Multi-modal non-invasive imaging techniques show improved vector-distribution of HSV-EBV-RV hybrid vectors within experimental glioma

One of the main problems in gene therapy of malignant gliomas is limited vector distribution within the target tissue. To serve a more wide-spread distribution of vector-mediated gene expression throughout the tumour glioma cells can be converted to vector producer cells onsite. Conversion of tumour cells to retrovirus packaging cells has been achieved using HSV-1/EBV/RV (HER) hybrid amplicons bearing retrovirus vector sequences and retroviral gag, pol and env genes1.

A HER vector (HER-LITG) has been constructed containing the optical imaging (OI) gene firefly luciferase (luc, L), the positron emission tomography (PET) reporter as well as therapeutic gene HSV-1 thymidine kinase (HSV-1tk, T) and the fluorescence gene gfp (G).

Activity of HSV-1TK and luciferase was analysed by MTT-assay, plate reader and OI, respectively. In vivo this vector was tested for functionality by using MR, PET- and optical imaging to i) localize i.c. tumours (MRI), ii) identify viable target tissue (18F-FLT PET), iii) assess tissue-dose of vector-mediated gene expression (18F-FHBG PET) and iv) measure luciferase activity (OI). Data were analysed for changes in gene expression over time and compared with those of a control vector (HSV-LITG).

In cell culture, HER-LITG-infected glioma cells showed expression of all three imaging genes. Furthermore, a gain in luciferase activity as well as in numbers of GFP-positive cells could be demonstrated over time. Finally, functionality of HSV-1-TK was proven by MTT assay. In vivo, optical and PET imaging data demonstrated luciferase and HSV-1 tk activity in all in vivo transduced tumours. Although HSV-LITG infected tumours showed a clear signal of luciferase and HSV-1 TK activity after infection this vanished in the following days. Only HER-infected gliomas showed an increase in luciferase signal intensity and 18F-FHBG accumulation over time.

An HSV/EBV/RV hybrid vector coding for different imaging and a therapeutic gene was constructed and positively tested for gene expression as well as functionality in vitro and in vivo. Furthermore, indirect evidence for generation of retroviral particles and thus improved vector-distribution was obtained by i) re-infection assays as well as increase in ii) gfp-positive cells, iii) luciferase and iv) HSV-1 TK activity over time.