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.

Adenovirus-mediated transfer of p53 and Fas ligand drastically enhances apoptosis in gliomas

“…17 This Adv for p53 (Adv-p53) is efficient for the transduction of genes into glioma cell lines. 17 Adv-mediated gene transduction was performed as described previously.…”

“…17 This Adv for p53 (Adv-p53) is efficient for the transduction of genes into glioma cell lines. 17 Adv-mediated gene transduction was performed as described previously. 18 The total MOI was adjusted to be the same in all experimental groups by supplementation with the appropriate amount of the Adv for lacZ (Adv-lacZ).…”

Conditionally replicative adenovirus expressing a targeting adapter molecule exhibits enhanced oncolytic potency on CAR-deficient tumors

“…A major obstacle for wider application of fiber-modified CRAds with other recognition specificities is, however, that the adenovirus fiber appears to impose not yet fully understood structural demands on the incorporated ligand. [23][24][25] The repertoire of genetically targeted CRAds could potentially be expanded tremendously if more complex, highaffinity ligands such as antibodies could be used. However, such molecules have so far not been successfully incorporated as a stable component of the adenovirus capsid.…”

Recombinant adenovirus vectors with knobless fibers for targeted gene transfer

“…3,4 This knowledge has directed several strategies for adenoviral vector targeting by genetic modification of the viral capsid. Peptide motifs with receptor binding specificity have been incorporated into the fiber protein by extension of the carboxy terminus of the fiber [5][6][7][8] ligands, a Myc-epitope and a 6His-tag, were attached via a flexible linker peptide. The targeted knobless fiber molecules were properly expressed and imported into the nucleus of adenovirus packaging cells, where they were incorporated as functional trimers into the adenovirus capsid.…”

“…However, it appears that incorporation of targeting ligands in the fiber knob is not always compatible with proper fiber folding, resulting in loss of fiber functions. 7,8 Deletions in the fiber knob as small as only two amino acids can result in loss of fiber trimerization, the second important function of this domain. 12,13 Since the trimeric fashion of the fiber is essential for its incorporation in the virus particle by binding to the penton base, 12,14 modifications that result in loss of trimerization function will prevent formation of viable viruses carrying the modified protein.…”