Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1999-05-18
2001-05-08
Schwartzman, Robert A. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S091100, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06228642
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to compositions and methods for modulating expression of the human tumor necrosis factor-&agr; (TNF-&agr;) gene, which encodes a naturally present cytokine involved in regulation of immune function and implicated in infectious and inflammatory disease. This invention is also directed to methods for inhibiting TNF-&agr; mediated immune responses; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the human TNF-&agr; gene.
BACKGROUND OF THE INVENTION
Tumor necrosis factor &agr; (TNF-&agr; also cachectin) is an important cytokine that plays a role in host defense. The cytokine is produced primarily in macrophages and monocytes in response to infection, invasion, injury, or inflammation. Some examples of inducers of TNF-&agr; include bacterial endotoxins, bacteria, viruses, lipopolysaccharide (LPS) and cytokines including GM-CSF, IL-1, IL-2 and IFN-&ggr;.
TNF-&agr; interacts with two different receptors, TNF receptor I (TNFRI, p55) and TNFRII (p75), in order to transduce its effects, the net result of which is altered gene expression. Cellular factors induced by TNF-&agr; include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-&ggr; (IFN-&ggr;), platelet derived growth factor (PDGF) and epidermal growth factor (EGF), and endothelial cell adhesion molecules including endothelial leukocyte adhesion molecule 1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (Tracey, K. J., et al.,
Annu. Rev. Cell Biol.,
1993, 9, 317-343; Arvin, B., et al.,
Ann. NY Acad. Sci.,
1995, 765, 62-71).
Despite the protective effects of the cytokine, overexpression of TNF-&agr; often results in disease states, particularly in infectious, inflammatory and autoimmune diseases. This process may involve the apoptotic pathways (Ksontini, R., et al.,
J. Immunol.,
1998, 160, 4082-4089). High levels of plasma TNF-&agr; have been found in infectious diseases such as sepsis syndrome, bacterial meningitis, cerebral malaria, and AIDS; autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease), sarcoidosis, multiple sclerosis, Kawasaki syndrome, graft-versus-host disease and transplant (allograft) rejection; and organ failure conditions such as adult respiratory distress syndrome, congestive heart failure, acute liver failure and myocardial infarction (Eigler, A., et al.,
Immunol. Today,
1997, 18, 487-492). Other diseases in which TNF-&agr; is involved include asthma (Shah, A., et al.,
Clinical and Experimental Allergy,
1995, 25, 1038-1044), brain injury following ischemia (Arvin, B., et al.,
Ann. NY Acad. Sci.,
1995, 765, 62-71), non-insulin-dependent diabetes mellitus (Hotamisligil, G. S., et al.,
Science,
1993, 259, 87-90), insulin-dependent diabetes mellitus (Yang, X.-D., et al.,
J. Exp. Med.,
1994, 180, 995-1004), hepatitis (Ksontini, R., et al.,
J. Immunol.,
1998, 160, 4082-4089), atopic dermatitis (Sumimoto, S., et al.,
Arch. Dis. Child.,
1992, 67, 277-279), and pancreatitis (Norman, J. G., et al.,
Surgery,
1996, 120, 515-521). Further, inhibitors of TNF-&agr; have been suggested to be useful for cancer prevention (Suganuma, M., et al. (
Cancer Res.,
1996, 56, 3711-3715). Elevated TNF-&agr; expression may also play a role in obesity (Kern, P. A.,
J. Nutr.,
1997, 127, 1917S-1922S). TNF-&agr; was found to be expressed in human adipocytes and increased expression, in general, correlated with obesity.
There are currently several approaches to inhibiting TNF-&agr; expression. Approaches used to treat rheumatoid arthritis include a chimeric anti-TNF-&agr; antibody, a humanized monoclonal anti-TNF-&agr; antibody, and recombinant human soluble TNF-&agr; receptor (Camussi, G.,
Drugs,
1998, 55, 613-620). Other examples are indirect TNF-&agr; inhibitors including phosphodiesterase inhibitors (e.g. pentoxifylline) and metalloprotease inhibitors (Eigler, A., et al.,
Immunol. Today,
1997, 18, 487-492). An additional class of direct TNF-&agr; inhibitors is oligonucleotides, including triplex-forming oligonucleotides, ribozymes, and antisense oligonucleotides.
