Use of Apoptosis-specific eIF-5A siRNA to Down Regulate Expression of Proinflammatory Cytokines to Treat Sepsis

Abstract

The present invention relates to apoptosis specific eucaryotic initiation factor 5A (eIF-5A), referred to as apoptosis-specific eIF-5A or eIF5-A1, nucleic acids and polypeptides and methods for down regulating pro-inflammatory cytokines in a mammal by administering siRNA against eIF-5A1 to the mammal to treat/prevent sepsis and/or hemorrhagic shock.

Description

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application 60/798,333, filed May 8, 2006 and U.S. provisional application 60/783,413, filed Mar. 20, 2006, both of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to apoptosis-specific eucaryotic initiation factor ("eIF-5A") or referred to as "apoptosis-specific eIF-5A" or "eIF-5A1" and the use of siRNA against eIF-5A1 to down regulate expression of pro-inflammatory cytokines.

BACKGROUND OF THE INVENTION

[0003] Apoptosis is a genetically programmed cellular event that is characterized by well-defined morphological features, such as cell shrinkage, chromatin condensation, nuclear fragmentation, and membrane blebbing. Kerr et al. (1972) Br. J. Cancer, 26, 239-257; Wyllie et al. (1980) Int. Rev. Cytol., 68, 251-306. It plays an important role in normal tissue development and homeostasis, and defects in the apoptotic program are thought to contribute to a wide range of human disorders ranging from neurodegenerative and autoimmunity disorders to neoplasms. Thompson (1995) Science, 267, 1456-1462; Mullauer et al. (2001) Mutat. Res, 488, 211-231. Although the morphological characteristics of apoptotic cells are well characterized, the molecular pathways that regulate this process have only begun to be elucidated.

[0004] One group of proteins that is thought to play a key role in apoptosis is a family of cysteine proteases, termed caspases, which appear to be required for most pathways of apoptosis. Creagh & Martin (2001) Biochem. Soc. Trans, 29, 696-701; Dales et al. (2001) Leuk. Lymphoma, 41, 247-253. Caspases trigger apoptosis in response to apoptotic stimuli by cleaving various cellular proteins, which results in classic manifestations of apoptosis, including cell shrinkage, membrane blebbing and DNA fragmentation. Chang & Yang (2000) Microbiol. Mol. Biol. Rev., 64, 821-846.

[0005] Pro-apoptotic proteins, such as Bax or Bak, also play a key role in the apoptotic pathway by releasing caspase-activating molecules, such as mitochondrial cytochrome c, thereby promoting cell death through apoptosis. Martinou & Green (2001) Nat. Rev. Mol. Cell. Biol., 2, 63-67; Zou et al. (1997) Cell, 90, 405-413. Anti-apoptotic proteins, such as Bcl-2, promote cell survival by antagonizing the activity of the pro-apoptotic proteins, Bax and Bak. Tsujimoto (1998) Genes Cells, 3, 697-707; Kroemer (1997) Nature Med., 3, 614-620. The ratio of Bax:Bcl-2 is thought to be one way in which cell fate is determined; an excess of Bax promotes apoptosis and an excess of Bcl-2 promotes cell survival. Salomons et al. (1997) Int. J. Cancer, 71, 959-965; Wallace-Brodeur & Lowe (1999) Cell Mol. Life Sci., 55, 64-75.

[0006] Another key protein involved in apoptosis is a protein that encoded by the tumor suppressor gene p53. This protein is a transcription factor that regulates cell growth and induces apoptosis in cells that are damaged and genetically unstable, presumably through up-regulation of Bax. Bold et al. (1997) Surgical Oncology, 6, 133-142; Ronen et al., 1996; Schuler & Green (2001) Biochem. Soc. Trans., 29, 684-688; Ryan et al. (2001) Curr. Opin. Cell Biol., 13, 332-337; Zornig et al. (2001) Biochem. Biophys. Acta, 1551, F1-F37.

[0007] Alterations in the apoptotic pathways are believed to play a key role in a number of disease processes, including cancer. Wyllie et al. (1980) Int. Rev. Cytol., 68, 251-306; Thompson (1995) Science, 267, 1456-1462; Sen & D'Incalci (1992) FEBS Letters, 307, 122-127; McDonnell et al. (1995) Seminars in Cancer and Biology, 6, 53-60. Investigations into cancer development and progression have traditionally been focused on cellular proliferation. However, the important role that apoptosis plays in tumorigenesis has recently become apparent. In fact, much of what is now known about apoptosis has been learned using tumor models, since the control of apoptosis is invariably altered in some way in tumor cells. Bold et al. (1997) Surgical Oncology, 6, 133-142.

[0008] Cytokines also have been implicated in the apoptotic pathway. Biological systems require cellular interactions for their regulation, and cross-talk between cells generally involves a large variety of cytokines. Cytokines are mediators that are produced in response to a wide variety of stimuli by many different cell types. Cytokines are pleiotropic molecules that can exert many different effects on many different cell types, but are especially important in regulation of the immune response and hematopoietic cell proliferation and differentiation. The actions of cytokines on target cells can promote cell survival, proliferation, activation, differentiation, or apoptosis depending on the particular cytokine, relative concentration, and presence of other mediators.

