Chimeric adenovirus fiber protein

Details Chimeric adenovirus fiber protein Chimeric adenovirus fiber protein

1999-06-04

2003-06-10

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

C530S324000, C530S350000, C435S320100, C536S023100, C536S023400, C536S023720

Reexamination Certificate

active

06576456

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to a recombinant adenovirus comprising a chimeric adenoviral fiber protein and the use of a recombinant adenovirus comprising a chimeric adenoviral fiber protein in gene therapy.
BACKGROUND OF THE INVENTION
Adenoviruses belong to the family Adenoviridae, which is divided into two genera, namely
Mastadenovirus
and
Aviadenovirus.
Adenoviruses are nonenveloped, regular icosahedrons 65-80 nm in diameter (Home et al.,
J. Mol. Biol.,
1, 84-86 (1959)). The capsid is composed of 252 capsomeres of which 240 are hexons and 12 are pentons (Ginsberg et al.,
Virology,
28, 782-783 (1966)). The hexons and pentons are derived from three different viral polypeptides (Maizel et al.,
Virology,
36, 115-125 (1968); Weber et al,
Virology,
76, 709-724 (1977)). The hexon comprises three identical polypeptides of 967 amino acids each, namely polypeptide II (Roberts et al.,
Science,
232, 1148-1151 (1986)). The penton contains a penton base, which is bound to the capsid, and a fiber, which is noncovalently bound to and projects from the penton base. The fiber protein comprises three identical polypeptides of 582 amino acids each, namely polypeptide IV. The adenovirus serotype 2 (Ad2) penton base protein is an 8×9 nm ring-shaped complex composed of five identical protein subunits of 571 amino acids each, namely polypeptide III (Boudin et al.,
Virology,
92, 125-138 (1979)). Proteins IX, VI, and III
a
are also present in the adenoviral coat and are thought to stabilize the viral capsid (Stewart et al.,
Cell,
67, 145-154 (1991); Stewart et al., EMBO J., 12(7), 2589-2599 (1993)).
Once an adenovirus attaches to a cell, it undergoes receptor-mediated internalization into clathrin-coated endocytic vesicles of the cell (Svensson et al., J. Virol., 51, 687-694 (1984); Chardonnet et al.,
Virology,
40, 462-477 (1970)). Virions entering the cell undergo a stepwise disassembly in which many of the viral structural proteins are shed (Greber et al,
Cell,
75, 477-486 (1993)). During the uncoating process, the viral particles cause disruption of the cell endosome by a pH-dependent mechanism (Fitzgerald et al.,
Cell,
32, 607-617 (1983)), which is still poorly understood. The viral particles are then transported to the nuclear pore complex of the cell (Dales et al.,
Virology,
56, 465-483 (1973)), where the viral genome enters the nucleus, thus initiating infection.
An adenovirus uses two separate cellular receptors, both of which must be present, to efficiently attach to and infect a cell (Wickham et al.,
Cell,
73, 309-319 (1993)). First, the Ad2 fiber protein attaches the virus to a cell by binding to an, as yet, unidentified receptor. Then, the penton base binds to &agr;
v
integrins, which are a family of a heterodimeric cell-surface receptors that mediate cellular adhesion to the extracellular matrix molecules fibronectin, vitronectin, laminin, and collagen, as well as other molecules (Hynes,
Cell,
69, 11-25 (1992)), and play important roles in cell signaling processes, including calcium mobilization, protein phosphorylation, and cytoskeletal interactions (Hynes, supra).
The fiber protein is a trimer (Devaux et al.,
J. Molec. Biol.,
215, 567-588 (1990)) consisting of a tail, a shaft, and a knob. The fiber shaft region is composed of repeating 15 amino acid motifs, which are believed to form two alternating b-strands and b-bends (Green et al., EMBO J., 2, 1357-1365 (1983)). The overall length of the fiber shaft region and the number of 15 amino-acid repeats differ between adenoviral serotypes. For example, the Ad2 fiber shaft is 37 nm long and contains 22 repeats, whereas the Ad3 fiber is 11 mn long and contains 6 repeats. The receptor binding domain of the fiber protein is localized in the knob region encoded by the last 200 amino acids of the protein (Henry et al.,
J. of Virology,
