Regulators of type-1 tumor necrosis factor receptor and other cytokine receptor shedding Number:7,135,303 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides compositions and methods for the regulation of cytokine signaling through the Tumor Necrosis Factor (TNF) pathway. Specifically, the invention provides a novel gene, polypeptide and related compositions and methods for the regulation of ectodomain shedding. In preferred embodiments, methods and compositions for the regulation of TNF Type-1 Receptor ectodomain shedding are provided. The present invention finds use in therapeutics, diagnostics, and drug screening applications.<H Regulators, shedding, Regulators, of, t, 7135303.html, Patent, pto, 7135303

Title: Regulators of type-1 tumor necrosis factor receptor and other cytokine receptor shedding

Abstract: The present invention provides compositions and methods for the regulation of cytokine signaling through the Tumor Necrosis Factor (TNF) pathway. Specifically, the invention provides a novel gene, polypeptide and related compositions and methods for the regulation of ectodomain shedding. In preferred embodiments, methods and compositions for the regulation of TNF Type-1 Receptor ectodomain shedding are provided. The present invention finds use in therapeutics, diagnostics, and drug screening applications.

Patent Number: 7,135,303 Issued on 11/14/2006 to Levine

Inventors: Levine; Stewart (North Potomac, MD)
Assignee: The United States of America as represented by the Department of Health and Human Services (Washington, DC)
N/A (

Appl. No.: 10/220,443

Filed: February 28, 2001

PCT Filed: February 28, 2001

PCT No.: PCT/US01/06464

371(c)(1),(2),(4) Date: December 19, 2002

PCT Pub. No.: WO01/64856

PCT Pub. Date: September 07, 2001

Current U.S. Class: 435/7.72 ; 435/320.1; 435/325; 435/6; 435/69.1; 435/7.1; 514/44; 530/350; 536/23.5

Current International Class: G01N 33/53 (20060101); A61K 48/00 (20060101); C12N 5/06 (20060101); C12P 21/02 (20060101); C12Q 1/68 (20060101)

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Foreign Patent Documents


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WO 99 58559	Nov., 1999	WO
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Primary Examiner: Andres; Janet L.
Assistant Examiner: Emch; Gregory S
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd

Government Interests

This invention was made during the course of work supported by the United States Government under the National Institutes of Health. As such, the United States Government may have certain rights to this invention.

Parent Case Text

This application is the national stage entry of PCT/US01/06464, filed Feb. 28, 2001, which claims priority benefit to U.S. Provisional Patent Application No. 60/185,586, filed Feb. 28, 2000.

Claims

What is claimed is:

1. A method for promoting the shedding of the extracellular domain of at least one cytokine receptor, comprising the steps of: a) providing: i) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, and ii) one or more cells expressing a cytokine receptor on their plasma membrane extracellular surface, wherein the cytokine receptor is selected from the group consisting of a type-1 tumor necrosis factor receptor, a type I interleukin-1 cytokine receptor, a type II interleukin-1 cytokine receptor, and an interleukin-6 cytokine receptor alpha-chain gp80; and b) delivering said polypeptide to said one or more cells, whereby said polypeptide promotes the shedding of the cytokine receptor from the surface of said cells.

2. The method of claim 1, wherein said cytokine receptor is a type-1 tumor necrosis factor receptor.

3. The method of claim 1, wherein said cytokine receptor is a type I interleukin-1 cytokine receptor.

4. The method of claim 1, wherein said cytokine receptor is a type II interleukin-1 cytokine receptor.

5. The method of claim 1, wherein said cytokine receptor is an interleukin-6 cytokine receptor alpha-chain gp80.

6. The method of claim 1, wherein the one or more cells are cultured cells.

7. The method of claim 1, wherein the one or more cells are human cells.

8. The method of claim 2, wherein the one or more cells are human cells.

9. The method of claim 3, wherein the one or more cells are human cells.

10. The method of claim 4, wherein the one or more cells are human cells.

11. The method of claim 5, wherein the one or more cells are human cells.

12. The method of claim 1, wherein the one or more cells are in a tissue.

13. The method of claim 2, wherein the one or more cells are in a tissue.

14. The method of claim 3, wherein the one or more cells are in a tissue.

15. The method of claim 4, wherein the one or more cells are in a tissue.

16. The method of claim 5, wherein the one or more cells are in a tissue.

17. The method of claim 7, wherein the one or more cells are in a tissue.

Description

FIELD OF THE INVENTION

The present invention provides compositions and methods related to regulation of cytokine signaling through the Tumor Necrosis Factor (TNF) pathway. Specifically, the invention provides novel genes, polypeptides and related compositions and methods for the regulation of TNF Type-1 Receptor ectodomain shedding. It is contemplated that the compositions and methods of the present invention will also find use in the regulation of ectodomain shedding of other cytokine receptors. It is further contemplated that these compositions and methods will find use in therapeutics for the treatment of diseases and disorders of the immune system.

BACKGROUND OF THE INVENTION

Aberrant regulation of cytokine signaling results in a wide variety of hyper-inflammatory, autoimmune and immune-deficiency pathological conditions. Cytokines are a large and diverse group of molecules which mediate interactions between cells, ultimately regulating the wide variety of cells of the immune repertoire. Cytokine signaling mediates numerous facets of normal immune system physiology, including development, response, activation, maintenance, memory and apoptosis (Roitt et al. (Eds.), Immunology, Fifth Edition, Mosby International Publishers [1998]).

