Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-11-23
2001-02-27
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S366000, C435S320100
Reexamination Certificate
active
06194212
ABSTRACT:
This invention relates to the use of scaffold attachment regions (SARs) to increase gene expression in primary non-proliferating cells i.e. in resting cells.
Eukaryotic chromosomes are organised into discrete chromatin domains which are thought to define independent units encompassing all required cis-regulatory elements for co-ordinated expression of the genes within the domain. These chromatin domains are bordered by sequences which specifically associate with the nuclear scaffold, or nuclear matrix, defining the boundaries of the chromatin domains. Such sequences are referred to as scaffold attachment region (SAR) or matrix attachment region (MAR). SAR elements are several hundred basepairs long and A/T rich (≧70%). Although cloned SAR and MAR elements share common structural features, no consensus sequence has been identified. SARs have been located upstream, downstream or within genes (introns) suggesting that they may represent functionally distinct classes (Bode J et al. 1995
Scaffold/Matrix
-
attachment regions
(
S/MAR
):
Structural properties creating transcriptionally active loci
Academic Press, Orlando). SAR elements can enhance expression of heterologous genes in transfection experiments in vitro and in transgenic mice. In some instances, it has been reported that SAR elements can confer position-independent expression to a linked transgene.
While transfected DNA integrates randomly into chromosomes, there is growing evidence that retroviral integration is not completely random (Shih, C. C 1988
Cell
53, 531-537, Rohdewohld, H et al 1987
J. Virol.
61, 336-343 and Mielke, C et al 1996
Biochemistry
35, 2239-2252). Notably, proviruses preferentially integrate into host SAR sequences (Mielke, C et al 1996) and into “open” chromatin characterised by sensitivity to DNaseI digestion (Rohdewohld, H et al 1987).
Our experience has shown that the regulation of gene expression is different for resting cells as opposed to proliferating cells. We have found that gene expression of transduced genes is significantly decreased in resting (i.e. not mitotically active) cells as compared to active cells. Low expression in resting cells is a problem when expression is desired in vivo, e.g., in gene therapy, because at any given time, most cells in the body (unlike most cells in cell cultures) are in a quiescent state. Thus, although methods are now available to permit and enhance integration of heterologous genetic material into normal resting cells, there are at present no established ways to enhance expression of the heterologous genetic material in such cells.
One might suppose that the difference in expression is due to limiting quantities of necessary transcription factors or to control by specific promoter/enhancer elements. Our research suggests, however, that this difference in expression between resting and proliferating cells is largely due to changes in chromatin structure mediated by the DNA SARs. Hereinafter, use of the term SAR will be understood to encompass scaffold and matrix attachment regions.
We have now discovered that SARs increase expression of heterologous genes in transduced eukaryotic resting primary cells, particularly in retrovirally transduced cells. The SAR sequence has no detectable influence on retroviral vector expression in transduced cell lines. In contrast, the SAR-containing vectors express at significantly higher levels compared to controls in resting primary T cells. For example, we have shown that in retrovirally transduced resting primary T cells, a SAR significantly increases expression of the heterologous gene, both in terms of percentage of cells expressing that gene and in terms of levels of expression per cell. This is the first demonstration that retroviral mediated transduction of a SAR and a heterologous gene in cis improves expression of that gene, and the first demonstration that co-transduction with a SAR and a heterologous gene improves expression of the gene in resting primary cells.
Vectors suitable for use in the present invention are chosen on the basis of their ability of causing integration with the host genetic material. Accordingly, retroviruses which include oncoviruses such as Moloney C type and lentiviruses are suitable for purposes of the present invention. The invention may also be practised by introducing the DNA by homologous recombination or by using artificial human chromosomes.
The invention thus provides, in a first embodiment
(i) Use of a SAR to increase gene expression in transduced cells for example resting cells, including resting progeny of transduced cells;
(ii) A method of increasing expression of a heterologous gene in a resting cell comprising transducing a cell, e.g., a non-immortal cell, with (i) the heterologous gene and (ii) one or more SARs.
A SAR for use in the present invention is not itself transcribed and translated to express a protein, nor is it a promoter or enhancer element for a gene; its effect on gene expression is mostly position-independent. By position-independent is understood that the SAR is placed within the vector and is not placed so as to destroy other functions required for gene transfer and expression for example the SAR should not be inserted in a position which blocks an essential LTR function. Preferably the SAR is at least 450 base pairs (bp) in length, preferably from 600-1000 bp, e.g., about 800 bp. The SAR is preferably AT-rich (i.e., more than 50%, preferably more than 70% of the bases are adenine or thymine), and will generally comprise repeated 4-6 bp motifs, e.g., ATTA, ATTTA, ATTTTA, TAAT, TAAAT, TAAAAT, TAATA, and/or ATATTT, separated by spacer sequences, e.g., 3-20 bp, usually 8-12 bp in length. The SAR may be from any eukaryote, preferably a mammal, most preferably a human. Suitably the SAR is the SAR for human IFN-&bgr; gene or fragment thereof, e.g., preferably derived from or corresponding to the 5′ SAR of human interpheron beta (IFN-&bgr;), Klehr, D et al. Scaffold-Attached Regions from the Human Interferon &bgr; domain Can Be Used To Enhance the Stable Expression of Genes under the Control of Various Promoters. Biochemistry 1991, 30, 1264-1270), e.g., a fragment of at least 450 base pairs (bp) in length, preferably from 600-1000 bp, e.g., about 800 bp, and being substantially homologous to a corresponding portion of the 5′ SAR of human IFN-&bgr; gene, e.g., having at least 80%, preferably at least 90%, most preferably at least 95% homology therewith. Especially preferred for use as a SAR in accordance with the present invention is the 800 bp Eco-RI-HindIII (blunt end) fragment of the 5′SAR element of IFN-&bgr; as described by Mielke, C et al. Biochemistry 1990 29: 7475-7485.
In a further embodiment, the invention provides:
(i) a retroviral vector comprising genetic material corresponding to (a) at least one SAR, and (b) at least one heterologous gene operatively linked to an expression control sequence, the heterologous gene (or at least one of the heterologous genes if there is more than one heterologous gene) being rev-M10 and the SAR or at least one SAR is derived from, obtainable from or corresponds to the 5′ SAR of the human interferon-&bgr; gene;
(ii) a packaging cell line transduced with a retroviral vector according to (i) ; and
(iii) a cellular composition comprising non-immortal eukaryotic cells (preferably a mammalian, e.g., human cell) transduced with a retroviral vector according to (i). Hereinafter (i), (ii) and (iii) above will be referred to as a retroviral vector of the invention, a packaging cell line of the invention and a cellular composition of the invention respectively.
Preferably, the retroviral vector is an amphotropic retroviral vector, preferably a vector characterized in that it has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV). murine stem cell virus (MSCV) or spleen focus forming virus(SFFV). Preferably, in the case of a vector according to
Agarwal Manju
Plavec Ivan
Veres Gabor
Karny Geoffrey M.
Novartis AG
Yucel Remy
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