Gene Transduction and Cell Entry Pathway of Fiber-Modified Adenovirus Type 5 Vectors Carrying Novel Endocytic Peptide Ligands Selected on Human Tracheal Glandular Cells

Abstract

Monolayers of cystic fibrosis transmembrane conductance regulator (CFTR)-deficient human tracheal glandular cells (CF-KM4) were subjected to phage biopanning, and cell-internalized phages were isolated and sequenced, in order to identify CF-KM4-specific peptide ligands that would confer upon adenovirus type 5 (Ad5) vector a novel cell target specificity and/or higher efficiency of gene delivery into airway cells of patients with cystic fibrosis (CF). Three different ligands, corresponding to prototypes of the most represented families of phagotopes recovered from intracellular phages, were designed and individually inserted into Ad5-green fluorescent protein (GFP) (AdGFP) vectors at the extremities of short fiber shafts (seven repeats [R7]) terminated by scissile knobs. Only one vector, carrying the decapeptide GHPRQMSHVY (abbreviated as QM10), showed an enhanced gene transduction of CF-KM4 cells compared to control nonliganded vector with fibers of the same length (AdGFP-R7-knob). The enhancement in gene transfer efficiency was not specific to CF-KM4 cells but was observed in other mammalian cell lines tested. The QM10-liganded vector was referred to as AdGFP-QM10-knob in its knobbed version and as AdGFP-QM10 in its proteolytically deknobbed version. AdGFP-QM10 was found to transduce cells with a higher efficiency than its knob-bearing version, AdGFP-QM10-knob. Consistent with this, competition experiments indicated that the presence of knob domains was not an absolute requirement for cell attachment of the QM10-liganded vector and that the knobless AdGFP-QM10 used alternative cell-binding domains on its capsid, including penton base capsomer, via a site(s) different from its RGD motifs. The QM10-mediated effect on gene transduction seemed to take place at the step of endocytosis in both quantitative and qualitative manners. Virions of AdGFP-QM10 were endocytosed in higher numbers than virions of the control vector and were directed to a compartment different from the early endosomes targeted by members of species C Ad. AdGFP-QM10 was found to accumulate in late endosomal and low-pH compartments, suggesting that QM10 acted as an endocytic ligand of the lysosomal pathway. These results validated the concept of detargeting and retargeting Ad vectors via our deknobbing system and redirecting Ad vectors to an alternative endocytic pathway via a peptide ligand inserted in the fiber shaft domain.Viruses have developed natural strategies to infect and grow in specific tissues of particular hosts, and their specificity and restricted host range make them theoretically the best-adapted vectors to transfer therapeutic genes into recipient cells that are their natural targets. However, when desired cell targets are not part of the natural host range of the virus, genetic modifications are required to adapt the virus and its derived vectors to these novel hosts. Since viral tropism is, in large part, under the control of primary virus-cell interaction occurring at the cell surface, the most obvious modification consists of...

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu

“…47 In addition to cellular binding, the fiber and its knob domain have been suggested to carry several functions important for intracellular trafficking, membrane lysis and virus maturation. [20][21][22][23][24][25] Fiber content of FibDCAR-HI-Link-ZHZH viruses was 12-29% of WT, which is an improvement from earlier studies where knobless virions have had fiber content of 2-10%. 10 Another improvement of Ad5/FibDCAR-HI-Link-ZHZH was the PP-to-PFU ratio, which was 140, as compared to 70 for the WT virus.…”

“…12,40 These growth defects can be explained by a low-fiber content caused by the lack of the fiber knob hampering virus entry and intracellular trafficking and a loss of other growth -or viral assembly functions carried by the fiber knob. [20][21][22][23][24][25] Only in the de-knobbed virus Ad5/R7-C2C2, containing the Ig binding C2 domain from Streptococcal protein G, we found a normal fiber content indicating that this fiber somehow compensates, as far as fiber content of the virus is concerned, for the loss of the fiber knob. 40 Therefore, a novel approach was also employed in the present study.…”

“…10,12 The poor performance of viruses with de-knobbed fibers is due to the fact that the knob has other important functions in the life cycle of the virus than just binding to the cellular receptor. [20][21][22][23][24][25] These facts make such viruses difficult to use for gene therapeutic purposes.…”

Transductional targeting of adenovirus vectors for gene therapy

“…A similar 'de-knobbing' strategy was employed by Magnussen et al, 111 wherein an integrin-binding RGD motif was utilized, resulting in selective infection of integrin-expressing cell lines in vitro, as well as human glandular cells. 112 Based on the feasibility of fiber replacement with T4 fibritin, an elegant system was devised wherein the trimeric CD40 ligand was fused to the C-terminus of this artificial fiber. 113 Notably, this vector provided CD40-specific gene delivery in vivo following systemic delivery.…”

Cystic Fibrosis - Renewed Hopes Through Research

“…The efficiency of Ad vectors in gene transfer has been demonstrated in numerous systems (Henning et al, 2002;Gaden et al, 2004;Toh et al, 2005) and the functional analysis of transgenes expressed by Ad can be tested in vitro in cell lines, ex vivo in tissues and in vivo in animal models. In vitro studies demonstrated that recombinant Ad vectors can express CFTR in cultured CF airway epithelial cells and correct the Cl -transport defect (Zabner et al, 1993).…”