WO2003057728A1 - A novel splice variant of myd88 and uses thereof - Google Patents
A novel splice variant of myd88 and uses thereof Download PDFInfo
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- WO2003057728A1 WO2003057728A1 PCT/EP2003/000381 EP0300381W WO03057728A1 WO 2003057728 A1 WO2003057728 A1 WO 2003057728A1 EP 0300381 W EP0300381 W EP 0300381W WO 03057728 A1 WO03057728 A1 WO 03057728A1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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Definitions
- the present invention relates to the field of infection and inflammation and more specifically to the field of pathogen-induced nuclear factor kappa B activation. More specifically, a novel splice variant of MyD88, (MyD ⁇ s), has been identified encoding a protein that inhibits TLR-induced NF-kappaB activation. MyD88s is a target to inhibit the phenomenon of endotoxin-tolerance that occurs in sepsis.
- the MyD88 gene was originally described as one of several myeloid differentiation primary response genes that are induced in murine M1 myeloblastic leukemia cells upon stimulation with IL-6. It is an exclusively cytosolic protein that functions as a unique adaptor for members of the type I interleukin-1 receptor (IL-1 R)/Toll like receptor (TLR) family.
- the MyD88 protein has a modular structure. At its N-terminus it has a 'death domain' (DD) similar to the cytoplasmic signalling domains found in many members of the tumour necrosis factor (TNF) receptor superfamily.
- DD 'death domain'
- TIR domain Toll/IL-1R' domain. Both domains are required for MyD88 homodimerization, and are separated by a short intermediate domain (ID) of unknown function.
- ID short intermediate domain
- the TIR domain of MyD88 forms a homophylic interaction with the TIR domain of IL-1 R and IL-1 Receptor accessory protein (IL-1 RacP), IL-18R, and several TLRs, whereas the DD binds with the DD of both IL-1 receptor associated kinase (IRAK) and IRAK-2. Interaction with MyD88 triggers IRAK phosphorylation.
- TNF receptor associated factor 6 TNF receptor associated factor 6
- JNK c-jun N-terminal kinase
- MyD ⁇ " ' " cells were resistant to LPS- induced endotoxic shock, but still showed delayed NF-kappaB translocation to the nucleus, which suggests redundancy at the level of MyD ⁇ in the LPS-pathway.
- MyD ⁇ mRNA expression has been found to be constitutively expressed in many adult human tissues as a 2.6 kb mRNA species.
- MyD ⁇ s a splice variant of MyD ⁇ , termed MyD ⁇ s, which encodes for a protein lacking the ID.
- MyD ⁇ s which encodes for a protein lacking the ID.
- a first aspect of the invention deals with endotoxin tolerance.
- Endotoxin tolerance is thought to be an adaptive response to protect the body from hyperactivation of the innate immune system during bacterial infections.
- endotoxin tolerance can also lead to a fatal blunting of the immune response to subsequent infections in survivors of sepsis (Kox et al. (2000) Intensive Care Med. 26, 124).
- therapies to treat patients from septic shock, so far most clinical trials yielded disappointing results (Kox et al. (2000) Intensive Care Med. 26, 124).
- Endotoxin tolerance is associated with monocyte deactivation which results in an impaired cytokine production or HLA-DR expression.
- the situation can be mimicked in vitro by pretreatment of monocytes with endotoxin which makes them refractory to subsequent LPS challenges (Adib-Conquy et al. (2000) Am. J. respir. Crit. Care Med. 164, 1 ⁇ 77).
- endotoxin which makes them refractory to subsequent LPS challenges
- Several mechanisms have been proposed to explain the impaired response e.g. diminished expression of TLR, changed p65/p50 ratio or expression of immuno- inhibitory factors such as IL10. Recently, a paper by Li et al. (2000) J. Biol. Chem.
- 275, 23340 shows that IRAK-phosphorylation upon LPS activation is impaired in endotoxin tolerant monocytes.
- MyD ⁇ s a splice variant of MyD ⁇ which blocks NF- ⁇ B activation, prevents IRAK-phosphorylation, and is upregulated in endotoxin tolerant monocytes.
- the present invention shows that MyD ⁇ s has a role in the induction of endotoxin tolerance, the transient, secondary downregulation of a subset of endotoxin-d riven responses after exposure to bacterial products. It is shown that MyD ⁇ s behaves as a dominant negative inhibitor of LPS-, but not of TNF-induced NF-kappaB activation.
- a second aspect of the invention deals with the so-called JNK and NF-kappaB signaling pathways, both of which consist of tiers of protein kinases, and are pivotal in determining whether cells die or survive.
- the JNKs are part of the evolutionarily conserved mitogen-activated protein kinase family, and are implicated in cell-death pathways stimulated by environmental stresses and TNF. Once activated, JNK proteins can move from the cytoplasm of the cell into the nucleus. There, they phosphorylate and activate numerous transcription factors. However, the exact mechanism by which JNKs contribute to cell death are still unknown, but mostly cell death requires a sustained activation of the JNK pathway.
- NF-kappaB enhances cell survival by switching on genes that dampen pro-apoptotic signals. It was shown that NF- ⁇ B can downregulate pro-apoptotic JNK signalling in response to TNF and chemotherapeutic drugs by shifting the sustained JNK activation to a more transient activation.
- the JNK inhibiting effect of NF- ⁇ B was mediated by the transcriptional upregulation of specific proteins (XIAP, Gadd45 ⁇ ).
- the present invention demonstrates that MyD ⁇ s allows specific activation of the JNK pathway and AP-1 dependent gene expression, while blocking NF-kappa B dependent gene expression.
- MyD ⁇ s can be used to stimulate apoptosis in for example cancer, or other situations where a lack of programmed cell death occurs.
- a third aspect of the invention deals with the expression of MyD ⁇ s in immune privileged tissues.
- Immune privilege is an example of regional immunity: immune effector cells are not only differentiated for maximum ability to eliminate pathogens, but also for minimum ability to interfere with a specific organ or tissue's physiological function.
- the present invention shows that MyD ⁇ s is specifically expressed in brain, testis, and eye of C57/BL6 mice, which is consistent with a role in immune privilege.
- the present invention also shows expression of MyD ⁇ s in the spleen under specific conditions (e.g. in Balb/c mice; in TNF-treated C57/BL6 mice).
- the spleen has a well-established role in immune regulation. As a lymphoid organ it provides a microenvironment where several immune cells come into close proximity, enabling a more efficient immune response (for review see Delves and Roitt (2000) New Engl. J. Med. 343, 108). Besides having a role in the initiation of an immune reaction, a more immunoregulatory and immunosuppressive role has also been attributed to the spleen. In several situations such as graft-versus-host disease (Wall et al. (198 ⁇ ) J. Immunol., 140, 2970), cyclophosphamide treatment (Angulo et al.
- MyD ⁇ s can be used to obtain immunosuppression (e.g. in the case of transplantation).
- the invention shows that modulation of the expression of MyD ⁇ s can regulate the cellular responses to LPS and other immunological stimuli.
- Fig. 1 Identification of a splice variant of MyD ⁇ , which lacks the intermediate domain.
- Fig. 2 MyD88 s specifically inhibits IL-1- and LPS-induced NF- ⁇ B activation.
- HEK293T cells (a)-(b) or Mf4/4 cells (c) were transiently transfected with the NF- ⁇ B dependent luciferase reporter plasmid pNFconluc and the ⁇ -galactosidase reporter plasmid pPGKneogal, each time combined with an expression plasmid for either MyD ⁇ s, MyD ⁇ L , MyD ⁇ -TIR or empty vector (EV) as indicated.
- 4 ⁇ h post-transfection cells were either left untreated or stimulated for 6 h with 100 ng/ml IL-1 ⁇ (a), 100 ng/ml TNF (b) or 100 ng/ml LPS (c).
- Luciferase activity in cell extracts is normalized for differences in transfection efficiency on the basis of ⁇ -galactosidase activity. Values are the mean (+ standard error) of three different transfections within a single experiment, and are expressed as fold induction relative to the unstimulated EV control, (a Insert) Electrophoretic mobility shift assay with nuclear extracts prepared from HEK293T cells that were transiently transfected with empty vector (EV), MyD ⁇ L or MyD ⁇ s. The shift in migration of a 32 P-labeled Ig KB DNA-oligo upon binding of NF- KB is shown. Proper expression of MyD ⁇ s and MyD ⁇ L was verified by western blot analysis (data not shown).
- Fig. 3 MyD88 s competes with MyD88 L for dimer formation (a) and recruitment to the IL-1R (b).
