Cell specific promoter mediated transgene expression for gene therapy purposes in the CNS

The aim of this project is specific gene-expression in neurons or glial cells mediated by cell type specific promoters in HSV-1 (Herpes simplex virus type-1) amplicon vectors.

Because of the overall goal of gene therapy in the central nervous system, the fusion gene tg17 consisting of the cell culture marker gene gfp and the PET marker gene HSV-1-tk (thymidine kinase) served as reporter. TK can be used for non-invasive in vivo imaging with PET and as therapeutic gene.

Methods: The glial specific glial-fibrillary-acidic-protein-promoter (GFAP), the neuron specific tyrosine-hydroxylase- (TH) and the platelet-derived-growth-factor-beta-promoter (PDGF-beta) were cloned in HSV-1 amplicon plasmids controlling the tg17-gene and packaged into HSV-1 virions.

Different cell lines were infected with amplicon vectors and the level of GFP-expression was quantified by a region of interest analysis. Promoter specificity was quantified by counting GFP-expressing cells in infected primary cocultures (containing glial and neuronal cells) that colocalised with glial (GFAP) or neuronal (microtubule associated protein 2, MAP-2) markers, respectively.

Results: In cell lines infected with amplicon vectors carrying the GFAP promoter, high GFP-expression was detected. In particular in Gli36 glioma cells there was no significant difference to the positive control (CMV-promoter). In coculture the GFAP-promoter resulted in specific expression of tg17 in glial cells (24–64% colocalisation with GFAP, 0–27% colocalisation MAP-2).

In constrast to this, GFP-quantification of cell lines transduced with tg17 under the control of the TH-promoter showed a weaker promoter activity with specific regulation only in SHSY5Y neuroblastoma cells. In cocultures the TH promoter was primarily active in neuronal cells (42–90% colocalised with MAP-2, 0–43% colocalised with GFAP).

Surprisingly, the PDGF promoter did not show neuronal specificity. Quantification in cell lines resulted in activation pattern similar to the GFAP-promoter and tg17-expression in cocultures mainly colocalised with GFAP (30–46% with GFAP, 0–28% with MAP-2).

In summary this work specifies the GFAP- and the TH-promoter as promising candidates for glial respectively neuronal expression of therapeutic genes for in vivo experiments with HSV-1 amplicons. It further reveals new aspects concerning PDGF-promoter activity in glial and glioma cells possibly due to the fact that PDGF plays an important role in the development of gliomas.