[0009] The use of anti-cytokines to treat autoimmune disorders such as psoriasis, rheumatoid arthritis, and Crohn's disease is gaining popularity. The pro-inflammatory cytokines IL-1 and TNF play a large role in the pathology of these chronic disorders. Anti-cytokine therapies that reduce the biological activities of these two cytokines can provide therapeutic benefits (Dinarello and Abraham, 2002).

[0010] Interleukin 1 (IL-1) is an important cytokine that mediates local and systemic inflammatory reactions and which can synergize with TNF in the pathogenesis of many disorders, including vasculitis, osteoporosis, neurodegenerative disorders, diabetes, lupus nephritis, and autoimmune disorders such as rheumatoid arthritis. The importance of IL-1β in tumour angiogenesis and invasiveness was also recently demonstrated by the resistance of IL-1β knockout mice to metastases and angiogenesis when injected with melanoma cells (Voronov et al., 2003).

[0011] Interleukin 18 (IL-18) is a recently discovered member of the IL-1 family and is related by structure, receptors, and function to IL-1. IL-18 is a central cytokine involved in inflammatory and autoimmune disorders as a result of its ability to induce interferon-gamma (IFN-γ), TNF-α, and IL-1. IL-1β and IL-18 are both capable of inducing production of TNF-α, a cytokine known to contribute to cardiac dysfunction during myocardial ischemia (Maekawa et al., 2002). Inhibition of IL-18 by neutralization with an IL-18 binding protein was found to reduce ischemia-induced myocardial dysfunction in an ischemia/reperfusion model of suprafused human atrial myocardium (Dinarello, 2001). Neutralization of IL-18 using a mouse IL-18 binding protein was also able to decrease IFN-γ, TNF-α, and IL-1β transcript levels and reduce joint damage in a collagen-induced arthritis mouse model (Banda et al., 2003). A reduction of IL-18 production or availability may also prove beneficial to control metastatic cancer as injection of IL-18 binding protein in a mouse melanoma model successfully inhibited metastases (Carrascal et al., 2003). As a further indication of its importance as a pro-inflammatory cytokine, plasma levels of IL-18 were elevated in patients with chronic liver disease and increased levels were correlated with the severity of the disease (Ludwiczek et al., 2002). Similarly, IL-18 and TNF-a were elevated in the serum of diabetes mellitus patients with nephropathy (Moriwaki et al., 2003). Neuroinflammation following traumatic brain injury is also mediated by pro-inflammatory cytokines and inhibition of IL-18 by the IL-18 binding protein improved neurological recovery in mice following brain trauma (Yatsiv et al., 2002).

[0012] TNF-α, a member of the TNF family of cytokines, is a pro-inflammatory cytokine with pleiotropic effects ranging from co-mitogenic effects on hematopoietic cells, induction of inflammatory responses, and induction of cell death in many cell types. TNF-α is normally induced by bacterial lipopolysaccharides, parasites, viruses, malignant cells and cytokines and usually acts beneficially to protect cells from infection and cancer. However, inappropriate induction of TNF-α is a major contributor to disorders resulting from acute and chronic inflammation such as autoimmune disorders and can also contribute to cancer, AIDS, heart disease, and sepsis (reviewed by Aggarwal and Natarajan, 1996; Sharma and Anker, 2002). Experimental animal models of disease (i.e. septic shock and rheumatoid arthritis) as well as human disorders (i.e. inflammatory bowel diseases and acute graft-versus-host disease) have demonstrated the beneficial effects of blocking TNF-α (Wallach et al., 1999). Inhibition of TNF-α has also been effective in providing relief to patients suffering autoimmune disorders such as Crohn's disease (van Deventer, 1999) and rheumatoid arthritis (Richard-Miceli and Dougados, 2001). The ability of TNF-α to promote the survival and growth of B lymphocytes is also thought to play a role in the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) and the levels of TNF-α being expressed by T cells in B-CLL was positively correlated with tumour mass and stage of the disease (Bojarska-Junak et al., 2002). Interleukin-1β (IL-1β) is a cytokine known to induce TNF-α production.

[0013] The amino acid sequence of eIF-5A is well conserved between species, and there is strict conservation of the amino acid sequence surrounding the hypusine residue in eIF-5A, which suggests that this modification may be important for survival. Park et al. (1993) Biofactors, 4, 95-104. This assumption is further supported by the observation that inactivation of both isoforms of eIF-5A found to date in yeast, or inactivation of the DHS gene, which catalyzes the first step in their activation, blocks cell division. Schnier et al. (1991) Mol. Cell. Biol., 11, 3105-3114; Sasaki et al. (1996) FEBS Lett., 384, 151-154; Park et al. (1998) J. Biol. Chem., 273, 1677-1683. However, depletion of eIF-5A protein in yeast resulted in only a small decrease in total protein synthesis suggesting that eIF-5A may be required for the translation of specific subsets of mRNA's rather than for protein global synthesis. Kang et al. (1993), "Effect of initiation factor eIF-5A depletion on cell proliferation and protein synthesis," in Tuite, M. (ed.), Protein Synthesis and Targeting in Yeast, NATO Series H. The recent finding that ligands that bind eIF-5A share highly conserved motifs also supports the importance of eIF-5A. Xu & Chen (2001) J. Biol. Chem., 276, 2555-2561. In addition, the hypusine residue of modified eIF-5A was found to be essential for sequence-specific binding to RNA, and binding did not provide protection from ribonucleases.

