COMPACT EPSTEIN-BARR VIRUS REPLICONS

Abstract: Disclosed herein are nucleic acid sequences which support episomal replication in a mammalian cell. These nucleic acid sequences, which have a length of less than 3 kb, include (a) a deleted OriP sequence and (b) a deleted EBNA1 sequence operably linked to a promoter.

Description: COMPACT EPSTEIN-BARR VTRUS REPLICONS Background of the Invention Epstein-Barr virus (EBV) is a human herpes virus with a latent phase characterized by stable episomal propagation of a circular form of the viral DNA. Two discontinuous DNA elements are required for latent phase replication, the cis-acting origin of replication, OriP, and the Epstein-Barr Nuclear Antigen 1 (EBNAl), the sole virally encoded protein necessary for replication (Yates et al., Proc. Natl. Acad. Sci. USA 81 : 3806-10, 1984; Yates et al., Nature 313: 812-5, 1985). Most EBV vectors are very large, on the order of 10 kb or more (without insert), because of the large size of the EBNAl and OriP segments. The size of most existing EBV EBNAl and OriP segments has interfered with the development of improved expression vectors and the creation of compact gene expression and persistence cassettes which can be embedded in other gene delivery vehicles, such as retroviruses or adenoviruses. Summary of the Invention In general, the invention features a nucleic acid sequence which supports episomal replication in a mammalian cell. This nucleic acid sequence includes (a) a deleted OriP sequence and (b) a deleted EBNAl sequence operably linked to a promoter, and the nucleic acid sequence has a length of less than 3 kb, preferably less than 2 kb, and, most preferably less than 1.8 kb. In preferred embodiments, the OriP sequence includes approximately residues 1-495 of SEQ ID NO: 1; the EBNAl sequence includes approximately residues 627- 1718 of SEQ ID NO: 1 ; the nucleic acid sequence further includes a polyadenylation consensus sequence (for example, approximately the sequence of SEQ ID NO: 2); and the promoter is a viral promoter. In related aspects, the invention also features vectors and cells (for example, mammalian cells, and preferably human cells) that include such nucleic acid sequences. Detailed Description The drawings will first be described. Figure 1 is the sequence of a shortened EBV OriP and EBNA-1 cassette. Figure 2 is the sequence of a compact synthetic bidirectional polyadenylation sequence which may be used in conjunction with a compact EBV replicon. After extensive deletion and mutagenesis, it has been found possible to embed the cis- and trans-acting functions necessary for EBV episomal replication into a fragment of less than 2 kb. In particular, an exemplary fragment of 1748 base pairs which acts as a compact EBV replicon is shown in Figure 1 (SEQ ID NO: 1). This fragment contains all sequences needed for efficient expression of the EBNA-1 protein, with the exception of a polyadenylation consensus sequence. The fragment is a Bgl2 to BamHl segment which contains the OriP element between residues 1 and 495, a modified promoter from the Herpes simplex virus 1 thymidine kinase gene between residues 496 to 616, and the coding sequence for a deleted and modified EBNAl gene between residues 627 and 1718. Plasmid vectors based on this sequence replicate as episomes in the nucleus of transfected cells of nonrodent origin (Yates et al., 1985. supra). To minimize overall sequence length, the above fragment was designed to be inserted upstream from a bidirectional polyadenylation sequence in an appropriate vector. An example of a compact synthetic bidirectional polyadenylation sequence is provided in Figure 2 (SEQ ID NO: 2). Compact EBV replicons find use in gene therapy vectors, for example, in gene delivery vehicles such as expression vectors. Other embodiments are within the claims.

Claims: Claims 1. A nucleic acid sequence which supports episomal replication in a mammalian cell, said nucleic acid sequence comprising (a) an OriP sequence and (b) an EBNAl sequence operably linked to a promoter, said nucleic acid sequence having a length of less than 3 kb. 2. The nucleic acid sequence of claim 1, said nucleic acid having a length of less than 2 kb. 2a. The nucleic acid sequence of claim 1 , said nucleic acid having a length of less than 1.8 kb. 3. The nucleic acid sequence of claim 1, wherein said OriP sequence comprises approximately residues 1-495 of SEQ ID NO: 1. 4. The nucleic acid sequence of claim 1, wherein said EBNAl sequence comprises approximately residues 627-1718 of SEQ ID NO: 1. 5. The nucleic acid sequence of claim 1, said nucleic acid sequence further comprising a polyadenylation consensus sequence. 6. The nucleic acid sequence of claim 5, wherein said polyadenylation consensus sequence comprises approximately SEQ ID NO: 2. 7. The nucleic acid sequence of claim 1, wherein said promoter is a viral promoter. 8. A vector comprising the nucleic acid sequence of claim 1, said vector being capable of replicating as an episome in the nucleus of a transfected cell. 9. A cell comprising the nucleic acid sequence of claim 1.

Inventors: SEED BRIAN, SEED, BRIAN
Applicants: GEN HOSPITAL CORP, SEED BRIAN, SEED, BRIAN, THE GENERAL HOSPITAL CORPORATION
Application year: 1998
Main classification: A61K39
IPC codes: A61K35, A61K48, C07K14, C12N1, C12N5, C12N15