Reduction of Natural Adenovirus Tropism to the Liver by both Ablation of Fiber-Coxsackievirus and Adenovirus Receptor Interaction and Use of Replaceable Short Fiber

Abstract

The initial recognition and binding of adenovirus vector to the host cell surface is mediated by interaction between the adenovirus fiber knob protein and its receptor, the coxsackievirus and adenovirus receptor (CAR). This natural tropism of adenovirus vector needs to be ablated in order to achieve targeted gene transfer. To this end, we noted that adenovirus serotype 40 (Ad40) contains two distinct long and short fibers; the short fiber is unable to recognize CAR, while the long fiber binds CAR. We generated adenovirus serotype 5-based mutants with chimeric Ad40-derived fibers, which were composed of either long or short shafts together with CAR binding or nonbinding knobs. The capacity of these adenovirus mutants for in vitro and in vivo gene transfer to liver cells was examined. In the case of primary human hepatocytes displaying a high expression level of CAR and alphav integrin, both CAR binding ability and fiber shaft length played important roles in efficient transduction. Most significantly, the high transduction efficiency observed in the liver and spleen following intravenous administration of adenovirus vector was dramatically reduced by both ablation of fiber-CAR interaction and the use of replaceable short fiber. In other tissues displaying a low level of transduction, no significant differences in transduction efficiency were observed among adenovirus vector mutants. Furthermore, incorporation of a 7-lysine-residue motif at the C-terminal end of CAR-nonbinding short fiber efficiently achieved transduction of target cells via the heparan-containing receptor. Our results demonstrated that the natural tropism of adenovirus in vivo is influenced not only by fiber-CAR interaction but also by fiber shaft length. Furthermore, our strategy may be useful for retargeting adenovirus to particular tumors and tissue types with specific receptors.

Full text

JOURNAL OF VIROLOGY, Feb. 2003, p. 2512–2521 Vol. 77, No. 4
0022-538X/03/$08.00⫹0 DOI: 10.1128/JVI.77.4.2512–2521.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Reduction of Natural Adenovirus Tropism to the Liver by both
Ablation of Fiber-Coxsackievirus and Adenovirus Receptor
Interaction and Use of Replaceable Short Fiber
Takafumi Nakamura,
1,2
* Kenzo Sato,
2
and Hirofumi Hamada
1
Department of Molecular Medicine, Sapporo Medical University, S1 W17, Chuo-ku, Sapporo 060-8556,
1
and
Department of Molecular Biology, Faculty of Medicine, Tottori University, Yonago 683-8503,
2
Japan
Received 3 June 2002/Accepted 15 November 2002
The initial recognition and binding of adenovirus vector to the host cell surface is mediated by interaction
between the adenovirus fiber knob protein and its receptor, the coxsackievirus and adenovirus receptor (CAR).
This natural tropism of adenovirus vector needs to be ablated in order to achieve targeted gene transfer. To
this end, we noted that adenovirus serotype 40 (Ad40) contains two distinct long and short fibers; the short
fiber is unable to recognize CAR, while the long fiber binds CAR. We generated adenovirus serotype 5-based
mutants with chimeric Ad40-derived fibers, which were composed of either long or short shafts together with
CAR binding or nonbinding knobs. The capacity of these adenovirus mutants for in vitro and in vivo gene
transfer to liver cells was examined. In the case of primary human hepatocytes displaying a high expression
level of CAR and ␣v integrin, both CAR binding ability and fiber shaft length played important roles in efficient
transduction. Most significantly, the high transduction efficiency observed in the liver and spleen following
intravenous administration of adenovirus vector was dramatically reduced by both ablation of fiber-CAR
interaction and the use of replaceable short fiber. In other tissues displaying a low level of transduction, no
significant differences in transduction efficiency were observed among adenovirus vector mutants. Further-
more, incorporation of a 7-lysine-residue motif at the C-terminal end of CAR-nonbinding short fiber efficiently
achieved transduction of target cells via the heparan-containing receptor. Our results demonstrated that the
natural tropism of adenovirus in vivo is influenced not only by fiber-CAR interaction but also by fiber shaft
length. Furthermore, our strategy may be useful for retargeting adenovirus to particular tumors and tissue
types with specific receptors.
Because adenovirus vectors are capable of efficiently deliv-
ering genes to a variety of cell types, they have been used in a
number of gene therapy approaches (14, 42). The initial rec-
ognition and binding of adenovirus vector to the host cell
surface is mediated by interaction between the adenovirus fiber
knob protein and the coxsackievirus and adenovirus receptor
(CAR) (3, 32, 40). However, the natural tropism of adenovirus
vector for CAR makes it impossible to restrict gene delivery to
specific cells in an efficient and safe manner. This natural
tropism may limit widespread application of adenovirus vec-
tors. Therefore, several studies have focused on ablation of
adenovirus vector tropism as a first step to redirection of ad-
enovirus vectors to specific cell and tissue targets (2, 4, 10, 18,
19, 20, 33).
A number of strategies to alter adenovirus vector tropism
have been based on modification of viral capsid protein, in
particular fiber protein (48). Chimeric vectors containing ade-
novirus serotype 3 (Ad3) (38), Ad7 (12), Ad11 (37), Ad17 (6),
or Ad35 (35, 36) fibers instead of Ad5 fiber, which recognize
receptors other than CAR, resulted in infection through CAR-
independent pathways. Furthermore, it was noted that the
natural tropism of Ad5 was removed by altering specific amino
acid residues in the fiber knob that are involved in CAR bind-
ing (2, 4, 18, 19, 20, 33).
It is well known that intravenous administration of an ade-
novirus vector results mostly in hepatocyte transduction (2, 10,
16, 20). This liver tropism prevents adenovirus vectors from
being used to target other cell types by intravenous injection.
Nevertheless, intravenously administered CAR interaction-de-
ficient adenovirus vectors primarily localized to the liver, sim-
ilar to the wild-type Ad5 vector (2, 20). Other factors apart
from CAR interaction may play a significant role in the liver
tropism displayed by adenovirus vectors. In this context, it has
recently been reported that removal of both CAR and integrin
interaction leads to effective reduction of liver tropism (10).
We noted that Ad40 contains two distinct long and short
fibers (7, 17, 22, 32, 39). The shaft domain from the Ad40 short
fiber contains 12 ␤sheets, while its knob region was hypothe-
sized not to bind to any receptor, including CAR. On the other
hand, the shaft from the Ad40 long fiber contains 21 ␤sheets,
while its equivalent knob region can bind CAR (32). Interest-
ingly, the N-terminal tail regions of the Ad40 short and long
fibers, which are involved in incorporating the fiber into the
penton base (7, 8), are more analogous to the corresponding
amino acid sequence regions of the Ad5 fiber than to other
adenovirus serotypes (7). As shown in Fig. 5, the adenoviral
fiber could be incorporated into the penton base in Ad5 capsid.
In addition, the CAR-binding knob from both Ad5 and Ad40
long fibers begin with a TLWT hinge sequence, whereas the
CAR-nonbinding knob from the Ad40 short fiber begins with
* Corresponding author. Present address: Molecular Medicine Pro-
gram, Mayo Foundation, Guggenheim 18, 200 First St. SW, Rochester,
MN 55905. Phone: (507) 538-0161. Fax: (507) 284-8388. E-mail:
Nakamura.Takafumi@mayo.edu.
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