Tumor Necrosis Factor

Tumor necrosis factor-.alpha. (commonly written as TNF, but also written as tumor necrosis factor or TNF.alpha.) is a multifunctional cytokine mediating pleiotropic biological functions in both health and disease states. TNF is secreted primarily by monocytes and macrophages, but can also be secreted by other cell types. The list of processes regulated by TNF is extensive, and includes inflammation, immunoregulation, cyytotoxicity and antiviral effects (See e.g., Vilcek et al, J. Biol. Chem., 266:7313 7316 [1991]). TNF plays an integral role in destroying tumors, mediating responses to tissue injury, and protecting hosts from infections by various microorganisms (Vassali, Ann. Rev. Immunol., 10:411 452 [1992]). TNF also induces the transcriptional activation of numerous genes, including NF-.kappa.B and AP-1, with the consequent expression of pro-inflammatory and immunoregulatory genes (Rothe et al., Cell 83:1243 124 [1995]; Varfolomeev et al., J. Exp. Med., 183:1271 1275 [1996]; Chinnaiyan et al., J. Biol. Chem., 271:4961 4965 [1996]; Hsu et al., Immunity 4:387 396 [1996]; and Hsu et al., Cell 84:299 308 [1996]). TNF-mediated NF-.kappa.B activation is also an important negative feedback mechanism regulating apoptosis (Beg and Baltimore, Science 274:782 784 [1996]; Van Antwerp et al., Science 274:787 789 [1996]; and Wang et al., Science 274:784 787 [1996]).

TNF in Disease

TNF has also been implicated in the pathogenesis of a variety of diseases and disorders. It is theorized that these pathologies result from the aberrant regulation of TNF activity, in which the pathologies manifest as a result of excessive or insufficient TNF activity. Among the activities for which TNF is most noted are its pro-inflammatory actions, sometimes termed the "acute phase immune response." Unfortunately, if not properly regulated, these proinflammatory responses can result in tissue injury and chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, septic shock, cachexia, autoirumune disorders, graft-versus-host disease and insulin resistance (Piguet et al., J. Exp. Med., 166:1280 [1987]; Pujol-Borrell et al., Nature 326:304 306 [1987]; Tracey et al., Nature 330:662 664 [1987]; Oliff, Cell 54:141 142 [1988]; Vilcek et al., J. Biol. Chem., 266:7313 7316 [1991]; and Eigler et al., Immunol. Today 18:487 92 [1997]). Excessive TNF activity results in the detrimental effects of an exaggerated immune response demonstrated in some of these diseases, exemplified by overstimulation of interleukin-6 and granulocyte/macrophage-colony stimulating factor (GM-CSF) secretion, enhanced cytotoxicity of polymorphonuclear neutrophils, prolonged expression of cellular adhesion molecules, induction of procoagulant activity on vascular endothelial cells, increased adherence of neutrophils and lymphocytes, and stimulation of the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Vassali, Ann. Rev. Immunol., 10:411 452 [1992]; Vilcek et al., J. Biol. Chem., 266:7313 7316 [1992]; and Barbara et al., Immunol. and Cell Biol., 74:434 443 [1996]).

Recent evidence also implicates TNF activity in the pathogenesis of many infections. TNF is thought to play a central role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock, including fever, malaise, anorexia, and cachexia (Beutler et al., Nature 316:552 554 [1985]; Bauss et al, Infect. Immun., 55:1622 1625 [1987]; Tracey et al., Nature 330:662 664 [1987]; and Vassali, Ann. Rev. Immunol., 10:411 452 [1992]). Because TNF can mimic many of the biological effects of endotoxin, it is theorized that TNF is a central mediator responsible for the clinical manifestations of endotoxin-related and other critical illnesses (Waage et al., Lancet 1:355 357 [1987]; Cerami et al., Immunol. Today 9:28 [1988]; Mitchie et al., N. Eng. J. Med., 318:1481 1486 [1988]; Revhaug et al., Arch. Surg., 123:162 170 [1988]; and Michie et al., Ann. Surg., 209:19 24 [1989]).

Tumor Necrosis Factor Receptor

The numerous biological effects of TNF are now known to be mediated by two transmembrane receptors, the 55 kilodalton Type I receptor (also written as "CD120a," and referred to herein as "TNFR1") and the 75 kilodalton Type II receptor (also written as "CD120b," and referred to herein as TNFR2). Although both TNFR1 and TNFR2 demonstrate strong affinity for TNF.alpha., these two receptors demonstrate no apparent homology in their cytoplasmic (i.e., intracellular) domains. This fact is consistent with the observation that these two receptors transduce different signals to the nucleus via distinct signaling intermediates (Lewis et al., Proc. Natl. Acad. Sci. USA 88:2830 2834 [1991]; Tartaglia and Goeddel, Immunol. Today 13:151 153 [1992]; and Barbara et al., Imunol. Cell Biol., 74:434 443 [1996]).

Soluble TNF inhibitors have been identified in normal human urine, as well as in sera and other body fluids of patients with infectious, neoplastic and immunologic disorders. This observation ultimately led to the revelation that these soluble TNF inhibitors were actually the extracellular domains of TNF receptors derived by proteolytic cleavage of the transmembrane forms (Engelmann et al., J. Biol. Chem., 264:11974 11980 [1989]; Olsson et al., Eur. J. Haematol., 42:270 275 [1989]; Seckinger et al., J Biol. Chem., 264:11966 11973 [1989]; Engelmann et al., J. Biol. Chem., 265:1531 1536 [1990]; and Aderka et al., J. Exp. Med., 175:323 329 [1992]). In the case of the TNFR1, this proteolytic activity results in the cleavage and shedding of the extracellular N-terminal domain (also called the ectodomain). These free, soluble TNFR1 ectodomains ("sTNFR1s") have an affinity for TNF that is similar to that of intact membrane receptors. Due to this affinity, the free receptors are able to bind and sequester TNF, thereby inhibiting the biological action of TNF. Furthermore, the generation of sTNFR1 is also likely to suppress TNF signaling by reducing the number of functional TNF receptors acting at the cell membrane. The sTNFR1 ectodomains are also theorized to serve a more complex buffering function in the regulation of TNF activity (Aderka et al., J. Exp. Med., 175:323 329 [1992]; and Werb and Yan, Science 282:1279 1280 [1998]). The complexity of TNF signaling is fuirther illustrated by the observation that many of the stimuli that result in TNF release also result in the release of the soluble TNF receptor, suggesting that these soluble TNF inhibitors may serve as part of a regulated feedback mechanism to control TNF activity (Adreke et al., J. Exp. Med., 175:323 329 [1992]; and Porteu and Nathan, J. Exp. Med., 172:599 607 [1990]).

