Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo

To evaluate the efficiency of gene delivery in gene therapy strategies for malignant brain tumors, it is important to determine the distribution and m agnitude of transgene expression in target tumor cells over time, Here, we assess the time- and vector dose-dependent kinetics of recombinant herpes s implex virus (HSV)-1 vector-mediated gene expression and vector replication in culture and irt vivo by a recently developed radiotracer method for non invasive imaging of gene expression (J. G. Tjuvajev et al., Cancer Res., 55 : 61.26-6132, 1995), The kinetics of viral infection of rat 91, gliosarcoma cells by the replica tion-conditional HSV-1 vector, hrR3, was studied by measuring the accumulat ion rate of 2-[C-14]-fluoro-5-iodo-1-beta -D-arabinofurano (FIAU), a select ive substrate for viral thymidine kinase (TK). The level of viral TR activi ty in 9L cells was monitored by the radiotracer assay to assess various vec tor doses and infection times, allowing vector replication and spread. In p arallel, viral yields and levels of Escherichia coil beta -galactosidase ac tivity were assessed quantitatively. To study vector replication, spread an d HSV-1-tk and lacZ gene coexpression in vivo, first- or second-generation recombinant HSV-1 vectors (hrR3 or MGH-1) were injected into s.c. growing m t 9L or human U87 Delta EGFR gliomas in nude rats at various times (8 h to 8 days) and at various vector doses [1 x 10(6) to 2 x 10(9) plaque-forming units (PFUs)] Drier to imaging. For noninvasive assessment of HSV-l-tk gene expression (I-124-labeled FIAU % dose/g), 0.15 mCi of I-124-labeled FIAU w as injected i.v. 8 h after the last vector administration, and FIAU positro n emission tomography (PET) was performed 48 h later, For the assessment of HSV-l-tk and lacZ gene coexpression, 0.2 mCi of I-131-labeled FIAU was inj ected i.v, 24 h after the last vector administration. Forty-eight h later, animals were killed, and tumors were dissected for quantitative autoradiogr aphical and histochemical assessment of regional distribution of radioactiv ity (TK expression measured as I-131-labeled FIAU % dose/g) and coexpressed lacZ gene activity. The rates of FIAU accumulation (Ki) in hrR3-infected 91, cells in culture, which reflect the levels of HSV-l-tk gene expression, ranged between 0.12 a nd 3.4 ml/g/min. They increased in a vector dose- and infection time-depend ent manner and correlated with the virus yield (PFUs/ml), where the PFUs:Ki ratios remained relatively constant over time, Moreover, a linear relation ship was observed between lacZ gene expression and FIAU accumulation 5-40 h after infection of 9L cells with a multiplicity of infection of 1.5, At la ter times (>52 h postinjection), high vector doses (multiplicity of infecti on, 1.5) led to a decrease of FIAU accumulation rates, viral yield, and cel l pellet weights, indicating vector-mediated cell toxicity, Various levels of HSV-l-tk gene expression could be assessed by FIAU-PET after in vivo inf ection of s.c. tumors. The levels of FIAU accumulation were comparatively l ow (similar to ranging from 0.00013 to 0.003% injected dose/g) and were spa tially localized; this may reflect viral-induced cytolysis of infected tumo r cells and limited lateral spread of the virus, Image coregistration of tu mor histology, HSV-l-tk related radioactivity (assessed by autoradiography) , and lacZ gene expression (assessed by beta -galactosidase staining) demon strated a characteristic pattern of gene expression around the injection si tes. A rim of lacZ gene expression immediately adjacent to necrotic tumor a reas was observed, and this zone was surrounded by a narrow band of HSV-l-t k-related radioactivity, primarily in viable-appearing tumor tissue. These results demonstrate that recombinant HSV-I vector-mediated HSV-l-tk g ene expression can be monitored noninvasively by PET, where the areas of FI AU-derived radioactivity identify the viable portion of infected tumor tiss ue that retains FIAU accumulation ability, and that the accumulation rate o f FIAU in culture, Ki, reflects the number of HSV-1 viral particles in the infected tumor cell population [4.1 +/- 0.6 x 106 PFUs/Ki unit PFUs + ml/mi n/g)l. Moreover, time-dependent and spatial relationships of HSV-l-tk and l acZ gene coexpression in culture and in vivo indicate the potential for ind irect in vivo imaging of therapeutic gene expression in tumor tissue infect ed with any recombinant HSV-1 vector where a therapeutic gene is substitute d for the lacZ gene.