A hypoxia-regulated adeno-associated virus vector for cancer-specific gene therapy

Abstract

The presence of hypoxic cells in human brain tumors is an important factor leading to resistance to radiation therapy. However, this physiological difference between normal tissues and tumors also provides the potential for designing cancer-specific gene therapy. We compared the increase of gene expression under anoxia (<0.01% oxygen) produced by 3, 6, and 9 copies of hypoxia-responsive elements (HRE) from the erythropoietin gene (Epo), which are activated through the transcriptional complex hypoxia-inducible factor 1 (HIF-1). Under anoxic conditions, nine copies of HRE (9XHRE) yielded 27- to 37-fold of increased gene expression in U-251 MG and U-87 MG human brain tumor cell lines. Under the less hypoxic conditions of 0.3% and 1% oxygen, gene activation by 9XHRE increased expression 11- to 18-fold in these cell lines. To generate a recombinant adeno-associated virus (rAAV) in which the transgene can be regulated by hypoxia, we inserted the DNA fragment containing 9XHRE and the LacZ reporter gene into an AAV vector. Under anoxic conditions, this vector produced 79- to 110-fold increase in gene expression. We believe this hypoxia-regulated rAAV vector will provide a useful delivery vehicle for cancer-specific gene therapy.

Figure 1

Structural map of plasmids pH3LacZ, pH6LacZ, pH9LacZ, and rAAV vectors. In pHnLacZ plasmids, the reporter gene LacZ was under the transcriptional control of different copy numbers of HRE and an SV40 minimal promoter. In pH9LacZ-AAV, the LacZ with nine copies of HRE and the SV40 minimal promoter was inserted between the two inverted repeat regions (ITR). The control vector pCMVLacZ-AAV contains a CMV promoter upstream of the LacZ gene.

Figure 2

Cell type-specific induction of HIF-1α under anoxic conditions. After exposure to either oxic or anoxic conditions for 16 hours, U-87 MG and U-251 MG cells were harvested and lysed. Equal amounts (25 µg) of proteins were analyzed by 7% SDS-PAGE and immunoblotting was performed using a monoclonal anti-human HIF-1α antibody (Novus Biologicals). U-87 MG cells showed minimal expression of HIF-1α under oxia and significantly increased HIF-1α under anoxia. In contrast, in U-251 MG cells HIF-1α showed considerable expression under oxic conditions and increased only slightly under anoxic conditions.

Figure 3

Anoxia-induced LacZ expression depends on HRE copy number. U-87 MG and U-251 MG cells were transiently transfected with plasmids containing the LacZ reporter gene and 3X, 6X, and 9XHRE. Transfected cells were incubated under either anoxic or oxic conditions for 16 hours before analysis for LacZ activity. The relative level of LacZ expression in oxic cells was designated as 1.0. The actual values obtained from two independent experiments are shown for U-87 MG cells (-●-, -▼-) and U-251 MG cells (-○-, -▽-).

Figure 4

Differential regulation of LacZ expression in pH9LacZ under intermediate oxygen concentrations. U-87 MG and U-251 MG cells were transiently transfected with the plasmid containing LacZ reporter gene and 9XHRE. Transfected cells were incubated under different oxygen concentrations for 16 hours before analysis for LacZ activity. The relative level of LacZ expression in oxic cells was designated as 1.0. The actual values obtained from two independent experiments are shown for U-87 MG cells (-●-, -▼-) and U-251 MG cells (-○-, -▽-).

Figure 5

Western blot showing BAX expression under Oxic (O) and Anoxic (A) conditions in pH9BAX-stably transfected U-251 MG clones. The intensity of the BAX protein was quantified using densitometry; the fold increase in BAX expression under anoxic conditions ranged from 10 to 39.

Figure 6

β-gal expression in U-251 MG cells. U-251 MG cells (3x10⁵) were infected with either AAVH9LacZ or AAVCMVLacZ at an MOI of 25 and incubated under either oxic or anoxic condition for 16 hours. At the end of incubation, cells were assayed for β-gal expression by the X-gal in situ staining method as described in the Materials and Methods section.