Several publications describe the use of oligonucleotides targeting TNF-&agr; by non-antisense mechanisms. U.S. Pat. No. 5,650,316, WO 95/33493 and Aggarwal, B. B. et al. (
Cancer Research,
1996, 56, 5156-5164) disclose triplex-forming oligonucleotides targeting TNF-&agr;. WO 95/32628 discloses triplex-forming oligonucleotides especially those possessing one or more stretches of guanosine residues capable of forming secondary structure. WO 94/10301 discloses ribozyme compounds active against TNF-&agr; mRNA. WO 95/23225 discloses enzymatic nucleic acid molecules active against TNF-&agr; mRNA.
A number of publications have described the use of antisense oligonucleotides targeting nucleic acids encoding TNF-&agr;. The TNF-&agr; gene has four exons and three introns. WO 93/09813 discloses TNF-&agr; antisense oligonucleotides conjugated to a radioactive moiety, including sequences targeted to the 5′-UTR, AUG start site, exon 1, and exon 4 including the stop codon of human TNF-&agr;. EP 0 414 607 B1 discloses antisense oligonucleotides targeting the AUG start codon of human TNF-&agr;. WO 95/00103 claims antisense oligonucleotides to human TNF-&agr; including sequences targeted to exon 1 including the AUG start site. Hartmann, G. et al. (
Mol. Med.,
1996, 2, 429-438) disclose uniform phosphorothioates and mixed backbone phosphorothioate/phosphodiester oligonucleotides targeted to the AUG start site of human TNF-&agr;. Hartmann, G. et al. (
Antisense Nucleic Acid Drug Devel.,
1996, 6, 291-299) disclose antisense phosphorothioate oligonucleotides targeted to the AUG start site, the exon 1/intron 1 junction, and exon 4 of human TNF-&agr;. d'Hellencourt, C. F. et al. (
Biochim. Biophys. Acta,
1996, 1317, 168-174) designed and tested a series of unmodified oligonucleotides targeted to the 5′-UTR, and exon 1, including the AUG start site, of human TNF-&agr;. Additionally, one oligonucleotide each was targeted to exon 4 and the 3′-UTR of human TNF-&agr; and one oligonucleotide was targeted to the AUG start site of mouse TNF-&agr;. Rojanasakul, Y. et al. (
J. Biol. Chem.,
1997, 272, 3910-3914) disclose an antisense phosphorothioate oligonucleotide targeted to the AUG start site of mouse TNF-&agr;. Taylor, M. F. et al. (
J. Biol. Chem.,
1996, 271, 17445-17452 and
Antisense Nucleic Acid Drug Devel.,
1998, 8, 199-205) disclose morpholino, methylmorpholino, phosphodiester and phosphorothioate oligonucleotides targeted to the 5′-UTR and AUG start codon of mouse TNF-&agr;. Tu, G.-C. et al. (
J. Biol. Chem.,
1998, 273, 25125-25131) designed and tested 42 phosphorothioate oligonucleotides targeting sequences throughout the rat TNF-&agr; gene.
Interestingly, some phosphorothioate oligodeoxynucleotides have been found to enhance lipopolysaccharide-stimulated TNF-&agr; synthesis up to four fold due to nonspecific immunostimulatory effects (Hartmann et al.
Mol. Med.,
1996, 2, 429-438).
Accordingly, there remains an unmet need for therapeutic compositions and methods for inhibiting expression of TNF-&agr;, and disease processes associated therewith.
BRIEF DESCRIPTION OF THE INVENTION
The present invention provides oligonucleotides which are targeted to nucleic acids encoding TNF-&agr; and are capable of modulating TNF-&agr; expression. The present invention also provides chimeric oligonucleotides targeted to nucleic acids encoding human TNF-&agr;. The oligonucleotides of the invention are believed to be useful both diagnostically and therapeutically, and are believed to be particularly useful in the methods of the present invention.
The present invention also comprises methods of modulating the expression of human TNF-&agr;, in cells and tissues, using the oligonucleotides of the invention. Methods of inhibiting TNF-&agr; expression are provided; these m
Baker Brenda F.
Bennett C. Frank
Butler Madeline M.
Shanahan, Jr. William R.
ISIS Pharmaceuticals Inc.
Licata & Tyrrell P.C.
Schmidt M
Schwartzman Robert A.
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