The importance of TNFR1 in the regulation of TNF activity in host defense, immunoregulation and development has been fuirther demonstrated in studies utilizing TNFR1 knockout mice. Mice deficient in TNFR1 show a variety of phenotypes, including phenotypes which mimic human immune disorders (Pfeffer et al., Cell 73:457 467 [1993]; Rothe, Nature 364:798 802 [1993]; Le Hir et al., J. Exp. Med., 183:2367 2372 [1996]; Matsumoto et al., Science 271:1289 1291[1996]; Mori et al. J. Immunol. 157:3178 3182 [1996]; Speiser et al., J. Immunol., 158:5185 5190 [1997]; Tkachuk et al., J. Exp. Med., 187:469 477 [1998]; and Kagi et al., J. Immunol., 162:4598 4605 [1999]).

The key role of TNFR1 shedding in the regulation of TNF bioactivity is highlighted by the association of germline mutations in TNFR1 extracellular domains with impaired TNFR1 shedding and autoinflammatory disease characterized by autosomal dominant periodic fever syndromes (McDermott et al., Cell 97:133 144 [1999]).

Other Mediators of Acute Phase Response

In addition to TNF, other cytokines have been implicated in the induction of the pro-inflammatory response (i.e., the acute phase immune response). These cytokines which demonstrate overlapping activities with TNF include the interleukins (e.g., IL-1 and IL-6) (Suffredini et al., J. Clin. Immunol., 19:203 214 [1999]).

IL-1 (consisting of both .alpha. and .beta. forms) is an important proinflammatory cytokine which regulates the expression of a wide variety of target genes and proteins in nearly every cell type (Dinarello, Blood 77:1627 1652 [1991]; and Dinarello, The Cytokine Handbook (ed. Angus W. Thomson), 3.sup.rd edition, Academic Press, San Diego, p. 35 72 [1998]). The spectrum of IL-1-mediated biologic effects includes inflammatory, metabolic, physiologic, hematopoietic, and immunologic functions. IL-1 is thought to play a role in the pathogenesis of several disease states, including septic shock, rheumatoid arthritis, inflammatory bowel disease, myelogenous leukemia, diabetes mellitus, and atherosclerosis (Dinarello et al., N. Engl. J. Med., 328:106 113 [1993]).

IL-6 is also a multifunctional cytokine with pleiotropic pro-inflammatory effects (DiCosmo, et al., J. Clin. Invest., 94:2028 2035 [1994]; and Kishimoto et al., Blood 86:1243 1254 [1995]). For example, IL-6 plays an important role in regulating B cell immunoglobulin production, T-cell activation, growth and differentiation, hematopoiesis, hepatic acute phase reactions and osteoclast development (Hirano, The Cytokine Handbook (ed. Angus W. Thomson), 3.sup.rd edition, Academic Press, San Diego, p. 197 228 [1998]). Dysregulated production of IL-6 may contribute to the pathogenesis of a variety of inflammatory, neoplastic and autoimmune disorders, such as plasma cell neoplasia and Castleman's disease (Yoshizaki, et al., Blood 74:1360 1367 [1989]; and Hirano, Int. J. Cell Cloning 9:166 184 [1991]).

The signal transduction pathways utilized by TNF, IL-1 and IL-6 also show shared signaling intermediates. For example, both TNF and IL-1 can activate both NF-.kappa.B and AP-1, which are important pro-inflammatory transcription factors (Ashkenazi et al., Science 281:1305 1308 [1998]; and Dinarello, The Cytokine Handbook (ed. Angus W. Thomson), 3.sup.rd edition, Academic Press, San Diego, p. 35 72 [1998]). Similarly, IL-6 signaling uses components of the JAK-STAT pathway, which has also been reported to be induced by TNF (Guo et al., J. Immunol., 160:2742 2750 [1998]; and Hirano, The Cytokine Handbook (ed. Angus W. Thomson), 3.sup.rd edition, Academic Press, San Diego, p. 197 228 [1998]).

The cognate receptors for the IL-1 and IL-6 cytokines are known. There are two IL-1 receptor forms, type I and type II. There is a single IL-6 receptor, consisting of gp80 alpha chain and gp130 beta chain subunits, where ligand binding is mediated by the alpha subunit. The IL-1 and IL-6 receptors are also present as soluble forms analogous to the soluble form of TNFR1. Furthermore, it has been suggested that these receptors play a role in the regulation of IL-1 and IL-6 activity and pro-inflammatory response (Dower et al., J. Immunol., 142:4314 [1989]; Novick et al., J. Exp. Med., 170:1409 [1989]; Eastgate et al., FEBS Lett., 260:213 [1990]; Giri et al., J. Biol. Chem., 265:17416 [1990]; Symons et al., Cytokine 2:190 [1990]; Symons et al., FEBS Lett., 272:133 [1990]; Symons et al., J. Exp. Med., 174:1251 1254 [1991]; Mullberg et al., Biochem. Biophys. Res. Commun., 189:794 [1992]; Mullberg et al., Eur. J. Immunol., 23:473 [1993]; Svenson et al., Cytokine 5:427 [1993]; and Arend et al., J. Immunol., 153:4766 4774 [1994]).