- HEK293T cells were transiently transfected with different expression plasmids for epitope-tagged proteins (amounts shown in ⁇ g on top of each figure).
- Cell extracts were immunoprecipitated (IP) with anti-Flag antibody, and co- immunoprecipitating proteins were revealed by western blotting (WB) with anti-E-tag antibody (upper panel). Immunoprecipitation of Flag-tagged proteins was confirmed by western blotting (middle panel). Expression of transfected proteins was confirmed by western blotting of total lysates (TL) with anti-E-tag antibody (lower panel).
- Fig. 4 MyD88 s still allows IRAK recruitment to the IL-1R complex (a), but no longer induces IRAK phosphorylation (b)
- Cell extracts were immunoprecipitated (IP) with anti-Flag antibody, and co-immunoprecipitating proteins were revealed by western blotting (WB) with an anti-IRAK antibody (upper panel). Immunoprecipitation of Flag-tagged proteins was confirmed by western blotting (second panel).
- HEK293T cells were transiently transfected with expression plasmids for epitope-tagged MyD ⁇ (amounts shown in ⁇ g on top of the figure), in combination with either an expression plasmid for IRAK(T66A) or IRAK-N(T66A).
- Cell lysates were analysed by western blotting and probed with anti- IRAK (upper and middle panel) or anti-E-tag antibody (lower panel).
- Fig. 5 Expression of endogenous MyD88 s in LPS-pretreated human monocytes is associated with a diminished LPS-response.
- HEK293T cells were transiently transfected with an expression plasmid for Flag-tagged JNK, together with increasing concentrations of different MyD ⁇ proteins. 2 days later, cells were treated for 30 min or 4h with IL-1 and analysed for JNK phosphorylation by immunoblotting with a phospho-JNK specific antibody (upper panel). Total expression of JNK was verified by western blotting with anti-Flag. This shows that IL-1 induces the phosphorylation of JNK after 30 min as well as after 4h treatment.
- MyD ⁇ -TIR deletion mutant of MyD ⁇ which only expresses the TIR domain
- MyD ⁇ -lpr point mutant in the death domain which disrupts the structure of the death domain
- Fig. 7 MyD ⁇ s induces AP-1 dependent gene expression
- HEK293T cells were transiently transfected with an AP-1 dependent luciferase reporter construct, together with increasing amounts of MyD ⁇ s, MyD ⁇ L or MyD ⁇ TIR expression plasmids. 2 days after transfection, cell extracts were prepared and luciferase activity determined. Differences in transfection efficiency were normalized by cotransfecting a constitutively expressed ⁇ gal expression plasmid and values are expressed as luc/gal.
- MyD ⁇ is an adaptor protein that is involved in signalling triggered by various members of the interleukin-1 receptor (IL-1R) / Toll-like receptor (TLR) superfamily.
- IL-1R interleukin-1 receptor
- TLR Toll-like receptor
- a role for MyD ⁇ has been shown in response to triggering of IL-1R, IL-1 ⁇ R, TLR2, TLR3, TLR4, TLR9.
- IL-1 and IL-18 are pleiotropic cytokines which play a central role in the immune response and in many inflammatory diseases such as rheumatoid arthritis or septic shock.
- TLRs behave as receptors for various microbial products (including bacterial, viral, yeast derived products).
- IL-1R/TLR superfamily As well as MyD ⁇ have been shown to play an important role in both innate and adaptive immune responses.
- MyD ⁇ a splice variant of MyD ⁇ , MyD ⁇ s, which lacks its intermediate domain, the domain in between the N-terminal death domain (necessary for interaction with IRAK) and the C-terminal TIR domain (necessary for interaction with the IL-1R or TLR). Deletion of the intermediate domain abolishes the ability of MyD ⁇ s to activate NF- ⁇ B.
- MyD ⁇ s acts as a dominant negative inhibitor of the IL-1 -and LPS-induced signaling pathway to NF- ⁇ B by interfering at the level of IRAK phosphorylation.
- MyD ⁇ s does not mediate phosphorylation of co-expressed IRAK.
- MyD ⁇ is also essential for the activation of JNK in response to IL-1 R and TLR triggering, we were interested to see if MyD ⁇ s still allows IL-1 induced JNK activation.
- MyD ⁇ s indeed allows activation of JNK and AP-1, whereas the NF- ⁇ B pathway is completely blocked.
- the invention provides an isolated polypeptide, designated as the splice form MyD ⁇ s, having the primary structural information of amino acids 1-251 as set forth in SEQ ID NO: 2, or a homologue or functional fragment thereof, possessing the biological properties of (1) down-regulating the TLR-induced nuclear factor kappa B activation and (2) activating the c-JUN N-terminal kinase pathway.
- said TLR functions as a receptor for lipopolysaccharide (LPS).
- LPS lipopolysaccharide
- TLRs recognize so-called pathogen-associated molecular patterns or PAMPs.
- TLRs are conserved motifs, unique to micro-organisms and essential for their metabolism and thus survival.
- TLRs have been identified in humans, which mediate recognition of diverse classes of pathogens.
- one group of pathogens is not exclusively recognized by one TLR (e.g. both TLR2 and TLR4 recognize Gram-positive derived PAMPs) and that one TLR can respond to many structurally unrelated ligands, which are often derived from different groups of pathogens (e.g. TLR4 recognizes both viral components as well as gram-negative LPS).
- TLR3 like TLR3, 5 and 9 seem to be more ligand-specific and at least up to now, appear to recognize only one type of ligand.
- TLR7 and TLR ⁇ have been shown to recognize synthetic anti-viral compounds with strong immunostimulatory capacity, belonging to the group of imidazoquinolines. The natural ligands of TLR7 and TLR ⁇ remain to be identified however.
- TLRs also recognize host derived ligands such as the extra domain A of the extracellular matrix protein fibronectin or heat shock proteins. Extracellular matrix proteins are often proteolytically cleaved during infection to facilitate access of macrophages and other immune effector cells to the site of infection.
- the extra domain A (EDA) of fibronectin is encoded by an alternatively spliced exon, which is induced only upon tissue injury.
- Heat shock proteins are normally expressed in the cytoplasm, thus not available for recognition by cell-surface receptors, but can be released by necrotic cells during tissue injury or viral infection. In this way, fragments of fibronectin containing the EDA region or heat shock proteins alert TLRs for an abnormal situation, e.g. tissue injury.
- Activation of TLRs by endogenous ligands implies that they do not only distinguish between self and non-self, but rather sense the presence of 'danger' which can be either non-self or harmful self.
- MyD ⁇ proved to be essential for cytokine induction after stimulation with a variety of ligands such as IL-1 beta, IL-18, LPS, mycoplasmal macrophage-activating lipopeptide-2 (MALP-2), bacterial CpG DNA, poly(l:C) and many others. All these ligands have been demonstrated to signal through different receptors of the TLR/IL-1R superfamily, suggesting that MyD8 ⁇ is a universal adaptor for the TLR/IL-1R superfamily.
- MALP-2 signals through TLR2, LPS through TLR4, Poly(l:C) double stranded RNA through TLR3, CpG DNA through TLR9.
- fragment refers to a polypeptide or polynucleotide of at least about 9 amino acids or 27 base pairs, typically 50 to 75, or more amino acids or base pairs, wherein the polypeptide contains an amino acid core sequence.
- a fragment may be for example a truncated MyD ⁇ s isoform, modified MyD ⁇ s isoform (as by amino acid substitutions, deletions, or additions outside of the core sequence), or other variant polypeptide sequence, but is not a naturally-occurring MyD ⁇ s isoform that is present in a human individual.
- the fragment may be fused at either terminus to additional amino acids or base pairs, which may number from 1 to 20, typically 50 to 100, but up to 250 to 500 or more.
- a “functional fragment” means a polypeptide fragment of MyD ⁇ s possessing the biological properties described above or a polynucleotide fragment encoding said MyD ⁇ s polypeptide fragment possessing the biological properties described above.
- Homology is determined using default parameters of a DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the University of Wisconsin. Also forming part of the invention are allelic variants.
- allelic variant means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
- allelic variant also means a protein encoded by an allelic variant of a gene.
- down-regulating the TLR-induced nuclear factor kappa B activation means that MyD ⁇ s down-regulates the NF-kappaB activation, with respect to the cell which was activated with LPS or other TLR ligands, with at least 50%, 60%, 70%, ⁇ 0% or preferentially with 90%, 95%, 99% or even 100%.
