Enhancer sequences for late T cell expressed genes

Details Enhancer sequences for late T cell expressed genes Enhancer sequences for late T cell expressed genes

1999-06-30

2002-09-10

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S070100, C435S320100, C536S024100

Reexamination Certificate

active

06448039

ABSTRACT:

TECHNICAL FIELD
The field of this invention concerns isolated nucleic acid sequences that functions as a transcription enhancer elements for heterologous promoters and methods for using it to identify potential compounds that inhibit expression of native RANTES gene product.
BACKGROUND OF THE INVENTION
In humans, inflammatory processes are orchestrated in part by a family of soluble mediators called chemokines. One branch of this gene family, the “CC or &agr; chemokines”, includes RANTES, I-309, the monocyte chemotactic proteins MCP-1, MCP-2, MCP-3 and MCP-4 and the macrophage inflammatory proteins MIP-1&agr; and MIP-1&bgr;. The CC chemokines are pro-inflammatory agents that function as potent and highly selective chemoattractants for specific subsets of hematopoietic cells. MIP-1&bgr; is a chemoattractant for naive helper T cells and MIP-1&agr; attracts cytotoxic T cells and B lymphocytes, while I-309, MCP-1, MCP-2 and MCP-3 are selective for monocytes. RANTES is chemotactic for monocytes, eosinophils, natural killer cells and the “memory” population (CD45RO
+
) of T lymphocytes. RANTES is released from activated platelets and activates basophils to release histamine.
These multiple activities suggest a role for RANTES in both acute and chronic inflammatory processes. Indeed, high concentrations of RANTES have recently been implicated in the control of the human immunodeficiency virus.
RANTES is expressed as an immediate early gene (6-20 hours) by TNF&agr; stimulated renal tubular epithelium and mesangial cells, TNF&agr; and IL-1&bgr; activated synovial fibroblasts, and lipopolysaccaride induced monocytes. By contrast, RANTES is expressed in T cells “late” (3-5 days) following activation by antigen or mitogen. This “late” expression of RANTES by T cells is coincident with the development of T cell effector function, including the expression of perforin and granzymes by cytotoxic T lymphocytes. This pattern of expression is in marked contrast to the immediate early expression (6-20 hours) found for other T cell expressed cytokines, such as IL-2, IL-3, IL-4, IL-5, IL-6, and &ggr;IFN or the chemokines I-309, MIP-1&agr;, and MIP-1&bgr;.
A general model has been proposed for the pivotal role of RANTES in the induction, amplification and propagation of an inflammatory response. In this scheme, RANTES and other chemokines are induced rapidly within an inflammatory site and bind to the endothelium, where they attract monocytes and T cells by haptotactic mechanisms. In combination with the induced expression of specific integrin and immunoglobulin superfamily members, chemokines lead to mononuclear cell extravasation. The infiltrating cells then follow a haptotactic/chemotactic trail of chemokines into the interstitium. Within days, T lymphocytes, attracted to the inflammatory site, encounter antigen, become activated, differentiate, and strongly upregulate RANTES protein expression. This production of RANTES by T cells then amplifies and propagates the inflammatory response.
Understanding the transcriptional control of an early lymphokine, interleukin-2, has proven a powerful probe into the mechanism of action of the most potent immunosuppressive drugs in clinical use, cyclosporine and FK506. This information has lead to the development of new drugs and the elucidation of pathways involved in the early stages of T cell activation. Understanding the transcriptional control of the late expressed cytokine, RANTES, may similarly provide insight into the molecular pathways involved in later stages of T cell differentiation and lead to the development of novel immunotherapeutics.
The transcriptional machinery controlling RANTES expression differs among the various tissue types capable of expressing this pro-inflammatory cytokine. The large number of potential consensus transcription factor binding sites found within the immediate upstream region of RANTES is unusual and corroborates the multiple points of control of RANTES expression indicated by functional analyses with reporter genes. This complex system for transcriptional control of RANTES expression, with both early and late kinetics in a variety of different tissue types, indicates that diverse activation signals can give rise to a single common pathway of RANTES release, which in turn leads to the attraction of effector cells into an inflammatory site. This single common pathway, therefore, represents an important potential target for inhibition of the inflammatory response.
This invention provides methods for readily identifying compounds that inhibit RANTES expression in T lymphocytes. These compounds can then be used to control undesired inflammatory responses.
RELEVANT LITERATURE
For a general introduction to RANTES, see Schall,
Cytokine
3:165 (1992), Schall, et al.,
J. Immunol
. 141:1018 (1988) and Nelson, et al.,
J. Immunol
. 151:2601 (1993). Wiedermann, et al.,
Current Biol
. 3:735 (1993) and Pattison, et al.,
Lancet
343:209 (1994) detail the pivotal role of RANTES in the induction, amplification and propagation of an inflammatory response. And, Nelson, et al.,
J. Immunol
. 151:2601 (1993) describes the genomic organization and transcriptional regulation of the human RANTES gene. Baldwin and Sharp,
Proc. Nat. Acad. Sci
. 85:723 (1988) and Baeuerle and Henkel,
Annu. Rev. Immunol
. 12:141 (1994) describe the function and activation of NF-&kgr;B. Danoff, et al.,
J. Immunol
. 152:1182 (1994) describes the murine RANTES.
SUMMARY OF THE INVENTION
This invention is based on the discovery of nucleic acid sequences present in the human RANTES promoter that mediate upregulation of the RANTES gene late in the T cell developmental pathway.
The present disclosure provides an isolated nucleic acid sequence consisting essentially of the sequence of SEQ ID NO:1, or an isolated nucleic acid sequence consisting essentially of the sequence of SEQ ID NO:2, or an isolated nucleic acid sequence consisting essentially of the sequence of SEQ ID NO:3, or an isolated nucleic acid sequence consisting essentially of the sequence of SEQ ID NO:4, or an isolated nucleic acid sequence consisting essentially of the sequence of SEQ ID NO:5.
In one embodiment, the invention provides an isolated nucleic acid sequence the human RANTES enhancer element R(A) (SEQ ID NO:2), said sequence defined by its ability to inducibly express a gene when positioned upstream of the CAAT and TATA boxes of a minimal promoter with the promoter operably linked to the gene to form an expression cassette where the expression cassette is transfected into an activated peripheral blood lymphocyte cell which has been contacted with an amount of anti-CD3 antibody sufficient to induce the expression of the gene with the proviso that the nucleic acid sequence is not operably linked to the RANTES gene product nor to the native RANTES promoter sequences. This R(A) element can be operably linked to a heterologous gene. It can be positioned downstream of a
CAAT box and upstream of a TATA box and further separated from said TATA box by a functional NF&kgr;B binding site. The NF&kgr;B binding site can be the native RANTES NF&kgr;B site which has been rendered non-functional.
The R(A) element can also be combined with additional transcriptional elements to form an artificial RANTES promoter. Three elements that can be operably linked to the R(A) element are: (1) the R(C) binding site which is SEQ ID NO:3 corresponding to −182 to −169 of the human Rantes promoter; (2) the Region “C” sequence corresponding to −195 to −144 of the human Rantes promoter which is SEQ ID NO:4; or (3) the Region E sequence corresponding to −115 to −91 of the human Rantes promoter which is SEQ ID NO:5.
This R(A) element as well as the above-identified transcriptional regions can be recombined with additional nucleic acid sequence to form a vector. The R(A) element can be operably linked to a DNA sequence which encodes a heterologous protein. The heterologous protein can be selected from the group consisting of: hormones, viral capsid proteins, bacterial enzymes and mamma

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