[0014] In addition, intracellular depletion of eIF-5A results in a significant accumulation of specific mRNAs in the nucleus, indicating that eIF-5A may be responsible for shuttling specific classes of mRNAs from the nucleus to the cytoplasm. Liu & Tartakoff (1997) Supplement to Molecular Biology of the Cell, 8, 426a. Abstract No. 2476, 37th American Society for Cell Biology Annual Meeting. The accumulation of eIF-5A at nuclear pore-associated intranuclear filaments and its interaction with a general nuclear export receptor further suggest that eIF-5A is a nucleocytoplasmic shuttle protein, rather than a component of polysomes. Rosorius et al. (1999) J. Cell Science, 112, 2369-2380.

[0015] The first cDNA for eIF-5A was cloned from human in 1989 by Smit-McBride et al., and since then cDNAs or genes for eIF-5A have been cloned from various eukaryotes including yeast, rat, chick embryo, alfalfa, and tomato. Smit-McBride et al. (1989) J. Biol. Chem., 264, 1578-1583; Schnier et al. (1991) (yeast); Sano, A. (1995) in Imahori, M. et al. (eds), Polyamines, Basic and Clinical Aspects, VNU Science Press, The Netherlands, 81-88 (rat); Rinaudo & Park (1992) FASEB J., 6, A453 (chick embryo); Pay et al. (1991) Plant Mol. Biol., 17, 927-929 (alfalfa); Wang et al. (2001) J. Biol. Chem., 276, 17541-17549 (tomato).

SUMMARY OF INVENTION

[0016] The present invention relates to apoptosis specific eucaryotic initiation factor 5A (eIF-5A), referred to as "apoptosis specific eIF-5A" or "eIF-5A1." The invention also relates to suppressing or inhibiting expression of pro-inflammatory cytokines in a subject, including a human, in vivo, (and in vitro in a cell) by inhibiting expression of apoptosis-specific eIF-5A through the use of eIF5A1 siRNAs or antisense polynucleotides. eIF5A1 siRNA and antisense constructs of eIF5A1 are administered to decrease expression of pro-inflammatory cytokines such as IL-1β, IL-2, IL-4, IL-5, IL-10, IFN-γ, TNF-α, IL-3, IL-6, IL-12(p40), IL-12(p70), G-CSF, KC, MIP-1a, and RANTES, which is useful in the treatment or prevention of sepsis and/or hemorrhagic induced shock.

[0017] The present invention also provides a pharmaceutical composition for decreasing expression of pro-inflammatory cytokines, comprising eIF5A1 siRNA and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention may be administered to treat or prevent the onset of sepsis in a subject, including a human. In certain embodiments, the pharmaceutical composition comprises the nucleotide sequence CGG AAU GAC UUC CAG CUG A.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows the effects of siRNA against eIF-5A1 on the effect of proinflammatory cytokines. FIG. 1 shows that siRNA against eIF-5A1 causes decreased expression of IL-10.

[0019] FIG. 2 shows that siRNA against eIF-5A1 causes decreased expression of IL-2.

[0020] FIG. 3 shows that siRNA against eIF-5A1 causes decreased expression of IL-4.

[0021] FIG. 4 shows that siRNA against eIF-5A1 causes decreased expression of IL-5.

[0022] FIG. 5 shows that siRNA against eIF-5A1 causes decreased expression of IL-10.

[0023] FIG. 6 shows that siRNA against eIF-5A1 causes increased expression of GM-CSF.

[0024] FIG. 7 shows that siRNA against eIF-5A1 causes decreased expression of IFN-γ.

[0025] FIG. 8 shows that siRNA against eIF-5A1 causes decreased expression of TNF-α.

[0026] FIG. 9 shows that siRNA against eIF-5A1 causes increased expression of IL-1α.

[0027] FIG. 10 shows that siRNA against eIF-5A1 causes decreased expression of IL-3.

[0028] FIG. 11 shows that siRNA against eIF-5A1 causes decreased expression of IL-6.

[0029] FIG. 12 shows that siRNA against eIF-5A1 causes decreased expression of IL-12(p40).

[0030] FIG. 13 shows that siRNA against eIF-5A1 causes decreased expression of IL-12(p70).

[0031] FIG. 14 shows that siRNA against eIF-5A1 causes increased expression of IL-17.

[0032] FIG. 15 shows that siRNA against eIF-5A1 causes decreased expression of G-CSF.

[0033] FIG. 16 shows that siRNA against eIF-5A1 causes decreased expression of KC.

[0034] FIG. 17 shows that siRNA against eIF-5A1 causes decreased expression of MIP-1α.

[0035] FIG. 18 shows that siRNA against eIF-5A1 causes decreased expression of RANTES.

