Enhanced transduction efficiency of fiber-substituted adenovirus vectors by the incorporation of RGD peptides in two distinct regions of the adenovirus serotype 35 fiber knob.

Fiber-substituted Ad serotype 5 vectors containing the fiber protein from Ad serotype 35 (Ad5F35) exhibit properties that render them suitable as a platform for targeted Ad vectors. Ad5F35 vectors do not show apparent tropism in certain organs, including the liver, and they elicit less innate immunity than other vectors after intravenous administration. In order to develop a targeted Ad vector, we previously developed fiber-mutant Ad5F35 vectors containing the integrin binding Arg-Gly-Asn (RGD) motif in the FG or HI loop of the Ad35 fiber knob. Mutant Ad5F35 vectors containing the RGD peptide in the FG or HI loop transduced CD46-negative cells more efficiently in an RGD-dependent manner, as compared to the efficiency achieved with unmodified Ad5F35 vectors (Matsui et al., 2009. Gene Therapy 16, 1050-1057). However, the transduction efficiency of the mutant Ad5F35 vectors in CD46-negative cells remained lower than had been expected. Ad5F35 vectors containing the RGD peptide in the HI or FG loop enabled a 6-fold higher transduction efficiency than that achieved with unmodified Ad5F35 vectors in CD46-negative cells, although this cell type abundantly expresses α(v)-integrins. In the present study, we aimed to enhance the transduction efficiency of fiber-mutant Ad5F35 vectors. To this end, we developed an Ad5F35-vector system in which foreign peptides could be incorporated into regions of FG and HI loops of the Ad35 fiber knob by means of in vitro ligation. Using this Ad5F35-vector system, firefly luciferase-expressing mutant Ad5F35 vectors containing the RGD peptides in both loops (Ad5F35-2xRGD-L2) were constructed. In CD46-negative cells, Ad5F35-2xRGD-L2 showed 12-fold and 3-fold greater transduction efficiency than unmodified Ad5F35 vectors and mutant Ad5F35 vectors containing only one copy of the RGD peptide in the FG or the HI loop. In addition, transduction with Ad5F35-2xRGD-L2 in CD46-negative cells was RGD peptide-dependent. These results indicate that fiber-mutant Ad5F35 vectors, by which foreign peptides can be simultaneously incorporated into both the FG and the HI loops of the Ad35 fiber knob, could be a promising gene delivery vehicle for various gene therapies, and could facilitate basic research efforts such as analyses of gene function.

Matsui H ，Sakurai F ，Katayama K ，Kurachi S ，Tashiro K ，Sugio K ，Kawabata K ，Mizuguchi H ... -  《-》

Development of fiber-substituted adenovirus vectors containing foreign peptides in the adenovirus serotype 35 fiber knob.

Matsui H ，Sakurai F ，Kurachi S ，Tashiro K ，Sugio K ，Kawabata K ，Yamanishi K ，Mizuguchi H ... -  《-》

Adenovirus serotype 35 vector-induced innate immune responses in dendritic cells derived from wild-type and human CD46-transgenic mice: Comparison with a fiber-substituted Ad vector containing fiber proteins of Ad serotype 35.

Recently, much attention has focused on replication-incompetent adenovirus (Ad) vectors containing fiber proteins derived from species B Ad serotype 35 (Ad35) (Ad5F35) and Ad vectors fully constructed from Ad35 as vaccine vectors expressing antigens. However, differences in the transduction properties, including the induction of innate immunity, of Ad5F35 and Ad35 vectors have not been properly and fully examined, partly because the transduction properties of these Ad vectors should be evaluated using nonhuman primates or human CD46-transgenic (CD46TG) mice, which ubiquitously express the primary receptor of Ad35, human CD46, in a pattern similar to that of humans. In the present study, we evaluated innate immune responses of mouse dendritic cells (mDCs) derived from bone marrow cells of wild-type (WT) and CD46TG mice following transduction with Ad serotype 5 (Ad5), fiber-substituted Ad5F35, or Ad35 vectors. Ad5F35 and Ad35 vectors mediated more efficient transduction in mDCs derived from CD46TG mice (CD46TG-mDCs) than did Ad5 vectors. Upregulation of costimulatory molecules and inflammatory cytokine induction by Ad5F35 and Ad35 vectors were significantly higher than those by Ad5 vectors in CD46TG-mDCs. However, the induction properties of the innate immune responses were different between Ad5F35 and Ad35 vectors. Ad35 vectors induced higher levels of costimulatory molecule expression and inflammatory cytokine production than did Ad5F35 vectors in CD46TG-mDCs. Furthermore, intravenous administration of Ad35 vectors in WT and CD46TG mice resulted in higher levels of serum interleukin (IL)-6 and IL-12 compared with administration of Ad5F35 vectors, which exhibited almost mock-transduced levels of these inflammatory cytokines. This study indicates that innate immune responses by Ad35 and Ad5F35 vectors are distinct even although both Ad vectors recognize human CD46 as a receptor.

Sakurai F ，Nakashima K ，Yamaguchi T ，Ichinose T ，Kawabata K ，Hayakawa T ，Mizuguchi H ... -  《-》

Generation of fiber-modified adenovirus vectors containing heterologous peptides in both the HI loop and C terminus of the fiber knob.

Fiber-modified adenovirus (Ad) vectors can be effective in overcoming the limitations of conventional Ad vectors, specifically their inefficient gene transfer into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Several types of fiber-modified Ad vectors have been developed. In this study, we evaluated the functionality of several fiber-modified Ad vectors. We developed a simple method based on in vitro ligation to construct Ad vectors containing heterologous foreign peptides in both the HI loop and C terminus of the fiber knob. A functional comparison of Ad vectors containing RGD and/or K7 (KKKKKKK) peptide in the HI loop or C terminus of the fiber knob was performed in several types of human, mouse, and rat cells, including CAR-positive and -negative cells, and tumor cells in mice in vivo. In the case of the in vitro experiment, Ad vectors containing RGD peptide in the HI loop of the fiber knob showed a higher level of gene transfer than vectors containing RGD peptide at the C terminus of the fiber knob. Ad vectors containing K7 peptide at the C terminus of the fiber knob showed levels of gene transfer similar to those of Ad vectors containing RGD peptide in the HI loop of the fiber knob, depending on the cell type. Ad vectors containing both peptides in the HI loop or C terminus of the fiber knob showed the highest levels of gene transfer and a broader tropism. For gene transfer into tumor cells in vivo, the Ad vectors containing RGD peptide were the most efficient. In the experiment using cultured cells, Ad vectors containing both RGD and K7 peptides were the most efficient with a broader tropism. In contrast, in the experiment in vivo, Ad vectors containing RGD peptide in the HI loop of the fiber knob were more efficient than the vectors containing K7 peptide (including double-modified vectors containing both the RGD and K7 peptides). These comparative analyses could provide a systemic reference for the use of fiber-modified Ad vectors. Our simple method, in which the peptide of interest can be expressed in Ad vectors in either the HI loop or the C terminus of the fiber knob, or both, could be a powerful tool for gene transfer into mammalian cells in studies of gene function as well as in gene therapy.

Koizumi N ，Mizuguchi H ，Utoguchi N ，Watanabe Y ，Hayakawa T ... -  《JOURNAL OF GENE MEDICINE》

Efficient gene delivery in human and rodent mast cells using adenovirus vectors.

Nakashima K ，Sakurai F ，Kawabata K ，Mizuguchi H ... -  《-》