WO2017197243A1 - Inhibition de cblb pour le traitement d'infections fongiques - Google Patents

Inhibition de cblb pour le traitement d'infections fongiques Download PDF

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Publication number
WO2017197243A1
WO2017197243A1 PCT/US2017/032370 US2017032370W WO2017197243A1 WO 2017197243 A1 WO2017197243 A1 WO 2017197243A1 US 2017032370 W US2017032370 W US 2017032370W WO 2017197243 A1 WO2017197243 A1 WO 2017197243A1
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Prior art keywords
cblb
dectin
albicans
subject
sirna
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PCT/US2017/032370
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English (en)
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Jian Zhang
Chad RAPPLEYE
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Ohio State Innovation Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • C. albicans is the most common cause of fungal infections in humans and has become one of the leading causes of hospital-acquired blood stream infections.
  • invasive candidiasis still has a high mortality rate ranging from 45 to 75% (Brown, G.D. et al. Sci. Transl. Med. 4, 165rvl3 (2012)).
  • the high morbidity and mortality associated with disseminated candidiasis are mainly due to the lack of early and accurate diagnostic tools, limited antifungal drugs, and the emergence of drug resistance.
  • Disclosed herein is a method for treating a fungal infection in a subject that involves administering to the subject a composition comprising a therapeutically effective amount of a casitas B lymphoma-b (CBLB) inhibitor.
  • CBLB casitas B lymphoma-b
  • the disclosed methods can in some embodiments be used to treat any fungal infection.
  • the disclosed methods can be used to treat a Candida spp. infection.
  • the fungal infection comprises Candida auris, aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof.
  • the CBLB inhibitor comprises functional nucleic acid that knocks down expression of the Cbl-b gene in the subject.
  • the functional nucleic acid comprises an siRNA.
  • the siRNA comprises the nucleic acid sequence 5'- AAAUUCUCGAAGUAUGCUCUU-3 ' (SEQ ID NO : 1 ), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.
  • the fungal infection comprises a Candida albicans infection.
  • the fungal infection comprises aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof.
  • the fungal infection comprises aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), or a combination thereof.
  • the CBLB inhibitor is an siRNA, a miRNA, a shRNA, a small molecule, an antisense molecule, a peptide, or a protein.
  • the CBLB inhibitor comprises a functional nucleic acid that knocks down expression of the Cbl-b gene in the subject.
  • the functional nucleic acid comprises an siRNA.
  • the functional nucleic acid is from about 15 to about 25 nucleotides.
  • the siRNA comprises the nucleic acid sequence 5'- UUUGCUAACGGACCAGUACUU-3 ' (SEQ ID NO:2) or 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3).
  • the siRNA comprises the nucleic acid sequence 5'- UUUGCUAACGGACCAGUACUU-3 ' (SEQ ID NO:2), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.
  • the siRNA comprises the nucleic acid sequence 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.
  • the subject is a mammal. In some embodiments, the subject is a human.
  • a method for treating or preventing a fungal infection in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of a casitas B lymphoma-b (CBLB) inhibitor, wherein the casitas B lymphoma-b (CBLB) inhibitor reduces the activity of a casitas B lymphoma-b (CBLB) protein in the subject.
  • CBLB casitas B lymphoma-b
  • the casitas B lymphoma-b (CBLB) inhibitor reduces the activity of a casitas B lymphoma-b (CBLB) protein in the subject comprises reducing the expression of the gene encoding the casitas B lymphoma-b (CBLB) protein.
  • the reducing the expression of the gene comprises RNA interference using a functional nucleic acid that knocks down expression of the gene in the subject.
  • FIGS. 1 A-1C CBLB inhibits pro-inflammatory cytokine production by macrophages after infection with C. albicans yeast cells or hyphae, and A. fumigatus conidia.
  • TNF-a left
  • IL-6 middle
  • IL- ⁇ right
  • production as determined by ELISA, in the culture supernatants of WT and Cblb _/ ⁇ BMDMs that were infected with C. albicans capl mutant yeast cell (top) and hyphal (bottom) forms (WT strain SC5314) (MOI: 1 : 1) for 1 h and 3 h.
  • IL- IRA production as measured by ELISA, in culture supernatants of WT and Cblb _/ ⁇ BMDMs that were infected with C. albicans yeast (left) and hyphal (right) forms.
  • FIGS. 2A-2G CBLB associates with dectin-1 and dectin-2 in macrophages after infection with C. albicans yeast cells or hyphae.
  • the numbers below the SYK (c) and CARD9 (d) blots indicate the extent of SYK expression in BMDMs of C57BL/6 mice that were treated with the control siRNA or the Syk-specific siRNA (c) or of CARD9 expression in
  • the numbers below the Flag blots in e,f indicate the extent of dectin-1 expression in Clec7a _/ ⁇ BMDMs reconstituted with constructs expressing WT dectin-1 or CLEC7A Y15F (e), or of dectin-2 expression in Fcerlg _/" BMDMs reconstituted with constructs expressing WT
  • FIGS. 3A-3H CBLB targets dectin-1 and dectin-2 for polyubiquitination and subsequent degradation in the lysosome.
  • FIGS. 4A-4D Loss of CBLB impairs dectin-1 and dectin-2 internalization and their downregulation at the cell surface.
  • DIC differential interference contrast
  • LAMP lysosomal-associated membrane protein
  • DAPI 4,6-diamidino-2-phenylindole dihydrochloride
  • FIGS. 7A-7B CBLB inhibits signaling via the dectin-1.
  • TLR ligands 1 to 9, zymosan, and curdlan for 48 h.
  • FIGS. 8A-8B CBLB inhibits TNF-a and IL-6 production by dendritic cells but has a limited role in the production of these cytokines by neutrophils upon infection with C. albicans yeast or hyphae.
  • (a) ELISA of TNF-a and IL-6 production in the supernatants collected from BMDCs of WT and Cblb-/- mice infected with C. albicans yeast and hyphae forms (MOI 1 : 1) for 1, 3, and 6 h.
  • MOI 1 : 1 : 1 : 1 for 1, 3, and 6 h.
  • FIGS. 9A-9D Human macrophages lacking CBLB fail to down-regulate dectin-1 and dectin-2 expression and produce more pro-inflammatory cytokines upon infection with C.
  • FIGS. 10A-10E CBLB deficiency impairs ubiquitination of dectin-1, dectin-2, and SYK in macrophages induced by C. albicans yeast and hyphal infection.
  • FIGS. 12A-12B CBLB C373 A mutation significantly reduces fungal burden in kidneys, livers, lungs, and spleens, and leads to less myeloid cell recruitment to the kidneys,
  • n 5 for WT or Cblb C 1 A mice. Data are shown as means ⁇ s.d. and analyzed using the Student's t test. *P ⁇ 0.05, **P ⁇ 0.01.