[0036] FIG. 19 provides an eIF-5A1 siRNA construct.

[0037] FIG. 20 shows the effect of cardiac puncture and bleeding on one hour post hemorrhagic lung. IL-10 expression significantly increases.

[0038] FIG. 21 shows that administration of eIF5A1 siRNA prior to inducement of hemorrhage shock, caused a decreased expression of Il-1B and TNF-α.

[0039] FIG. 22 provides the nucleotide sequence of human eIF5A1 aligned against eIF5A2.

[0040] FIG. 23 provides the amino acid sequence of human eIF5A1 aligned against eIF5A2.

[0041] FIG. 24 provides the nucleotide sequence of human eIF5A1 with exemplary antisense oligonucleotides.

[0042] FIG. 25 provides the nucleotide sequence of human eIF5A1 with exemplary antisense oligonucleotides.

[0043] FIGS. 26A and B provide the nucleotide sequence of human eIF5A1 with exemplary siRNAs.

[0044] FIG. 27 provides the nucleotide sequence of human eIF5A1 with exemplary siRNAs.

DETAILED DESCRIPTION OF THE INVENTION

[0045] Several isoforms of eukaryotic initiation factor 5A ("eIF-5A") have been isolated and present in published databanks. It was thought that these isoforms were functionally redundant. The present inventors have discovered that one isoform is upregulated immediately before the induction of apoptosis, which they have designated apoptosis-specific eIF-5A or eIF-5A1. The subject of the present invention is apoptosis-specific eIF-5A and the down regulation of its expression to down regulate expression of pro-inflammatory cytokines.

[0046] Sepsis is a process of malignant intravascular inflammation causing ˜210,000 deaths annually. Accordingly, adjunctive therapies are needed. Sepsis is also known as systemic inflammatory response syndrome ("SIRS"). Sepsis is caused by bacterial infection that can originate anywhere in the body. Sepsis can be simply defined as a spectrum of clinical conditions caused by the immune response of a patient to infection that is characterized by systemic inflammation and coagulation. It includes the full range of response from systemic inflammatory response (SIRS) to organ dysfunction to multiple organ failure and ultimately death.

[0047] Sepsis is a very complex sequence of events and much work still needs to be done to completely understand how a patient goes from SIRS to septic shock. Patients with septic shock have a biphasic immunological response. Initially they manifest an overwhelming inflammatory response to the infection. This is most likely due to the pro-inflammatory cytokines Tumor Necrosis Factor (TNF), IL-1, IL-12, Interferon gamma (IFN-γ), and IL-6. The body then regulates this response by producing anti-inflammatory cytokines (IL-10), soluble inhibitors (TNF receptors, IL-1 receptor type II, and IL-1RA (an inactive form of IL-1)), which is manifested in the patient by a period of immunodepression. Persistence of this hypo-responsiveness is associated with increased risk of nosocomial infection and death.

[0048] This systemic inflammatory cascade is initiated by various bacterial products. These bacterial products such as gram-negative bacteria=endotoxin, formyl peptides, exotoxins, and proteases; gram-positive bacteria=exotoxins, superantigens (toxic shock syndrome toxin (TSST), streptococcal pyrogenic exotoxin A (SpeA)), enterotoxins, hemolysins, peptidoglycans, and lipotechoic acid, and fungal cell wall material, which bind to cell receptors on the host's macrophages and activate regulatory proteins such as Nuclear Factor Kappa B (NFkB). Endotoxin activates the regulatory proteins by interacting with several receptors. The CD receptors pool the LPS-LPS binding protein complex on the surface of the cell and then the TLR receptors translate the signal into the cells.

[0049] As mentioned above, the pro-inflammatory cytokines produced are tumor necrosis factor (TNF), Interleukins 1, 6 and 12 and Interferon gamma (IFN-γ). These cytokines can act directly to affect organ function or they may act indirectly through secondary mediators. The secondary mediators include nitric oxide, thromboxanes, leukotrienes, platelet-activating factor, prostaglandins, and complement. TNF and IL-1 (as well as endotoxin) can also cause the release of tissue-factor by endothelial cells leading to fibrin deposition and disseminated intravascular coagulation (DIC).

[0050] These primary and secondary mediators then cause the activation of the coagulation cascade, the complement cascade and the production of prostaglandins and leukotrienes. Clots lodge in the blood vessels which lowers profusion of the organs and can lead to multiple organ system failure. In time, this activation of the coagulation cascade depletes the patient's ability to make a clot resulting in DIC and ARDS.

[0051] The cumulative effect of this cascade is an unbalanced state, with inflammation dominant over anti-inflammation and coagulation dominant over fibrinolysis. Microvascular thrombosis, hypoperfusion, ischemia, and tissue injury result. Severe sepsis, shock, and multiple organ dysfunction may occur, leading to death.

