Generation of fiber-mutant recombinant adenoviruses for gene therapy of malignant glioma.
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.
Yoshida
Y
Y
Department of Molecular Biotherapy Research, Cancer Chemotherapy Center, Cancer Institute, Tokyo, Japan.
Sadata
A
A
Zhang
W
W
Saito
K
K
Shinoura
N
N
Hamada
H
H
D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D001932:Brain Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D002460:Cell Line; D017931:DNA Primers; D015316:Genetic Therapy; D005910:Glioma / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics; D007376:Interleukin-2 / Q000235:genetics; D008854:Microscopy, Electron; D010957:Plasmids; D011995:Recombination, Genetic; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007376:Interleukin-2; D018664:Interleukin-12