- activating in the wording "activating the c-JUN N-terminal kinase pathway” means that the c-JUN N-terminal kinase pathway is still active while the NF-kappaB-pathway is shut down.
- the IKK-pathway in turn activates the transcription factor NF- ⁇ B
- JNK activates several other transcription factors, including AP-1 , Elk-1, NF-AT, ATF-2, as well as a number of other substrates (e.g. bcl-X L ) (Barr et al. (2001) Int J. Biochem. Cell Biol. 33:1047.
- the transcription factor AP-1 by phosphorylating the c-jun and ATF-2 component. Both, NF- ⁇ B and AP-1, coordinate the induction of various genes encoding inflammatory mediators, anti-apoptotic proteins, and many others.
- the invention provides a polynucleotide encoding a polypeptide designated as the splice form MyD ⁇ s, having the primary structural information of amino acids 1-251 as set forth in SEQ ID NO: 2, or any homologue or functional fragment thereof, possessing the biological properties of (1) down-regulating the TLR- induced nuclear factor kappa B activation and (2) activating the c-JUN N-terminal kinase pathway.
- said TLR-induced nuclear factor kappa B activation is an LPS-induced nuclear factor kappa B activation.
- the words "polynucleotide” may be interpreted to mean the DNA and cDNA sequence as detailed by Yoshikai et al.
- the invention provides a polynucleotide as described herein before as set forth in SEQ ID NO: 1.
- the invention provides a molecule which comprises a region specifically binding to the intermediate domain of MyD ⁇ or nucleic acids encoding said intermediate domain of MyD ⁇ , and modulates NF-kappaB activation and/or IRAK phosphorylation and/or activation of the c-JUN N-terminal kinase pathway.
- modulates can either mean activates (meaning also for example stimulates or enhances) or inhibits (meaning also for example downregulates or suppresses).
- the invention provides a molecule which comprises a region specifically binding to the intermediate domain of MyD ⁇ or nucleic acids encoding said intermediate domain of MyD ⁇ , and suppresses or prevents MyD ⁇ expression but not MyD ⁇ s expression, and activates the c-JUN N-terminal kinase pathway and inhibits the TLR-mediated nuclear factor kappa B pathway.
- the molecule which comprises a region specifically binding to the intermediate domain of MyD ⁇ or nucleic acids encoding said intermediate domain of MyD ⁇ , and suppresses or prevents MyD ⁇ expression but not MyD ⁇ s expression, and activates the c-JUN N-terminal kinase pathway is chosen from the group comprising an antibody or any fragment thereof, a small molecule, a ribozyme, an oligonucleotide, a peptide or a peptidomimetic.
- the term 'antibody' or 'antibodies' relates to an antibody characterized as being specifically directed against the intermediate domain of MyD ⁇ , with said antibodies being preferably monoclonal antibodies; or an antigen-binding fragment thereof, of the F(ab') 2 , F(ab) or single chain Fv type, or any type of recombinant antibody derived thereof.
- the monoclonal antibodies of the invention can for instance be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat immunized against the intermediate domain of MyD ⁇ , and of cells of a myeloma cell line, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the intermediate domain of MyD ⁇ which have been initially used for the immunization of the animals.
- the monoclonal antibodies according to this embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.
- the monoclonal antibodies according to this embodiment of the invention may be human monoclonal antibodies.
- Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice as described in PCT/EP 99/03605 or by using transgenic non-human animals capable of producing human antibodies as described in US patent 5,545,606.
- PBL peripheral blood lymphocytes
- SCID severe combined immune deficiency
- fragments derived from these monoclonal antibodies such as Fab, F(ab)' 2 and scFv ("single chain variable fragment"), providing they have retained the original binding properties, form part of the present invention.
- Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses.
- the antibodies involved in the invention can be labeled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
- the antibody against the intermediate domain of MyD ⁇ can also be camel antibody or a functional fragment thereof.
- Camel antibodies are fully described in WO94/25591 , WO94/0467 ⁇ and in WO97/49 ⁇ 05.
- oligonucleotide sequences that include anti- sense RNA and DNA molecules and ribozymes that function to inhibit the translation of MyD ⁇ but not the translation of MyD ⁇ s.
- Anti-sense RNA and DNA molecules act to directly block the translation of the part of the mRNA that encode the intermediate domain of MyD ⁇ by binding to the targeted mRNA and preventing protein translation.
- Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
- the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, thus to the mRNA encoding the ID-domain of MyD ⁇ , followed by a endonucleolytic cleavage within this region.
- engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage within the intermediate domain sequence of MyD ⁇ .
- Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which include the following sequences, GUA, GUU and GUC.
- RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable.
- the suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
- Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules.
- RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
- DNA sequences may be incorporated into a wide variety of vectors, which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
- antisense cDNA constructs that synthesize anti-sense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
- RNA inhibition RNA inhibition
- siRNA short interference RNA oligo ' s
- the siRNA duplexes should be shorter than 30 nucleotides, because longer stretches of dsRNA activate the PKR pathway in mammalian cells, which results in a global a-specific shutdown of protein synthesis.
- Target regions should be AA(N19)TT or AA(N21), should be specific for the gene of interest and should have a GC content of appr. 50%.
- the siRNAs duplexes can for example be transfected in the cells of interest by oligofectamin (Life Technologies) and the transfection efficiency reaches 90-95%.
- anti-sense oligonucleotides can also alter mRNA structure by modulating splicing of pre-mRNA.
- Oligonucleotide-induced alteration of splicing includes shifting of alternative splicing pathways, skipping of exons or inclusion of introns. Since splicing is a nuclear process, the anti-sense molecules must be active in the nuclei of the cells. It has been shown in the art how anti-sense oligonucleotides can be used to manipulate the splicing 'equilibrium' and redirect alternative splicing routes (Kole R. and Sazani P.K (2001) Curr. Opinion Mol. Therapeutics 3, 229.
- the invention provides a peptide or a peptidomimetic thereof which is derived from a region of MyD ⁇ , amino acids 95 till 172, depicted in SEQ ID NO: 14.
- SEQ ID NO: 14 comprises the ID domain of MyD ⁇ .
- Said ID domain is depicted in SEQ ID NO: 15 (which is defined as a region encompassing amino acids 110 till 154 of MyD ⁇ ).
- said peptide or peptidomimetic comprises at least 5 residues, 10 residues, 15 residues, 20 residues or more derived from the sequence depicted in SEQ ID NO: 15.
- the wording 'peptidomimetic' is described further.
- the term 'peptidomimetic' means a molecule able to mimic the biological activity of a peptide but is no longer peptidic in chemical nature.
- a peptidomimetic is a molecule that no longer contains any peptide bonds (that is, amide bonds between amino acids).
- the term peptide mimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudo- peptides, semi-peptides and peptoids.
- peptidomimetics provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the peptide on which the peptidomimetic is based.
- the peptidomimetic has effects on biological systems, which are similar to the biological activity of the peptide.
- the peptidomimetic of this invention are preferably substantially similar in both three-dimensional shape and biological activity to the peptides set forth above. Substantial similarity means that the geometric relationship of groups in the peptide that react with for example the ID region of MyD ⁇ or SEQ ID NO: 14 is preserved.
- peptides described in the present invention have utility in the development of such small chemical compounds with similar biological activities and therefore with similar therapeutic utilities.
- the techniques of developing peptidomimetics are conventional.
- peptide bonds can be replaced by non- peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original peptide.
- Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.
- the development of peptidomimetics can be aided by determining the tertiary structure of the original peptide, either free or bound to a substrate, e.g. the ID region of MyD ⁇ , by NMR spectroscopy, crystallography and/or computer-aided molecular modelling.
- the invention provides a molecule which comprises a region specifically binding to nuclear pre-RNA encoding MyD ⁇ or mRNA encoding MyD ⁇ s, and suppresses or prevents MyD ⁇ s expression but not MyD ⁇ expression, and inhibits the down-regulation of TLR-induced nuclear factor kappa B activation.
- said molecule is an oligonucleotide.
- TLR functions as a receptor for LPS.
- the invention provides a molecule which comprises a region specifically binding to nuclear pre-RNA encoding MyD ⁇ or mRNA encoding MyD ⁇ s, and induces or stimulates MyD ⁇ s expression but not MyD ⁇ expression, and induces the down-regulation of TLR-induced nuclear factor kappa B activation and wherein said molecule is an oligonucleotide.
