Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-11-16
2003-05-06
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S069100, C435S070100, C435S366000, C435S372000, C435S320100, C424S233100
Reexamination Certificate
active
06558948
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a permanent amniocytic cell line comprising at least one nucleic acid which brings about expression of the gene products of the adenovirus E1A and E1B regions. The present invention further relates to the production of a permanent amniocytic cell line and to its use for producing gene transfer vectors and/or adenovirus mutants. Further aspects are the use of amniocytes and of the adenoviral gene products of the E1A and E1B regions for producing permanent amniocytic cell lines.
Adenoviruses
Adenoviruses are a relatively homogeneous group of viruses characterized by an icosahedral capsid which consists mainly of the virally encoded hexon, penton and fiber proteins, and of a linear, double-stranded DNA genome with a size of about 36 kilobases (kb). The viral genome contains at the ends the inverted terminal repeat sequences (ITRs) which comprise the viral origin of replication. There is furthermore at the left-hand end of the genome the packaging signal which is necessary for packaging of the viral genome into the virus capsids during an infection cycle. Adenoviruses have been isolated from many species. There are more than 40 different human serotypes based on parameters which discriminate between the various serotypes, such as hemagglutination, tumorigenicity and DNA sequence homology (Wigand et al., in: Adenovirus DNA, Doerfler ed., Martinus Nijoff Publishing, Boston, pp. 408-441, 1986). Adenoviral vectors to date are usually derived from serotypes 2 (Ad2) and 5 (Ad5). Infections by Ad2 and Ad5 are endemic in humans. Ad2 and Ad5 are not oncogenic in humans and have good safety documentation because vaccinations have been performed on military personnel successfully and without complications in the USA (Pierce et al., Am. J. Epidemiol. 87, 237-246, 1968). The biology of adenoviruses is relatively well understood because adenoviruses have played an essential part in molecular biology as experimental tool for elucidating various fundamental biological principles such as DNA replication, transcription, RNA splicing and cellular transformation. Adenoviral particles enter the cell during an infection through receptor-mediated endocytosis in which, according to the current view, interaction of the knob domain of the fiber protein with the coxsackie adenovirus receptor (CAR) mediates adhesion of the virus particle to the cell surface (Bergelson et al., Science 275, 1320-1323, 1997). In a second step there is internalization of the virus particle, for which interaction of the penton base with integrins plays an essential part (Wickham et al., Cell 73, 309-319, 1993). After the particle has entered the cell, the viral genome gets into the cell nucleus as DNA-protein complex. The adenoviral infection cycle is divided into an early and a late phase which are separated by the start of adenoviral replication (Shenk, in: Virology, Fields ed., Lippincott-Raven Publishing, Philadelphia, pp. 2111-2148, 1996). In the early phase there is expression of the early viral functions E1, E2, E3 and E4. The late phase is characterized by transcription of late genes which are responsible for the expression of viral structural proteins and for the production of new viral particles.
E1A is the first viral gene to be expressed by the viral chromosome after the cell nucleus is reached. The E1A gene codes for the 12S and 13S proteins which are formed by alternative splicing of the E1A RNA. The E1A proteins activate the transcription of a number of cellular and viral genes by interacting with transcription factors. The main functions of E1A are a) activation of the other early viral functions E1B, E2, E3 and E4 and b) inducing resting cells to enter the S phase of the cell cycle. Expression of E1A on its own leads to programmed cell death (apoptosis).
E1B is one of the early viral genes activated by E1A. The E1B gene codes for the E1B 55 kD protein and the E1B 19 kD protein, which result through alternative splicing of the E1B RNA. The 55 kD protein modulates the progression of the cell cycle by interacting with the p53 tumor suppressor gene, is involved in preventing the transport of cellular mRNA in the late phase of the infection, and prevents E1A-induced apoptosis of cells. The E1A 19 kD protein is likewise important for preventing E1A-induced apoptosis of cells.
All human adenoviruses are able to transform rodent cells in cell culture. As a rule, coexpression of E1A and E1B is necessry for oncogenic transformation.
The protein IX gene which codes for a structural component of the viral capsid is embedded in the E1B transcription unit.
The E2A and E2B genes code for various proteins which are essential for replication of the viral genome. These comprise the precursor protein of the terminal protein (pTP), the DNA polymerase (Pol) and the single strand-binding protein (SSBP). On replication, pTP binds to the ITRs of the viral genome. There it acts as protein primer for DNA replication, which is initiated by Pol together with cellular factors. Pol, SSBP and the cellular factor NFII, and presumably other factors, are necessary for DNA chain extension.
E4 codes for various proteins. Inter alia, the E4 34 kD protein blocks, together with the E1B 55 kD protein, the accumulation of cellular mRNAs in the cytoplasm, and at the same time it facilitates the transport of viral RNAs from the cell nucleus into the cytoplasm.
After the start of replication of the viral genome there is expression of viral structural proteins which are necessary for establishment of the viral capsid and for complexation of the viral DNA with virally encoded DNA-binding proteins. There is evidently initial formation of an empty capsid, into which the viral genome subsequently enters. A cis element on the viral genome is necessary for this process, the so-called packaging signal which is located at the left-hand end of the viral genome and, in the case of Ad5, extends over a region from base pair 260 to base pair 460 (Hearing et al., J. Virol. 62, 2555-2558, 1987; Graeble and Hearing, J. Virol. 64, 2047-2056, 1990). The packaging signal overlaps with the E1A enhancer which is essential for activity of the E1A promoter. The exact mechanism of the packaging of the viral genome into the virus capsids is not clear but it is probable that interaction of cellular and/or viral proteins with the packaging signal is necessary for this.
Adenovirus Vectors
Adenoviral vectors are particularly important as expression vectors, especially for the purpose of gene therapy. There are several reasons for this: the biology of adenoviruses has been thoroughly investigated. The virus particles are stable and can be produced relatively simply and in high titers. Genetic manipulation of the adenoviral genome is easy. Adenovirus vectors are able efficiently to transduce replicating and nonreplicating cells in vitro and in vi vo.
a) First-generation Adenoviral Vectors
First-generation adenoviral vectors (Gilardi et al., FEBS Letters 267, 60-62, 1990; Stratford-Perricaudet et al., Hum. Gene Ther. 1, 241-256, 1990) are characterized by deletions of the E1A and E1B genes. E1A and E1B have transforming and transactivating properties. In addition, E1A is necessary for activating viral genes and E1B is necessary for the accumulation of viral transcripts. In some vectors in addition E3 is deleted in order to increase the capacity for uptake of foreign DNA. E3 is dispensable for producing adenoviruses in cell culture. The capacity for uptake of foreign DNA is about 8 kb. First-generation adenovirus vectors have to date been produced mainly in 293 cells (see below) which complement the E1A and E1B deficit of the vectors.
b) Second-generation Adenoviral Vectors
Second-generation adenoviral vectors are characterized by deletions of E2 and/or E4 in addition to deletions of E1A and E1B (Engelhardt et al., Proc. Natl. Acad. Sci., USA 91, 6196-6200, 1994; Yang et al., Nature Genet., 7, 362-367, 1994; Gorziglia et al., J. Virol. 70, 4173-4178, 1996; Krougliak and Graham, Hum. Gene Ther. 6, 1575-1586, 1995; Zhou et al., J.
Kochanek Stefan
Schiedner Gudrun
Brown Stacy S.
Clark & Elbing LLP
Park Hankyel T.
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