Genome Size and Structure Determine Efficiency of Postinternalization Steps and Gene Transfer of Capsid-Modified Adenovirus Vectors in a Cell-Type-Specific Manner

Abstract

Adenovirus serotype 5 (Ad5) vectors containing Ad B-group fibers have become increasingly popular as gene transfer vectors because they efficiently transduce human cell types that are relatively refractory to Ad5 infection. So far, most B-group fiber-containing vectors have been first-generation vectors, deleted of E1 and/or E3 genes. Transduction with these vectors, however, results in viral gene expression and is associated with cytotoxicity and immune responses against transduced cells. To circumvent these problems, we developed fiber-chimeric Ad vectors devoid of all viral genes that were produced either by the homologous recombination of first-generation vectors or by using the Cre/lox-based helper virus system. In this study we compared early steps of infection between first-generation (35-kb genome) and Ad vectors devoid of all viral genes with genome sizes of 28 kb and 12.6 kb. All vectors possessed an Ad35-derived fiber knob domain, which uses CD46 as a primary attachment receptor. Using immortalized human hematopoietic cell lines and primary human CD34-positive hematopoietic cells, we found that the Ad genome size did not affect the efficiency of virus attachment to and internalization into cells. Furthermore, independently of the genome length and structure, all vectors migrated to the nucleus through late endosomal and lysosomal cellular compartments. However, the vector containing the short 12.6-kb genome was unable to efficiently escape from endosomes and deliver its DNA into the nucleus. Moreover, compared to other vectors, these Ad particles were less stable and had an abnormal capsid protein composition, including a lack of capsid-stabilizing protein IX. Our data indicate that the size and structure of the packaged viral genomes can affect the integrity of Ad particles, which in turn results in lower infectivity of Ad vectors.

Effect of Adenovirus-Mediated Heat Shock Protein Expression and Oncolysis in Combination with Low-Dose Cyclophosphamide Treatment on Antitumor Immune Responses

“…The resulting mgp96 fragment was released and cloned into pAd.RSV to generate pAd.mgp96, or in replacement of the lacZ cassette, into pAd.IR.E1A/bGal, in order to generate pAd.IR-E1a/mgp96. Adenoviral vectors were produced in 293 cells by recombination with pBHG10 and titrated by OD 260 spectrometry as well as by quantitative Southern blotting for viral genomes using an 8 kb probe corresponding to a region in the backbone of all adenoviral vectors as described elsewhere (33). The infectious titers were determined by plaque titering on 293 cells.…”

A Capsid-Modified Helper-Dependent Adenovirus Vector Containing the β-Globin Locus Control Region Displays a Nonrandom Integration Pattern and Allows Stable, Erythroid-Specific Gene Expression

“…The Ad.HCA-CMV-GFP vector was generated earlier and described elsewhere (47). It contains an Ad5/35 capsid and a cytomegalovirus (CMV) promoter-GFP gene expression cassette flanked by stuffer DNA derived from human X-chromosomal (ChX) DNA (Fig.…”

“…Cellular DNA was isolated from transduced MO7e cell clones by a standard protocol and analyzed by Southern blotting as described elsewhere (47). The blots were hybridized with a CTPlabeled GFP probe.…”

“…To generate the HD-Ad vectors Ad.LCR-␤-GFP and Ad.LCR-CMV-GFP (Fig. 1A), C7.Cre cells (2), which express Cre recombinase, and Ad5/35 helper virus (47) were used. Ad5/35 helper virus is an E1-deleted Ad35 fiber-modified chimeric vector that contains a packaging signal flanked by loxP sites.…”

Gene therapy for malignant mesothelioma: beyond the infant years

“…4,29 These viruses can infect neighboring cells, and spread the transgenes. A potential limitation is the cellular receptor of the recombinant virus: most adenovirus vectors are based on the group C adenovirus serotype 5 strain (Ad5), 30 which uses the coxsackie and adenovirus receptor (CAR) as its receptor. This receptor is poorly expressed on dendritic cells (DC) and primary tumor cells, thus limiting spread of the recombinant virus.…”