Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-09-28
2002-11-12
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S455000, C435S456000, C435S457000, C536S023100, C536S023720, C536S024100
Reexamination Certificate
active
06479279
ABSTRACT:
FIELD OF THE INVENTION
The invention relates in general to episomal vectors.
BACKGROUND OF THE INVENTION
In lower organisms, such as prokaryotes and budding yeast, replication origins contain welldefined cis-sequences called “replicators” and interaction of these sequences with a specific initiator protein complex leads to the initiation of DNA synthesis in these cells (Jacob et al., 1963; Stillman, 1994 and references therein; DePamphilis, 1993). Extrachromosomal replicators, generally, in addition to their origin function, encode functions that assure equal distribution of replicated molecules (i.e., partitioning) between daughter cells at cell division. For prokaryotic plasmids these partitioning functions are well studied and can be provided by several different mechanisms in bacterial cells (Nordström, 1990). In higher organisms, less is known about mechanisms for partitioning of extrachromosomal replicators. For artificial plasmids in yeast, chromosomal centromeres can provide this function. In metazoan cells, one well studied example of a stable extrachromosomal replicator exists—the latent origin oriP from Epstein-Barr Virus (EBV). The maintenance function of EBV requires the viral replication factor EBNA-1 and a series of binding sites for EBNA-1 termed the family of repeats (FR). A model that has been suggested for the function of the EBNA-1/FR combination is that EBNA-1 bound to FR provides physical retention of the oriP plasmids in the cell nucleus (Krysan et al., 1989).
Papillomaviruses are also capable of stable extrachromosomal replication. Infection and transformation of the cells by papillomaviruses follows single hit kinetics. (Dvoretzky et al., 1980). Papillomavirus genomes are maintained as multicopy nuclear plasmids in transformed cells. The viral life-cycle can be viewed as three stages (Botchan et al., 1986). First, following initial entry, the papillomaviral genome is amplified in the cell nucleus, i.e., viral DNA is synthesized faster than chromosomal DNA and the copy-number is increased. The second stage represents maintenance of the viral DNA at a constant copy-number and latent phase of the viral infection is established. During the third, vegetative, stage of the viral life-cycle viral DNA amplification is initiated again, late proteins are synthesized and viral particles are assembled.
The E1 and E2 proteins are the only viral factors required for initiation of papillomavirus DNA replication (Ustav and Stenlund 1991; Ustav et al., 1991; Yang et al., 1991; Chiang et al., 1992; Kuo et al., 1994). A similar, if not identical, set of cellular replication factors and enzymes, in addition to viral initiator proteins, is utilized by SV40 (Tsurimoto et al., 1990; Weinberg et al., 1990) and BPV-1 (Muller et al., 1994) at the origin of replication to initiate DNA synthesis. Analysis of the essential cis-sequences shows that the BPV-1 minimal origin (Ustav et al., 1993) resembles a typical eukaryotic origin of replication (DePamphilis, 1993) and it has been suggested that this similarity could also be extended to the mechanisms of replication of all papovaviruses (Nallaseth and DePamphilis, 1994; Bonne-Andrea et al., 1995). However, the ability of the papillomaviruses to persist as plasmids distinguishes papillomaviruses from other papovaviruses. It has been known for more than 10 years that BPV-1 replicates in transformed cells as a multicopy nuclear plasmid, which can persist in the tissue culture cells over long periods of time (Law et al., 1981). This indicates that papillomaviruses have efficient mechanisms for segregation, i.e., control of copy-number and partitioning, in the transformed cells.
The role of viral factors, cis-acting sequences and cellular factors in long-term persistence of papillomaviruses, which relates to the segregation functions of viral DNA, is not clearly understood. That is, the regions of the viral genome which specify copy number are not identified in the prior art; nor are the regions of the viral genome which participate with the host cell to ensure proper segregation of the viral genome during partitioning. Much more is understood with respect to the initial amplification stage of the papillomavirus life-cycle.