- MyD ⁇ s there occurs a high expression of MyD ⁇ s in immune privileged tissues. Indeed it is shown that MyD ⁇ s is specifically expressed in brain, testis, and eye of C57/BL6 mice, which is consistent with a role in immune privilege. Therefore in another embodiment molecules able to modulate the expression of MyD ⁇ s can be used to modulate immunosuppression (e.g. in the case of transplantation it is desired that there occurs a stimulation of immunosuppression).
- oligonucleotide is intended to include both naturally occurring and non-naturally occurring (i.e., “synthetic") oligomers of linked nucleosides. Although such linkages generally are between the 3' carbon of one nucleoside and the 5' carbon of a second nucleoside (i.e., 3'-5' linkages), other linkages (such as 2'-5' linkages) can be formed.
- Naturally occurring oligonucleotides are those which occur in nature; for example ribose and deoxyribose phosphodiester oligonucleotides having adenine, guanine, cytosine, thymine and uracil nucleobases.
- non-naturally occurring oligonucleotides are oligonucleotides that contain modified sugar, internucleoside linkage and/or nucleobase moieties. Such oligonucleotide analogs are typically structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic wild type oligonucleotides. Thus, non-naturally occurring oligonucleotides include all such structures which function effectively to mimic the structure and/or function of a desired RNA or DNA strand, for example, by hybridizing to a target.
- nucleobases include adenine, guanine, cytosine, uridine, and thymine, as well as other non-naturally occurring and natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5- uracil (pseudo uracil), 4-thiouracil, ⁇ -halo, oxa, amino, thiol, thioalkyl, hydroxyl and other ⁇ -substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine.
- nucleobases include aden
- nucleobases include those disclosed in U.S. Pat. No. 3,637, ⁇ 0 ⁇ (Merigan, et al.), in chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al., Angewandte Chernie, International Edition, 1991 , 30, 613-722 (see especially pages 622 and 623, and in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed., John Wiley & Sons, 1990, pages 35 ⁇ - ⁇ 59, Cook, Anti-Cancer Drug Design 1991 , 6, 5 ⁇ 5-607, each of which are hereby incorporated by reference in their entirety).
- nucleosidic base is further intended to include heterocyclic compounds that can serve as like nucleosidic bases including certain "universal bases” that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases.
- a universal base is 3-nitropyrrole.
- Sugars having O-substitutions on the ribosyl ring are also amenable to the present invention. Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
- one additional modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
- Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues; a phospholipid, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety.
- lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues; a
- Oligonucleotides according to the present invention that are hybridizable to a target nucleic acid preferably comprise from about 5 to about 50 nucleosides. It is more preferred that such compounds comprise from about ⁇ to about nucleosides, with 15 to 25 nucleosides being particularly preferred.
- a target nucleic acid is any nucleic acid that can hybridize with a complementary nucleic acid-like compound.
- hybridization shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleobases.
- adenine and thymine are complementary nucleobases, which pair through the formation of hydrogen bonds.
- “Complementary” and “specifically hybridizable,” as used herein, refer to precise pairing or sequence complementarity between a first and a second nucleic acid-like oligomers containing nucleoside subunits. For example, if a nucleobase at a certain position of the first nucleic acid is capable of hydrogen bonding with a nucleobase at the same position of the second nucleic acid, then the first nucleic acid and the second nucleic acid are considered to be complementary to each other at that position.
- the first and second nucleic acids are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases, which can hydrogen bond with each other.
- “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. It is understood that an oligomeric compound of the invention need not be 100% complementary to its target RNA sequence to be specifically hybridizable.
- An oligomeric compound is specifically hybridizable when binding of the oligomeric compound to the target RNA molecule interferes with the normal function of the target RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed.
- Phosphorothioate linkages in the oligonucleotides of the invention are prepared using standard phosphoramidite chemistry on, for example, an automated DNA synthesizer (e.g., Applied Biosystems model 380B) and oxidation with 0.2 M solution of 3H-1 ,2-benzodithiole-3-one 1 ,1- dioxide in acetonitrile for the stepwise thiation of the phosphite linkages.
- Phosphorothioate linkages that have Sp configuration can be prepared generally according to the procedures described in U.S. Pat. Nos. 5,212,295, 5,587,361 and 5,599,797.
- 2'-modified amidites are used to synthesize compounds of the invention according to standard phosphoramidite regimes.
- the amidites have a 2'-methoxyethoxy ("MOE") substituent.
- MOE 2'-methoxyethoxy
- this invention concerns oligonucleotides that exhibit increased stability relative to their naturally occurring counterparts. Extracellular and intracellular nucleases generally do not recognize (and, therefore, do not bind to) the compounds of the invention.
- the modified internucleoside linkages of this invention preferably replace naturally-occurring phosphodiester-5'-methylene linkages to confer nuclease resistance.
- a molecule comprising a region specifically binding to the intermediate domain of MyD ⁇ or nucleic acids encoding said intermediate domain of MyD ⁇ , and (1) suppresses or prevents MyD88 expression but not MyD ⁇ s expression, and (2) activates the c-JUN N-terminal kinase pathway or alternatively in another embodiment the invention provides a molecule which comprises a region specifically binding to nuclear pre-RNA encoding MyD ⁇ or mRNA encoding MyD ⁇ s, and (1) suppresses or prevents MyD ⁇ s expression but not MyD ⁇ expression, and (2) inhibits the down-regulation of TLR-induced nuclear factor kappa B activation and wherein said molecules comprise an antibody or any fragment thereof, a small molecule, a ribozyme, anti-sense nucleic acids or an oligonucleotide, a peptide or a peptidomimetic thereof for use as a medicament.
- the above described molecules that can suppress or prevent MyD ⁇ s expression but not MyD ⁇ expression, and inhibit the down-regulation of TLR-induced nuclear factor kappa B activation can be used for the manufacture of a medicament to treat endotoxin tolerance. Since endotoxin tolerance is a manifestation that occurs during sepsis said molecules can be used for the manufacture of a medicament to treat sepsis.
- the term 'medicament to treat' relates to a composition comprising molecules as described above and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to treat diseases as indicated above.
- Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives.
- Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers.
- the 'medicament' may be administered by any suitable method within the knowledge of the skilled man. The preferred route of administration is parenterally.
- the medicament of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with the pharmaceutically acceptable excipients as defined above.
- the dosage and mode of administration will depend on the individual.
- the medicament is administered so that the protein, polypeptide, peptide of the present invention is given at a dose between 1 ⁇ g/kg and 10 mg/kg, more preferably between 10 ⁇ g/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg.
- it is given as a bolus dose.
- Continuous infusion may also be used and includes continuous subcutaneous delivery via an osmotic minipump. If so, the medicament may be infused at a dose between 5 and 20 ⁇ g/kg/minute, more preferably between 7 and 15 ⁇ g/kg/minute.
- the molecules that comprise a region specifically binding to the intermediate domain of MyD ⁇ or nucleic acids encoding said intermediate domain of MyD ⁇ , and suppress or prevent MyD ⁇ expression but not MyD ⁇ s expression and activate the c-JUN N-terminal kinase pathway can be used for the manufacture of a medicament to activate the c-JUN N-terminal pathway. Since the c-JUN N-terminal kinase stimulation is involved in the induction of apoptosis the molecules of the present invention can be used for the manufacture of a medicament to treat insufficiency of apoptosis. Insufficiency of apoptosis is a manifestation that frequently occurs in cancer growth and hence the MyD ⁇ s of the present invention can be used for treatment of cancer.
- a polynucleotide encoding MyD ⁇ s or any homologue or functional fragment thereof, possessing the biological properties of (1) down-regulating the TLR-induced nuclear factor kappa B activation and (2) activating the c-JUN N- terminal kinase pathway can be used as a medicament. Therefore this aspect of administration for treatment involves the use of gene therapy to deliver the polynucleotide encoding MyD ⁇ s or a functional fragment thereof or a homologue thereof for the treatment of insufficiency of apoptosis.
- a polynucleotide encoding MyD ⁇ s or a functional fragment thereof or a homologue thereof can be used in a gene therapeutic method for the inhibition of diseases where TLR-induced nuclear factor kappa B activation occurs as for example in the case of an infection (for example an infection from a pathogen such as a virus or bacterium) and for example in the case of rheumatoid arthritis since recently it has been shown in the art that TLR-induced nuclear factor kappa B activation is involved in rheumatoid arthritis.
- the present invention provides the nucleic acids of MyD ⁇ s or a functional fragment thereof or a homologue thereof for the transfection of cells in vitro and in vivo.
- nucleic acids can be inserted into any of a number of well-known vectors for the transfection of target cells and organisms as described below.