Analogy between regulation of TNF and other cytokines is further illustrated by studies utilizing peptide-hydroxamate metalloprotease inhibitors. Specifically, the protease inhibitors TAPI (TNF-.alpha. protease inhibitor) and RU36156 have been reported to inhibit the proteolytic cleavage and shedding of both TNFR1 and IL-6R (Mullberg et al., J. Immunol., 155:5198 5205 [1995]; and Gallea-Robache et al., Cytokine 9:340 346 [1997]).

As discussed above, in view of the importance of TNF, IL-1 and IL-6 in both health and disease states, there exists a need for methods and compositions for the regulation of TNF, IL-1 and IL-6 cytokine activity. These methods and compositions will find use as therapeutic agents for the treatment of disease states.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods related to regulation of cytokine signaling through the Tumor Necrosis Factor (TNF) pathway, as well as other signaling pathways controlled by other cytokines, including IL-1 and IL-6. It is contemplated that these compositions and methods will find use in therapeutics for the treatment of diseases and disorders of the immune system.

The present invention also provides novel polypeptides and a nucleic acid sequences. In particular, the present invention provides isolated nucleic acids comprising the nucleotide sequence set forth in SEQ ID NO:1, which encodes a polypeptide referred to as "ARTS-"1 (i.e., aminopeptidase regulator of type I, 55 kDa tumor necrosis factor receptor ectodomain shedding) which has the ability to promote the shedding of the extracellular domain of Type I Tumor Necrosis Factor Receptor (TNFR1). Thus, the present invention also provides the isolated polypeptides comprising the amino acid sequence set forth in SEQ ID NO:2, as well as isolated nucleic acids encoding the polypeptide set forth in SEQ ID NO:2.

The present invention further provides recombinant vectors comprising the ARTS-1 gene and host cells comprising these vectors. In one embodiment, the host cell is eukaryotic, while in an alternative embodiment, the host cell is prokaryotic. The present invention also provides antibodies raised against at least a portion of the ARTS-1 polypeptide of SEQ ID NO:2. In some embodiments, the antibodies are monoclonal, while in alternative embodiments, the antibodies are polyclonal.

In other embodiments, the present invention provides isolated nucleic acids that are substantially homologous to the nucleic acid of SEQ ID NO:1, wherein the nucleic acid is capable of hybridizing under high stringency conditions to the nucleic acid of SEQ ID NO:1. In a preferred embodiment, the nucleic acid substantially homologous to the ARTS-1 gene encodes a polypeptide having the ability to regulate the shedding of the extracellular domain of at least one cytokine receptor. In particularly preferred embodiments, the cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80. In some embodiments, the nucleic acid substantially homologous to the nucleic acid of the ARTS-1 gene is identified using PCR methods. In alternative embodiments, the nucleic acid substantially homologous to the ARTS-1 gene is identified using hybridization screening methods.

The present invention further provides methods for the isolation of amplifiable nucleic acid substantially homologous to the nucleic acid of SEQ ID NO:1 comprising: providing a sample comprising template nucleic acid suspected of encoding a gene substantially homologous to the nucleic acid of SEQ ID NO:1; and at least two primers; annealing the primers to the template nucleic acid; extending the primers (e.g., with reiterated DNA synthesis) under conditions such that the template nucleic acid is amplified, to produce an amplified product; and visualizing the amplified product. In some preferred embodiments, the amplified product is isolated. The present invention further provides the product of these amplification methods. In preferred embodiments, the amplified product encodes a polypeptide having the ability to regulate the shedding of the extracellular domain of at least one cytokine receptor. In particularly preferred embodiments, the cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80.

The present invention also provides methods for the use of these compositions to regulate the shedding of sTNFR1. It is contemplated that methods which regulate the shedding of the sTNFR1 also regulate the activity of TNF. In a most preferred embodiment, the invention provides methods for use of these compositions in therapeutic applications in the treatment of immune system diseases and disorders resulting from aberrant cytokine activity.

The present invention provides methods for regulating the shedding of the extracellular domain of at least one cytokine receptor, comprising the steps of: providing a recombinant vector comprising SEQ ID NO:1 in the sense orientation, a first tissue containing one or more cells expressing at least one cytokine receptor, and a second tissue comprising one or more cells capable of expressing the polypeptide encoded by the recombinant vector; delivering the vector to the cells of the second tissue in the presence of the first tissue, under conditions which result in regulation of shedding of the cytokine receptor(s) from cells of the first tissue. In some preferred embodiments, the cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80. In alternative preferred embodiments, the delivery of the vector to the second tissue comprises a means of intracellular delivery selected from the group consisting of direct nucleic acid administration, liposome administration, viral vector delivery, and ex vivo gene delivery followed by transplantation.

In other embodiments, the present invention provides compositions and methods suitable for regulating TNFR1 ectodomain shedding by overexpressing or suppressing the activity of the ARTS-1 polypeptide. In some of these embodiments, TNFR1 ectodomain shedding is regulated by the intracellular delivery of a vector which results in overexpression of the ARTS-1 polypeptide (e.g., SEQ ID NO:2). In another embodiment, TNFR1 ectodomain shedding is regulated by delivering purified ARTS-1 polypeptide (e.g., SEQ ID NO:2) to tissues.