68(8), 5239-5246 (1994)). The regions necessary for trimerization are also located in the knob region of the protein (Henry et al. (1994), supra). A deletion mutant lacking the last 40 amino acids does not trimerize and also does not bind to penton base (Novelli et al.
Virology,
185, 365-376 (1991)). Thus, trimerization of the fiber protein is necessary for penton base binding. Nuclear localization signals that direct the protein to the nucleus to form viral particles following its synthesis in the cytoplasm are located in the N-terminal region of the protein (Novelli et al. (1991), supra). The fiber, together with the hexon, are the main antigenic determinants of the virus and also determine the serotype specificity of the virus (Watson et al.,
J. Gen. Virol.,
69, 525-535 (1988)). The fiber protein is glycosylated with single N-acetyl-glucosamine residues; however, the functional significance of the glycosylation remains unclear (Caillet-Boudin et al.,
Eur. J. Biochem.,
184, 205-211 (1989)).
Over ten fiber proteins from different adenoviral serotypes have been sequenced, only to reveal a larger sequence diversity than that observed among other adenoviral proteins. For example, the knob regions of the fiber proteins from the closely related Ad2 and Ad5 serotypes are only 63% similar at the amino acid level (Chroboczek et al.,
Virology,
186, 280-285 (1992)), whereas their penton base sequences are 99% identical. Ad2 and Ad5 fiber proteins, however, both likely bind to the same cellular receptor, since they cross-block each other's binding. In contrast, Ad2 and Ad3 fibers are only 20% identical (Signas et al.,
J. of Virology,
53, 672-678 (1985)) and presumably bind to different receptors, since each fails to cross-block the other's binding (Defer et al.,
J. of Virology,
64(8), 3661-3673 (1990)). Ad3 fiber utilizes sialic acid as its receptor, whereas Ad2 fiber does not. Pretreatment of cells with neuraminidase or periodate abrogates Ad3, but not Ad2, binding. Also, soluble analogues of sialic acid block Ad3, but not Ad2, binding. However, sequence comparisons of the Ad2 and Ad3 fiber genes do show distinct regions of conservation. Most of these regions are also conserved in the other human adenoviral fiber genes. Nonhuman adenoviral fiber genes show less homology to human serotypes but still trimerize. The receptors used by nonhuman serotypes are unknown.
Recombinant adenoviral vectors have been used for the cell-targeted transfer of one or more recombinant genes to diseased cells or tissue in need of treatment. Such vectors are characterized by the advantage of not requiring host cell proliferation for expression of adenoviral proteins (Horwitz et al., In
Virologv
, Raven Press, New York, vol. 2, pp. 1679-1721 (1990); and Berkner,
BioTechniques,
6, 616 (1988)), and, if the targeted tissue for somatic gene therapy is the lung, these vectors have the added advantage of being normally trophic for the respiratory epithelium (Straus,
In Adenoviruses
, Plenan Press, New York, pp. 451-496 (1984)).
Other advantages of adenoviruses as potential vectors for human gene therapy are as follows:
(i) recombination is rare; (ii) there are no known associations of human malignancies with adenoviral infections despite common human infection with adenoviruses; (iii) the adenoviral genome (which is a linear, double-stranded DNA) can be manipulated to accommodate foreign genes that range in size; (iv) an adenoviral vector does not insert its DNA into the chromosome of a cell, so its effect is impermanent and unlikely to interfere with the cell's normal function; (v) the adenovirus can infect non-dividing or terminally differentiated cells, such as cells in the brain and lungs; and (vi) live adenovirus, having as an essential characteristic the ability to replicate, has been safely used as a human vaccine (Horwitz et al. (1990), supra; Berkner et al. (1988), supra; Straus et al. (1984), supra; Chanock et al., JAMA, 195, 151 (1966); Haj-Ahmad et al.,
J. Virol.,
57, 267 (1986); and Ballay et al., EMBO, 4, 3861 (1985)).
A drawback to adenovirus-mediated gene therapy is that significant decreases in gene expression are observed afte

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