  • FIGS. 13A-13E CBLB C373 A mutation facilitates fungal killing by enhancing ROS activity in the blood and within spleens and kidneys by monocytes/macrophages, (a) Fungal burden of WT and Cblb C373A in the blood at 2 and 6 h after systemic C. albicans infection, and killing capacity of WT or Cblb C373A mice as assessed by 24 h co-culture with C. albicans, (b) Killing capacity of splenic monocytes and neutrophils of WT or Cblb C313A mice as assessed by 24 h co-culture with C.
  • mice 5 mice per group and are representative data from three independent experiments. Error bars, mean ⁇ s.e.m. Data were analyzed using the Student's t test. *P ⁇ 0.05, **P ⁇ 0.01. FIGS. 14A-14B.
  • FIGS. 15A-15D Autoantibody, IgG, and IgE titers in the sera and IL-17/IFN-Y in the sera and kidneys of WT, Cblb _/" , and Cblb C373A mice before and after C. albicans infection, (a-c) Serum titers of anti-dsDNA and anti-ssDNA antibodies, IgG, IgE, IL-17, and IFN- ⁇ in WT, Cblb " /_ , and Cblb C373A mice before or 48 h after C. albicans infection (1 x 10 6 CFU).
  • FIG. 16 Loss of CBLB stabilizes dectin-3, but not MR, Mincle and DC-SIGN.
  • Disseminated C. albicans infection in patients who have a weakened immune system is life-threatening.
  • 40% of bloodstream infections (candidemia) are caused by Candida spp.
  • invasive candidiasis still has a high mortality rate ranging from 45 to 75%.
  • the high morbidity and mortality associated with disseminated candidiasis are mainly due to the lack of early and accurate diagnostic tools, the limited antifungal drugs, and the emergence of drug resistance, thus highlighting the need to further understand host-pathogen interactions and the mechanisms of immune resistance to fungal spread, and to develop alternative immune-based strategies to combat candidemia.
  • albicans is controlled after activation of innate immune cells via cell surface pattern recognition receptors (PRRs) such as TLR2 and C-type lectin receptors (CLRs) that detect the infecting fungi.
  • PRRs cell surface pattern recognition receptors
  • CLRs C-type lectin receptors
  • the CLRs Dectinl and Dectin2/3 recognize C. albicans yeast cells and hyphae by binding to the surface ⁇ -glucans and a-mannans of the two fungal forms, respectively. Recognition of these molecules results in release of inflammatory cytokines from dendritic cells and macrophages, which is critical for antifungal immunity.
  • the mechanisms that control this CLR-mediated pro-inflammatory response to fungal infection are completely unknown.
  • E3 ubiquitin ligase Cbl-b targets K48-linked poly-ubiquitination of Dectinl and 2, two key pattern recognition receptors for sensing C albicans, leading to Dectin internalization and degradation.
  • Loss of Cbl-b function protects mice from systemic infection with a lethal dose of C albicans and deficiency of Dectin- 1, -2, or both in Cbl-b "/_ mice negates this protection.
  • silencing Cbl-b gene in vivo protects mice from lethal systemic C albicans infection.
  • subject refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • terapéuticaally effective refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • an “effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect.
  • the amount of beneficial agent that is “effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an "effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.
  • an "effective amount" of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a "therapeutically effective amount” of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result
  • a “prophylactically effective amount” of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition.
  • Therapeutically effective and prophylactically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject.
  • terapéuticaally effective amount can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect.
  • the precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and/or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • carrier means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose.
  • a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • treating or “treatment” of a subject includes the administration of a drug to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder.
  • the terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage.
  • the term "preventing" a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and/or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event.
  • small interfering RNA refers to a double stranded RNA duplex of any length, with or without single strand overhangs, wherein at least one strand is homologous to the target mRNA to be degraded.
  • an antisense molecule is a single stranded oligonucleotide which is complementary to a section of the target RNA and must hybridize or bind to it in a 1 : 1 ratio in order to cause its degradation.
  • siRNA provides a substrate for the RNA-induced silencing complex (RISC), and unlike antisense, is inactive until incorporated into this macromolecular complex.
  • RISC RNA-induced silencing complex
  • siRNA comprises a double-stranded RNA duplex of at least about 15, or at least about 19, nucleotides with no overhanging nucleotides. In another embodiment, the siRNA has nucleotide overhangs.
  • the siRNA may have two nucleotide overhangs, thus the siRNA can comprise a 21 nucleotide sense strand and a 21 nucleotide antisense strand paired so as to have a 19 nucleotide duplex region.
  • the number of nucleotides in the overhang can be in the range of about 1 to about 6, about 2 to about 4, or about 3 homologous nucleotide overhangs at each of the 5' and 3' ends.
  • the nucleotide overhang can be modified, for example to increase nuclease resistance.
  • the terms "about” and “approximately” are defined as being "close to" as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.
  • a method for treating a fungal infection in a subject that involves administering to the subject a composition comprising a therapeutically effective amount of a casitas B lymphoma-b (CBLB) inhibitor.
  • CBLB casitas B lymphoma-b
  • the disclosed methods can in some embodiments be used to treat any fungal infection.
  • the disclosed methods can be used to treat a Candida spp. infection.
  • the fungal infection comprises Candida auris, aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof.
  • the CBLB inhibitor comprises functional nucleic acid that knocks down expression of the Cbl-b gene in the subject.
  • the functional nucleic acid comprises an siRNA.
  • the siRNA comprises the nucleic acid sequence 5'- AAAUUCUCGAAGUAUGCUCUU-3 ' (SEQ ID NO: 1), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.
  • the fungal infection comprises a Candida albicans infection.
  • the fungal infection comprises aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof.
  • the fungal infection comprises aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), or a combination thereof.
  • the CBLB inhibitor is an siRNA, a miRNA, a shRNA, a small molecule, an antisense molecule, a peptide, or a protein.
  • the CBLB inhibitor comprises a functional nucleic acid that knocks down expression of the Cbl-b gene in the subject.
  • the functional nucleic acid comprises an siRNA.
  • the functional nucleic acid is from about 15 to about 25 nucleotides.
  • the siRNA comprises the nucleic acid sequence 5'- UUUGCUAACGGACCAGUACUU-3 ' (SEQ ID NO:2) or 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3).
  • the siRNA comprises the nucleic acid sequence 5'- UUUGCUAACGGACCAGUACUU-3 ' (SEQ ID NO:2), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.
  • the siRNA comprises the nucleic acid sequence 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.
  • the subject is a mammal. In some embodiments, the subject is a human.
  • a method for treating or preventing a fungal infection in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of a casitas B lymphoma-b (CBLB) inhibitor, wherein the casitas B lymphoma-b (CBLB) inhibitor reduces the activity of a casitas B lymphoma-b (CBLB) protein in the subject.
  • CBLB casitas B lymphoma-b
  • the disclosed methods can in some embodiments be used to treat any fungal infection.
  • the disclosed methods can be used to treat a Candida spp. infection.
  • the fungal infection comprises Candida auris, aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof.
  • the CBLB inhibitor comprises functional nucleic acid that knocks down expression of the Cbl-b gene in the subject.
