic information and animal studies can be directly extended into human applications with clinical PET. This, however, is at a greater expense and requires the ability to produce the radiolabeled reporter probe. Also, the background signal in the optical approach is low, as the reporter probe does not become active until it comes into contact with the reporter protein. In contrast, a somewhat higher background signal is observed in PET imaging as the radioactive probe is detected while being cleared from various tissues, including the blood pool. The specificity of [ 18 F]F- HBG for HSV1-sr39tk is very high relative to mammalian tk s [23] and this is not a significant reason for the decreased sensitivity of the PET approach. Improved substrates with greater affinity for HSV1-sr39tk may help PET 688

9 sensitivity, but the mass of substrate that can be introduced will still hinder sensitivity. For optical imaging, mass levels (mg) of D-Luciferin are injected into the animal, leading to greater production of photons. In contrast, PET imaging involves the use of trace levels (ng) of [ 18 F]FHBG. Further studies involving the above mentioned issues would be helpful to compare the absolute sensitivities of the two modalities in a bicistronic approach. The limitations of the number of detectable cells at different depths for the optical CCD technique are under active investigation. The micropet has a spatial resolution of 8 mm 3 so that a significantly greater number of cells (tens of millions) compared to optical CCD imaging are likely needed for sufficient signal. For many applications it will therefore be useful to have both an optical and a PET reporter gene present in the lentiviral vector. Stable expression of both HSV1-sr39tk and Fluc reporter genes in the N2a cells over several months and the highly correlated expression of both reporter genes should be useful for many applications. The lentivirus should allow stable marking of many cell types, including stem cells, that can subsequently be imaged with both optical CCD and micropet in living subjects for cell trafficking studies. Another potential application of this lentivirus is to replace one of the two reporter genes with a therapeutic gene and then to image noninvasively the expression of the reporter gene to infer expression of the therapeutic gene indirectly. We tested this approach by utilizing HSV1-sr39tk as a suicide gene and Fluc as the reporter gene. Utilizing mice implanted with N2a cells stably expressing both HSV1-sr39tk and Fluc, we could image the mice prior to and during GCV prodrug therapy. As expected, in the experimental tumor the Fluc signal decreases due to destruction of tumor cells expressing HSV1- sr39tk as GCV treatment started, whereas Fluc signal in the control tumor keeps on increasing during the study period. The FDG micropet scanning before and after GCV treatment helps to detect the actual viable tumor mass on both sides [25]. As the control tumor continues to grow in the absence of sr39tk expression, the central necrotic area of tumor with less or no metabolic activity appears with a lot less central activity and much higher signal at the periphery. The continued tumor growth on the right shoulder can likely be explained by the fact that cells expressing low levels of HSV1-sr39tk survived, while the other cells did not survive, as we have previously reported [31]. These results illustrate that Fluc can be used to follow indirectly those cells expressing HSV1-sr39tk and therefore allow indirect monitoring of the suicide gene and has potential to be generalized to other genes. Future studies in which the lentivirus is injected directly into the tumor site followed by systemic GCV therapy may also be useful in characterizing the potential of utilizing this vector for suicide gene therapy. The data based on both CCD and FHBG micropet imaging suggest that the lentivirus can infect tumor cells grown in nude mice. However, the signal yield with both CCD and FHBG micropet imaging is significantly less in comparison to those mice in which tumors were grown with the same number of ex vivo virus-infected cells. From our previous experience with direct adenovirus injection at a tumor xenograft site [32], we have observed liver reporter gene expression likely due to escape of virus into the blood from the tumor site. In the present study, intratumoral injection of lentivirus may have also caused viral spread, but as VSVG-pseudotyped lentivirus is not restricted to predominantly liver infection (as in adenovirus), and as they likely exert much lower signal in comparison to transient expression with adenovirus infection, no significant signal was detected outside the tumor site. We are currently performing studies in which the bicistronic lentivirus is systemically administered via mouse tail vein and intraperitoneally to characterize noninvasively the biodistribution of gene delivery and expression as a function of route of administration. We are also developing lentivirus with tissue-specific promoters replacing CMV and lentivirus with two-step transcriptional amplification schemes [33,34] to test the utility of imaging tissue-specific reporter gene expression. The use of lentivirus for stable transfection of cells ex vivo as well as in living subjects will likely grow. The approaches developed should help to facilitate the imaging of tumor xenografts, metastases, trafficking of various cell populations, and optimization of preclinical and clinical gene therapy models. MATERIALS AND S HIV-1-based vector plasmid construct. The lentivector used in this study is a second-generation vector with the Tat element included in the packaging construct [4] (a gift from Dr. C. Sawyers, UCLA). A bicistronic cassette with two reporter genes, HSV1-sr39tk and Fluc, flanked by an EMCV IRES was built into the pcdna 3.1( ) mammalian expression vector. After checking the proper expression of each element by in vitro assays, we transferred the sr39tk-ires-fluc cassette to the lentivector (CS-CG) [4] under the internal CMV promoter by replacing the GFP element between the 5 NheI and the 3 XhoI restriction sites. The final vector was named CS-CMVsr39tk-I-Fluc. As a control for the Fluc gene, another lentiviral vector (CS-CMVFluc) carrying only Fluc was also constructed and used in this study. This virus vector was constructed by replacing GFP with a 1.7-kb PCR-derived Fluc fragment under the same (CS-CG) backbone. Cell lines. 