Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
1999-02-09
2004-09-28
Falk, Anne-Marie (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C435S320100, C435S455000
Reexamination Certificate
active
06797494
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to stable self-replicating episomal expression vectors for expressing a gene of interest in a host cell.
BACKGROUND OF THE INVENTION
The expression of a foreign gene in a host cell is generally achieved by transferring the gene into the host cell using a gene transfer vector. Gene transfer vectors are available in the art and include for example retrovirus vectors, adenoviral vectors and adenoassociated viral vectors. Many transfer vectors operate by integrating at least the transferred gene, if not the complete gene transfer vector, into the host cell chromosome, although non-integrating transfer vectors are known in the art. The efficiency of stable integration of transfected gene constructs is generally very inefficient (1: 10
3
-10
6
). The disadvantage of using viral vector-based gene transfer is that the amount of genetic material that the vector is able to accommodate is limited by the genetic packaging limitations of the virus. Gene transfer vectors which include large fragments of inserted genetic material are difficult to produce at a sufficiently high titre to be of practical value. Therefore, it is difficult to include additional genetic material in a virus-based vector, for example, gene regulatory elements, without deleteriously affecting stable gene transfer.
Locus Control Regions (LCRs) (Grosveld et al.,
Cell
51:975-985 (1987)), also known as Dominant Activator Sequences, Locus Activating Regions or Dominant Control Regions, confer tissue-specific, integration-site independent copy number-dependent expression on a linked gene that has been integrated into the chromosome of a host cell. LCRs were originally discovered in the human globin gene system, which exhibited strong position effects when integrated into a chromosome of a host cell in a tissue of a transgenic mouse or a mouse erythro-leukaemia (MEL) cell (see, for example, Magram et al.,
Nature
315:338-340 (1985); Townes et al.,
EMBOJ
. 4:1715-1723 (1985); Kollias et al.,
Cell
46:89-94(1986); Antoniou et al.,
EMBO J
. 7:377-384 (1988)). Position effects were overcome when the LCRs were linked directly to such transgenes (Grosveld et al., supra). Other LCRs have since been identified, including the &bgr;-globin LCR (&bgr;LCR) which promotes gene expression in erythroid tissue and the CD2 LCR which promotes gene expression in T cells (see, for example, Greaves et al.,
Cell
56:979 (1989), European Patent Application EP-A-0 668 357), themacrophage-specific lysozyme LCR (Boniferet al., (1985, 1990)), and a class II MHC LCR (Carson et al.,
Nucleic Acids Res
. 21,9:2065-2072 (1993)).
The present invention provides a stable gene transfer system which, when present in a host cell, confers stable and tissue-restricted expression of a gene of interest carried in the vector.
SUMMARY OF THE INVETNION
This invention provides self-replicating, LCR-containing, episomal expression vectors into which a gene of interest is inserted for expression of the gene in cells of a specific tissue-type.
The invention therefore encompasses a self-replicating episomal DNA expression vector for expressing a gene of interest in a host cell in a tissue-restricted manner, the vector comprising: (a) an origin of replication capable of directing replication of the DNA expression vector in cells of the specific type of tissue; and (b) an LCR, or component thereof, which when operatively linked to a gene of interest and present in a host cell directs expression of the gene in a tissue-restricted manner.
A vector according to the invention also may include the gene of interest inserted into a cloning site and operatively linked to the LCR.
The term gene is used to define any DNA sequence capable of being expressed. The gene of interest may be a foreign or heterologous gene, that is, a gene that is either not normally found in the genomic DNA of the host cell or is not normally expressed in that host cell. The gene of interest also may be an artificial DNA sequence.
The tissue-restricted expression of a gene of interest is mediated in vectors of the invention using an appropriate LCR, or components or portions thereof, which confer tissue-type specific expression on the gene of interest.
As used herein, a locus control region (LCR) is defined as a genetic element which is obtained from a tissue-specific locus of a eukaryotic host cell and which, when linked to a gene of interest and integrated into a chromosome of a host cell, confers tissue-specific, integration-site independent (position independent), copy number-dependent expression on the gene of interest. An LCR that is usefil according to the invention possesses these characteristics when integrated into chromosomal DNA, and will retain the ability to confer tissue-type restricted expression of a linked gene when present in a self-replicating episomal vector according to the invention. A component of an LCR refers to a portion of an LCR that also confers tissue-restricted gene expression when linked to a gene and integrated into a self-replicating episomal vector. An LCR may be identified structurally in that it is associated with one or more DNase I hypersensitive sites in its natural chromosomal context, and a component of an LCR useful according to the invention will also encompass at least one DNase I hypersensitive site.
An enhancer is defined herein as a genetic element which increases the level of transcription of a linked gene when present on a self-replicating episomal vector, but which does not confer tissue-specific gene expression when present on the vector.
It is preferred that the vector comprises a component of an LCR which confers tissue-specific expression and contains at least one DNase I hypersensitive site. In the human &bgr;-globin LCR, the preferred component of the LCR consists essentially of HS3; also preferred is the human &bgr;-globin LCR excluding site HS2; also preferred are sites HS3 and HS4 together without site HS2.
The invention also encompasses a pair of vectors comprising a self-replicating episomal DNA expression system for expressing a gene of interest in a host cell in a tissue-restricted manner, the pair of vectors comprising: a first vector comprising (a) an origin of replication; (b) an LCR, or component thereof, which when operatively linked to a gene of interest and present in a host cell directs expression of the gene in a tissue-restricted manner; and (c) a cloning site for a gene of interest; and a second vector comprising (d) an origin of replication; and (e) a sequence encoding a replication protein, the replication protein being necessary for replication of the origin of replication.
In another embodiment, the second vector also may include an LCR, which may be the same LCR as is present on the first vector or may be a different LCR which specifies the same or at least an overlapping tissue specificity as the first LCR such that the gene of interest and the viral replication gene are expressed in some of the same cells.
It is preferred that for red cell-restricted gene expression, the &bgr;-globin LCR from the &bgr;-globin locus be used. As used herein, red cells refer to cells of erythroid lineage.
It is preferred that for T-cell restricted gene expression, the CD2 LCR from the CD2 locus be used, or a component thereofcontaining at least one DNase I hypersensitive site that directs T-cell restricted gene expression in an episomal context. It is preferred that for class II MHC-bearing cell restricted gene expression the class II MHC LCR be used, or a component thereof containing at least one DNase I hypersensitive site that directs MHC-bearing cell restricted gene expression in an episomal context. It also is preferred that for macrophage cell restricted gene expression the macrophage/lysozyme LCR be used, or a component thereof containing at least one DNase I hypersensitive site that directs macrophage-cell restricted gene expression in an episomal context.
The term episomal vector refers to a nucleic acid vector which may be linear or circular, and which is usually double-stranded in f
Antoniou Michael
Grosveld Frankin G.
Falk Anne-Marie
Medical Research Council
O'Connor P.C. Cozen
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