RCA-free adenoviral vector system and propagation method

Details RCA-free adenoviral vector system and propagation method RCA-free adenoviral vector system and propagation method

1999-05-27

2002-11-19

Priebe, Scott D. (Department: 1632)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S320100, C435S325000, C435S366000, C435S456000

Reexamination Certificate

active

06482616

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to recombinant, multiply deficient adenoviral vectors and complementing cell lines and to the therapeutic use of such vectors.
BACKGROUND OF THE INVENTION
During the winter and spring of 1952-1953, Rowe and his colleagues at the National Institutes of Health (NIH) obtained and placed in tissue culture adenoids that had been surgically removed from young children in the Washington, D.C. area (Rowe et al.,
Proc. Soc. Exp. Biol. Med.,
84, 570-573 (1953)). After periods of several weeks, many of the cultures began to show progressive degeneration characterized by destruction of epithelial cells. This cytopathic effect could be serially transmitted by filtered culture fluids to established tissue cultures of human cell lines. The cytopathic agent was called the “adenoid degenerating” (Ad) agent. The name “adenovirus” eventually became common for these agents. The discovery of many prototype strains of adenovirus, some of which caused respiratory illnesses, followed these initial discoveries (Rowe et al., supra; Dingle et al.,
Am. Rev. Respir. Dis.,
97, 1-65 (1968); reviewed in Horwitz, “Adenoviridae and their replication,”
In Virology,
Fields et al., eds., 2nd ed., Raven Press Ltd., New York, N.Y., pp. 1679-1721 (1990)).
Over 40 adenoviral subtypes have been isolated from humans and over 50 additional subtypes have been isolated from other mammals and birds (reviewed in Ishibashi et al., “Adenoviruses of animals,”
In The Adenoviruses,
Ginsberg, ed., Plenum Press, New York, N.Y., pp. 497-562 (1984); Strauss, “Adenovirus infections in humans,”
In The Adenoviruses,
Ginsberg, ed., Plenum Press, New York, N.Y., pp. 451-596 (1984)). All these subtypes belong to the family Adenoviridae, which is currently divided into two genera, namely
Mastadenovirus
and
Aviadenovirus
. All adenoviruses are morphologically and structurally similar. In humans, however, adenoviruses show diverging immunological properties and are, therefore, divided into serotypes. Two human serotypes of adenovirus, namely Ad2 and Ad5, have been studied intensively and have provided the majority of information about adenoviruses in general.
Adenoviruses are nonenveloped, regular icosahedrons, 65-80 nm in diameter, consisting of an external capsid and an internal core. The capsid is composed of 20 triangular surfaces or facets and 12 vertices (Horne et al.,
J. Mol. Biol.,
1, 84-86 (1959)). The facets are comprised of hexons and the vertices are comprised of pentons. A fiber projects from each of the vertices. In addition to the hexons, pentons, and fibers, there are eight minor structural polypeptides, the exact positions of the majority of which are unclear. One minor polypeptide component, namely polypeptide IX, binds at positions where it can stabilize hexon-hexon contacts at what is referred to as the group-of-nine center of each facet (Furcinitti et al.,
EMBO,
8, 3563-3570 (1989)). The minor polypeptides VI and VIII are believed to stabilize hexon-hexon contacts between adjacent facets, and the minor polypeptide IIIA, which is known to be located in the regions of the vertices, is suggested to link the capsid and the core (Stewart et al.,
Cell,
67, 145-154 (1991)).
The viral core contains a linear, double-stranded DNA molecule with inverted terminal repeats (ITRs), which vary in length from 103 bp to 163 bp (Garon et al.,
PNAS USA
69, 2391-2394 (1972); Wolfson et al.,
PNAS USA,
69, 3054-3057 (1972); Arrand et al.,
J. Mol. Biol.,
128, 577-594 (1973); Steenberg et al.,
Nucleic Acids Res.,
4, 4371-4389 (1977); and Tooze,
DNA Tumor Viruses,
2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory. pp. 943-1054 (1981)). The ITRs harbor origins of DNA replication (Garon et al., supra; Wolfson et al., supra; Arrand et al., supra; Steenberg et al., supra). The viral DNA is associated with four polypeptides, namely V, VII, &mgr;, and terminal polypeptide (TP). The 55 kd TP is covalently linked to the 5′ ends of the DNA via a dCMP (Rekosh et al.,
