Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity.

Abstract

Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.

Effects of ectopic decorin in modulating intracranial glioma progression in vivo, in a rat syngeneic model

“…We have also previously demonstrated that the GFAP promoter driving transgenes encoded within recombinant adenovirus vectors exerts a several fold lower translational activity when compared to the hCMV promoter. 35 To test the therapeutic effects of adenovirus-mediated decorin gene therapy in our brain tumor model, 3 days after the implantation of tumor cells, adenovirus vectors bearing the therapeutic gene were stereotactically injected into the tumor. The intratumoral delivery of 4 Â 10 7 iu of RAd/GFAP/decorin and RAd/hCMV/decorin resulted in no significant difference in the survival of CNS-1 tumorbearing rats as compared with RAd/hCMV/b-gal-injected animals (data not shown).…”

Inhibition of glioma cells in vitro and in vivo using a recombinant adenoviral vector containing an astrocyte-specific promoter

“…9 Thus, the results obtained after infection of HepG2 hepatocellular carcinoma cells by Adgfa2TK are of particular interest. After infection with Adgfa2TK, this cell line showed no sensitivity to up to 50 g/mL GCV, whereas infection with AdCMVTK resulted in 80% toxicity after exposure to 50 g/mL GCV.…”

“…7 Several recent studies have in fact demonstrated that a GFAP promoter fragment can induce specific expression of a transgene in glial cells compared with non-glial cells (neuroblastoma, fibroblast, epithelial cell line) and can limit expression of a toxic protein in glial cells after systemic administration. 8,9 In this study, we investigated the use of a 2.2-kb human GFAP promoter fragment, gfa2, to target HSV-TK expression in gliomas using a high-titer recombinant adenovirus. The gfa2 promoter was selected for use as it has previously been shown to effectively express genes specifically in astrocytes, 10 including expression of HSV-TK in cultured glioma cells 11 and in astrocytes in transgenic mice.…”

Adenovirus-mediated expression of HSV1-TK or Fas ligand induces cell death in primary human glioma-derived cell cultures that are resistant to the chemotherapeutic agent CCNU

“…Cells were prepared for FACS analysis as described previously. 28 Secretion of mFasL into the culture supernatants was also verified. To prepare the supernatants, the culture medium was centrifuged at 200Âg for 10 minutes to remove any apoptotic cells.…”

“…The construction of RAds expressing Escherichia coligalactosidase (RAd35 ), 24,25 HSV1 -TK ( RAd128 ) , 15,26 and mFasL ( RAdhCMV-mFasL ), 27,28 all under the control of the major immediate early human cytomegalovirus promoter ( mIE -hCMV ) , has been described in detail previously. The RAds were grown and purified as previously described.…”