Sustained production of recombinant gamma interferon using an Epstein-Barr virus replicon

Claim 2

An Epstein-Barr virus based expression vector having the identifying characteristics of p222 (pEBv-gamma-IFN), p239 (pEBV-gamma-IFN), or p147 (pEBV-gamma-IFN).

Claim 3

A host cell transformed by a vector of claim 1.

Claim 4

A recombinant DNA expression vector comprising: an Epstein-Barr virus origin of replication; an EBNA-1 gene; and a human gamma interferon expression cassette comprising a promoter region, an open reading frame encoding human gamma interferon and a polyadenylation region.

Claim 5

A recombinant DNA expression vector comprising: an Epstein-Barr virus origin of replication; an EBNA-1 gene; and a human gamma interferon expression cassette comprising a promoter region, an open reading frame encoding human gamma interferon and a polyadenylation region.

Claim 6

The vector of claim 3, wherein said human gamma interferon expression cassette and said EBNA-1 gene are oriented in the same transcriptional direction.

Claim 7

The vector of claim 4, comprising a selectable marker expression cassette, and wherein said human gamma interferon expression cassette includes an enhancer region.

Claim 8

The vector of claim 5, wherein said enhancer region is selected from the group consisting of M-MSV enhancer and H-CMV enhancer.

Claim 9

The vector of claim 6, wherein said promoter region is selected from the group consisting of M-MT promoter and SV40 early promoter.

Claim 10

The vector of claim 7, wherein said selectable marker expression cassette comprises a hygromycin phosphotransferase expression cassette

Claim 11

The vector of claim 8, comprising a multiple cloning site.

Claim 12

The vector of claim 9, wherein said multiple cloning site is derived from pUC12.

Claim 13

The vector of claim 10, comprising a prokaryotic replicon.

Claim 14

The vector of claim 11, wherein said prokaryotic replicon is derived from pBR322.

Claim 15

A mammalian host cell transformed by the vector of claim 3.

Claim 16

A mammalian host cell transformed by the vector of claim 12.

Claim 17

The host cell of claim 13, wherein said host cell is capable of expressing and secreting human gamma interferon at a rate between about 4500 and about 8500 U/ml/day.

Claim 18

The hoet cell of claim 14, wherein said host cell is capable of expressing and secreting human gamma interferon at a rate between about 4500 and about 8500 U/ml/day.

Claim 19

A process for producing human gamma interferon comprising: providing a stable, extrachromosomally replicable vector capable of directing the expression of a DNA sequence encoding human gamma interferon in mammalian cells, said vector comprising a cis-acting Epstein-Barr virus origin of replication; a trans-acting EBNA-1 gene; a pUC12 multiple cloning site; a selectable marker expression cassette oriented in an opposite transcriptional direction as said EBNA-1 gene and comprising a hygromycin phosphotransferase expression cassette; a human gamma interferon expression cassette oriented in the same transcriptional direction as said EBNA-1 gene and comprising, in order of transcription, an enhancer region selected from the group consisting of M-MSV and H-CMV enhancer, a promoter region selected from the group consisting of M-MT promoter and SV40 early promoter, a nucleotide sequence encoding human gamma interferon and an SV40-derived polyadenylation sequence; transforming said mammalian cells with said vector; and maintaining said transformed mammalian cells under conditions permitting the expression of said human gamma interferon.

Claim 20

A process for producing human gamma interferon comprising: providing a stable, extrachromosomally replicable vector capable of directing the expression of a DNA sequence encoding human gamma interferon in mammalian cells, said vector comprising a cis-acting Epstein-Barr virus origin of replication; a trans-acting EBNA-1 gene; a pUC12 multiple cloning site; a selectable marker expression cassette oriented in an opposite transcriptional direction as said EBNA-1 gene and comprising a hygromycin phosphotransferase expression cassette; a human gamma interferon expression cassette oriented in the same transcriptional direction as said EBNA-1 gene and comprising, in order of transcription, an enhancer region selected from the group consisting of M-MSV and H-CMV enhancer, a promoter region selected from the group consisting of M-MT promoter and SV40 early promoter, a nucleotide sequence encoding human gamma interferon and an SV40-derived polyadenylation sequence; transforming said mammalian cells with said vector; and maintaining said transformed mammalian cells under conditions permitting the expression of said human gamma interferon.

Claim 21

The process of claim 17, wherein said vector is selected from the group consisting of p147, p222 and p239.

Claim 22

The process of claim 17, further comprising the step of recovering said human gamma interferon.

Claim 23

The process of claim 18, further comprising the step of recovering said human gamma interferon.