[0052] Because the present inventors had previously determined that eIF5A1 siRNA (delivered intranasaly as naked siRNA) decreased the production or expression of multiple potential mediators of sepsis (e.g. IL-1β, TNF-α, IL-8, iNOS, TLR-4 expression) in cell systems and a few proinflammatory cytokines in blood following intranasal lipopolysaccharide (LPS) challenge in vivo, the impact on survival and cytokine expression in endotoxemic mice was studied. See co-pending U.S. application Ser. Nos. 11/134,445 (filed May 23, 2005), 11/184,982 (filed Jul. 20, 2005), 11/293,391 (filed Nov. 28, 2005), and 11/595,990 (filed Nov. 13, 2006), which are all herein incorporated by reference in their entirety.

[0053] BALB/C mice were inoculated with E. coli O111:B4 LPS intraperitoneally (IP), causing death in 93% of controls. Animals received either eIF5A1 siRNA (N=5) (3'-GCC UUA CUG AAG GUC GAC U-5') or scrambled RNA as a control (N=15). A 50 μg dose of eIF5A1 siRNA was given IP in conjunction with 100 μg of transfection micelle comprised of DOTAP. The siRNA-liposome complex was dosed at t=-48 and -24 hrs prior to LPS administration. Survival experiments were conducted and under similar conditions mice were sacrificed at 90 min or 8 hours after LPS administration and blood sampled. A bead-based multiplex sandwich immunoassay quantified circulating cytokines. The results indicate that treatment of BALB/C mice with eIF5A1 siRNA conferred 60% protection (p<0.01). With treatment, IL-113 dropped from 5909 to 658 pg/mL at 90 min and from 2478 to 1032 pg/mL at 8 hrs. Treatment also decreased TNF-α from 33649 to 3696 pg/mL at 90 min and from 1272 to 901 at 8 hrs. MIP-1α also decreased from 10499 to 3475 pg/mL at 90 min and from 680 to 413 pg/mL at 8 hrs with treatment. At 8 hrs, treatment reduced IFN-γ from 142 to 86 pg/mL and IL-12(p40) from 46570 to 14261 pg/mL. The anti-inflammatory cytokine IL-10 was increased from 719 to 898 pg/mL at 90 min with treatment. These studies show that targeting inflammatory mediators with siRNA confers protection in endotoxemic mice and suggests this may be a useful approach in the treatment of septic patients.

[0054] In addition, to the septic model discussed above, the inventors also developed a novel murine model for studying hemorrhagic shock. In this model, male mice C-57BL/6J (8-12 weeks old) were induced into hemorrhage shock by withdrawal of 30% of the calculated blood volume (0.55 ml) by cardiac puncture over a 60-sec period (under methoxyflurane anesthesia). Lungs were harvested at 1 h after bleeding and were homogenized in 1 ml of ice-cold extraction buffer containing 20 mM HEPES (pH 7.4), 20 mM glycerophosphate, 20 mM sodium pyrophosphate, 0.2 mM Na3VO4, 2 mM EDTA, 20 mM sodium fluoride, 10 mM benzamidine, 1 mM DTT, 20 ng/ml leupeptin, 0.4 mM Pefabloc SC, and 0.01% Triton X-100. The homogenate was centrifuged at 14,000 g for 15 min at 4° C. The supernatant was collected, and the protein concentration was determined with the bicinchoninic acid assay. The resulting supernatant was used for determination of TNF, IL-1, and IL-6 by ECL (liquid phase ELISA), according to the manufacturer's suggestions. Final results were expressed as picograms cytokine protein per milligram of protein.

[0055] In another hemorrhagic model, the inventors showed that providing eIF5A siRNA, they could reducing expression of TNFα and IL-1β. 5 Mice C-57BL/6J, male induced i.p. were treated with 50 μg of eF5A1 siRNA 24 hours prior to hemorrhage. In the control, 5 Mice C-57BL/6J, male induced i.p. were treated with 50 ug of scrambled siRNA 24 hours prior to hemorrhage. Hemorrhage shock was developed by withdraw of 0.55 mL by cardiac puncture over a 60-sec period (under methoxyflurane-anesthesia). FIG. 21 shows that administration of siRNA prior to inducement of hemorrhage shock, provided a protective benefit by decreasing expression of Il-1β and TNF-α.

[0056] Thus, one embodiment of the present invention provides a method for decreasing expression of pro-inflammatory cytokines in vivo in a subject, comprising administering eIF5A1 siRNA to the subject, whereby the eIF5A1 siRNA decreases expression of pro-inflammatory cytokines. The subject may be any animal including a human.

[0057] The pro-inflammatory cytokine is any cytokine that is involved in the inflammation cascade, such as IL-1β, IL-2, IL-4, IL-5, IL-10, IFN-γ, TNF-α, IL-3, IL-6, IL-12(p40), IL-12(p70), G-CSF, KC, MIP-1α, and RANTES. FIGS. 1-18 and 21-22 show that treatment with eIF5A1 siRNA resulted in a decreased amount of proinflammatory cytokines as compared to animals not having received the eIF5A1 siRNA.

[0058] As shown above, the inventors demonstrated that eIF5A siRNA confers protection in endotoxemic mice when pro-inflammatory cytokine expression was reduced. Hence, one embodiment of the invention also provides a method of treating sepsis in a subject by administering eIF5A1 siRNA to the subject, whereby administration of eIF5A1 siRNA decreases expression of eIF5A1 and results in decreased expression of pro-inflammatory cytokines. Decreased expression means reduced expression as well as decreased or reduced levels of a particular protein as compared to levels of expression or amounts of a protein in a subject not having been treated with eIF5A1 siRNA other eIF5A1 antisense constructs.