  • the functional nucleic acid comprises an siRNA.
  • the siRNA comprises the nucleic acid sequence 5'- AAAUUCUCGAAGUAUGCUCUU-3 ' (SEQ ID NO : 1 ), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.
  • the fungal infection comprises a Candida albicans infection.
  • the fungal infection comprises aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, or a combination thereof.
  • the fungal infection comprises aspergillosis, Pneumocystis carinii pneumonia (PCP), coccidioidomycosis (valley fever), or a combination thereof.
  • the CBLB inhibitor is an siRNA, a miRNA, a shRNA, a small molecule, an antisense molecule, a peptide, or a protein.
  • the CBLB inhibitor comprises a functional nucleic acid that knocks down expression of the Cbl-b gene in the subject.
  • the functional nucleic acid comprises an siRNA.
  • the functional nucleic acid is from about 15 to about 25 nucleotides.
  • the siRNA comprises the nucleic acid sequence 5'- UUUGCUAACGGACCAGUACUU-3 ' (SEQ ID NO:2) or 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3).
  • the siRNA comprises the nucleic acid sequence 5'- UUUGCUAACGGACCAGUACUU-3 ' (SEQ ID NO:2), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.
  • the siRNA comprises the nucleic acid sequence 5'- UAAUACCCAAAAUUCGACCUU-3 ' (SEQ ID NO:3), or a variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3.
  • the subject is a mammal. In some embodiments, the subject is a human.
  • the casitas B lymphoma-b (CBLB) inhibitor reduces the activity of a casitas B lymphoma-b (CBLB) protein in the subject comprises reducing the expression of the gene encoding the casitas B lymphoma-b (CBLB) protein.
  • the reducing the expression of the gene comprises RNA interference using a functional nucleic acid that knocks down expression of the gene in the subject.
  • the Cbl-b gene sequence is the human Cbl-b gene sequence is SEQ ID NO:4 (Homo sapiens Cbl proto-oncogene B (CBLB), transcript variant 1, mRNA; NCBI Reference Sequence: NM 001321786.1).
  • the Cbl-b gene sequence is the mouse Cbl-b gene sequence Accession Number NM_001033238.1 (Mus musculus Casitas B- lineage lymphoma b (Cblb), mRNA; NCBI Reference Sequence: NM_001033238.1).
  • the siRNA comprises a nucleic acid sequence from about 15 to about 25 nucleotides targeting the human Cbl-b gene sequence. In some embodiments, the siRNA comprises a nucleic acid sequence from about 15 to about 25 nucleotides targeting SEQ ID NO:4.
  • the shRNA comprises a nucleic acid sequence from about 15 to about 25 nucleotides (length of the duplex region is from 15 to 25 nucleotides) targeting the human Cbl-b gene sequence. In some embodiments, the shRNA comprises the nucleic acid sequence from about 15 to about 25 nucleotides targeting SEQ ID NO:4.
  • a method for treating a Candida albicans fungal infection in a subject that involves administering to the subject a composition comprising a therapeutically effective amount of an siRNA, wherein the siRNA comprises a nucleic acid sequence from about 15 to about 25 nucleotides targeting SEQ ID NO:4.
  • a method for treating a Candida albicans fungal infection in a subject that involves administering to the subject a composition comprising a therapeutically effective amount of an siRNA, wherein the siRNA is selected from the SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3.
  • CBLB inhibitors are well known in the prior art.
  • siRNA against cbl-b for treating cancer are disclosed in U.S. Patent No. 9,186,373, which is incorporated by reference in its entirety for the teaching of these siRNA.
  • CBLB inhibitors have also been described for example in Loeser et al. (JEM (2007) doi: 10.1084/iem.20061699; Chiang et al. (Journal of Clinical Investigation 117 (4) (2007): 1033-1034); Lametschwandtner et al. (Journal of
  • US 2007/0087988 relates to a method for regulating HPKl, whose expression may be enhanced by increasing the expression of Cbl-b, and vice versa (e. g. by Cbl-b siRNA inhibition).
  • CBLB inhibitors are disclosed, for example, in US20070054355; WO 2005007141; US 9334522; which are incorporated by reference in their entirety.
  • the CBLB inhibitor reduces or inhibits the function of CBLB by reducing or inhibiting the expression of CBLB or Cbl-b.
  • reduce/reduction or “inhibit/inhibition” relate to a reduction or inhibition of the function (or expression) of CBLB or Cbl-b as compared to the unmodified natural function, optionally including the complete inhibition of said function.
  • the function (or expression) is reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.
  • the reduction or inhibition of the function of CBLB or Cbl-b is transient, i. e. the function is only temporarily reduced as described in the above and can therefore recover again, e. g.
  • Cbl-b siRNA a transient reduction of Cbl-b in immune cells can also be performed in a repetitive manner, e. g. until a therapeutic success has been achieved.
  • the expression of CBLB or Cbl-b is reduced or inhibited by the use of Cbl-b antisense RNA or siRNA.
  • Cbl-b antisense RNA or siRNA short DNA and/or RNA sequences that are complementary to one of the regions of the target ⁇ Cbl-b) mRNA sequence are employed, so that hybridization and inactivation of the corresponding sequences will occur.
  • These sequences preferably have a length of at least 15, 18, 20, 22, 25, 28, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or up to 200 bases until the length of the complete target sequence is reached, preferably up to 2500, 2000, 1500, 1000, 500 or 300 bases.
  • the sequence of SEQ ID No. 1 is used.
  • the CBLB inhibitor of the provided method can be a functional nucleic acid.
  • Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction.
  • Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting.
  • functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, RNAi, and external guide sequences.
  • the functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.
  • Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains.
  • functional nucleic acids can interact with the mRNA of Cbl-b or the genomic DNA of Cbl-b or they can interact with the polypeptide CBLB.
  • nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule.
  • the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.
  • Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing.
  • the interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation.
  • the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication.
  • Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC.
  • antisense molecules bind the target molecule with a dissociation constant (Kd)less than or equal to 10 "6 , 10 "8 , 10 "10 , or 10 "12 .
  • Kd dissociation constant
  • Aptamers are molecules that interact with a target molecule, preferably in a specific way.
  • aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
  • Aptamers can bind small molecules, such as ATP (U. S. Patent No. 5,631, 146) and theophiline (U.S. Patent No. 5,580,737), as well as large molecules, such as reverse transcriptase (U. S. Patent No. 5,786,462) and thrombin (United States patent 5,543,293).
  • Aptamers can bind very tightly with K d 's from the target molecule of less than 10-12 M.
  • the aptamers bind the target molecule with a Kd less than 10 "6 , 10 "8 , 10 "10 , or 10 "12 .
  • Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (U. S. Patent No.
  • the aptamer have a Kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the Kd with a background binding molecule. It is preferred when doing the comparison for a polypeptide for example, that the background molecule be a different polypeptide. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in U.S. Patent Nos.
  • Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions.
  • ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (U.S. Patent Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998, 193, 5,998,203;
  • Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates.
  • Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in U.S. Patent Nos.
  • Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single- stranded nucleic acid.
  • triplex molecules When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a Kd less than 10-6, 10-8, 10-10, or 10-12. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in U.S. Patent Nos.
  • EGSs External guide sequences
  • RNase P RNase P
  • RNAse P aids in processing transfer RNA (tRNA) within a cell.
  • Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate.
  • RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukarotic cells.
  • WO 93/22434 by Yale
  • WO 95/24489 by Yale
  • Yuan and Altman EMBO J 14: 159-168 (1995)
  • Carrara et al. Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)
  • Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules be found in U.S. Patent Nos. 5, 168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.
  • RNAi RNA interference
  • dsRNA double stranded small interfering RNAs 21- 23 nucleotides in length that contains 2 nucleotide overhangs on the 3 ' ends
  • siRNA double stranded small interfering RNAs
  • RISC RNAi induced silencing complex
  • Short Interfering RNA is a double-stranded RNA that can induce sequence- specific post-transcriptional gene silencing, thereby decreasing or even inhibiting gene expression.
  • an siRNA triggers the specific degradation of homologous RNA molecules, such as mRNAs, within the region of sequence identity between both the siRNA and the target RNA.
  • WO 02/44321 discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with 3' overhanging ends, herein incorporated by reference for the method of making these siRNAs.
  • Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer (Elbashir, S.M., et al. (2001) Nature, 411 :494 498) (Ui- Tei, K., et al. (2000) FEBS Lett 479:79-82).
  • siRNA can be chemically or in vitro-synthesized or can be the result of short double-stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the cell.
  • Synthetic siRNAs are generally designed using algorithms and a conventional DNA/RNA synthesizer.
  • siRNA can also be synthesized in vitro using kits such as Ambion's SILENCER® siRNA Construction Kit. Disclosed herein are any siRNA designed as described above based on the sequences for Cbl-b.
  • siRNA from a vector is more commonly done through the transcription of a short hairpin RNAs (shRNAs).
  • Kits for the production of vectors comprising shRNA are available, such as, for example, Imgenex's GENESUPPRESSORTM Construction Kits and Invitrogen's BLOCK-ITTM inducible RNAi plasmid and lentivirus vectors.
  • Disclosed herein are any shRNA designed as described above based on the sequences for the herein disclosed inflammatory mediators.
  • composition comprising a CBLB inhibitor in a pharmaceutically acceptable carrier.
  • the composition comprises a pharmaceutically acceptable carrier that is suitable for the intracellular administration in a patient.
  • the composition comprises vehicles such as liposomal or microsomal formulations which are particularly preferred for the administration of nucleic acids.
  • compositions may comprise pharmaceutically suitable salts as well as additional buffers, tonicity components or pharmaceutically acceptable carriers.
  • inhibitory nucleic acids such as antisense nucleic acids, siRNA and shRNA
  • Pharmaceutical carrier substances are provided to improve the tolerability of the composition and to improve the solubility and bioavailability of the active ingredients. Examples include emulsifying agents, thickening agents, redox components, starch, alcohol solutions, polyethylene glycol or lipids.
  • the selection of a suitable pharmaceutical carrier strongly depends on the mode of administration. For oral administration, liquid or solid carriers can be used, whereas final compositions in liquid form are advantageous for injections.
  • the pharmaceutical composition comprises buffer substances or tonic substances.
  • a buffer By using a buffer, it is possible to adjust the pH value of the pharmaceutical composition to physiological conditions and to attenuate or buffer pH variations.
  • An example for such a substance is a phosphate buffer.
  • Tonicity agents are used to adjust the osmolarity and may comprise ionic substances, such as inorganic salts, e. g. NaCl, or non-ionic substances, e. g. glycerol, or carbohydrates.
  • the composition is provided to be suitable for a systemic, topical, oral or intranasal administration.
  • the pharmaceutical composition can be suitable for intravenous, intraarterial, intramuscular, intravascular, intraperitoneal or subcutaneous administration.
  • injection or transfusions are suitable for this purpose.
  • Administering the pharmaceutical composition directly into the bloodstream will have the advantage that the active ingredients of the pharmaceutical composition are distributed throughout the body and are thus capable of reaching their target tissues quickly.
  • topical applications are provided. The administration either directly to or in the vicinity of a site at which an immune response is to be induced or enhanced, e. g. the site of an infection.
  • Example 1 Targeting CBLB as a therapeutic approach for disseminated candidiasis.
  • CLRs The fungi-responsive C-type lectin receptors
  • CLRs have a central role in the detection of Candida during bloodstream infection.
  • Candida albicans is controlled by activation of innate immune cells via cell surface pattern-recognition receptors (PRRs) such as toll-like receptor 2 (TLR2) and CLRs that detect the infecting fungus.
  • PRRs cell surface pattern-recognition receptors
  • TLR2 toll-like receptor 2
  • CLRs dectin-1 encoded by Clec7a in mice
  • dectin-2 encoded by Clec4n in mice
  • C. albicans yeast cells and hyphae by binding to surface ⁇ -glucans and a-mannans on the two fungal forms, respectively (Taylor, P.R. et al. Nat.
  • dectin-mediated signaling pathways including those involving spleen tyrosine kinase (SYK), that control the pro-inflammatory response to fungal infection, is completely unknown.
  • SYK spleen tyrosine kinase
  • CBLB Casitas B lymphoma-b
  • CBLB functions as a negative regulator of fungal recognition during systemic C albicans infection by targeting dectin-1, dectin -2, and SYK for Lys48 (K48)-linked polyubiquitination.
  • Negative regulation of dectin-1- and dectin-2-mediated signaling by CBLB is crucial for restraining the magnitude of the innate immune responses against C albicans infection, but it leads to suboptimal protection of the host.
  • Systemic in vivo delivery of Cblb-speciiic siRNA protects C57BL/6 mice from systemic C albicans infection. Therefore, the disclosed data show that CBLB is a drug target for systemic candidiasis.
  • CBLB inhibits signaling via dectin receptors
  • wild-type (WT) and Cblb ⁇ h bone marrow (BM)-derived macrophages (BMDMs) and BM-derived dendritic cells (BMDCs) were stimulated with ligands for TLRs 1-9 or with zymosan (a ligand for TLR2 and dectin-1).
  • TLR ligand-induced production of tumor necrosis factor (TNF)-a and interleukin (IL)-6 was comparable between WT and Cblb ⁇ h BMDMs and BMDCs, zymosan-induced TNF-a and
  • IL-6 production was substantially higher in Cblb ⁇ h BMDMs and BMDCs than in WT cells (Fig.
  • Curdlan stimulation induced a markedly higher level of TNF-a and IL-6 in Cblb ⁇ ' ⁇ BMDMs and BMDCs than in WT cells (Fig. 7a,b).
