Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-09-10
2002-09-24
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S235100, C435S320100, C435S325000, C435S069700, C435S069100, C530S350000, C530S300000, C530S402000, C536S023100, C536S023400, C536S024100
Reexamination Certificate
active
06455314
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an alternately targeted adenovirus and includes methods for producing and purifying such viruses as well as protein modifications mediating alternate targeting.
BACKGROUND OF THE INVENTION
The various physiological responses of a host animal to the presence of a virus depend on the different ways such viruses interact with the host animal, each of which is first mediated by the surface of the virus (“the virion”). The adenoviral virion is a non-enveloped icosahedron about 65-80 nm in diameter (Horne et al.,
J. Mol. Biol
., 1, 84-86 (1959)). It comprises 252 capsomeres—240 hexons and 12 pentons (Ginsberg et al.,
Virology
, 28, 782-83 (1966))—derived from three viral proteins (proteins II, III, and IV) (Maizel et al.,
Virology
, 36, 115-25 (1968); Weber et al.,
Virology
, 76, 709-24 (1977)). Proteins IX, VI, and IIIa, also present, stabilize the virion (Stewart et al.,
Cell
, 67, 145-54 (1991); Stewart et al.,
EMBO J
., 12(7), 2589-99 (1993)).
The hexon provides structure and form to the capsid (Pettersson, in
The Adenoviruses
, pp. 205-270, Ginsberg, ed., (Plenum Press, New York, N.Y., 1984)), and is a homotrimer of the protein II (Roberts et al.,
Science
, 232, 1148-1151 (1986)). The hexon provides the main antigenic determinants of the virus, and it also contributes to the serotype specificity of the virus (Watson et al.,
J. Gen. Virol
., 69, 525-35 (1988); Wolfort et al.,
J. Virol
., 62, 2321-28 (1988); Wolfort et al.,
J. Virol
., 56, 896-903 (1985); Crawford-Miksza et al.,
J. Virol
., 70, 1836-44 (1996)).
The hexon trimer is comprised of a pseudohexagonal base and a triangular top formed of three towers (Roberts et al., supra; Athappilly et al.,
J. Mol. Biol
., 242, 430-455 (1994)). The base pedestal consists of two tightly packed eight-stranded antiparallel beta barrels stabilized by an internal loop. The predominant antigenic and serotype-specific regions of the hexon appear to be in loops 1 and 2 (i.e., LI or l1, and LII or l2, respectively), within which are seven discrete hypervariable regions (HVR1 to HVR7) varying in length and sequence between adenoviral serotypes (Crawford-Miksza et al., supra).
The penton contains a base, which is bound to the capsid, and a fiber, which is non-covalently bound to and projects from, the penton base. The penton base, consisting of protein III, is highly conserved among serotypes of adenovirus, and (except for the enteric adenovirus Ad40 and Ad41) it has five RGD tripeptide motifs (Neumann et al.,
Gene
, 69, 153-57 (1988)). These RGD tripeptides apparently mediate adenoviral binding to &agr;
v
integrins, a family of a heterodimeric cell-surface receptors that also interact with the extracellular matrix and play important roles in cell signaling (Hynes,
Cell
, 69, 11-25 (1992)). These RGD tripeptides also play a role in endocytosis of the virion (Wickham et al. (1993), supra; Bai et al.,
J. Virol
., 67, 5198-3205 (1993)).
The adenoviral fiber is a homotrimer of the adenoviral polypeptide IV (Devaux et al.,
J. Molec. Biol
., 215, 567-88 (1990)), which has three discrete domains. The amino-terminal “tail” domain attaches non-covalently to the penton base. A relatively long “shaft” domain, comprising a variable number of repeating 15 residue &bgr;-sheets motifs, extends outwardly from the vertices of the viral particle (Yeh et al.,
Virus Res
., 33, 179-98 (1991)). Lastly, about 200 residues at the carboxy-terminus form the “knob” domain. Functionally, the knob mediates both primary viral binding to cellular proteins and fiber trimerization (Henry et al.,
J. Virol
., 68(8), 5239-46 (1994)). Trimerization also appears necessary for the amino terminus of the fiber to properly associate with the penton base (Novelli et al.,
Virology
, 185, 365-76 (1991)). In addition to recognizing cell receptors and binding the penton base, the fiber contributes to serotype integrity and mediates nuclear localization. Moreover, adenoviral fibers from several serotypes are glycosylated (see, e.g., Mullis et al.,
J. Virol
., 64(11), 5317-23 (1990); Hong et al.,
J. Virol
., 70(10), 7071-78 (1996); Chroboczek et al.,
Adenovirus Fiber
, p. 163-200 in “The Molecular Repertoire of Adenoviruses I. Virion Structure and Function,” W. Doerfler and P. Böhm, eds. (Springer, N.Y. 1995)).