[0059] Another embodiment of the present invention further provides a method of preventing hemorrhagic shock in a subject, including a human, comprising administering an eIF5A1 siRNA or antisense polynucleotide to decrease expression of IL-1β and/or TNF-α.

[0060] Any eIF5A1 siRNA that inhibits expression of eIF5A1 may be used. The term "inhibits" also means reduce or decrease. One exemplary eIF5A1 siRNA comprises the sequence: CGG AAU GAC UUC CAG CUG A. Co-pending U.S. application Ser. Nos. 11/134,445 (filed May 23, 2005), 11/184,982 (filed Jul. 20, 2005), 11/293,391 (filed Nov. 28, 2005), and 11/595,990 (filed Nov. 13, 2006) (which are herein incorporated by reference in its entirety) provides additional exemplary eIF5A1 siRNAs and other antisense constructs that have been used to inhibit expression of eIF5A1 in other cell types and were also shown to inhibit expression of pro-inflammatory cytokines. One skilled in the art could design other eIF5A1 siRNAs given the eIF51A sequence and can easily test for the siRNAs ability to inhibit expression without undue experimentation. FIGS. 22-27 provide sequences of eIF5A1, exemplary eIF5A1 siRNAs and antisense constructs.

[0061] The preset invention also provides pharmaceutical compositions comprising eIF-5A1 siRNA or antisense polynucleotides discussed above useful for decreasing expression of pro-inflammatory cytokines. The composition may comprising eIF5A1 siRNA or antisense polynucleotides and a pharmaceutically acceptable carrier. Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[0062] Generally, an effective amount of the eIF5A1 siRNA or eIF5A1 antisense nucleotides described above will be determined by the age, weight and condition or severity of disease of the recipient. Dosing may be one or more times daily, or less frequently. It should be noted that the present invention is not limited to any dosages recited herein.

[0063] Pharmaceutical compositions may be prepared as medicaments to be administered in any method suitable for the subject's condition, for example, orally, parenterally (including subcutaneous, intramuscular, and intravenous), rectally, transdermally, buccally, or nasally, or may be delivered to the eye as a liquid solution.

[0064] The siRNA or antisense construct can be delivered as "naked" siRNA or antisense nucleotide or may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0065] The antisense polynucleotides and/or siRNA may be chemically modified. This may enhance their resistance to nucleases and may enhance their ability to enter cells. For example, phosphorothioate oligonucleotides may be used. Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-O-alkyl analogs and 2'-O-methylribonucleotide methylphosphonates.

[0066] Alternatively mixed backbone oligonucleotides (MBOs) may be used. MBOs contain segments of phosphothioate oligodeoxynucleotides and appropriately placed segments of modified oligodeoxy- or oligoribonucleotides. MBOs have segments of phosphorothioate linkages and other segments of other modified oligonucleotides, such as methylphosphonate, which is non-ionic, and very resistant to nucleases or 2'-O-alkyloligoribonucleotides.