  • BMDMs, BMDCs, and BM neutrophils from WT and Cblb ⁇ mice were infected with a C albicans yeast-only mutant (capl; hereafter referred to as yeast), in which the adenylate-cyclase-associated protein-1 (Capl) gene was disrupted, causing the failure of yeast- hypha transition due to the lack of cAMP (Bru, Y.S. et al. J. Bacterid. 183, 3211-3223
  • Dectin-1 and dectin-2 recognize the yeast and hyphal forms of C albicans, respectively, by binding to the surface ⁇ -glucans (dectin-1) and a-mannans (dectin-2) of the two fungal forms (Taylor, P R. et al. Nat. Immunol. 8, 31-38 (2007); Saijo, S. et al. Immunity 32, 681-691 (2010); Zhu, L.L. et al. Immunity 39, 324-334 (2013)).
  • CBLB deficiency resulted in increased production of TNF-a and IL-6 by BMDMs and BMDCs in response to signaling via both the yeast and hyphal forms of C.
  • Cblb ⁇ h neutrophils produced comparable amounts of TNF-a and IL-6 to those produced in WT neutrophils, except at the 3-h time point after infection (Fig. 8b), suggesting that CBLB may have a limited role in affecting the inflammatory response of neutrophils to C. albicans infection.
  • Cblb ⁇ h BMDMs also produced more TNF-a and IL-6 than WT BMDMs infected with
  • Aspergillus fumigatus conidia Fig. lb
  • Aspergillus fumigatus conidia Fig. lb
  • dectin-1 is a major PRR recognizing A. fumigatus (Steele, C. et al. PLoS Pathog. 1, e42 (2005); Gersuk, G.M. et al. J. Immunol. 176, 3717-3724 (2006); Rivera, A. et al. J. Exp. Med. 208, 369-381 (2011)).
  • CBLB has the potential to regulate the dectin family of CLRs in response to some fungal pathogens. Because several studies have indicated that either the NLRP3 inflammasome or a noncanonical, caspase-8-mediated inflammasome participates in host defense to C. albicans infection (Hise, A.G. et al. Cell Host Microbe 5, 487-497 (2009); Gringhuis, S.I. et al. Nat. Immunol. 13, 246-254 (2012)), IL- ⁇ production by WT and Cblb _/ ⁇ BMDMs after infection with C. albicans yeast and hyphae was measured. Both WT and Cblb _/ ⁇ BMDMs produced comparable levels of IL- ⁇ (Fig. la), suggesting that CBLB does not regulate the inflammasome activation that is mediated by dectin- 1 or dectin-2.
  • human monocyte-derived macrophages were generated (Kang, P.B. et al. J.
  • Dectin family CLRs have a major role in fungal recognition and host innate responses against fungal infection (Brown, G.D. et al. Nat. Rev. Immunol. 6, 33-43 (2006); Brown, G.D. et al. Annu. Rev. Immunol. 29, 1-21 (2011); Hardison, S.E. et al. Nat. Immunol. 13, 817-822 (2012)).
  • Dectin-l 's cytoplasmic tail contains an immunoreceptor tyrosine-based activation motif (IT AM) that can be phosphorylated by Src family kinases.
  • IT AM immunoreceptor tyrosine-based activation motif
  • dectin-1 recruits and activates SYK, thereby initiating downstream signaling via the CARD9-BCL10- MALT1 complex (Brown, G.D. et al. Nat. Rev. Immunol. 6, 33-43 (2006); Hardison, S.E. et al. Nat. Immunol. 13, 817-822 (2012)).
  • dectin-2 lacks this ITAM-like motif, it binds FcR- ⁇ (Saijo, S. et al. Immunity 32, 681-691 (2010)), which contains ITAMs (Osorio, F. et al.
  • CBLB was infected with C. albicans yeast cells or hyphae for different amounts of time.
  • co-IP co- immunoprecipitation
  • CBLB binds to SYK in B cells after B cell receptor (BCR) stimulation (Sohn, H.W. et al. J. Exp. Med. 197, 1511-1524 (2003)) or to CARD11 (also known as CARMA1), a homolog of CARD9, in NKT cells (Kojo, S. et al. Proc. Natl. Acad. Sci. USA 106, 17847-17851 (2009)).
  • BCR B cell receptor
  • CARD11 also known as CARMA1
  • SYK and CARD9 are potential binding partners of CBLB in the signaling pathways downstream of dectin-1 and dectin-2
  • Syk gene expression was silenced in WT BMDMs by using a -S ⁇ -specific siRNA.
  • the tyrosine of the hemi-ITAM was mutated to phenylalanine in dectin-l 's cytoplasmic tail (CLEC7A Y15F ) and the tyrosines within the ITAMs of FcR- ⁇ were mutated to phenylalanine (FCER1G Y65F Y76F ), then Clec7a ⁇ ' ⁇ BMDMs and Fcergl ⁇ h BMDMs with these mutated alleles were reconstituted and infected with C. albicans yeast and hyphae, respectively.
  • dectin-1 at Tyrl5, or FcR- ⁇ at Tyr65 and Tyr76 completely abrogated the binding of CBLB to dectin-1 or dectin-2 (Fig. 2e,f), indicating that phospho-Tyrl5 of dectin-1 or phospho-Tyr65 and phospho-Tyr76 of FcR- ⁇ is critical for their binding to CBLB. Indeed, CBLB bound to FcR- ⁇ in WT BMDMs after infection with C.
  • Dectin-1, dectin-2, and SYK are targets of CBLB
  • dectin-1 and dectin-2 are the targets of CBLB
  • protein stability of dectin-1, dectin-2, SYK, and CARD9 was first examined in macrophages infected with C albicans yeast cells or hyphae.
  • dectin-1 and dectin-2, but not SYK or CARD9 underwent degradation in WT BMDMs after infection with C. albicans yeast cells and hyphae, but not in BMDMs lacking CBLB (Fig. 3a).
  • CBLB is the E3 ubiquitin ligase for dectin-1 or dectin-2.
  • BMDMs generated from WT mice, Cblb ⁇ mice, or mice expressing an E3-ligase-dead mutant of CBLB (Cblb C373A ) (Oksvold, M.P. et al. Mol. Immunol. 45, 925-936 (2008)) were infected with C albicans yeast cells or hyphae.
  • CBLB C373A mutant abrogated ubiquitination of dectin-1 and dectin-2 (Fig. 3c,d, upper panel, Fig. 1 la,b).
  • K48-ubiquitin- or K63-ubiquitin-specific antibodies were used. It was confirmed that both dectin-1 and dectin-2 underwent K48-linked polyubiquitination, and that this K48-linked polyubiquitination of dectin-1- and dectin-2 was abrogated in BMDMs lacking CBLB or expressing the CBLB C373A mutant (Fig. 3c,d, bottom, Fig. l la,b).
  • BMDMs after infection with C. albicans yeast cells or hyphae after infection with C. albicans yeast cells or hyphae.
  • SYK underwent K48- linked polyubiquitination after infection with both C. albicans yeast and hyphae, but this ubiquitination was greatly reduced in BMDMs expressing CBLB C373A (Fig. 1 lc,d). Therefore, data suggest that dectin-1, dectin-2, and SYK are targets of CBLB and that CBLB keeps the expression of these CLRs in check. Consistent with these data, SYK and the transcription factor F-KB were highly activated in BMDMs lacking CBLB after infections with C. albicans yeast and hyphae (Fig. l ie).