- the nucleic acids are transfected into cells, ex vivo or in vivo, through the interaction of the vector and the target cell.
- Said nucleic acids under the control of a promoter, then expresses MyD ⁇ s or a functional fragment thereof or a homologue thereof, thereby mitigating the effects of absent, partial inactivation, or abnormal expression of MyD ⁇ s or a functional fragment thereof or a homologue thereof.
- Such gene therapy procedures have been used in the art to correct acquired and inherited genetic defects, cancer, and viral infection in a number of contexts.
- Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome.
- Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
- Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
- Lipofection is described in, e.g., US Pat. No. 5,049,386, US Pat No. 4,946,787; and US Pat. No. 4,697,355 and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
- Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Flegner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
- the preparation of lipid: nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Then 2:291-297 (1995); Behr et al., Bioconjugate Chem.
- RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
- Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).
- Conventional viral based systems for the delivery of nucleic acids could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.
- Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
- Lentiviral vectors are retroviral vector that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised on c/s-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum c/s-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
- Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); PCT/US94/05700.
- MiLV murine leukemia virus
- GaLV gibbon ape leukemia virus
- SIV simian immunodeficiency virus
- HV human immunodeficiency virus
- adenoviral based systems are typically used.
- Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division.
- Adeno-associated virus (AAV) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., U.S. Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-301 (1994); Muzyczka. Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No.
- rAAV Recombinant adeno-associated virus vectors
- All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system (Wagner et al., Lancet 351:9117 1702-3 (1998).
- Ad vectors Replication-deficient recombinant adenoviral vectors (Ad) are predominantly used in transient expression gene therapy, because they can be produced at high titer and they readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaced the Ad E1a, E1b, and E3 genes; subsequently the replication deficient vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, kidney and muscle system tissues. Conventional Ad vectors have a large carrying capacity.
- Ad vector An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Sterman et al., Hum. Gene Ther. 9:7 1083-1069 (1998); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998)). Packaging cells are used to form virus particles that are capable of infecting a host cell.
- Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or PA317 cells, which package retrovirus.
- Viral vectors used in gene therapy are usually generated by producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line.
- AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
- Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
- the cell line is also infected with adenovirus as a helper.
- the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
- the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
- a viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface.
- the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest.
- a receptor known to be present on the cell type of interest.
- Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
- This principle can be extended to other pairs of virus expressing a ligand fusion protein and target cell expressing a receptor.
- filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
- Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intra-peritoneal, intra-muscular, sub-dermal, or intra- cranial infusion) or topical application, as described below.
- systemic administration e.g., intravenous, intra-peritoneal, intra-muscular, sub-dermal, or intra- cranial infusion
- topical application as described below.
- vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
- Ex vivo cell transfection for diagnostics, research, or for gene therapy e.g., via re-infusion of the transfected cells into the host organism
- cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient).
- stem cells are used in ex vivo procedures for cell transfection and gene therapy.
- the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
- Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM- CSF, IFN- ⁇ and TNF- ⁇ are known (see Inaba et al., J. Exp. Med. 176: 1693-1702 (1992)).
- cytokines such as GM- CSF, IFN- ⁇ and TNF- ⁇ are known (see Inaba et al., J. Exp. Med. 176: 1693-1702 (1992)).
- Stem cells are isolated for transduction and differentiation using known methods.
- stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells) (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).
- Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
- therapeutic nucleic acids can be also administered directly to the organism for transduction of cells in vivo.
- naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells.
- Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells.
- the nucleic acids are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
- JNK is also required for T-cell differentiation (Dong et al. (2000) Nature 405:91 ; Sabapathy et al. (2001) J. Exp. Med. 193:317), and is involved in T helper 1 (Th1) versus Th2 cell polarization during infection (Rincon et al. (2000) Free Radical Biology and Medicine 28:1328; Jankovic et al. (2001) Trends in Immunology 22:450).
- the present invention demonstrates that MyD88s is constitutively expressed in spleen of Balb/c mice, whereas in the spleen of C57/BL6 mice MyD ⁇ s is not expressed.
- mice of the Balb/c strain produce a polarized Th2 response and fail to promote resistance
- mice of the C57/BL6 strain produce a polarized Th1 response and are resistant (Reiner et al. (1995) Annu. Rev. Immunol. 13 :151 ; Guler et al. (1996) Science 271 : 984).
- a polynucleotide encoding MyD88s or any homologue or functional fragment thereof can be used for the manufacture of a medicament to modulate Th1/Th2 cell polarization.
- a polynucleotide encoding MyD ⁇ s or any homologue or functional fragment thereof can be used for the manufacture of a medicament to modulate immunosuppression.
- the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising molecules that (1) comprise a region specifically binding to the intermediate domain of MyD ⁇ or nucleic acids encoding said intermediate domain of MyD ⁇ , and wherein said molecules can suppress or prevent MyD ⁇ expression but not MyD ⁇ s expression, and activate the c-JUN N-terminal kinase pathway.
- said molecules comprise an antibody or any fragment thereof, a small molecule, a ribozyme, oligonucleotides, peptides or peptidomimetics.
- the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising molecules that (1) comprise a region specifically binding to nuclear pre- RNA encoding MyD ⁇ or mRNA encoding MyD ⁇ s, and wherein said molecules can suppress or prevent MyD ⁇ s expression but not MyD ⁇ expression, and inhibit the down-regulation of TLR-induced nuclear factor kappa B activation.
- said molecules comprise at least one oligonucleotide.
- the invention provides a polypeptide, designated as MyD ⁇ s or any homologue or functional fragment thereof, possessing the biological properties of (1) down-regulating the TLR-induced nuclear factor kappa B activation and (2) activating the c-JUN N-terminal kinase pathway, for use as a medicament.
- the invention provides a method to identify molecules comprising (a) exposing the intermediate domain of MyD ⁇ or nucleic acids encoding said intermediate domain of MyD ⁇ to at least one molecule whose ability to activate the c-JUN N-terminal kinase pathway and down-regulate the TLR-induced nuclear factor kappa B activation is sought to be determined, (b) determining binding or hybridising of said molecule(s) to the intermediate domain of MyD ⁇ or nucleic acids encoding said intermediate domain, and monitoring said activation of the c-JUN N- terminal kinase pathway and down-regulation of the TLR-induced nuclear factor kappa B activation when administering at least one of said molecule(s).
- the invention provides a method to identify molecules comprising (a) exposing the nuclear pre-RNA encoding MyD ⁇ or mRNA encoding MyD ⁇ to at least one molecule whose ability to inhibit the down-regulation of TLR- induced nuclear factor kappa B activation is sought to be determined, (b) determining binding or hybridising of said molecule(s) to the nuclear pre-RNA encoding MyD ⁇ or mRNA encoding MyD ⁇ s, and (c) monitoring said inhibition of down-regulation of TLR- induced nuclear factor kappa B activation when administering at least one of said molecule(s).
- the invention provides a method to identify molecules comprising exposing a region comprising the intermediate domain of MyD ⁇ (SEQ ID NO 14 (amino acid 95 till 172) or nucleic acids encoding SEQ ID NO: 14 of MyD ⁇ to at least one molecule whose ability to activate the c-JUN N-terminal kinase pathway and to activate the nuclear factor kappa B activation is sought to be determined, determining binding or hybridising of said molecule(s) to the intermediate domain of MyD ⁇ or nucleic acids encoding said intermediate domain and, monitoring said activation of the c-JUN N-terminal kinase pathway and nuclear factor kappa B activation when administering at least one of said molecule(s).
- the invention provides methods for identifying compounds or molecules (both words can be used interchangeable in this invention) which bind on the ID domain of MyD ⁇ or nucleic acids encoding said ID domain or the nuclear pre-RNA encoding MyD ⁇ or mRNA encoding MyD ⁇ .
- These methods are also referred to as 'drug screening assays' or 'bioassays' and typically include the step of screening a candidate/test compound or agent for the ability to interact with the ID domain of MyD ⁇ or nucleic acids encoding said ID domain or the nuclear pre-RNA encoding MyD ⁇ or mRNA encoding MyD ⁇ .
- 'Compound' in relation to the screening methods described herein above means any anorganic or organic compound, including simple or complex inorganic or organic molecules, oligonucleotides, peptides, peptidomimetics, proteins, antibodies, carbohydrates, nucleic acids or derivatives thereof.
- Candidate/test compounds such as small molecules, e.g. small organic molecules, and other drug candidates can be obtained, for example, from combinatorial and natural product libraries.