Sequence CWU 1

1

39119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1cggaaugacu uccagcuga 19219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 2ucagcuggaa gucauuccg 19323DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic construct 3aacggaauga cuuccagcug att 23421DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic construct 4cggaaugacu uccagcugat t 21521DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic construct 5ucagcuggaa gucauuccgt t 216465DNAHomo sapiens 6atggcagatg acttggactt cgagacagga gatgcagggg cctcagccac cttcccaatg 60cagtgctcag cattacgtaa gaatggcttt gtggtgctca aaggccggcc atgtaagatc 120gtcgagatgt ctacttcgaa gactggcaag cacggccacg ccaaggtcca tctggttggt 180attgacatct ttactgggaa gaaatatgaa gatatctgcc cgtcaactca taatatggat 240gtccccaaca tcaaaaggaa tgacttccag ctgattggca tccaggatgg gtacctatca 300ctgctccagg acagcgggga ggtacgagag gaccttcgtc tccctgaggg agaccttggc 360aaggagattg agcagaagta cgactgtgga gaagagatcc tgatcacggt gctgtctgcc 420atgacagagg aggcagctgt tgcaatcaag gccatggcaa aataa 4657462DNAHomo sapiens 7atggcagacg aaattgattt cactactgga gatgccgggg cttccagcac ttaccctatg 60cagtgctcgg ccttgcgcaa aaacggcttc gtggtgctca aaggacgacc atgcaaaata 120gtggagatgt caacttccaa aactggaaag catggtcatg ccaaggttca ccttgttgga 180attgatattt tcacgggcaa aaaatatgaa gatatttgtc cttctactca caacatggat 240gttccaaata ttaagagaaa tgattatcaa ctgatatgca ttcaagatgg ttacctttcc 300ctgctgacag aaactggtga agttcgtgag gatcttaaac tgccagaagg tgaactaggc 360aaagaaatag agggaaaata caatgcaggt gaagatgtac aggtgtctgt catgtgtgca 420atgagtgaag aatatgctgt agccataaaa ccctgcaaat aa 4628154PRTHomo sapiens 8Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala 1 5 10 15 Thr Phe Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val 20 25 30 Leu Lys Gly Trp Pro Cys Lys Ile Val Glu Met Ser Ala Ser Lys Thr 35 40 45 Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe 50 55 60 Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp 65 70 75 80 Val Pro Asn Ile Lys Arg Asn Asp Phe Gln Leu Ile Gly Ile Gln Asp 85 90 95 Gly Tyr Leu Ser Leu Leu Gln Asp Ser Gly Glu Val Pro Glu Asp Leu 100 105 110 Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu Ile Glu Gln Lys Tyr Asp 115 120 125 Cys Gly Glu Glu Ile Leu Ile Thr Leu Leu Ser Ala Met Thr Glu Glu 130 135 140 Ala Ala Val Ala Ile Lys Ala Met Ala Lys 145 150 9153PRTHomo sapiens 9Met Ala Asp Glu Ile Asp Phe Thr Thr Gly Asp Ala Gly Ala Ser Ser 1 5 10 15 Thr Tyr Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val 20 25 30 Leu Lys Gly Arg Pro Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr 35 40 45 Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe 50 55 60 Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp 65 70 75 80 Val Pro Asn Ile Lys Arg Asn Asp Tyr Gln Leu Ile Cys Ile Gln Asp 85 90 95 Gly Tyr Leu Ser Leu Leu Thr Glu Thr Gly Glu Val Arg Glu Asp Leu 100 105 110 Lys Leu Pro Glu Gly Glu Leu Gly Lys Glu Ile Glu Gly Lys Tyr Asn 115 120 125 Ala Gly Glu Asp Val Gln Val Ser Val Met Cys Ala Met Ser Glu Glu 130 135 140 Tyr Ala Val Ala Ile Lys Pro Cys Lys 145 150 1020DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 10cctgtctcga agtccaagtc 201120DNAHomo sapiens 11gacttggact tcgagacagg 201220DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 12ggaccttggc gtggccgtgc 201320DNAHomo sapiens 13gcacggccac gccaaggtcc 201420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 14ctcgtacctc cccgctctcc 201519DNAHomo sapiens 15ggacagcggg gaggtacga 19161309DNAHomo sapiens 16ggcacgaggg tagaggcggc ggcggcggcg gcagcgggct cggaggcagc ggttgggctc 60gcggcgagcg gacggggtcg agtcagtgcg ttcgcgcgag ttggaatcga agcctcttaa 120aatggcagat gacttggact tcgagacagg agatgcaggg gcctcagcca ccttcccaat 180gcagtgctca gcattacgta agaatggctt tgtggtgctc aaaggccggc catgtaagat 240cgtcgagatg tctacttcga agactggcaa gcacggccac gccaaggtcc atctggttgg 300tattgacatc tttactggga agaaatatga agatatctgc ccgtcaactc ataatatgga 360tgtccccaac atcaaaagga atgacttcca gctgattggc atccaggatg ggtacctatc 420actgctccag gacagcgggg aggtacgaga ggaccttcgt ctccctgagg gagaccttgg 480caaggagatt gagcagaagt acgactgtgg agaagagatc ctgatcacgg tgctgtctgc 540catgacagag gaggcagctg ttgcaatcaa ggccatggca aaataactgg ctcccaggat 600ggcggtggtg gcagcagtga tcctctgaac ctgcagaggc cccctccccg agcctggcct 660ggctctggcc cggtcctaag ctggactcct cctacacaat ttatttgacg ttttattttg 720gttttcccca ccccctcaat ctgtcgggga gcccctgccc ttcacctagc tcccttggcc 780aggagcgagc gaagctgtgg ccttggtgaa gctgccctcc tcttctcccc tcacactaca 840gccctggtgg gggagaaggg ggtgggtgct gcttgtggtt tagtcttttt tttttttttt 900tttttttttt aaattcaatc tggaatcaga aagcggtgga ttctggcaaa tggtccttgt 960gccctcccca ctcatccctg gtctggtccc ctgttgccca tagcccttta ccctgagcac 1020caccccaaca gactggggac cagccccctc gcctgcctgt gtctctcccc aaaccccttt 1080agatggggag ggaagaggag gagaggggag gggacctgcc ccctcctcag gcatctggga 1140gggccctgcc cccatgggct ttacccttcc ctgcgggctc tctccccgac acatttgtta 1200aaatcaaacc tgaataaaac tacaagttta atatgaaaaa aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 13091719DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 17gcacggccac gccaaggtc 191820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 18ggacagcggg gaggtacgag 20191299DNAHomo sapiens 19ggcacgaggg cggcggcggc ggtagaggcg gcggcggcgg cggcagcggg ctcggaggca 60gcggttgggc tcgcggcgag cggacggggt cgagtcagtg cgttcgcgcg agttggaatc 120gaagcctctt aaaatggcag atgacttgga cttcgagaca ggagatgcag gggcctcagc 180caccttccca atgcagtgct cagcattacg taagaatggc tttgtggtgc tcaaaggccg 240gccatgtaag atcgtcgaga tgtctacttc gaagactggc aagcacggcc acgccaaggt 300ccatctggtt ggtattgaca tctttactgg gaagaaatat gaagatatct gcccgtcaac 360tcataatatg gatgtcccca acatcaaaag gaatgacttc cagctgattg gcatccagga 420tgggtaccta tcactgctcc aggacagcgg ggaggtacga gaggaccttc gtctccctga 480gggagacctt ggcaaggaga ttgagcagaa gtacgactgt ggagaagaga tcctgatcac 540ggtgctgtct gccatgacag aggaggcagc tgttgcaatc aaggccatgg caaaataact 600ggctcccagg atggcggtgg tggcagcagt gatcctctga acctgcagag gccccctccc 660cgagcctggc ctggctctgg cccggtccta agctggactc ctcctacaca atttatttga 720cgttttattt tggttttccc caccccctca atctgtcggg gagcccctgc ccttcaccta 780gctcccttgg ccaggagcga gcgaagctgt ggccttggtg aagctgccct cctcttctcc 840cctcacacta cagccctggt gggggagaag ggggtgggtg ctgcttgtgg tttagtcttt 900tttttttttt tttttttttt tttaaattca atctggaatc agaaagcggt ggattctggc 960aaatggtcct tgtgccctcc ccactcatcc ctggtctggt cccctgttgc ccatagccct 1020ttaccctgag caccacccca acagactggg gaccagcccc ctcgcctgcc tgtgtctctc 1080cccaaacccc tttagatggg gagggaagag gaggagaggg gaggggacct gccccctcct 1140caggcatctg ggagggccct gcccccatgg gctttaccct tccctgcggg ctctctcccc 1200gacacatttg ttaaaatcaa acctgaataa aactacaagt ttaatatgaa aaaaaaaaaa 1260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 12992021DNAHomo sapiens 20aaaggaatga cttccagctg a 212123DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 21aaaggaauga cuuccagcug att 232223DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 22ucagcuggaa gucauuccuu utt 232321DNAHomo sapiens 23aagatcgtcg agatgtctac t 212423DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 24aagaucgucg agaugucuac utt 232523DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 25aguagacauc ucgacgaucu utt 232621DNAHomo sapiens 26aaggtccatc tggttggtat t 212723DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 27aagguccauc ugguugguau utt 232823DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 28aauaccaacc agauggaccu utt 232921DNAHomo sapiens 29aagctggact cctcctacac a 213023DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 30aagcuggacu ccuccuacac att 233123DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 31uguguaggag gaguccagcu utt 233221DNAHomo sapiens 32aaagtcgacc ttcagtaagg a 213323DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 33aaagucgacc uucaguaagg att 233423DNAArtificial SequenceDescription of Combined DNA/RNA Molecule Synthetic oligonucleotide 34uccuuacuga aggucgacuu utt 233523DNAHomo sapiens 35aaaggaatga cttccagctg att 233623DNAHomo sapiens 36aagatcgtcg agatgtctac ttc 233723DNAHomo sapiens 37aaggtccatc tggttggtat tga 233823DNAHomo sapiens 38aagctggact cctcctacac aat 233923DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 39aaagtcgacc ttcagtaagg att 23