The present invention also provides methods for regulating the shedding of the extracellular domain of at least one cytokine receptor, comprising the steps of: providing a recombinant vector comprising at least a transcribeable portion of the nucleic acid of SEQ ID NO:1 in an antisense orientation, a first tissue comprising one or more cells expressing at least one cytokine receptor, and a second tissue comprising one or more cells expressing the endogenous polypeptide of SEQ ID NO:2, and one or more cells capable of transcribing the antisense nucleic acid; and delivering the vector to the second tissue in the presence of the first tissue, under conditions that result in regulation of shedding of the cytokine receptor(s) from the cells of the first tissue. In preferred embodiments, the cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80. In alternative preferred embodiments, the delivery of the vector to the second tissue comprises a means of intracellular delivery selected from the group consisting of direct nucleic acid administration, liposome administration, viral vector delivery, and ex vivo gene delivery followed by transplantation.

The present invention further provides methods for regulating the shedding of the extracellular domain of at least one cytokine receptor, comprising the steps of: providing a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, and a tissue comprising one or more cells expressing at least one cytokine receptor on their plasma membrane extracellular surface; and delivering the polypeptide to the tissue under conditions such that the polypeptide regulates the shedding of the cytokine receptor(s) from the surface of the cells of the tissue. In some preferred embodiments, the cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80. In alternative preferred embodiments, the delivery of the polypeptide to the tissue comprises a means of delivery selected from the group consisting of oral administration, intra-arterial injection, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, local surgical administration, systemic surgical administration, catheter, and any combination of these means of delivery.

The present invention further provides methods for regulating the shedding of the extracellular domain of at least one cytokine receptor, providing an antibody raised against the ARTS-1 polypeptide, and a tissue comprising one or more cells expressing at least one cytokine receptor and the endogenous polypeptide of SEQ ID NO:2; and delivering the antibody to the tissue under conditions such that the antibody regulates the shedding of the cytokine receptor(s) from the surface of the cells of the tissue. In some preferred embodiments, the cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type I interleukin-1 cytokine receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80. In alternative preferred embodiments, the means of delivery is selected from the group consisting of oral administration, intra-arterial injection, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, local surgical administration, systemic surgical administration, catheter, and any combination of these means of delivery.

In view of the overlapping activities between TNF and other proinflammatory cytokines, the present invention also provides compositions and methods suitable for regulating the shedding of other cytokine receptors in addition to TNFR1, including, but not limited to IL-1 and IL-6 cytokine receptors.

In some particularly preferred embodiments, the present invention provides compositions and methods to treat subjects displaying pathology, as well as subjects suspected of displaying or at risk of displaying pathology resulting from abnormal cytokine activity. The compositions provided for use in the most preferred embodiment include vectors capable of expressing the ARTS-1 polypeptide, vectors capable of transcribing at least a portion of the ARTS-1 gene in an antisense orientation, the ARTS-1 polypeptide set forth in SEQ ID NO:2, and antibodies raised against at least a portion of the ARTS-1 polypeptide.

The present invention also provides methods for treating a subject, comprising the steps of: providing a composition selected from the group consisting of a recombinant vector comprising at least a portion of SEQ ID NO:1 in the sense orientation, a recombinant vector comprising at least a portion of SEQ ID NO:1 in the antisense orientation, at least a portion of the ARTS-1 polypeptide, at least a portion of SEQ ID NO:2, and antibody directed against at least a portion of the ARTS-1 polypeptide, as well as a subject, and a means of delivery of the composition to at least one tissue of the subject; and delivering the composition to the subject using the means of delivery. In preferred embodiments, the subject is selected from the group consisting of a subject displaying pathology resulting from abnormal cytokine activity, a subject suspected of displaying pathology resulting from abnormal cytokine activity, and a subject at risk of displaying pathology resulting from abnormal cytokine activity. In some preferred embodiments, the cytokine activity is mediated by a cytokine selected from the group consisting of tumor necrosis factor .alpha., interleukin-1 alpha, interleukin-1 beta, and interleukin-6. In some particularly preferred embodiments, the subject is a human. In alternative preferred embodiments, the means of delivery is selected from the group consisting of oral administration, intra-arterial injection, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, local surgical administration, systemic surgical administration, catheter, and any combination of these means of delivery. In further preferred embodiments, the means of delivery is further selected from the group consisting of direct nucleic acid administration, liposome administration, viral vector delivery, and ex vivo gene delivery followed by transplantation.

In other embodiments, the present invention provides ARTS-1 markers, including those selected from the group consisting of the ARTS-1 mRNA transcript and the ARTS-1 polypeptide. Furthermore, the invention also provides compositions and means for detecting the ARTS-1 mRNA and polypeptide markers. In preferred embodiments, these compositions are selected from the group consisting of nucleic acid complementary to the ARTS-1 mRNA, and antibodies specific for at least a portion of the ARTS-1 polypeptide.

The present invention also provides means for detecting an ARTS-1 mRNA in a sample, wherein the means comprises at least a portion of the nucleic acid of SEQ ID NO:1 complementary to at least a portion of ARTS-1 mRNA, and further wherein the nucleic acid is a probe. In alternative embodiments, the means comprises Northern blotting. In preferred embodiments, the sample is a tissue sample from a subject. In additional preferred embodiments, the methods provide means for detecting an ARTS-1 polypeptide in a sample, wherein the means comprises an antibody directed against at least a portion of the ARTS-1 polypeptide. In some preferred embodiments, the means comprises Western immunoblotting, while in alternative preferred embodiments, the means comprises an enzyme-linked immunosorbent assay. In alternative preferred embodiments, the sample is a tissue sample from a subject.