  • Clec7a ⁇ mice were reconstituted with constructs expressing either WT dectin-1 or the dectin-1 lysine-to arginine mutants and BMDMs lacking dectin-2 (from Clec4n ⁇ mice) with a construct expressing either WT dectin-2 or the CLEC4N K10R mutant. These reconstituted BMDMs were then infected with C. albicans yeast cells or hyphae.
  • Clec7a ⁇ BMDMs expressing the dectin-1 triple mutant (CLEC7A K2R K27R K34R ) or Clec4n ' ⁇ BMDMs reconstituted with CLEC4N K10R produced significantly higher amounts of TNF-a and IL-6 after infection with C. albicans yeast cells or hyphae (Fig. 3g,h).
  • CBLB regulates the internalization of dectin-1 and dectin-2, and their trafficking to the lysosome
  • Cell surface receptor internalization can occur when receptors are mono- or
  • CBLB negatively regulates ROS production and fungal killing but not phagocytosis of C. albicans
  • ROS highly reactive oxygen species
  • Cblb ⁇ and Cblb C373A - expressing BMDMs produced more ROS than WT controls at a multiplicity of infection (MOI) of 5: 1 or 2: 1 (Fig. 12a).
  • MOI multiplicity of infection
  • Enhanced ROS activity in Cblb ⁇ BMDMs correlated with an increase in their fungal-killing potency (Fig. 12b).
  • CBLB inhibits dectin-mediated innate immune responses to systemic C. albicans infection
  • Cblb- Clec7a-'-, Clec4n , Cblb- Clec4n , Clec7a-'-Clec4n , and Cblb- Clec7a- Clec4n l - mice were infected with C. albicans. Dectin-1 or dectin-2 single deficiency rendered Cblb _/ ⁇ mice susceptible to C. albicans infection, and a deficiency in both dectin-1 and dectin-2 greatly increased the sensitivity of Cblb ⁇ mice to systemic C. albicans infection.
  • Cblb ⁇ h or Cblb C313A mice at 8-12 weeks of age did not show signs of autoimmunity, as revealed by comparable amounts of autoantibody titers to double-stranded (ds) DNA and single- stranded (ss) DNA, and of IL-17 and IFN- ⁇ levels, in the sera of WT and Cblb ⁇ or Cblb C313A mice, as well as no elevated IL-17 and IFN- ⁇ in the kidneys of Cblb ⁇ or Cblb C313A mice, as compared to that of WT mice (Fig. 15a-d).
  • CBLB may regulate an additional CLR(s), such as the mannose receptor (MR), dectin-3, or Mincle, which have been shown to be involved in host defense against C. albicans infection (Zhu, L.L. et al. Immunity 39, 324-334 (2013); Wells, C.A. et al. J. Immunol. 180, 7404-7413 (2008); van de Veerdonk, F.L. et al. Cell Host Microbe 5, 329-340 (2009); Cambi, A. et al. Eur. J. Immunol. 33, 532-538 (2003)).
  • MR mannose receptor
  • Mincle Mincle
  • CBLB is a therapeutic target for antifungal infection
  • CBLB downregulates dectin family CLR signaling and host innate immune responses
  • decreasing CBLB expression may enhance phagocyte antifungal responses, providing evidence for a new therapeutic approach.
  • Cblb-specific siRNA to knock down Cblb.
  • WT mice were first infected with C. albicans by intravenous (i.v.) injection, and 24 h later the Cblb-specific siRNA or a nonsense siRNA was injected via the tail vein. Mortality of the mice was monitored for 7 d. Although all of the WT mice that were treated with the nonsense siRNA died within 7 d after infection, seven of nine WT mice that were treated with the siRNA to Cblb survived. There was a significantly higher fungal burden in the kidneys of WT mice that received the nonsense siRNA than those that received the Cblb-specific siRNA (Fig. 6). These data indicate that CBLB can serve as a potent therapeutic target for enhancing host defense against fungal infections.
  • mice C57BL/6 mice and Rag ⁇ mice were purchased from the Jackson Laboratory. Fcerlg ⁇ h mice were purchased from Taconic (Hudson, NY). Cblb ⁇ h mice7 were provided. Cblb C373A mice and Clec7a ⁇ h were described previously (Taylor, P.R. et al. Nat. Immunol. 8, 31-38 (2007); Oksvold, M.P. et al. Mol. Immunol. 45, 925-936 (2008)). Clec4rf' ⁇ mice were described previously (Saijo, S. et al. Immunity 32, 681-691 (2010)).
  • Cblb ⁇ h mice on a C57BL/6 background were crossed with Clec7a ⁇ or Clec4n _/" mice to generate C£/£ ⁇ / ⁇ Clec7a ⁇ / ⁇ and Cblb ⁇ Clec4n ⁇ mice, or Cblb ⁇ Clec7a ⁇ Clec4n ⁇ mice.
  • Cblb ⁇ mice were also crossed with Ragl ⁇ h mice to generate Cblb ⁇ h Rag mice. The mice were used at 8-12 weeks of age, and both male and female mice were used in this study. All animal experimentation involving systemic C. albicans infection and in vivo delivery of control and Cblb-specific siRNA was approved by the Institutional Animal Care and Use Committees (IACUCs) of the Ohio State University and the Xiangya School of Medicine, Central South University.
  • IACUCs Institutional Animal Care and Use Committees
  • Antibodies against CBLB (G-l), SYK (N-19), CARD9 (H-90), DC-SIGN (T- 13), CD206 (H-300), and ubiquitin (P4D1) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
  • Anti-dectin-1 (GE2; Ab82888) was obtained from Abeam (Cambridge, MA).
  • Anti-dectin-2 (217611) and mouse IL-1RA/IL-1F3 Quantikine ELISA Kit (MRA00) was purchased from R&D Systems (Minneapolis, MN).
  • PE-conjugated anti- dectin-2 (MCA2415PE) was obtained from AbD Serotec (Raleigh, NC). Anti-Mincle (D292-3) was purchased from MBL Life Science (Woburn, MA). ELISA kits for mouse IgG (88-50400) and IgE (88-50460) were purchased from eBioscience (San Diego, CA). ELISA kits for anti- ssDNA (5310) and anti-dsDNA (total (A + G + M) (5110) were purchased from Alpha
  • Clec4n (dectin-2) (pCMV2-Flag) were purchased from Sino Biologicals, Inc. (Beijing, P.R. China).
  • Anti-dectin-3 was provided.
  • Mouse neutrophil isolation kit, monocyte isolation kit, and CD45 microbeads (mouse) were purchased from Miltenyi Biotec (San Diego, CA). Histopaque 1 1 19 (Sigma 1 1 191), Histopaque 1077 (Sigma 10771), and anti- Flag (M2) were obtained from Sigma-Aldrich (St. Louis, MO).
