MXPA99011726A

MXPA99011726A - Compact epstein-barr virus replicons

Info

Publication number: MXPA99011726A
Application number: MXPA/A/1999/011726A
Authority: MX
Inventors: Seed Brian
Original assignee: Seed Brian; The General Hospital Corporation
Priority date: 1997-06-19
Filing date: 1999-12-15
Publication date: 2000-06-01

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

REPLICONES OF EPSTEIN-BARR COMPACT VIRUSES to Antecedent-bes of the Invention Epstein-Barr virus (EBV) is a human herpes virus with a latent phase characterized by the stable episomal spread of a circular form of viral DNA. Two discontinuous DNA elements are required for replication of the latent phase, the origin of replication acting in cis, OriP, and the nuclear antigen Epstein-Barr 1 (EBNAl), the only virally encoded protein necessary for replication (Yates et al., Proc. Nati, Acad. Sci. USA 81: 3806-10, 1984; Yates et al., Nature 313: 812-5, 1985). Most of the EBV vectors are extremely large, of the order of 10 kb or more (without insert), due to the large size of the EBNAl and OriP segments. The size of most existing EBNAl and OriP EBV segments has interfered with the development of improved expression vectors and the creation of compact, persistent gene and persistence expression cassettes that can be attached to other gene delivery vehicles. , such as retroviruses or adenoviruses. SUMMARY OF THE INVENTION In general, the invention relates to a nucleic acid sequence that 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 1-95 of SEQ ID NO: 1; the sequence of EBNAl includes approximately residues 627-1,718 of SEQ ID NO: 1; the nucleic acid sequence further includes a consensus polyadenylation sequence (eg, approximately the sequence of SEQ ID NO: 2); and the promoter is a viral promoter. In related aspects, the invention also relates to vectors and cells (e.g., mammalian cells, and preferably human cells) that include such nucleic acid sequences. Detailed Description First, the drawings will be described. Figure 1 is the sequence of a cassette of OriP and EBNA-1 of EBV. Figure 2 is the sequence of a compact, synthetic bi-directional polyadenylation sequence that can be used in conjunction with a compact EBV replicon. After extensive deletion and mutagenesis, it has been found possible to attach the cis- and trans-acting functions necessary for EBV episomal replication in a fragment of less than 2 kb. In particular, an exemplary fragment of 1,748 base pairs, which acts as a compact EBV replicon, is shown in Figure 1 (SEQ ID NO: 1). This fragment contains all the sequences necessary for efficient expression of the EBNA-1 protein, with the exception of a polyadenylation consensus sequence. The fragment is a Bgl2 to BamH1 segment that contains the OriP element between residues 1 and 495, a modified promoter of the herpes simplex 1 virus thymidine kinase gene between residues 496 to 616, and the coding sequence for a EBNAl deleted and modified between residues 627 and 1,718. Plasmid vectors based on this sequence replicate as episomes in the nucleus of transfected cells of non-rodent origin (Yates et al., 1985, supra). To minimize the overall sequence length, the above fragment was designed to be inserted upstream of a bi-directional polyadenylation sequence into an appropriate vector, an example of a compact, synthetic bi-directional polyadenylation sequence is provided in the 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 that supports episomal replication in a mammalian cell, said nucleic acid sequence comprising (a) a sequence of OriP and (b) a sequence of EBNAl operably linked to a promoter, said nucleic acid sequence having a length less than 3 kb.
2. The nucleic acid sequence of claim 1, wherein said nucleic acid has a length of less than 2 kb.
3. The nucleic acid sequence of claim 1, wherein said nucleic acid has a length of less than 1.8 kb.
The nucleic acid sequence of claim 1, wherein the OriP sequence comprises approximately residues 1-495 of SEQ ID NO: 1.
5. The nucleic acid sequence of claim 1, wherein said EBNAl sequence comprises approximately residues 627-1,718 of SEQ ID NO: 1.
6. The nucleic acid sequence of claim 1, said nucleic acid sequence further comprising a polyadenylation consensus sequence.
7. The nucleic acid sequence of claim 6, wherein said consensus polyadenylation sequence comprises approximately SEQ ID NO: 2.
8. The nucleic acid sequence of claim 1, wherein said promoter is a viral promoter.
9. 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.
10. A cell comprising the nucleic acid sequence of claim.