- the assays are cell-free assays which include the steps of combining for example the ID domain of MyD ⁇ protein or a nucleic acid encoding said ID domain and a candidate/test compound, e.g., under conditions which allow for interaction of (e.g. binding of) the candidate/test compound with for example the ID domain of MyD ⁇ protein or a nucleic acid encoding said ID domain to form a complex, and detecting the formation of a complex, in which the ability of the candidate compound to interact with for example the ID domain of MyD ⁇ protein or a nucleic acid encoding said ID domain is indicated by the presence of the candidate compound in the complex.
- Formation of complexes between for example the ID domain of MyD ⁇ protein or a nucleic acid encoding said ID domain and the candidate compound can be quantitated, for example, using standard immunoassays.
- the ID domain of MyD ⁇ protein or a nucleic acid encoding said ID domain in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
- Interaction e.g., binding of
- a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
- the ID domain of MyD ⁇ that is tagged can be adsorbed onto Ni-NTA microtiter plates, or a particular heterotetrameric channel -ProtA fusions adsorbed to IgG, which are then combined with the cell lysates (e.g., 35 S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the plates are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated.
- the cell lysates e.g., 35 S-labeled
- the candidate compound e.g., 35 S-labeled
- the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the plates are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or
- the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of the ID domain of MyD ⁇ - binding protein found in the bead fraction quantitated from the gel using standard eiectrophoretic techniques.
- Other techniques for immobilizing protein on matrices can also be used in the drug screening assays of the invention.
- the ID domain of MyD ⁇ can be immobilized utilizing conjugation of biotin and streptavidin.
- Biotinylated particular ID domain of MyD ⁇ can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, III.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
- biotinylation kit Pierce Chemicals, Rockford, III.
- antibodies reactive with the ID domain of MyD ⁇ can be derivatized to the wells of the plate, and the ID domain of MyD ⁇ can be trapped in the wells by antibody conjugation.
- preparations of the ID domain of MyD ⁇ and a candidate compound are incubated in particular MyD ⁇ ID domain -presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.
- Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the ID domain of MyD ⁇ -target molecule, or which are reactive with the ID domain of MyD ⁇ and compete with the target molecule; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
- Another technique for drug screening which provides for high throughput screening of compounds having suitable binding affinity to the ID domain of MyD ⁇ s is described in detail in "Determination of Amino Acid Sequence Antigenicity" by Geysen HN, WO 64/03564, published on 13/1584.
- a solid substrate such as plastic pins or some other surface.
- the protein test compounds are reacted with the ID domain of MyD ⁇ and washed. Bound ID domain of MyD ⁇ is then detected by methods well known in the art.
- a purified ID domain of MyD ⁇ can also be coated directly onto plates for use in the aforementioned drug screening techniques.
- non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
- This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the ID domain of MyD ⁇ specifically compete with a test compound for binding the ID domain of MyD ⁇ .
- MyD ⁇ mRNA was the major species in splenic extracts from C57/BL6 mice.
- MyD ⁇ s mRNA levels in the spleen of these mice were strongly upregulated 1 h after injection of mice with TNF.
- MyD ⁇ s expression in spleen of TNF-treated mice was also studied at the protein level.
- C57/BL6 mice were injected with 20 ⁇ g TNF, and spleen homogenates were prepared 1h, 4h, or 13h after TNF injection, respectively. Immunoblotting of these extracts with MyD ⁇ specific antibodies showed that MyD ⁇ was constitutively expressed in untreated and TNF-treated mice. In contrast, MyD ⁇ s expression was not detectable in untreated mice or mice treated for 1h with TNF. However, after 4h and 13h TNF treatment, specific induction of a 27 kDa band corresponding to MyD88s was observed. In conclusion, these results clearly demonstrate the mouse strain-specific and inducible expression of MyD ⁇ s in the spleen.
- MyD ⁇ s does not activate NF-kappaB but behaves as a dominant negative inhibitor
- MyD3 ⁇ s activates NF- ⁇ B
- HEK293T cells were transiently transfected and analysed for NF- ⁇ B activation by reporter gene (Figure 2a) or gel shift assay ( Figure 2a insert).
- MyD ⁇ b MyD ⁇ s overexpression did not induce DNA binding of NF- ⁇ B or NF- ⁇ B reporter gene activation.
- MyD ⁇ s competes with MyD ⁇ L for binding to the IL-1R/IL-1RacP complex.
- MyD ⁇ has previously been shown to be recruited as a homodimer to the activated IL-1R (3). Both the DD and the TIR domain are required for homodimerization (3). Therefore, we first tested if MyD3 ⁇ s was still able to form dimers with MyD ⁇ upon expression in HEK293T cells.
- MyD ⁇ binds IRAK primarily through DD-DD interactions, therefore it was unlikely that removal of the ID would interfere with IRAK binding. Indeed, MyD ⁇ s still binds IRAK (data not shown) and has no effect on its recruitment into the IL-1 R complex ( Figure 4a).
- MyD ⁇ triggers IRAK phosphorylation (4). This can simply be demonstrated by co-expression of MyD ⁇ and a mutant version of IRAK, IRAK(T66A) which, in contrast to wild type IRAK, is not rapidly degraded by co- expression with MyD ⁇ .
- IRAK(T66A) migrates as a series of phosphospecies, which are converted to a hyperphosphorylation state (corresponding to the slower migrating species) by co-expressing MyD ⁇ ( Figure 4b upper panel and (4)). We therefore wished to test whether MyD ⁇ s could similarly induce phosphorylation of IRAK(T66A) and found that MyD ⁇ s did not induce IRAK phosphorylation. Similar results were obtained when we used an N-terminal deletion construct of IRAK(T66A) (IRAKN(T66A), residues 1-20 ⁇ (Burns K et al. (2000) Nat. Cell. Biol.
- MvD ⁇ is required for IRAK-4-induced IRAK-1 phosphorylation
- IRAK-1 KD a kinase dead mutant of IRAK-1
- a second kinase was postulated to phosphorylate IRAK-1 and perhaps to activate IRAK-1 's own kinase activity.
- IRAK-4 so called for its homology to other members of the IRAK-1 family (other members include the kinase inactive IRAK-2 and IRAK-M/3), was identified as a candidate for the IRAK-1 kinase (Wesche H.
- IRAK-4 is a kinase for IRAK-1
- IRAK-4 was cotransfected together with IRAK-1 KD (IRAK-1 D340N) (used because it cannot self-phosphorylate like overexpressed wildtype IRAK-1) and phosphorylation monitored by the appearance of a slower migrating species in SDS- PAGE.
- IRAK-4 induced phosphorylation of IRAK-1 KD. That phosphorylation was specifically induced by IRAK-4 was confirmed by the observation that coexpression of IRAK-1 KD with two different IRAK-4 kinase dead mutants, IRAK- 4KD (IRAK-4KK213AA or IRAK-4D311N), did not similarly induce IRAK-1 KD phosphorylation. Although coexpression of IRAK-4 clearly induced IRAK-1 phosphorylation, only a partial conversion to the phosphorylated species was observed. Addition of MyD ⁇ however, significantly enhanced IRAK-4 induced IRAK- 1KD phosphorylation, suggesting that MyD ⁇ stimulates IRAK-4's activity.
- IRAK-4 and IRAK-1 KD do not directly associate.
- addition of MyD ⁇ but not MyD ⁇ s permitted assembly of a complex containing both IRAKs.
- MyD ⁇ thereby appears to act like a hinge inducing the proximity of IRAK-1 and IRAK-4.
- phosphorylated IRAK-1 is stably detected together with MyD ⁇ , IRAK-1 and IRAK-4, suggesting that MyD ⁇ /IRAK-1 interactions are destabilized only after multiple sites are phosphorylated on IRAK-1.
- MvD ⁇ s blocks IRAK-4-induced IRAK-1 phosphorylation.
- MyD ⁇ s does not stimulate IRAK-4 -induced IRAK-1 KD phosphorylation.
- MyD ⁇ s inhibited MyD ⁇ 's stimulatory effect on IRAK-4-induced IRAK-1 KD phosphorylation in a dose dependent manner.
- MvD ⁇ s does not bind to IRAK-4 and blocks recruitment of IRAK-4 to the IL-1 Rs
- MyD ⁇ s acts as a negative regulator by its incapacity to bind to IRAK-4 and thus to prevent IRAK-4-induced IRAK-1 phosphorylation.
- MyD ⁇ s acts as a negative regulator by its incapacity to bind to IRAK-4 and thus to prevent IRAK-4-induced IRAK-1 phosphorylation.
