Recombinant cell line produces adenoviral gene products E1...

Details Recombinant cell line produces adenoviral gene products E1... Recombinant cell line produces adenoviral gene products E1...

2000-12-22

2002-10-01

Salimi, Ali R. (Department: 1648)

Chemistry: molecular biology and microbiology

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

C435S325000, C435S069100, C435S320100, C424S233100, C424S199100

Reexamination Certificate

active

06458578

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to a recombinant cell line, specifically one that produces adenoviral E1 and DEF-A and/or DEF-B gene products.
BACKGROUND OF THE INVENTION
Modified adenoviruses have proven convenient vector systems for investigative and therapeutic gene transfer applications, and adenoviral viral systems present several advantages for such uses. The development of adenoviral vectors rests on an understanding of viral genetics and molecular biology. Structurally, all adenoviral virions are nonenveloped capsids with a number of surface proteins. Through interactions of these capsid proteins with the surface of a host cell, the virus is internalized and encased in an clathrin-coated organelle resembling an endocytotic vessel (Pastan et al.,
Concepts in Viral Pathogenesis
, Notkins and Oldstone, eds.
Springer-Verlag, New York. pp. 141-46 (1987)). The acidic condition within the vesicle alters the surface configuration of the virus, resulting in vesicle rupture and release of the virus into the cytoplasm of the cell, where it is partially freed of associated proteins while being transported to the nucleus. Once an adenoviral genome is within the host cell nucleus, its expression proceeds through a highly ordered and well characterized cascade. Groups of adenoviral genes (i.e., translation units) are typically organized into common transcription units (“regions”), each having at least one distinct promoter. The transcript from each region is processed after transcription to generate the multiple mRNA species corresponding to each viral gene. Generally, the separate regions are either “early” or “late,” although some genes are expressed as early and late.
Cellular transcription factors first bind to the upstream enhancer of the first early (E1A) region of the adenoviral genome. The E1A gene products, in turn, regulate the expression of other early promoters, one of which (E2B) drives the expression of the transcription unit including three early genes involved in adenoviral DNA replication (Doefler, pages 1-95, in
Adenovirus DNA, the Viral Genome, and Expression
, Nojhoff, Boston, (1986)). These three proteins (the precursor terminal protein (pTP), the single-stranded DNA binding protein (ssDBP), and the DNA polymerase (pol)) form a tight unit with at least three cellular proteins to drive priming and elongation of the viral genome (Bodnar et al.,
J. Virol
., 63, 4344-53 (1989); Schnack et al.,
Genes Devel
., 4, 1197-1208 (1990); Pronk et al.,
Clomosoma
, 102, S39-S45 (1992); Kelly et al., pages 271-308, in
The Adenoviruses
(H. S. Ginsberg, ed.), Plenum Press, New York (1984)).
Once viral DNA replication commences, the activity of the early promoters declines (Sharp et al., pages 173-204, in
The Adenoviruses
(H. S. Ginsberg, ed.), Plenum Press, New York (1984)), as does the expression of cellular genes, due to the activity of the viral host shut-off early gene products. Conversely, promoters controlling the expression of the late genes become active beginning with the onset of viral DNA replication (Thomas et al.,
Cell
, 22, 523-33 (1980)). Indeed, DNA replication appears necessary for the expression of some late genes. For example, while the major late promoter (MLP) exhibits some activity at early times, only the promoter proximal genes are expressed (Shaw et al.,
Cell
, 22, 905-16 (1980); Winter et al.,
J. Virol
., 65, 5250-59 (1991)). However, the activity of the MLP sharply increases following the onset of viral DNA replication (Shaw et al., supra), resulting in the expression of all the MLP gene products (Doeller et al., supra; Thomas et al., supra; Nevins et al.,
Nature
, 290, 113-18 (1981)). The structure of this promoter has been extensively characterized (see, e.g., Lu et al.,
J. Virol
