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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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
Naoya
N
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo 158-8501, Japan.
Mizuguchi
Hiroyuki
H
Utoguchi
Naoki
N
Watanabe
Yoshiteru
Y
Hayakawa
Takao
T
D000256:Adenoviridae / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D019763:Heparin Lyase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
D036022:Capsid Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C018315:hexon capsid protein, Adenovirus; C047981:arginyl-glycyl-aspartic acid; D019763:Heparin Lyase