Claims

1. A method for decreasing expression of pro-inflammatory cytokines in vivo in a subject, comprising administering eIF5A1 siRNA to the subject, whereby the eIF5A1 siRNA decreases expression of pro-inflammatory cytokines in the subject.

2. The method of claim 1 wherein the subject is a human.

3. The method of claim 1 wherein the pro-inflammatory cytokine is selected from the group consisting of IL-1.beta., IL-2, IL-4, IL-5, IL-10, IFN-.gamma., TNF-.alpha., IL-3, IL-6, IL-12(p40), IL-12(p70), G-CSF, KC, MIP-1.alpha., and RANTES.

4. The method of claim 1 wherein the pro-inflammatory cytokine is TNF-.alpha..

5. The method of claim 1 wherein the pro-inflammatory cytokine is IL-6.

6. The method of claim 1 wherein the pro-inflammatory cytokine is KC.

7. The method of claim 1 wherein the pro-inflammatory cytokine is MIP-1.alpha..

8. The method of claim 1 wherein said decreased expression of pro-inflammatory cytokines further provides treatment of sepsis.

9. The method of claim 1 wherein the sense strand of the eIF5A1 siRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29 and SEQ ID NO:32.

10. A pharmaceutical composition for decreasing expression of pro-inflammatory cytokines, comprising eIF5A siRNA and a pharmaceutically acceptable carrier.

11. The composition of claim 11 wherein the sense strand of the siRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29 and SEQ ID NO:32.

12. A method of preventing hemorrhagic shock in a subject comprising administering an eIF5A1 siRNA or eIFA1 antisense polynucleotide to decrease expression of IL-1.beta. and/or TNF-.alpha. in the subject, thereby preventing hemorrhagic shock.

13. The method of claim 1 wherein the sense strand of siRNA comprises SEQ ID NO: 30 and the antisense strand comprises SEQ ID NO: 31.

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