., 71(1), 102-09 (1997); Lutz et al.,
J. Virol
., 70(3), 1396-1405 (1996); Reach et al.,
EMBO J
., 10(11), 3439-46 (1991); Reach et al.,
J. Virol
., 64(12), 5851-60 (1990); Brunet et al.,
Mol. Cell. Biol
., 7(3), 1091-1100 (1987); Miyamoto et al.,
EMBO J
., 4, 3563-70 (1985)). In particular, the MLP of the Ad5 serotype has three upstream promoter elements, two downstream elements and an initiator element (INR, SEQ ID NO:1) located at the start site. The three upstream elements are an inverted CAAT box (i.e., GTTA) located 76 base pairs upstream of the start site, an upstream promoter element (UPE, SEQ ID NO:2) located 63 base pairs upstream of the start site, and the TATA box (sequence: TATAAAA), located 31 base pairs upstream of the start site. The two downstream elements are DE1 (SEQ ID NO:3), located 86 base pairs downstream of the start site, and DE2 (SEQ ID NO:4), located 101 base pairs downstream of the start site. These various promoter elements interact with viral and cellular proteins to drive late transcription of the MTLU. For example, two proteins (DEF-A and DEF-B) bind to the downstream elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (Tribouley et al.,
J. Virol
, 68, 4450-57 (1994)), which has been cloned (van Beveren et al.,
Gene
, 16, 179-89 (1981)). In addition, as mentioned, E1A gene products drive some MLP activity during the early stage of infection
Post-transcriptional processing of the major late transcription unit (MTLU) gives rise to five families of late mRNA, designated respectively as L1 to L5 which encode structural components of the viral capsid (Shaw et al.,
Cell
, 22, 905-916 (1980)). These proteins are highly toxic to cells, and they can potentiate immune responses against infected cells (see, e.g., Yang et al.,
Proc. Nat. Acad. Sci
. (
USA
), 91, 4407-11 (1994)). This immune response leads to tissue swelling and destruction of the transduced cells, shortening the period of time transgenes are expressed in the cells. “First generation” adenoviral vectors have been engineered to silence the adenoviral genome with the aim of reducing these deleterious effects. Because, as mentioned, the E1A gene products begin the cascade of viral gene expression, the earliest adenoviral vectors lacked functional E1A regions. For example, insertion of an exogenous gene into the E1 region results in recombinant vectors that can express the exogenous gene but not the E1A gene. The recombinant adenoviruses must be propagated either in complementary cells or in the presence of a helper virus to supply the impaired or absent essential E1 products (Davidson et al.,
J. Virol
., 61, 1226-39 (1987); Mansour et al.,
Mol. Cell Biol
., 6, 2684-94 (1986)).
While such first generation viruses have proven effective in several gene transfer applications, they are not optimal for all uses. In particular, because they must be grown in the presence of E1 complementing DNA, at some frequency recombination events can generate a replication competent adenovirus (RCA). RCA contamination of viral stocks is problematic because RCAs can outgrow recombinant stocks and transform host cells. Moreover, at higher multiplicity of infections (m.o.i.s), several adenoviral promoters are active even in the absence of the E1A gene products, which can lead to the production of cytotoxic adenoviral proteins (Nevins,
Cell
, 26, 213-20 (1981); Nevins et al.,
Curr. Top. Microbiol. Immunol
., 113, 15-19 (1984)). An additional disadvantage of first generation vectors is largely attributable to this background expression of late gene products. For example, such residual late gene expression can potentiate host immune responses eliminating virally transduced cells (see, e.g., Yang et al., supra; Gilgenkrantz et al.,
Hum. Gene. Ther
., 6, 1265-74(1995); Yang et al.,
J. Virol
., 69, 2008-15(1995); Yang et al.,
J. Virol
., 70, 7209-12 (1996)).
One approach for blocking late gene expression is to selectively block viral replication by mutating the virus such that it fails to express one or more of the three E2B enzymes involved in viral DNA replication. However, while E1A-deficient viruses lacking E2B function can be generated, the approach requires the use of complementing

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