The present invention also provides diagnostic kits comprising a means to measure ARTS-1 expression, wherein the means comprises at least a portion of SEQ ID NO:1 that is complementary to at least a portion of ARTS-1 mRNA, and further wherein the nucleic acid is a probe. In alternative embodiments, the diagnostic kits of the present invention provides means for detecting an ARTS-1 polypeptide in a sample, wherein the means comprises antibody directed against at least a portion of the ARTS-1 polypeptide. In some preferred embodiments, the means comprises Western immunoblotting, while in alternative preferred embodiments, the means comprises an enzyme-linked immunosorbent assay.

The present invention further provides compositions and methods for the screening for drugs with the ability to regulate ARTS-1 peptidase activity and cytokine receptor shedding regulatory activity. In some embodiments, the present invention provides methods for drug screening to identify drugs having the ability to regulate ARTS-1 expression comprising the steps of: providing a drug, cultured cells, and a means to measure ARTS-1 expression, wherein the means comprises at least a portion of SEQ ID NO:1 that is complementary to at least a portion of ARTS-1 mRNA, and further wherein the nucleic acid is a probe; exposing the cells to the drug; and using the means to measure ARTS-1 expression. In alternative embodiments, the diagnostic kits of the present invention provide means for detecting an ARTS-1 polypeptide in a sample, wherein the means comprises an antibody directed against at least a portion of the ARTS-1 polypeptide. In some preferred embodiments, the cultured cells are human NCI-H292 pulmonary mucoepidermoid carcinoma cells.

The present invention further provides methods for drug screening to identify drugs capable of regulating the peptidase activity of ARTS-1, comprising the steps of: providing purified ARTS-1 polypeptide, an amino acid p-nitroaniline, a means to measure amino acid p-nitroaniline cleavage, and a drug; exposing the purified ARTS-1 polypeptide to the amino acid p-nitroaniline in the absence and presence of the drug; and measuring amino acid p-nitroaniline cleavage in the absence and presence of the drug. In some preferred embodiments, the purified ARTS-1 polypeptide comprises glutathione-S-transferase. In alternative preferred embodiments, the amino acid p-nitroaniline is selected from the group consisting of isoleucine p-nitroanilide, phenylalanine p-nitroanilide and glycine p-nitroanilide. In still further preferred embodiments, the means to measure amino acid p-nitroaniline cleavage comprises measuring absorbance at 380 nm.

The present invention also provides methods for drug screening to identify drugs capable of regulating the shedding of a cytokine receptor, comprising the steps of: providing cultured cells expressing at least one cytokine receptor, a means to quantitate the concentration of the soluble form of the cytokine receptor(s) in the supernatants of the cultured cells, and a drug; culturing the cells in the absence and presence of the drug; quantitating the concentration of the soluble form of the cytokine receptor(s) in the supernatants of the cultured cells; and comparing the concentrations of the soluble cytokine receptor(s) in the supernatants of the cell cultures in the absence and presence of drug. In some preferred embodiments, the cytokine receptor is selected from the group consisting of type-1 tumor necrosis factor receptor, type II interleukin-1 cytokine receptor, and interleukin-6 cytokine receptor alpha-chain gp80. In alternative preferred embodiments, the means to quantitate the concentration of the soluble form of a cytokine receptor comprises an enzyme-linked immunosorbent assay. In further embodiments, the cultured cells are cultured human NCI-H292 pulmonary mucoepidermoid carcinoma cells.

DESCRIPTION OF THE FIGURES

FIG. 1 presents the ARTS-1 transcription unit and open reading frame translation.

FIG. 2 provides a schematic representation of the ARTS-1 protein, indicating domains of homology with the aminopeptidase family of gluzincin zinc metalloproteases.

FIG. 3 provides a Northern blot analysis of multiple human tissues using a .sup.32p-labelled ARTS-1 cDNA probe. The upper panel shows the blot probed with the ARTS-1 cDNA probe. The lower panel shows the same blot following stripping and rehybridization to a probe specific for the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a reference for RNA loading normalization.

FIG. 4 provides Western immunoblots using pre-immune and polyclonal anti-ARTS-1 antisera in conjunction with crude whole cell homogenates, and membrane and cytosolic fractions made from cultured NCI-H292 cells. The top panel provides a Western immunoblot using the immune sera. The next panel provides a Western immunoblot using the pre-immune sera. The bottom two panel provide results from Western immunoblot competition experiments using bovine serum albumin or ARTS-1 cognate peptide, respectively.

FIG. 5 provides Western immunoblots using polyclonal anti-ARTS-1 antisera and membrane and cytosolic fractions made from primary cells and cell lines. Panel A provides a Western immunoblot using human bronchial brush cells collected from human subjects. Panel B provides a Western immunoblot using the NCI-H292, BEAS-2B, BET-1A and A549 cultured cell lines. Panel C provides a Western immunoblot using primary cultures of normal human bronchial epithelial cells (NHBE), human umbilical vein endothelial cells (HUVEC) and human fibroblasts.

FIG. 6 provides results of an analysis of recombinant GST-ARTS-1 fusion protein purification and analysis of the aminopeptidase activity of this protein following purification. Panel A provides a Coomassie stained gel of protein samples obtained during the production and purification of the GST-ARTS-1 fusion protein. Panel B provides an FPLC elution profile of the purified GST-ARTS-1 fusion protein. Panel C provides the results of an assay of phenylalanine p-nitroaniline substrate aminopeptidase activity of the FPLC eluted fractions.

FIG. 7 provides a Western immunoblot using anti-ARTS-1 polyclonal antiserum as the primary antibody. Samples analyzed in the blot are from membrane protein fractions derived from stably transfected NCI-H292 cells which were untransfected (WT), control transfected (Mock), ARTS-1 sense-overexpressing (ARTS-1) or ARTS-1 anti-sense expressing (AS). Two independent clones each from the ARTS-1 and AS cell lines were analyzed.

