Vectors and methods for gene transfer to cells

Details Vectors and methods for gene transfer to cells Vectors and methods for gene transfer to cells

1999-01-29

2002-10-15

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

Introduction of a polynucleotide molecule into or...

C435S320100, C435S325000, C435S455000, C530S330000, C530S329000, C530S328000, C530S327000, C530S326000, C530S324000, C530S350000

Reexamination Certificate

active

06465253

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention pertains to a chimeric adenovirus coat protein which is able to direct entry into cells of a vector comprising the coat protein that is more efficient than a similar vector having a wild-type adenovirus coat protein. Such a chimeric coat protein is a fiber, hexon, or penton protein. The present invention also pertains to a recombinant vector comprising such a chimeric adenoviral coat protein, and to methods of constructing and using such a vector.
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 of about 65 to 80 nanometers in diameter (Horne et al.,
J. Mol. Biol
., 1, 84-86 (1959)). The adenoviral 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 a 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 IIIa 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, as well as other molecules (Hynes,
Cell
, 69, 11-25 (1992)).
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 &bgr;-strands and &bgr;-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 nanometers long and contains 22 repeats, whereas the Ad3 fiber is 11 nanometers 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. 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)).
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 Virology
, Raven Press, New York, vol. 2, pp. 1679-1721 (1990); and Berkner,
BioTechniques
, 6, 616 (1988)). Moreover, 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: (i) recombination is rarely observed with use of such vectors; (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); PCT patent application WO 94/17832).
A drawback to adenovirus-mediated gene therapy is that significant decreases in gene expression are observed after two weeks following administration of the vector. In many therapeutic applications, the loss of expression requires re-administration of the viral vector. However, following re-administration, neutralizing antibodies are raised against both the fiber and hexon proteins of the viral vector (Wohlfart,
J. Virology
, 62, 2321-2328 (1988); Wohlfart et al.,
J. Virology
, 56, 896-903 (1985)). This antibody response against the virus can prevent effective re-administration of the viral vector.
Another drawback of using recombinant adenovirus in gene therapy is that certain cells are not readily amenable to adenovirus-mediated gene delivery. For instance, lymphocytes, which lack the &agr;
v
integrin adenoviral receptors, are impaired in the uptake of adenoviruses (Silver et al.,
Virology
165, 377-387 (1988); Horvath et al.,
J. Virology
, 62(1), 341-345 (1988)). This lack of ability to infect all cells has lead researchers to seek out ways to introduce adenovirus into cells that cannot be infected by adenovirus, e.g. due to lack of adenoviral receptors. In particular, the virus can be coupled to a DNA-polylysine complex containing a ligand (e.g., transferrin) for mammalian cells (e.g., Wagner et al.,
Proc. Natl. Acad. Sci
., 89, 6099-6103 (1992); PCT patent application WO 95/26412). Similarly, adenoviral fiber protein can be sterically blocked with antibodies, and tissue-specific antibodies can be chemically linked to

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