Fiber proteins from different adenoviral serotypes differ considerably. For example, the number of shaft repeats differs between adenoviral serotypes (Green et al.,
EMBO J
., 2, 1357-65 (1983)). Moreover, the knob regions from the closely related Ad2 and Ad5 serotypes are only 63% similar at the amino acid level (Chroboczek et al.,
Virology
, 186, 280-85 (1992)), and Ad2 and Ad3 fiber knobs are only 20% identical (Signas et al.,
J. Virol
., 53, 672-78 (1985)). In contrast, the penton base sequences of Ad5 and Ad2 are 99% identical. Despite these differences in the knob region, a sequence comparison of even the Ad2 and Ad3 fiber genes demonstrates distinct regions of conservation, most of which are also conserved among the other human adenoviral fibers (see, e.g., FIGS.
1
and
2
).
One interaction between the adenoviral virion and the host animal is the process of cellular infection, during which the wild-type virion first binds the cell surface by means of a cellular adenoviral receptor (AR) (e.g., the coxsackievirus and adenovirus receptor (CAR), the MHC class I receptor, etc. (Bergelson et al.,
Science
, 275, 1320-23 (1997); Tanako et al.,
Proc. Nat. Acad. Sci
. (
USA
), 94, 3352-56 (1997)), Hong et al.,
EMBO J
., 16(9), 2294-06 (1997)). After attachment to an AR, the virus binds &agr;
v
integrins. Following attachment to these cell surface proteins, infection proceeds by receptor-mediated internalization of the virus into endocytotic vesicles (Svensson et al.,
J. Virol
., 51, 687-94 (1984); Chardonnet et al.,
Virology
, 40, 462-77 (1970)). Within the cell, virions are disassembled (Greber et al.,
Cell
, 75, 477-86 (1993)), the endosome disrupted (Fitzgerald et al.,
Cell
, 32, 607-17 (1983)), and the viral particles transported to the nucleus via the nuclear pore complex (Dales et al.,
Virology
, 56, 465-83 (1973)). As most adenoviral serotypes interact with cells through broadly disseminated cell surface proteins, the natural range of host cells infected by adenovirus is broad.
In addition to cellular infection, host animals react defensively to the presence of adenoviral virions through mechanisms that reduce the effective free titer of the virus. For example, host immune systems, upon exposure to a given adenoviral serotype, can efficiently develop neutralizing antibodies, greatly reducing the effective free titer of the virus upon repeat administration (see, e.g., Setoguchi et al.,
Am. J. Respir. Cell. Mol. Biol
., 10, 369-77 (1994); Kass-Eisler et al.,
Gene Ther
., 1, 395-402 (1994); Kass-Eisler et al.,
Gene Ther
., 3, 154-62 (1996)). Interestingly, such antibodies typically are directed against the same determinants of adenoviral serotype specificity, and are primarily directed to the hypervariable hexon regions and, to some extent, fiber and penton base domains (Watson et al., supra; Wolfort et al. (1988), supra; Wolfort et al. (1985), supra; Crawford-Miksza et al., supra). Of course, the presence of adenoviruses agglutinates red blood cells in humans in a serotype-dependent manner (Hierholzer,
J. Infect. Diseases
, 123(4), 541-50 (1973)). Additionally, adenoviral virions are actively scavenged from the circulation by cells of the reticuloendothelial system (RES) (see, e.g., Worgall et al.,
Hum Gene Ther
., 8, 1675-84 (1997); Wolff et al.,
J. Virol
., 71(1), 624-29 (1997)). In such a response, Kupffer cells, endothelial liver cells, or other RES cells scavenge the virus from the circulation (see generally, Moghini et al.,
Crit. Rev. Ther. Drug Carrier Sys
., 11(1), 31-59 (1994); Van Rooijen et al.,
J. Leuk. Biol
., 62, 702-09 (1997)). For example, virions can become opsonized, possibly though interaction between collectins and glycocylated viral proteins, triggering r
Bruder Joseph T.
Kovesdi Imre
Roelvink Petrus W.
Wickham Thomas J.
GenVec, Inc.
Guzo David
Leffers, Jr. Gerald G.
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