- MyD ⁇ s acts as a negative regulator of IL-1beta/LPS -induced NF-kB activation by preventing IRAK-4's access to its substrate.
- MvD ⁇ s is involved in endotoxin tolerance
- tissue-specific and inducible expression of a splice variant of MyD ⁇ that acts as a dominant-negative inhibitor of IL-1 and LPS-induced NF- ⁇ B activation implicates an important role for alternative splicing of MyD ⁇ in the regulation of the cellular response to IL-1 , LPS and possibly other triggers of the IL-1R/TLR superfamily.
- MyD ⁇ not only functions as a passive adaptor protein, but also plays an active role in the phosphorylation and activation of IRAK through its lD.
- PBMC peripheral blood mononuclear cells
- MyD ⁇ 8s which inhibits IL-1 induced NF- ⁇ B activation, does not inhibit IL-1 induced activation of JNK
- HEK 293T cells which stably express the IL-1R, were transiently transfected with an expression plasmid for Flag-tagged JNK and increasing concentrations of different MyD ⁇ proteins.
- Cells were treated for 30 min or 4h with IL-1 and analysed for JNK phosphorylation by immunoblotting with a phospho-JNK specific antibody (upper panel of figure 6). Total expression of JNK was verified by western blotting with anti-Flag. This shows that IL-1 induces the phosphorylation of JNK after 30 min as well as after 4h treatment.
- MyD88s which is ineffective to activate NF- ⁇ B dependent gene expression, still activates JNK and induces AP-1 (c-fos/c-iun) dependent gene expression MyD ⁇ s is no longer able to activate NF- ⁇ B dependent gene expression.
- AP-1 dependent luciferase reporter construct In order to analyze whether MyD ⁇ s is still able to activate AP-1 dependent gene expression, we transiently transfected HEK293 cells with an AP-1 dependent luciferase reporter construct, together with increasing amounts of MyD ⁇ s, MyD ⁇ L or MyD ⁇ TIR expression plasmids. Cell extracts were prepared and luciferase activity determined.
- NF- ⁇ B and JNK/AP- 1 pathways diverge at the level of MyD ⁇ .
- MyD ⁇ s allows specific activation of the JNK pathway and AP-1 dependent gene expression, while blocking NF- ⁇ B dependent gene expression.
- Obtaining specificity might be important in view of specifically modulating gene expression linked with inflammation/immunity and T cell differentiation, but also to modulate the role of JNK in cell survival (e.g. IL-1 induced apoptosis of islet cells; TNF-induced apoptosis of cancer cells; neuronal apoptosis), without modulating the anti-apoptotic NF- ⁇ B pathway.
- RNA inhibition RNA inhibition
- siRNA duplexes are transfected in the cells of interest by oligofectamin (Life Technologies) and the transfection efficiency reaches 90-95%. Efficient knock-down of the gene of interest is verified by western blot analysis or RT-PCR.
- MyP8 ⁇ s which lacks the IP, will not be affected by these siRNA duplexes. This allows to study the effect of MyP ⁇ s expression in a MyP ⁇ i . negative background. Such cells should only respond to LPS or IL-1 by JNK and AP-1 activation, whereas NF- B activation is blunted. Moreover, JNK activation will be sustained because of the inhibiting effect of NF- ⁇ B on the JNK pathway is no longer occurring.
- RNAi with a siRNA duplex that is specifically directed to the sequence that forms the boundary between the death domain and TIR domain of MyP ⁇ s.
- MyP3 ⁇ _ which lacks this exon 1-exon 3 boundary, will not be affected by these siRNA duplexes. This allows to study the response of cells in the absence of MyP ⁇ s expression, and will prevent the negative regulation of NF- ⁇ B activation in tolerant cells. Interference with alternative splicing of MvP ⁇
- MyP ⁇ s expression is induced by alternative splicing of MyP ⁇ .
- Alternative splicing of MyP ⁇ to MyP ⁇ s can be prevented by the use of antisense oligo's, which target a particular 3' intron-exon junction, and make it less likely that this site shall be recognized as acceptor site in the splicing process (Lim and Hertel (2001) J Biol Chem 276: 14476).
- MyP ⁇ s is formed by exon skipping of exon 2. This means that the 3' acceptor splice sites of exon 2 and exon 3 compete with each other for the 5' donor site from exon 1.
- antisense oligo to prevent alternative splicing of human MyP88 to MyP ⁇ s: 5'-GGCAUAUGCCCUGGGUGCAGA-3'
- administration of antisense oligo's designed against the intron-exon junction of the 3' acceptor splice site of exon 2 the balance may be tilted to favor exon2 exclusion and formation of MyP ⁇ s.
- differential splicing of MyP ⁇ can also be modulated by overexpression of specific splicing factors (e.g. SR proteins, hnRNP proteins; Chabot (1996) Trends in Genetics 12:472).
- specific splicing factors e.g. SR proteins, hnRNP proteins; Chabot (1996) Trends in Genetics 12:472.
- RPMI 1640 in case of mouse macrophage Mf4/4 and human monocyte THP-1 cells
- PMEM in case of human embryonic kidney (HEK) 293T cells
- 10% fetal calf serum 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 1 mM sodium pyruvate, and 50 ⁇ M 2-mercaptoethanol.
- Recombinant mouse TNF-alfa. and IL-1 beta was provided by Apotech (San Piego, USA) and Sigma, respectively.
- Lipopolysaccharide (LPS) from Salmonella abortus equi was purchased from Sigma (Saint Louis, USA).
- the source of the various antibodies used in this study is as follows: anti-Flag/M2 (Eastman Kodak Company), anti-VSV (Sigma), anti-IRAK-1 (Alexis or Santa Cruz), anti-MyP ⁇ (ProSci Incorporated) and anti-E (Pharmacia) antibodies.
- PCR amplification of MyD ⁇ Total RNA was reverse transcribed with Superscript II RNase H " reverse transcriptase (Gibco BRL, Life Technologies, Paisley, UK)) and oligo (dT) primer.
- a Balb/c mouse multiple tissue cPNA panel was purchased from Clontech (Palo Alto, CA, USA). The quality of the cPNA was verified by PCR amplification of ⁇ -actin.
- _ and pCAGGS-E-MyP ⁇ s were obtained by in frame cloning of RT-PCR fragments of MyP ⁇ with an N-terminal E-tag into the eukaryotic expression vector pCAGGS.
- pCPNA3-AU1-MyP88-TIR(152-296) was a generous gift of Pr. M. Muzio (Mario Negri Institute, Milan) and was described in Muzio M. et al (1997) Science 27 ⁇ : 1612.
- pPCRIII-Flag-MyP ⁇ pPCRIII-Flag-IL1 R, pPCRIII-Flag-IL1RAcP, pcPNA3-IRAK(T66A) and pcPNA3-IRAK-N(T66A) were described in Burns K. et al (2000) Nat. Cell Biol. 2:346.
- pNFconluc containing the luciferase reporter gene driven by a minimal NF- ⁇ B responsive promoter, was a gift of Pr. A. Israel (Institut Pasteur, Paris).
- pPGK-neogal containing the ⁇ -galactosidase gene after the PGK promotor, was obtained from Pr. P. Soriano (Fred.
- IRAK-4 was PCR amplified from an EST clone and inserted into pCRIII containing an N-terminal Flag or VSV tag or into pGAPIO.
- Kinase dead mutants of IRAK-4 (IRAK-4KK213AA or IRAK-4P311 N) were generated by double PCR and inserted into a pCRIII vector with an N-terminal tag.
- pGBT9 MyP88, pGBT9 MyP ⁇ -N (aa 1-172), pGBT9 MyP ⁇ -TIR (aa 161-296), expressing pGALPB (the GAL4 PNA- binding domain) fused to full-length MyP ⁇ or the indicated deletion mutants have been described previously (Burns et al (1998) J. Biol. Chem. 273: 12203-12209).
- pGBT9 MyP88-IP (aa 110-157 ) and pGBT9 MyP88-PP (aa 1-110) were prepared by inserting PCR generated fragments into pGBT9. The sequence of all PCR generated cPNAs were confirmed by PNA sequencing.
- pGAPIO IRAK-4 expressing Gal4AP- IRAK4 (a fusion protein of GAL4 transcription activation with full-length IRAK-4) was made by inserting IRAK-4 cPNA as an EcoRI fragment into pGAPIO.
- An AP-1 dependent luciferase reporter construct, pAP-1luc was purchased from Stratagene (PathPetect System).