  • Collagenase type IV (021951 10) was purchased from MP Biomedicals (Santa Ana, CA).
  • CellRox Deep Red (C I 0422) was purchased from ThermoFisher Scientific (Waltham, MA). The validation of the antibodies used is provided on the manufacturers' websites.
  • K-to-R Single and triple lysine-to-arginine (K-to-R)-encoding mutations of dectin-1 (CLEC7A K2R , CLECTA 1 "TM, CLEC7A K34R , and CLEC7A K2R K27R K34R ) and dectin-2 (CLEC4N K10R ), CLEC7A Y15F , and FCR-Y Y65F Y76F were generated by site-directed mutagenesis at Mutagenex Inc. (Piscataway, NJ).
  • BM cells were harvested from the femurs and tibias of mice. Cells were cultured in Dulbecco' s modified Eagle' s medium (DMEM) (Sigma-Aldrich, St. Louis, MO) containing 10% FBS and 30% conditioned medium from L929 cells expressing macrophage colony stimulating factor (M- CSF). After 1 week of culture, non-adherent cells were removed, and adherent cells were 80- 90%) F4/80 + CDl lb + , as determined by flow cytometric analysis.
  • DMEM Dulbecco' s modified Eagle' s medium
  • M- CSF macrophage colony stimulating factor
  • Mouse BMDCs were generated using granulocyte-macrophage colony stimulating factor (GM-CSF) and purified from bulk cultures by magnetic selection with anti-CD 1 lc microbeads. This routinely gave purities of >98%>.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • For isolation of BM neutrophils total BM cells were recovered from the femurs and tibias by flushing with RPMI medium (Sigma-Aldrich) with an 18-gauge needle; erythrocytes were lysed with red blood cell (RBC) lysis buffer (eBioscience) and BM neutrophils were isolated by neutrophil isolation kit (Milteny); neutrophil purity (>98%>) was confirmed by flow cytometry.
  • mice Isolation of mouse PBMCs and neutrophils from blood, and splenic monocytes, neutrophils, and kidney CD 45+ cells.
  • WT and Cblb C313A mice were anesthetized, and blood was collected from the tail vein.
  • the RBC were lysed using RBC lysis buffer (eBioscience).
  • PBMCs and neutrophils were isolated by gradient centrifugation over Histopaque 1 1 19 (density, 1.1 19 g/ml) and Histopaque 1077 (density, 1.077 g/ml), according to the manufacturer's instructions, at
  • PBMCs were collected from the interface between the plasma and Histopaque 1077. Neutrophils were recovered at the interface of the interface of the Histopaque 1 1 19 and Histopaque 1077 layers, and they were 80-90% pure and >95% viable, as determined by flow cytometry. PBMCs and neutrophils were washed twice and resuspended in RPMI 1640 medium supplemented with 10%) FBS. Splenic monocytes and neutrophils of WT and Cblb C313A mice were isolated by monocyte isolation and neutrophil isolation kits (Milteny). Monocyte and neutrophil purities (>98%) were confirmed by flow cytometry.
  • mice were killed at 48 h after infection with C. albicans (by tail vein injection) at a dose of 1 ⁇ 10 6 c.f.u.
  • Kidneys were perfused, minced, and placed in 2 ml of Hank' s balanced salt solution (HBSS) (50 mM HEPES, 12 mM Dextrose, 280 mM NaCl, 10 mM KC1, 1.5 mM Na2HP04, pH to 7.05) containing 2 mg/ml collagenase IV, and incubated at 37 °C for 30 min with gentle agitation.
  • HBSS Hank' s balanced salt solution
  • Digested kidney tissues were passed through a 40- ⁇
  • Clec7a ⁇ BMDMs were transfected with constructs expressing Flag-tagged dectin-1, CLEC7A K2R , CLEC7A K27R , CLEC7A K34R , CLEC7A K2R K27R K34R , or
  • Clec4n ' ⁇ or Fcergl ⁇ ' ⁇ BMDMs were transfected with Flag-tagged dectin-2, CLEC4N K10R , FcR- ⁇ , and FCR-Y Y65F Y76F , respectively.
  • ROS assay phagocytosis of C. albicans and fungal killing assay.
  • RLU Relative light units
  • C. albicans yeast were labeled with Alexa Fluor 488 (Invitrogen) in 100 mM HEPES buffer (pH 7.5) (diluted to 1 :500) and then co-cultured with WT or Cblb ⁇ BMDMs for 45 min at 37 °C.
  • Adherent fungal cells were quenched with trypan blue, and the rate of phagocytosis was determined by flow cytometry (Wirnsberger, G. et al. Nat. Genet. 46, 1028-1033 (2014)).
  • WT or Cblb ⁇ BMDMs (1 x 10 5 /well) were incubated with C. albicans at an MOI of 1 :500 for 24 h.
  • C. albicans To determine the fungal killing capacity of PBMCs, blood neutrophils, splenic monocytes, and neutrophils and of kidney CD45 + cells, WT and Cblb C373A mice were infected with C. albicans by tail vein injection (1 x 10 6 c.f.u.).
  • PBMCs, blood neutrophils, and splenic monocytes, neutrophils and kidney CD45 + cells were co-cultured with a C. albicans form at an MOI of 1 : 10 for 24 h.
  • the cell lysates were immunoprecipitated with anti-CBLB (1 : 100) and blotted with anti-dectin-1 (1 : 1,000) or anti-dectin-2 (1 :5,000), anti-SYK (1 : 1,000), and anti-
  • CARD9 (1 : 1,000).
  • BMDMs from WT and either Cblb ⁇ or Cblb C373A mice were infected with C. albicans yeast capl mutant or hyphae
  • albicans yeast cells or hyphae 1 : 1) at the indicated times and lysed for immunoblotting with antibodies against dectin-1 (1 : 1,000), dectin-2 (1 : 1,000), dectin-3 (1 : 1,000), MR (1 : 1,000), Mincle (1 : 1,000), DC-SIGN (1 : 1,000), SYK, and CARD9, respectively.
  • Detection of serum and kidney cytokines, serum IgG and IgE, and autoantibodies by ELISA 10 5 BMDMs from WT, Cblb ⁇ or Cblb C313A mice were infected with live C. albicans capl mutant cells or hyphae at MOI 1 : 1 for the times indicated, and cytokine production in the supernatant was measured by ELISA.
  • mice For detection of serum IL-17, IFN- ⁇ , IL-6, TNF-a and IL- ⁇ , WT, Cblb ⁇ ' ⁇ or Cblb C373A mice were infected with C. albicans (5 x 10 4 , or 1 x 10 6 c.f.u. for some experiments), sera were collected at different time points and subjected for ELISA analysis. The kidneys harvested at 48 h after infection were homogenized, and the supernatant was recovered following centrifugation at 15,000g for 20 min at 4 °C. The cytokines, including IL-17, IFN- ⁇ , and IL-6, in the kidney homogenates were determined by using ELISA kits according to the manufacturer's instructions.
  • the ELISA results were expressed as 'pg per g of kidney' .
