Regulated protein expression in stably transfected mammalian cel

Details Regulated protein expression in stably transfected mammalian cel Regulated protein expression in stably transfected mammalian cel

1998-06-29

2000-10-31

Schwartzman, Robert A.

Chemistry: molecular biology and microbiology

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

4353201, 435325, 435363, 536 231, 536 2372, 536 241, C07H 2104, C12N 510, C12N 701, C12N 15864

Patent

active

06140103&

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to recombinant DNA molecules encoding products of interest, as well as to methods employing said molecules. In particular, it relates to recombinant DNA molecules capable of regulated expression of one or more genes in a host cell. In particular, the invention relates to expression of products which are in some way toxic when expressed in a host cell. A good example of such a product or products are the products of non-structural genes of parvoviruses (PV). Because these products are toxic to their host cells it is not feasible to arrange for constitutive expression of said products in host cells.
High level PV-protein expression in stably transfected mammalian cells is very often an elusive goal in trying to make host cells express PV-proteins genes introduced therein by recombinant DNA technology. For instance, high levels of PV-protein synthesis in human cells are of importance for the production of packaging cells for recombinant PV vectors. In such packaging cells, the recombinant PV. vector DNA is replicated and packaged into PV-capsids when PV-proteins are supplied in trans by the packaging cell.
The parvovirus Adeno-Associated Virus (AAV) is a non-pathogenic human parvovirus (reviewed in .sup.1, 2). The virus replicates to a single stranded DNA of approximately 4.6 kb. Both the plus and the minus strand are packaged and infectious. Efficient replication of AAV requires the co-infection of the cell by a helper virus such as Adenovirus or Herpes Simplex Virus. In the absence of a helper virus, no substantial replication of AAV is observed. AAV is therefore also classified as a "Dependovirus". When no helper virus is present, the AAV genome integrates into the host cell genome.
The wild-type virus has a strong preference (70%) for an integration site on the long arm of chromosome 19 (19 q13.3) .sup.3-5.
Following integration, the expression of the virus genes is not detectable. The integrated provirus replicates as a normal part of the host cell genome upon division of the transduced cell and ends up in both daughter cells. This stage of the virus life cycle is known as the latent stage. This latent stage is very stable, but can be interrupted upon infection of the transduced cell by a helper virus. Following infection by the helpervirus, AAV is excised from the host cell genome, expresses its genes and starts to replicate. During the early phase of this lytic cycle, the REP-genes are expressed. After approximately 16 hours, the capsid proteins VP1, VP2 en VP3 are expressed and the replicated virus is packaged into virions (structure of the AAV-genome and its genes are depicted in FIG. 1). The virions accumulate in the nucleus of the cell and are released when the cell lyses as a result of the accumulation of AAV and the helpervirus.
The AAV-genome consists of two genes rep and cap (FIG. 1). Three promoters (P5, P19 and P40) drive the synthesis of mRNAs coding for 4 Rep-proteins (Rep78, Rep68, Rep52 and Rep40) and three capsid proteins (VP1, VP2 and VP3). Both the Rep and the VP-proteins are encoded in overlapping reading frames. Therefore the Rep proteins and also the VP-proteins share a common carboxyterminal end but differ in their N-termini. The AAV-genome is flanked on both sides by a 145 bp sequence, called the Inverted Terminal Repeat (ITR). The ITR contains all information required in cis for replication, packaging and integration of the AAV-genome. During a productive infection, the P5-promoter is activated first and directs the production of Rep78 and Rep68. These proteins are essential for AAV-replication and trans regulation of viral genes. The large Rep-proteins activate the P19 and the P40 promoter. In a latent infection, however, Rep78 and Rep68 downregulate expression of the P5 promoter and help to maintain the latency of AAV (for a review see1). The smaller Rep-proteins, Rep52 and Rep40 are encoded by transcripts from the P19 promoter and are important for the formation of infectious virus .sup.6. The P40 promoter is the last promoter to become activated

REFERENCES:
patent: 5464758 (1995-11-01), Gossen et al.
Chang et al. (1989) Adeno-associated virus p5 promoter contains an adenovirus E1A-inducible element and a binding site for the major late transcription factor. J. Virol. 63:3479-3488, Aug. 1989.
Shi et al. (1991) Transcriptional repression by YY1, a human GLI-Kruppel-related protein, and relief of repression by adenovirus E1A protein. Cell 67:377-388, Oct. 1991.
Antoni et al. (1994) Adeno-associated virus rep protein inhibits human immunodeficiency virus type 1 production in human cells. J. Virol. 65:396-404, Jan. 1991.
Paulweber et al. (1993) The mechanism by which the human apolipoprotein B gene reducer operates involves blocking of transcriptional activation by hepatocyte nuclear factor 3. Mol. Cell. Biol. 13:1534-1546, Mar. 1993.
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