Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes
Introduction
Recombinant adenovirus (Ad) vectors have been extensively used to deliver foreign genes to a variety of cell types in vitro and in vivo (Kovesdi et al., 1997, Yeh and Perricaudet, 1997, Benihoud et al., 1999). So far, more than 49 serotypes of human Ad have been identified and classified in six distinct subgroups (A–F) (Shenk, 1996). Among them, Ad type 5 (Ad5), which belongs to subgroup C, has been commonly used to prepare recombinant Ad vectors due to the extensive knowledge of the genetic and biological characteristics of the virus.
Ad5 requires the coxsackievirus and adenovirus receptor (CAR) on the cell membrane as a initial receptor for infection (Bergelson et al., 1997, Tomko et al., 1997). After binding Ad fiber with CAR, interaction between the RGD motif of the penton bases with secondary host cell receptors, avb3 and avb5 integrins, facilitates internalization via receptor-mediated endocytosis (Wickham et al., 1993). The interaction of the fiber knob with CAR on the cell is the key mediator by which the Ad enters the cells. Ad-mediated gene transfer is inefficient to the cells lacking in sufficient CAR expression, such as differentiated airway epithelium, skeletal muscle, smooth muscle, peripheral blood cells, hematopoietic stem cells, etc. (Wickham, 2000).
Modification of fiber protein overcomes this obstacle. There are two approaches to modify the fiber protein: insertion (addition) of foreign peptides into the fiber knob (Wickham et al., 1997, Dmitriev et al., 1998, Krasnykh et al., 1998, Yoshida et al., 1998, Bouri et al., 1999, Koizumi et al., 2001, Mizuguchi et al., 2001), and substitution of fiber protein (knob or knob plus shaft) into that of other serotypes (Gall et al., 1996, Stevenson et al., 1997, Chillon et al., 1999, Shayakhmetov et al., 2000). In the former approaches, we and others reported expanded tropism of the Ad vector containing a stretch of lysine residues (Wickham et al., 1997, Yoshida et al., 1998, Bouri et al., 1999) or a RGD motif (Dmitriev et al., 1998, Koizumi et al., 2001, Mizuguchi et al., 2001), which target the heparan sulfate or αv integrin on the cellular surface, respectively. In the latter approaches, altered vector tropism was reported by the substitution of Ad5 fiber protein into that of Ad3 (Stevenson et al., 1997), Ad7 (Gall et al., 1996), Ad17 (Chillon et al., 1999) and Ad35 (Shayakhmetov et al., 2000). Most Ad serotypes belonging to subgroups A, C, D, E, and F use CAR as the initial receptor for the virion (Roelvink et al., 1998), although Ad8, Ad19, and Ad37 (serotype D) use other molecules for infection (Roelvink et al., 1998, Arnberg et al., 2000a, Arnberg et al., 2000b). Cellular receptor(s) of Ad belonging to the subgroup B are as yet unknown. Ad11 and Ad35 (serotype B), however, show high binding and infective efficiencies for committed hematopoietic cell lines (Segerman et al., 2000). Ad vectors containing chimeric fiber protein composed of Ad5 and Ad35 show efficient gene transfer into human CD34+ cells (Shayakhmetov et al., 2000).
In this study we examined the gene transfer activity of the Ad vector containing Ad5/35 chimeric fiber protein by using several types of human cells (CAR-positive or -negative), rodent cells, and human blood cells (a total of 20 type of cells), and the systemic gene delivery properties of the vector in mice after intravenous injection, in comparison with conventional Ad5-based vector. Data suggest that the Ad vector containing the Ad5/35 chimeric fiber protein had altered and expanded tropism, compared with the Ad5-based vector. We also show that the Ad vector containing Ad5/35 chimeric fiber protein allows the packaging of approximately 8.8 kb of foreign DNA, due to the smaller size of the fiber shaft-coding sequence, without affecting the viral growth rate and titer. This overcomes the packagable size limit, 8.1–8.2 kb, of foreign DNA by the conventional Ad5-based vector (Bett et al., 1994).
Section snippets
Cells
SK HEP-1 (endothelial cell line derived from the human liver) (Heffelfinger et al., 1992) and HeLa cells (human epitheloid carcinoma of the cervix) were cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). Human glioma cell lines, LN319 (anaplastic astrocytoma), LN444 (glioblastoma multiforme), LNZ308 (glioblastoma multiforme), and SF295 cells (glioblastoma multiforme) (kindly provided by Dr M. Tada, Hokkaido University, Hokkaido, Japan) were
Construction of vector plasmids for generation of Ad vector containing Ad5/35 chimeric fiber protein
First, a simple system to generate the Ad vector containing the Ad5/35 chimeric fiber protein was developed. Previously, we developed the simple in vitro ligation method to generate the Ad vector containing the Ad5 wild-type fiber protein or the RGD peptide motif in the fiber knob (Mizuguchi and Kay, 1998, Mizuguchi and Kay, 1999, Mizuguchi et al., 2001). Three unique restriction sites, I-CeuI, SwaI, and PI-SceI, were introduced into the E1 deletion region of the Ad genome cloned in the plasmid
Acknowledgements
We would like to thank Mr Jun Murai, Mr Naoya Koizumi, and Ms Nobuko Heishi for technical assistance. We would also like to thank Dr R. Shibayama for her help in determining GFP expression. This work was supported by grants from the Ministry of Health, Labour, and Welfare in Japan.
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