Glioma-specific 4optHBS-Msi1 / GFAP Promoter To Construct Evaluation And Preliminary Application In Gene Therapy

Glioma is a common and highly malignant neoplasm of the central nervous system (CNS) with poor prognosis. None of the current treatments could significantly improve the patients’ prognosis. Glioma is characterized by in situ recurrence, which makes gene therapy an appropriate choice. Our previous experiments have proved that recombined adeno-associated virus1(rAAV1) had a significant tropism for astrocytes in brain and highest transfection efficiency. Construction of cell type-specific and regulatable promoters in rAAVs could optimize a more selective treatment of gliomas.Musashil and Glial Fibrillary Acidic Protein (GFAP) are proteins expressed specifically in glioma cells and astrocytes, respectively. Their promoters, Musashil-promoter and GFAP-promoter, could allow the expression of downstream exogenous genes. The glioma cells are usually under hypoxic microenviroment, while optHBS (optimized HIF binding site) sequence is sensitive to hypoxic environment. Therefore, we first presumed the potential P/Msil sequences(M1/M2/M3/M4) by using bioinformatic approaches, DNA fragments containing M1/M2/M3/M4, p/GFAP, and4optHBS—Msil/GFAP promoters respectively were obtained by gene recombination. Consequently we linked them to the pGL3—Basic vectors and compared their activity in cell lines such as U251, BT325,SH-SY5Y,HeLa,PC12,293T by measuring their relative luciferase activity.The results demonstrated that M1(chr12:119,291,292-119,292,123) was the active sequence of P/Msil; in U251, BT325cell line, the activity of P/Msil was weaker compared with the P/GFAP; in the non-glioma tumor cells SH-SY5Y, HeLa cell line, only P/Msil had prominent activities; while in PC12(a model of neuronal cell),293T cell line, both above two promoters were of no activities. The results demonstrated the cell specificity and high efficiency of P/Msil and p/GFAP.The activities of4optHBS—Msil/GFAP promoters in U251cell lines, increased by4.01and2.67-fold respectively, under0.3%O2condition compared with those under19.6%O2condition, while in BT325cell lines, their activities increased by4.96and3.09-fold, respectively.Finally,4optHBS-Msil/GFAP—Bax a vectors were constructed to introducing Bax gene expression, the results of Western Blot, TUNEL stain, PI stain and FCM detection showed that4optHBS-Msil/GFA-promoters up-regulated the bax gene expression and elicited glioma specific apoptosis in U251and BT325cells. Thus the4optHBS Msil/GFAP-promoters were apparently strong and tumor-selective promoters with potential application in targeted glioma gene therapy. Further studies will be carried out regarding the apoptotic effects of4optHBS-Msil/GFAP—Bax a under hypoxic condition; the transfer of4optHBS-Msil/GFAP—Bax a into rAAVl vector and confirming its pro-apoptotic effects; and in vivo studies with glioma transplanted nude mice.