FIG. 8 provides results of an ELISA to determine the levels of sTNFR1 resulting from TNFR1 ectodomain shedding in cell culture supernatants from cultures of the same cell lines as indicated for FIG. 7.

FIG. 9 provides a graph depicting the ability of ARTS-1 overexpression to potentiate the cleavage and shedding of TNFR ectodomain from the surface of NCI-H292 cells in response to PMA stimulation, as determined by an ELISA measuring sTNFR1 in the cell culture supernatants.

FIG. 10 provides a histogram showing the results of an ELISA analysis. The ELISA determined the levels of sTNFR1 (a measure of TNFR1 ectodomain shedding) in cell culture supernatants for stably transfected NCI-H292 cell lines expressing various ARTS-1 mutants.

FIG. 11 provides a Western immunoblot using an anti-TNFR1 antibody as the primary antibody to detect membrane bound TNFR1. Samples analyzed in the blot include membrane protein fractions derived from stably transfected NCI-H292 cells which were untransfected (WT), control transfected (Mock), ARTS-1 sense-overexpressing (ARTS-1), or ARTS-1 anti-sense expressing (AS). Two independent clones of each cell line were analyzed.

FIG. 12 provides two Western immunoblots following two in vivo immunoprecipitation experiments using membrane protein fractions isolated from cultured NCI-H292 cells. In the top panel, an anti-TNFR1 antibody was used in the immunoprecipitation step (indicated as "IP"), and anti-ARTS-1 antiserum was used as the primary antibody in the immunoblotting (indicated as "IB"). Conversely, in the lower panel, the anti-ARTS-1 antiserum was used in the immunoprecipitation, while the anti-TNFR1 antibody was used as the primary antibody in the immunoblotting.

FIG. 13 provides a Western immunoblot following an in vivo immunoprecipitation experiment using an anti-TNFR1 monoclonal antibody for the immunoprecipitation and anti-ARTS-1 antiserum as the primary antibody in the blot. The immunoprecipitations used cell membrane protein fractions derived from stably transfected NCI-H292 cells overexpressing ARTS-1 (ARTS-1), expressing an anti-sense ARTS-1 message (AS), as well as control-transfected (Mock) and non-transfected (WT) cell lines.

FIG. 14 provides results of two drug screening assays. Panel A provides a Western immunoblot using anti-ARTS-1 polyclonal antiserum as the primary antibody, tested with membrane protein fractions from NCI-H292 cells following exposure to 4b-phorbol 12-myristate 13-acetate (PMA) (over a time course). Panel B provides the results of an ELISA to determine the levels of sTNFR1 in cell culture supernatants from NCI-H292 cell cultures following exposure PMA, over time.

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined or clarified below:

The terms "peptide," "polypeptide" and "protein" all refer to a primary sequence of amino acids that are joined by covalent "peptide linkages." In general, a peptide consists of a few amino acids, typically from 2 25 amino acids, and is shorter than a protein. Polypeptides may encompass either peptides or proteins. Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms, such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

As used herein, the term "nucleic acid" refers to any sequence of the bases adenine, thymine, cytosine and guanine, and various analogs of these bases. A nucleic acid is characterized by a specific nucleotide sequence (i.e., the sequence of the bases and base analogs in the molecule). A "nucleic acid" is not limited to DNA or RNA, and is not limited in any way by the size of the molecule. A nucleic acid may be double stranded or single stranded. The term "nucleic acid" encompasses sequences that include any of the bases adenine, thymine, guanine and cytosine, as well as known analogs of these bases, including but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

As used herein, the term "oligonucleotide," refers to a short length of single-stranded polynucleotide chain. Oligonucleotides are typically less than 100 residues long (e.g., between 15 and 50), however, as used herein, the term is also intended to encompass longer polynucleotide chains. Oligonucleotides are often referred to by their length. For example a 24 residue oligonucleotide is referred to as a "24-mer." Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes.

As used herein, "recombinant nucleic acid," "recombinant gene" or "recombinant DNA molecule" indicate that the nucleotide sequence or arrangement of its parts is not a native configuration, and has been manipulated by molecular biological techniques. The term implies that the DNA molecule is comprised of segments of DNA that have been artificially joined together. Protocols and reagents to manipulate nucleic acids are common and routine in the art (See e.g., Maniatis et al.(eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, [1982]; Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Volumes 1 3, Cold Spring Harbor Laboratory Press, NY, [1989]; and Ausubel et al. (eds.), Current Protocols in Molecular Biology, Vol. 1 4, John Wiley & Sons, Inc., New York [1994]).

Similarly, a "recombinant protein" or "recombinant polypeptide" refers to a protein molecule that is expressed from a recombinant DNA molecule. Use of these terms indicates that the primary amino acid sequence, arrangement of its domains or nucleic acid elements which control its expression are not native, and have been manipulated by molecular biology techniques. As indicated above, techniques to manipulate recombinant proteins are also common and routine in the art.

The terms "exogenous" and "heterologous" are sometimes used interchangeably with "recombinant." An "exogenous nucleic acid," "exogenous gene" and "exogenous protein" indicate a nucleic acid, gene or protein, respectively, that has come from a source other than its native source, and has been artificially supplied to the biological system. In contrast, the terms "endogenous protein," "native protein," "endogenous gene," and "native gene" refer to a protein or gene that is native to the biological system, species or chromosome under study. A "native" or "endogenous" polypeptide does not contain amino acid residues encoded by recombinant vector sequences; that is, the native protein contains only those amino acids found in the polypeptide or protein as it occurs in nature. A "native" polypeptide may be produced by recombinant means or may be isolated from a naturally occurring source. Similarly, a "native" or "endogenous" gene is a gene that does not contain nucleic acid elements encoded by sources other than the chromosome on which it is normally found in nature.