  • sera were collected from WT, Cblb ⁇ h or Cblb C313A mice before C. albicans infection and at 48 h after infection and were subjected to ELISA analysis.
  • dectin-1 and dectin-2 Internalization of dectin-1 and dectin-2 in macrophages after infection with C. albicans yeast and hyphae.
  • WT and Cblb ⁇ BMDMs were infected with C. albicans yeast capl mutant (MOI: 1 : 1) for the times indicated.
  • Flow cytometry was then used to determine the surface expression of dectin-1 and dectin-2.
  • BMDMs from WT and Cblb ⁇ mice were labeled with PE-conjugated anti-dectin-1 (1 :200) or anti-dectin-2 (1 :200). Cells were then incubated at 37 °C for 5, 15, and 30 min.
  • the cells were fixed in 1% paraformaldehyde, permeabilized in 0.05% saponin and stained with FITC-conjugated anti-LAMP-1. Imaging was performed on a Leica TCS-SP2 confocal microscope (1 : 100). Imaging was performed on a laser- scanning confocal microscope (Flowview 1000, Olympus).
  • mice were infected with C. albicans i.v. at 1-5 x 10 5 c.f.u. and monitored daily. After infection, mice were weighed and monitored daily. Mice were euthanized if they lost >20% of their body weight. In a separate group, the kidneys were harvested 2 d after infection. The left kidneys were photographed and homogenized for enumeration of fungal burden. The right kidneys were fixed for histological analysis. The fungal burden in the kidneys, spleens, livers, and lungs was determined by c.f.u. in kidney, spleen, liver, and lung homogenates. The fungal burden in the blood at 2 and 6 h after infection was also determined. Mice were allocated to experimental groups based upon their genotypes and randomized within their sex- and age-matched groups. No blinding was done in this study.
  • MDM human monocyte-derived macrophages
  • human MDMs were transfected with control siRNA or Cblb-specific siRNA (100 or 200 nM; Dharmacon RNA Technologies) by using Lonza nucleofector reagent, and they were then plated in RPMI 1640 containing 20% autologous serum. After 36 h, the MDMs were washed and infected with yeast cells or hyphae of C. albicans. The protocol was approved by The Ohio State University Institutional Review Board.
  • AAAUUCUCGAAGUAUGCUCUU-3 ' (SEQ ID NO: 1) or a nonsense siRNA (2 mg/kg/mouse) (Dharmacon RNA Technologies) in In vivo-j etPEI-FluoF (Polyplus-transfection, Inc.; New York, NY) via tail vein injection.
  • spleen cells were collected and lysed in RIPA buffer. The cell lysates were subjected to SDS-PAGE, transferred and blotted with anti- CBLB and anti-actin, respectively.
  • the fungal cell wall consists mainly of carbohydrates, including mannose-based structures (the mannoproteins), ⁇ -glucan, and chitin. Recognition of ⁇ -glucans and a-mannans by dectin-1 and dectin-2 is essential for antifungal immunity (Brown, G.D. et al. Annu. Rev.
  • CBLB functions as a negative regulator of the dectin-1 and dectin-2 CLRs, which initiate innate immune responses to fungal pathogens in human and mouse macrophages.
  • CBLB targets dectin-1 and dectin-2, and SYK for K48-linked polyubiquitination, which inhibits dectin-1- or dectin-2-mediated signaling pathways.
  • CBLB deficiency or inactivation leads to increased pro-inflammatory responses that decrease dissemination of C. albicans and bolster host defense.
  • CLEC7A K2R ' K34R and CLEC4N K10R mutants in which ubiquitination is abrogated, result in increased production of TNF-a and IL-6 by macrophages infected with C. albicans yeast cells or hyphae (Fig. 3g,h), thus mirroring the data obtained from Cblb ⁇ and Cblb C313A mice.
  • the disclosed data therefore provide evidence that ubiquitination of dectin-1 and dectin-2 is a key mechanism for terminating innate immune responses during fungal infection, thereby avoiding excessive inflammation and subsequent tissue damage, while at the same time dampening optimal host-defense properties.
  • Phagocytosis is a key cellular process, both during homeostasis and after infection or tissue damage, and dectin-1 has been shown to be a phagocytic receptor (Goodridge, H.S. et al.
  • ROS production by phagocytes is associated with pathogen killing (Dupre-Crochet, S. et al. J. Leukoc. Biol. 94, 657-670 (2013)), and it was reported that dectin-1 activates SYK in macrophages and is important for dectin-1 -stimulated ROS
  • CBLB is critical for T cell activation, tolerance induction, and TH2 and TH9 cell differentiation (Liu, Q. et al. Cell Cycle 13, 1875-1884 (2014)), it is possible that the enhanced antifungal immune response in the absence of CBLB may result in heightened adaptive T cell responses.
  • this possibility was excluded by the observation that the phenotype of Cblb ⁇ h Rag mice, which do not have T and B cells, phenocopies that of Cblb ⁇ h mice after C. albicans infection (Fig. 5e), supporting the notion that CBLB is crucial for controlling innate immune responses against systemic C. albicans infection. The heightened innate immune responses observed during systemic C.
  • albicans infection is mediated by dectin-1 and dectin-2, because introducing mutants of dectin-1, dectin-2, or both into Cblb ⁇ h mice abrogates these heightened responses and renders Cblb ⁇ h mice susceptible to C. albicans infection (Fig. 5f). More notably, systemic in vivo delivery of a Cblb-speciiic siRNA to C57BL/6 mice protects them from lethal systemic C. albicans infection (Fig. 6). These data show that CBLB is a therapeutic target for controlling disseminated candidiasis. Of note, inhibition of CBLB may have detrimental effects due to unchecked inflammation, particularly on patients in intensive care. However, inhibition of Cblb by using an siRNA in vivo may be a more viable approach because the siRNA would have a limited half-life and dosages could be modulated to minimize the degree of inflammation.
  • the disclosed data provide the first evidence that CBLB has an essential role in regulating dectin-mediated innate immune responses to fungal pathogens following inflammatory responses to fungi in immunocompetent hosts.
  • One consequence of this dampening of inflammatory responses is the creation of a less-than-optimal host defense program.
  • Targeting CBLB therefore serves as a new and important therapeutic strategy in fighting fungal infections.
  • CBLB Homo sapiens Cbl proto-oncogene B
  • NCBI Reference Sequence NM_001321786.1

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Abstract

L'invention concerne un procédé de traitement d'une infection fongique chez un sujet, qui consiste à administrer au sujet une composition comprenant une quantité thérapeutiquement efficace d'un inhibiteur de lymphome CBL-b (casitas B lymphoma-b). Plus précisément, l'inhibiteur de lymphome CBL-b est un ARNsi. L'invention concerne en outre des séquences d'acides nucléiques spécifiques d'inhibiteurs de CBL-b ARNsi.
PCT/US2017/032370 2016-05-13 2017-05-12 Inhibition de cblb pour le traitement d'infections fongiques WO2017197243A1 (fr)

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