293T (human embryonic kidney fibroblast) cells were used to develop virus particles. 293T cells were cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin (100 g/ml)/streptomycin (292 g/ml). After harvest, the virus was used to infect mouse neuroblastoma (Neuro-2a or N2a) cells (a gift from Dr. Vincent Mauro, Scripps Research Institute, La Jolla, CA). N2a cells were cultured in high-glucose Dulbecco s minimal Eagles medium supplemented with 10% FBS and 1% penicillin (100 g/ml)/streptomycin (292 g/ml). The virus was also used to infect several other cell lines such as RG2 (rat glioma cell line; ATCC, Manassas, VA) and C6 (rat glioma cells; a gift from Dr. M. Black, Washington State University, Pullman, WA). The culturing medium for RG2 was the same as that for N2a. C6 cells were cultured in high-glucose deficient MEM supplemented with 10% FBS and 689

10 doi: /s (03) % penicillin (100 g/ml)/streptomycin (292 g/ml) and glutamine (100 mm), histidinol (27 g/ml) by volume. All cell culture was done using a 37 C incubator maintained with 5% CO 2. Virus production. Virus was developed by transfecting CS-CMVsr39tk-I- Fluc or CS-CMVFluc plasmid together with a defective packaging construct (pcmv.r8.2) encoding HIV-1 gag, pol, rev, and tat genes and the plasmid (pmd.g) coding for VSVG envelope protein into 293T cells using the standard calcium phosphate method [4]. The transfected plates were incubated in a 37 C incubator with 5% CO 2 for 12 h and then the medium was replaced with fresh medium. Forty-eight to seventy-two hours after transfection, virus-containing medium was harvested, centrifuged at a low speed (3000 rpm for 5 min), and filter purified by passing through a m filter. To make a high-titer concentrated stock of the virus particles, the virus-containing medium was ultracentrifuged for 150 min at 50,000g using an SW41 (Beckman Coulter) rotor. After centrifugation the virus pellets were dissolved in 100 l of serum-free medium and stored frozen at 70 C in aliquots. Viral titer. To determine the relative viral titer, equal numbers ( )of N2a cells were plated in six-well plates (Falcon) and grown overnight and then serially diluted concentrated virus stocks were added to the wells. Cells with added virus were incubated for 24 h as usual and after the virus was removed the cells were lysed using 250 l of lysis buffer provided with the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was measured in a luminometer (Lumat 9507; Berthold, Germany) with an integration time of 10 s and the amount of protein was also determined from the optical density of the respective protein samples used for measuring the RLU. The relative viral titers are expressed as RLU/ g protein/s/ml of viral stock. In vitro assays. To measure the TK enzyme activity, the cells were harvested 48 h after infection and assayed for HSV1-sr39TK enzyme activity as described previously [18], except that [8-3 H]penciclovir (PCV) obtained from Moravek Biochemicals, Inc. (Brea, CA), was used as a substrate for increased sensitivity (instead of [8-3 H]ganciclovir). For FLUC activity, the cells were harvested and assayed for FLUC activity by using the Dual- Luciferase kit and a luminometer (specified above). Negative controls for both the assays were performed using the parental N2a cells. Animal experiments. Six- to eight-week-old adult male nude (athymic nude-nu) mice (source: Harlan Sprague Dawley, Indianapolis, IN) were used for this study. All animal procedures were performed with the approval of the UCLA Chancellor s Animal Research Committee. Unless otherwise stated, four mice were used for each of the following study conditions. Lentiviral CS-CMVsr39tk-I-Fluc-infected N2a cells as well as parental N2a cells ( ) were implanted sc on the right and left shoulder, respectively, of each of nude mouse. For serial monitoring of Fluc expression over time, tumors were implanted with 10 times fewer ( ) cells, to ensure the tumor size remained below 15 mm in diameter within a span of 14 days. For suicide therapy experiment, tumors were grown to a size of 5 6 mm diameter by implanting the same cells along with the control cells in six mice (i.e., N2a cells infected with lentivirus CS-CMVFluc), followed by treating the mice with 2.5 mg/mouse/day ganciclovir (GCV). For in vivo transduction experiments, only parental N2a cells were implanted on both shoulders. Once the tumors reached a palpable size of 5 6 mm, 50 l of concentrated virus stock of titer 138 RLU/ g protein/s/ml was injected once directly into the right tumor. Optical CCD imaging. In vivo optical imaging for FLUC was done 20 min after intraperitoneal injection of 3 mg D-Luciferin/animal using a Xenogen-IVIS cooled CCD optical system (Xenogen IVIS, Alameda, CA). Photons emitted from FLUC-expressing cells implanted in the mice were collected and integrated for a period of 1 5 min. Images were obtained by superimposed gray-scale photographic images and FLUC color images using the overlay option of the Living Image software (Xenogen) and Igor Image analysis software (Wave Metrics, Lake Oswego, OR). For quantitation of measured light, regions of interest were drawn over the tumor region and maximum photons/s/cm 2 /sr were obtained as previously validated [21]. Final values were expressed by averaging the maximum values obtained from all the mice scanned in each experiment. These are reported along with the standard error of the mean. MicroPET imaging. 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