Generation of Fiber-Mutant Recombinant Adenoviruses for Gene Therapy of Malignant Glioma

Abstract

Recombinant adenovirus (Adv)-mediated gene transduction is a powerful technology for cancer gene therapy. In this article, we report the generation of a fiber-mutant Adv vector, using the Adv genomic DNA-terminal protein complex (DNA-TPC) cotransfection method. First, a fiber-mutant construct in a plasmid carrying the right-side two-thirds of the human adenovirus type 5 (Ad5) genome (pTR) was cotransfected with Ad5 DNA-TPC, yielding the recombinant Adv with the desired fiber mutation. The DNA-TPC from the mutant Adv was then utilized to produce a second-step recombinant Adv with an expression cassette in the place of E1. By this procedure, we generated a fiber mutant, F/K20, that has a linker and a stretch of 20 lysine residues added at the C terminus of the fiber. By using Adv carrying a reporter lacZ gene (AxCAZ2) with either F/K20 or wild-type fiber (F/wt), we examined the transduction efficiency of F/K20-Adv. No significant difference in the transduction efficiency between F/K20 and F/wt-Adv was observed for a human fibroblast line, WI-38, or various tumor cell lines, including melanoma, prostate, esophageal, and pancreatic cancer lines. In clear contrast, F/K20-Adv showed a remarkably enhanced efficiency in genetic transduction of human glioma cells. In all four human glioma lines tested, the multiplicities of infection (MOIs) for transduction of 50% of the population (ED50) were decreased with F/K20-Adv compared with F/wt-Adv: 7-fold for T98G, 14-fold for U251, 9-fold for U373, and 42-fold for U87 cells. Therefore, we attempted to apply F/K20-Adv for gene therapy of malignant glioma. Glioma cells infected with F/K20-Adv carrying genes for interleukin 2 or interleukin 12 produced a high level of each cytokine at a much lower MOI than did cells infected with F/wt-Adv. Infection with F/K20-Adv carrying the wild-type p53 tumor suppressor gene resulted in an enhanced level of p53 protein expression and an increased incidence of F/K20-Adv in transduction efficiency for malignant glioma, providing promising tools for gene therapy.

Enhanced osteoinduction by mesenchymal stem cells transfected with a fiber-mutant adenoviral BMP2 gene

“…Replication‐deficient adenoviral vectors (AxCAhBMP2‐F/RGD and AxCAhBMP2‐F/wt) carrying the human bone morphogenetic protein 2 (BMP2) gene with RGD‐mutated fibers or wild‐type fibers, and containing a CAG promoter (CMV‐IE enhancer, chicken β‐actin promoter), were constructed as described previously 12, 19–21. The human BMP2 gene was harvested from SaOS2 cells.…”

“…Adenovirus‐mediated gene transduction was performed as described previously 20. Briefly, the transduction efficiencies of Adv with wild‐type fibers and of those with F/RGD fibers were tested by a 1‐h infection with AxCAZ3‐F/wt or AxCAZ3‐F/RGD, followed by a 48‐h incubation.…”

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

“…Further studies followed with the incorporation of a heparin-binding polylysine motif (pK7 or K20) or an RGD motif [140–143]. These strategies were successful in enhancing the in vitro infection of a panel of CAR-deficient cell lines, including CF-KM4 and MM-39 cells (serous cell lines from submucosal tracheal glands) [140], glioma cells [143], endothelial and smooth muscle cells [142].…”

“…Further studies followed with the incorporation of a heparin-binding polylysine motif (pK7 or K20) or an RGD motif [140–143]. These strategies were successful in enhancing the in vitro infection of a panel of CAR-deficient cell lines, including CF-KM4 and MM-39 cells (serous cell lines from submucosal tracheal glands) [140], glioma cells [143], endothelial and smooth muscle cells [142]. More recently, the 11 amino acid (aa) protein transduction domain of HIV-1 TAT was incorporated into the C-terminus of the Ad5 knob, resulting in improved CAR-independent transduction in vitro and enhanced tumor transduction in vivo [144].…”

Maternal IL‐6 can cause T‐cell‐mediated juvenile alopecia by non‐scarring follicular dystrophy in mice

“…The mammalian expression vectors pCA‐mIL6 (RDB1520) and pCA‐LacZ (RDB1870), which use the cytomegalovirus (CMV) promoter to drive the expression of mouse IL‐6 or β ‐galactosidase, respectively, were obtained from the Riken BRC DNA bank through the national bio‐resource project of the MEXT, Japan . Plasmid DNA was isolated using an Endofree Plasmid Maxi Kit (Qiagen, Valencia, CA, USA) and brought to 1 mg/ml in Tris–EDTA (TE) buffer.…”