Bovine Papillomavirus (13BPV) and Human Papillomaviruses (HPVs) persist as stably maintained plasmids in mammalian cells. Transient assays, i.e., on the order of several hours to 3-4 days, have been used to define the minimal origin of replication (MO) which is required for transient replication in BPV (Ustav et al., EMBO J, 10, 4231-4329, 1991) and for several HPV subtypes. Two trans-acting factors encoded by BPV and HPVs, namely E1 and E2, have been identified in transient assays which are necessary to mediate replication in many cell types via MO (Ustav et al., EMBO J., 10, 449-457 (1991); Ustav et al., EMBO J, 10, 4231-4329, (1991); Ustav et al., PNAS, 90, 898-902 (1993).) E1 and E2 from BPV will replicate via the BPV MO and via the MO of many HPV subtypes. (Chiang et al., PNAS, 89, 5799-5803 (1992). E1 and E2 from HPV will replicate via the BPV MO and via the MO of many HPV subtypes. (Chiang et al., PNAS, 89, 5799-5803 (1992). Replication of plasmids containing the above elements is high level but transient in eukaryotic cells. Plasmid loss is rapid in the presence and absence of selective pressure.
The papillomavirus life cycle has been the subject of much research. Different portions of the viral genome have been tested in short-term, i.e., transient, transcription or replication assays. See, for example, Szymanski et al., 1991, Jour. Virol. 11:5710; Vande Pol et al., 1990, Jour. Virol 64:5420; Sowden et al., 1989, Nucl. Acids Res. 17:2959; Stenlund, 1987, Science 236:1666; Sedman et al., 1995, Eur. Jour. Mol. Biol. 14:6218; Haugen et al., 1988, Eur. Jour. Mol. Biol. 7:4245; and Kuo et al., 1994, Jour. Biol. Chem. 269:24058.
The BPV 69% transforming region has been used to introduce the rat preproinsulin gene into mouse cells (Sarver et al., 1981, Mol. Cell. Biol. 6:486).
The PMS1 and PMS2 regions of BPV have been reported to “independently support” extrachromosomal replication of the Tn5 neomycin gene in cells that provide viral factors in trans. PMS-1 (plasmid maintenance sequence-1) is localized within a 521 bp region mapping at positions 6945-7476 of the BPV genome, and PMS-2 has been localized to a 140 bp region within the putative open reading frame for the E1 protein, which maps at positions 1515-1655 of the BPV genome. It has been reported that recombinant plasmids carrying either of the PMS elements are unrearranged and stably maintained at a constant copy number. In addition, E1, E6 and E7 are identified as candidate factors for trans regulation of the plasmid state. See Lusky et al., 1984, Cell 36:391, and Lusky et al., 1986, Jour. Virol. 11:729.
Woo et al., W094/12629 report a vector containing a papilloma virus origin of replication, the “vector maintenance sequence” described in Lusky et al., 1984, supra, a therapeutic nucleic acid, and an E2 gene sequence or an E1/E2 chimeric gene. Woo et al. suggest that such a vector may be tested for stable episomal maintenance over a period of 2-30 days post-transfection. The “vector maintenance sequence” of Lusky et al., 1984, which is described in Woo et al., is shown herein not to be capable of providing long-term vector persistence.
Mutations in the E2 gene have a pleiotropic effect on viral gene functions, including oncogenic transformation. These effects may be the result of the requirement for E2 expression to regulate viral transcription (see DiMaio and Neary, 1989, The Genetics of bovine papillomavirus type 1 papillomaviruses and human cancer. (Ed. N. Pfister), CRC Press, Boca Raton, Fla.). The BPV-1 E2 protein has been shown to activate viral enhancers in trans (Spalholz et al., 1985, Cell 42:183). The E2 open reading frame has been shown to encode a site-specific DNA binding protein that can bind to several sites within the E2 responsive enhancers 1 and 2 (Androphy et al., 1987, Nature 325:70; Moskaluk et al., 1987, Proc. Nat. Aca. Sci. 84:1215).
Estonian Biocentre
Guzo David
Kathleen Madden Williams (Palmer & Dodge, LLP)
LandOfFree
Episomal vectors and uses thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Episomal vectors and uses thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Episomal vectors and uses thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2969881