As used herein, the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

Nucleic acid molecules (e.g., DNA or RNA) are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide or polynucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand. The promoter and enhancer elements that direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element or the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

The term "gene" refers to a nucleic acid (e.g., DNA) sequence comprised of parts, that when appropriately combined in either a native or recombinant manner, provide some product or function. Genes may or may not comprise coding sequences necessary for the production of a polypeptide. Examples of genes which do not encode polypeptide sequences include ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. Genes can encode a polypeptide or any portion of a polypeptide within the gene's "coding region" or "open reading frame." The polypeptide produced by the open reading frame of a gene may or may not display functional activity or properties of the full-length polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.).

In addition to the coding region of the nucleic acid, the term "gene" also encompasses the transcribed nucleotide sequences of the full-length mRNA adjacent to the 5' and 3' ends of the coding region. These noncoding regions are variable in size, and typically extend for distances up to or exceeding 1 kb on both the 5' and 3' ends of the coding region. The sequences that are located 5' and 3' of the coding region and are contained on the mRNA are referred to as 5' and 3' untranslated sequences (5' UT and 3' UT). Both the 5' and 3' UT may serve regulatory roles, including translation initiation, post-transcriptional cleavage and polyadenylation. The term "gene" encompasses mRNA, cDNA and genomic forms of a gene.

It is contemplated that the genomic form or genomic clone of a gene may contain the sequences of the transcribed mRNA, as well as other non-coding sequences which lie outside of the mRNA. The regulatory regions which lie outside the mRNA transcription unit are sometimes called "5' or 3' flanking sequences." A functional genomic form of a gene must contain regulatory elements necessary for the regulation of transcription. The term "promoter/enhancer region" is usually used to describe this DNA region, typically but not necessarily 5' of the site of transcription initiation, sufficient to confer appropriate transcriptional regulation. The word "promoter" alone is sometimes used synonymously with "promoter/enhancer." A promoter may be constitutively active, or alternatively, conditionally active, where transcription is initiated only under certain physiological conditions or in the presence of certain drugs. The 3' flanking region may contain additional sequences which regulate transcription, especially the termination of transcription. "Introns" or "intervening regions" or "intervening sequences" are segments of a gene which are contained in the primary transcript (i.e., hetero-nuclear RNA, or hnRNA), but are spliced out to yield the processed mRNA form. Introns may contain transcriptional regulatory elements such as enhancers. The mRNA produced from the genomic copy of a gene is translated in the presence of ribosomes to yield the primary amino acid sequence of the polypeptide.

As used herein, the term "regulatory element" refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that enables the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.

Transcriptional control signals in eukaryotes comprise "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 [1987]). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells, as well as viruses. Analogous control elements (i.e., promoters and enhancers) are also found in prokaryotes. The selection of a particular promoter and enhancer to be operably linked in a recombinant gene depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional only in a limited subset of cell types (for review see, Voss et al., Trends Biochem. Sci., 11:287 [1986] and Maniatis et al., Science 236:1237 [1987]). For example, the SV40 early gene enhancer is very active in a wide variety of mammalian cell types (Dijkema et al., EMBO J., 4:761 [1985]). Two other examples of promoter/enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor 1.alpha. gene (Uetsuki et al., J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene 91:217 [1990]; Mizushima and Nagata, Nuc. Acids. Res., 18:5322 [1990]), the long terminal repeats of the Rous sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 [1982]), and human cytomegalovirus (Boshart et al., Cell 41:521 [1985]). Some promoter elements serve to direct gene expression in a tissue-specific manner.

As used herein, the term "promoter/enhancer" denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (i.e., the functions provided by a promoter element and an enhancer element). For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The promoter/enhancer may be "endogenous," or "exogenous," or "heterologous." An "endogenous" promoter/enhancer is one which is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" promoter/enhancer is one placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques such as cloning and recombination) such that transcription of the gene is controlled by the linked promoter/enhancer.

The presence of "splicing signals" on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, New York [1989], pp. 16.7 16.8). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cells requires the presence of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term "poly A site" or "poly A sequence" as used herein denotes a nucleic acid sequence that directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be "heterologous" or "endogenous." An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is one that is isolated from one gene and placed 3' of another gene. A commonly used heterologous poly A signal is the SV40 poly A signal. The SV40 poly A signal is contained on a 237 bp BamHI/BclI restriction fragment and directs both termination and polyadenylation (See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, New York [1989], pp. 16.6 16.7).

The terms "in operable combination," "in operable order," "operably linked" and similar phrases when used in reference to nucleic acid herein are used to refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.

As used herein, the terms "an oligonucleotide having a nucleotide sequence encoding a gene," "polynucleotide having a nucleotide sequence encoding a gene," and similar phrases are meant to indicate a nucleic acid sequence comprising the coding region of a gene (i.e., the nucleic acid sequence which encodes a gene product). The coding region may be present in either a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide, polynucleotide or nucleic acid may be single-stranded (i.e., the sense strand or the antisense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" and similar phrases refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (e.g., protein) chain. The DNA sequence thus codes for the amino acid sequence.

As used herein, the term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through "translation" of the mRNA. Gene expression can be regulated at many stages. "Up-regulation" or "activation" refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while "down-regulation" or "repression" refers to regulation that decreases mRNA or protein production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called "activators" and "repressors," respectively.

As used herein, the terms "complementary" or "complementarity" are used in r