Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob

Abstract

Targeting of gene transfer at the level of cell entry is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success because proper targeting ligand-receptor systems on the cells of interest are generally unknown. Systematic approaches to generate adenovirus vectors targeting any given cell type need to be developed to achieve this goal. Here, we constructed an adenovirus library that was generated by a Cre-lox-mediated in vitro recombination between an adenoviral fiber-modified plasmid library and genomic DNA to display random peptides on a fiber knob. As proof of concept, we screened the adenovirus display library on a glioma cell line and observed selection of several particular peptide sequences. The targeted vector carrying the most frequently isolated peptide significantly enhanced gene transduction in the glioma cell line but not in many other cell lines. Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. This vector-screening system can facilitate the development of a targeted adenovirus vector for a variety of applications in medicine.

Oncolytic virus therapy for pancreatic cancer using the adenovirus library displaying random peptides on the fiber knob

“…Although there was a possibility to isolate peptide sequences that can generally enhance gene transduction in many cells, a selected peptide showed cell typedirected infection, which was consistent with our previous results, 15 suggesting that library screening based on the binding to target cells may enable identification of tumor-specific targeting ligands. In addition to the positive selection followed by a validation of the tumor cell specificity, negative selection can be incorporated in the early screening phase.…”

“…15 Amplified and expanded adenoviruses on AsPC-1 cells were recovered after three rounds of selection, and the DNA region containing oligonucleotide inserts was amplified by PCR. DNA sequencing of the PCR products revealed enrichment of mainly two peptides, IVRGRVF (58% of the sequenced PCR fragments) and SYENFSA (38%; Table 1).…”

“…The engineering of a capsid-coding region often disturbs viral production and replication by impaired viral assembly and maturation, possibly due to the conformational change of the fiber knob. 15 A library approach with a replication-competent adenovirus may be highly useful to isolate a targeted oncolytic adenovirus, because the most efficient adenovirus should be selected from the library based on its high infectivity and replication capacity through the process of several rounds of virus amplification and spread through target cells.…”

“…15,22,23 In pancreatic cancer, a regional therapy is also particularly relevant, because locally advanced cases are surgically unresectable but can be accessible by ultrasound-or CT-guided percutaneous injection. In addition to locally advanced cases, distant metastasis at the liver and in the peritoneal cavity may be a clinical target of local oncolytic virus therapy.…”

“…14 We have recently developed a novel system for producing adenoviral libraries displaying a variety of peptides on the HI-loop of the fiber knob and established a procedure to select an adenoviral vector with high infectivity in target cells. 15 As the binding affinity might be determined by the overall conformation of a modified fiber and not by inserted peptides alone, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. In this study, first, the adenovirus library was screened for high affinity to a pancreatic cancer cell line (AsPC-1).…”