WO2019018662A1 - Inhibition de tgf-bêta pour traiter des symptômes hématologiques du syndrome de shwachman-diamond - Google Patents

Inhibition de tgf-bêta pour traiter des symptômes hématologiques du syndrome de shwachman-diamond Download PDF

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Publication number
WO2019018662A1
WO2019018662A1 PCT/US2018/042913 US2018042913W WO2019018662A1 WO 2019018662 A1 WO2019018662 A1 WO 2019018662A1 US 2018042913 W US2018042913 W US 2018042913W WO 2019018662 A1 WO2019018662 A1 WO 2019018662A1
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sds
tgfp
subject
cells
bone marrow
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PCT/US2018/042913
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English (en)
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Carl NOVINA
Cailin JOYCE
Akiko SHIMAMURA
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Dana-Farber Cancer Institute, Inc.
Children's Medical Center Corporation
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Publication of WO2019018662A1 publication Critical patent/WO2019018662A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • 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
    • A61K31/69—Boron compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor

Definitions

  • SDS Shwachman-Diamond Syndrome
  • the present invention is based upon the surprising discovery that transforming growth factor beta (TGFP) inhibitors treat Shwachman-Diamond Syndrome (SDS). Accordingly, described herein are methods of treating SDS in a subject comprising administering a TGFP inhibitor. Methods of treating or preventing bone marrow failure in a subject, e.g., a human subject, having or at risk of developing bone marrow failure are carried out by administering a TGFP inhibitor to the subject, thereby treating or preventing bone marrow failure in the subject.
  • TGFP transforming growth factor beta
  • Exemplary types of bone marrow failure include SDS (also known as Shwachman-Bodian- Diamond syndrome (SBDS)), Fanconi anemia (FA), dyskeratosis congenita (DC), congenital amegakaryocytic thrombocytopenia (CAMT), Blackfan-Diamond anemia (BDA), and reticular dysgenesis (RD).
  • SDS Shwachman-Bodian- Diamond syndrome
  • FA Fanconi anemia
  • DC dyskeratosis congenita
  • AVT congenital amegakaryocytic thrombocytopenia
  • BDA Blackfan-Diamond anemia
  • RD reticular dysgenesis
  • the bone marrow failure comprises (or manifests as) SDS.
  • the TGFP inhibitor reduces or inhibits a symptom or sequelae associated with SDS.
  • Exemplary symptoms or sequelae associated with SDS are selected from the group consisting of neutropenia (e.g., exibiting an absolute neutrophil count ⁇ 1500/mL), anemia, thrombocytopenia (e.g., exibiting a platelet count below 50,000/mm 3 ), exocrine pancreatic dysfunction, growth retardation, chronic steatorrhea, metaphyseal dysplasia, myelodysplasia, megakaryocyte dysplasia, erythroid dysplasia, acute myeloid leukemia (AML), and generalized osteopenia.
  • neutropenia e.g., exibiting an absolute neutrophil count ⁇ 1500/mL
  • anemia thrombocytopenia (e.g., exibiting a platelet count below 50,000/mm 3 )
  • the TGFp inhibitor reduces or inhibits a symptom or sequelae associated with SDS by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • the TGFp inhibitor reduces hematological symptoms in the subject.
  • the TGFP inhibitor increases hematopoietic colony formation in the bone marrow of the subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • the TGFP inhibitor increases hematopoiesis in bone marrow hematopoietic stem or progenitor cells (HSPCs) by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • HSPCs bone marrow hematopoietic stem or progenitor cells
  • Exemplary TGFP inhibitors for use in the methods described herein include AVID200, SD208, LY2157299, TEW-7197, bortezomib, P144, pirfenidone, LY333531, and LY2109761.
  • the TGFp inhibitor is administered at a dose of from about 0.1 picomolar (pM) to about 1 molar (M), e.g., about 1 pM to about 1 nanomolar (nM), about InM to about 1 micromolar ( ⁇ ), about 1 ⁇ to about 1 millimolar (mM), about 1 mM to about 1 M.
  • the TGFP inhibitor is administered at a frequency of from about once per hour to about once per year, e.g., once every six hours, twice per day, once per day, twice per week, once per week, twice per month, once per month, once every two months, once every six months, or once per year.
  • the TGFp inhibitor is administered for a duration of between one day and one year, e.g., the TGFP inhibitor is administered for a duration of one week, two weeks, one month, two months, three months, six months, or one year. In some cases, the TGFP inhibitor is
  • TGFp receptor 1 expression or activity is inhibited by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%), at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • the TGFP inhibitor is administered orally. In another example, the TGFP inhibitor is administered systemically.
  • the method further comprises administering existing therapeutic modalities in combination with the therapies described herein.