- Transient transfections and NF- ⁇ B or AP-1 reporter gene assays HEK 293T cells were seeded in 6-well plates at 2 x 10 5 cells/well and transiently transfected by the PNA-calcium phosphate precipitation method with 100 ng pNFconluc or 100 ng pAP-1luc, 100 ng pGK-neogal and different concentrations of specific MyP ⁇ expression plasmids. The total amount of PNA was kept constant by adding empty vector up to 1 ⁇ g PNA per well. Transfections were done in triplicate. 6 h post transfection, cells were trypsinized and seeded at a density of 2 x 10 4 cells/well in 24-well plates.
- 3 x 10 6 Mf4/4 cells were transiently transfected by electroporation (conditions: 300 V and 1350 ⁇ F) with 1 ⁇ g pPGKneogal, 4 ⁇ g pNFconluc and 5 ⁇ g of a MyP ⁇ expression plasmid.
- Transfected cells were seeded at a density of 2.5 x 10 5 c/well in a 24-well plate.
- 4 ⁇ h post transfection cells were stimulated for 6 h with 100 ng/ml IL1- ⁇ , 100 ng/ml TNF, 100 ng/ml LPS or left untreated.
- NF-KB and AP-1 activity was determined by measuring the luciferase activity present in cell extracts. Luciferase values were normalized for differences in transfection efficiency on the basis of ⁇ - galactosidase activity in the same extracts, and expressed as fold induction values relative to the unstimulated empty vector control.
- HEK 293T cells were seeded at 1.5 x 10 6 cells/10 cm petridish and transfected with pCAGGS-E-MyP ⁇ s , pCAGGS-E-Myd ⁇ L or empty vector.
- Nuclear fractions were prepared as described by Pignam JP et al (19 ⁇ 3) Nucleic Acid Res. 11:1475.
- NF- ⁇ B PNA binding activity was analyzed by incubating 3 ⁇ g nuclear proteins for 30 min with the 32 P-end-labeled oligonucleotide 5'-agctATGTGGGATTTTCCCATGGAGCagct-3', corresponding to the NF- ⁇ B recognition sequence of the Ig KB promotor.
- PNA/nucleoprotein complexes were separated from free probe on a 4% polyacrylamide gel.
- 2 x 10 6 HEK293T cells were plated on 10-cm petridishes and transiently transfected with 1 ⁇ g of the indicated expression plasmids.
- the total amount of PNA was kept at 5 ⁇ g per petridish by adding empty vector.
- 24 h post-transfection cells were washed with PBS, and lysed in 500 ⁇ l lysis buffer (50 mM HEPES pH7.6, 250 mM NaCI, 0.1% NP- 40, 5 mM EPTA, supplemented with protease and phosphatase inhibitors). Lysates were incubated for 16h with 5 ⁇ g anti-Flag (Sigma).
- Immunocomplexes were immobilized on protein A-Trisacryl beads (Pierce Chemical Co., Rockford, USA). The beads were washed twice with lysis buffer and twice with lysis buffer containing 1 M NaCI. Bound proteins were eluted by boiling in Laemli buffer and analyzed by 10% SPS-PAGE and Western blotting. Western blots were blocked with 5% milk and incubated overnight with primary antibodies. Antibodies raised against the C-terminal part of MyP ⁇ were purchased from Immucor (Roedermark, Germany).
- E-tag antibody was purchased from Amersham Pharmacia Biotech (Rainham, UK), an IRAK- antibody from Alexis (San Piego, CA, USA) and an l ⁇ B- ⁇ antibody from Santa Cruz Biotechnology lnc.(Santa Cruz, CA, USA).
- HRP-conjugated anti-mouse or HRP- conjugated anti-rabbit secondary antibodies were purchased from Amersham Pharmacia Biotech and incubated with the blots for 1h. Immunoreactive bands were revealed by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
- cell lysates were prepared in RIPA buffer (25 mM Tris pH ⁇ .2, 50 mM NaCI, 0.5% NP-40, 0.5% deoxycholic acid, 0.1% SPS, 10 ⁇ M iodoacetate) supplemented with protease and phosphatase inhibitors. Lysates were analyzed by 10% SPS-PAGE and Western blotting with anti-JNK or anti-phospoJNK (Cell Signaling, Beverly, MA, USA). TNF bio-assay
- TNF levels in the supernatans of THP-1 cells were measured in a bioassay for TNF which is based on TNF cytotoxicity for the mouse fibroblast cell line L929 in the presence of 1 ⁇ g/ml actinomycin P, as described in Schotte P. et al (2001) J. Biol. Chem. 276: 25939. Yeasr 2-hybrid interaction studies.
- yeast-two-hybrid interaction studies performed as described previously (Pe Valck et al (1996) FEBS Lett. 384: 61-64. Briefly, yeast cells of the S. cerevisiae strain HF7c were cotransformed with the pGAPIO IRAK-4 and pGBT9 MyP ⁇ or pGBT9 fused to different MyP ⁇ deletion mutants. Transformation efficiency was verified by growth on appropriate synthetic media using Trp and Leu selection markers. Protein interaction was revealed by His auxotrophy and assessed by b- galactosidase expression filter assays. All pGBT9 MyP ⁇ fusion proteins were negative for autoactivation.
- Transfected 293T cells were lysed in lysis buffer (1 %NP-40, 20 mM HEPES, pH 7.9, 250 mM NaCI, 20 mM b-glycerophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 2 mM dithiothreitol, 1 mM EPTA and a protease inhibitor cocktail).
- lysis buffer 1 %NP-40, 20 mM HEPES, pH 7.9, 250 mM NaCI, 20 mM b-glycerophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 2 mM dithiothreitol, 1 mM EPTA and a protease inhibitor cocktail.
- the cell extracts were incubated with one of the following antibodies for 2 hrs at 4°C: (1 ⁇ g) anti-M2, anti-VSV, anti-IRAK-1, or anti-E that were preincubated with protein G Sepharose.
- transiently transfected HEK 293T cells were lysed in 500 ⁇ l of 20 mM Tris pH 7.5, 50 mM KCl, 5 mM MgCI2 , 400 mM NaCI, 2 mM PTT, 1% Triton-X-100, 20% glycerol and protease and phosphatase inhibitors.
- IRAK- 1KP was immunoprecipitated for 2 h at 4°C with an anti-IRAK-1 antibody (Alexis), followed by addition of protein A trisacryl (Pierce).
- Immune complexes were washed twice with lysis buffer and twice with kinase buffer containing 20 mM Tris-HCI, pH 7.5, 50 mM KCl, 2 mM MgCI2 , 2 mM MnCI2 , 5% glycerol and protease inhibitors. After the last wash, immune complexes were resuspended in 40 ⁇ l kinase buffer. For each kinase reaction 10 ml of the respective immune complexes were mixed with 5 ⁇ Ci of gamma- 32 P] ATP (3000 Ci/mmol) in total volume of 25 ⁇ l. Reactions were allowed to proceed for 15 min at 30°C and then directly analysed by SPS-PAGE and autoradiography. A reaction without ATP added was set up in parallel and analysed by western blot to estimate the input. References
- Li L, Cousart S, Hu J, McCall CE Characterization of interleukin-1 receptor- associated kinase in normal and endotoxin-tolerant cells. J Biol Chem 2000 275:23340-23345.
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WO2011043740A1 (en) * | 2009-10-09 | 2011-04-14 | Kemijski Institut | Peptides for inhibition of myd88-dependent signaling pathways |
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US7879992B2 (en) * | 2005-01-31 | 2011-02-01 | Isis Pharmaceuticals, Inc. | Modification of MyD88 splicing using modified oligonucleotides |
WO2012047175A1 (en) | 2010-10-05 | 2012-04-12 | Kemijski inštitut | Fusion polypeptides comprising tir and dimerization domain for modulation of tlr/innate immunity signaling |
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US9707235B1 (en) | 2012-01-13 | 2017-07-18 | University Of Kentucky Research Foundation | Protection of cells from degeneration and treatment of geographic atrophy |
WO2013169382A1 (en) * | 2012-05-07 | 2013-11-14 | DePuy Synthes Products, LLC | Methods and devices for treating intervertebral disc disease |
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Cited By (3)
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US7122656B2 (en) | 2002-01-10 | 2006-10-17 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Splice variant of MyD88 and uses thereof |
WO2011043740A1 (en) * | 2009-10-09 | 2011-04-14 | Kemijski Institut | Peptides for inhibition of myd88-dependent signaling pathways |
AT510991A5 (en) * | 2009-10-09 | 2015-02-15 | Kemijski Inst | Peptides for the inhibition of MyD88-dependent signaling pathways |
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