Cell,
11, 283-295 (1977); Robinson et al.,
Virology,
56, 54-69 (1973)). The other three polypeptides are noncovalently bound to the DNA and fold it in such a way as to fit it into the small volume of the capsid. The DNA appears to be packaged into a structure similar to cellular nucleosomes as seen from nuclease digestion patterns (Corden et al.,
PNAS USA,
73, 401-404 (1976); Tate et al.,
Nucleic Acids Res.,
6, 2769-2785 (1979); Mirza et al.,
Biochim. Biophys. Acta,
696, 76-86 (1982)).
The overall organization of the adenoviral genome is conserved among serotypes, such that specific functions are similarly positioned. The Ad2 and Ad5 genomes have been completely sequenced and sequences of selected regions of genomes from other serotypes are available.
Adenovirus begins to infect a cell by attachment of the fiber to a specific receptor on the cell membrane (Londberg-Holm et al.,
J. Virol.,
4, 323-338 (1969); Morgan et al.,
J. Virol.,
4, 777-796 (1969); Pastan et al., “Adenovirus entry into cells: some new observations on an old problem,”
In Concerts in Viral Pathogenesis,
Notkins et al., eds., Springer-Verlag, New York, N.Y., pp. 141-146 (1987)). Then, the penton base binds to an adenoviral integrin receptor. The receptor-bound virus then migrates from the plasma membrane to clathrin-coated pits that form endocytic vesicles or receptosomes, where the pH drops to 5.5 (Pastan et al.,
Concepts in Viral Pathogenesis,
Notkins and Oldstone, eds. Springer-Verlag, N.Y. pp. 141-146 (1987)). The drop in pH is believed to alter the surface configuration of the virus, resulting in receptosome rupture and release of virus into the cytoplasm of the cell. The viral DNA is partially uncoated, i.e., partially freed of associated proteins, in the cytoplasm while being transported to the nucleus.
When the virus reaches the nuclear pores, the viral DNA enters the nucleus, leaving most of the remaining protein behind in the cytoplasm (Philipson et al.,
J. Virol.,
2, 1064-1075 (1968)). However, the viral DNA is not completely protein-free—at least a portion of the viral DNA is associated with at least four viral polypeptides, namely V, VII, TP and &mgr;, and is converted into a viral DNA-cell histone complex (Tate et al.,
Nucleic Acids Res.,
6, 2769-2785 (1979)).
The cycle from cell infection to production of viral particles lasts 1-2 days and results in the production of up to 10,000 infectious particles per cell (Green et al.,
Virology,
13, 169-176 (1961)). The infection process of adenovirus is divided into early (E) and late (L) phases, which are separated by viral DNA replication, although some events which take place during the early phase also take place during the late phase and vice versa. Further subdivisions have been made to fully describe the temporal expression of viral genes.
During the early phase, viral mRNA, which constitutes a minor proportion of the total RNA present in the cell, is synthesized from both strands of the adenoviral DNA present in the cell nucleus. At least five regions, designated E1-4 and MLP-L1, are transcribed (Lewis et al.,
Cell,
7, 141-151 (1976); Sharp et al.,
Virology,
75, 442-456 (1976); Sharp, “Adenovirus transcription,”
In The Adenoviruses
, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 173-204 (1984)). Each region has a distinct promoter(s) and is processed to generate multiple mRNA species, and, therefore, each region may be thought of as a gene family.
The products of the early (E) regions serve regulatory roles for the expression of other viral components, are involved in the general shut-off of cellular DNA replication and protein synthesis, and are required for viral DNA replication. The intricate series of events regulating early mRNA transcription begins with expression of immediate early regions E1A, L1 and the 13.5 kd gene (reviewed in Sharp (1984), supra; Horwitz (1990), supra). Expression of the delayed early regions E1B, E2A, E2B, E3 and E4 is dependent on the E1A gene products. Three promoters, the E2 promoter at 7

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