  • the method further comprises performing an allogeneic bone marrow transplant or an allogeneic hematopoietic stem cell transplant in the subject.
  • the method further comprises administering a granulocyte-colony stimulating factor (G-CSF) polypeptide to the subject.
  • G-CSF granulocyte-colony stimulating factor
  • SBDS Shwachman-Bodian-Diamond Syndrome
  • methods for regenerative medicine i.e., the process of replacing, engineering, or regenerating human cells, tissues, or organs to restore or establish normal function.
  • methods of treating SDS in a subject having or at risk of developing SDS are carried out by contacting a CD34 + hematopoietic stem or progenitor cell (HSPC) of the subject with a TGFP inhibitor.
  • HSPC hematopoietic stem or progenitor cell
  • the CD34+ HSPC is isolated from the subject prior to contacting the TGFP inhibitor.
  • the CD34 + HSPC is isolated from the bone marrow of the subject and subsequently contacted with the TGFP inhibitor.
  • the method further comprises administering the CD34 + HSPC to the subject as autologous cell transplant therapy.
  • a suitable CD34 + HSPC includes a hematopoietic stem cell (HSC) or a multipotent progenitor cell (MPP).
  • Also provided are methods of treating SDS in a subject having or at risk of developing SDS comprising contacting a CD34 + HSPC (or a plurality of CD34 + HSPC) of the subject with an inhibitor or agonist of a polypeptide or nucleotide in the TGFP signaling pathway. That is, a TGFP signaling pathway gene that is aberrantly expressed in a CD34 + HSPC is contacted with an inhibitor or agonist of the gene, as needed.
  • TGFB family ligands TGFP3, growth/differentiation factor 15 (GDF15), and bone morphogenic protein 1 (BMPl)
  • GDF15 growth/differentiation factor 15
  • BMPl bone morphogenic protein 1
  • Methods of determining whether a subject has SDS are carried out by obtaining a test sample from a subject at risk of developing SDS, determining an expression level of a gene in the TGFp signaling pathway in the test sample, comparing the expression level of the gene in the test sample with the expression level of the gene in a reference sample, and determining that the subject has SDS if the expression level of the gene in the test sample is differentially expressed as compared to the level of the gene in the reference sample.
  • the gene comprises TGFP3, GDF15, or BMPl .
  • Exemplary test samples are those obtained from blood, serum, or plasma.
  • agent any small compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
  • an alteration is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art-known methods such as those described herein.
  • an alteration includes at least a 1% change in expression levels, e.g., at least a 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%), 80%), 90%), or 100% change in expression levels.
  • an alteration includes at least a 5%-10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
  • ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • control or “reference” is meant a standard of comparison.
  • "changed as compared to a control" sample or subject is understood as having a level that is statistically different than a sample from a normal, untreated, or control sample.
  • Control samples include, for example, cells in culture, one or more laboratory test animals, or one or more human subjects. Methods to select and test control samples are within the ability of those in the art.
  • An analyte can be a naturally occurring substance that is characteristically expressed or produced by the cell or organism (e.g., an antibody, a protein) or a substance produced by a reporter construct (e.g, ⁇ -galactosidase or luciferase). Depending on the method used for detection, the amount and measurement of the change can vary. Determination of statistical significance is within the ability of those skilled in the art, e.g., the number of standard deviations from the mean that constitute a positive result.
  • Detect refers to identifying the presence, absence, or amount of the agent (e.g., a nucleic acid molecule, for example deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) to be detected.
  • the agent e.g., a nucleic acid molecule, for example deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)
  • detectable label is meant a composition that when linked (e.g., joined - directly or indirectly) to a molecule of interest renders the latter detectable, via, for example, spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • Direct labeling can occur through bonds or interactions that link the label to the molecule, and indirect labeling can occur through the use of a linker or bridging moiety which is either directly or indirectly labeled.
  • Bridging moieties may amplify a detectable signal.
  • useful labels may include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent labeling compounds, electron-dense reagents, enzymes (for example, as commonly used in an enzyme- linked immunosorbent assay (ELISA)), biotin, digoxigenin, or haptens.
  • ELISA enzyme- linked immunosorbent assay
  • biotin digoxigenin
  • digoxigenin or haptens.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, p- phthaldehyde and fluorescamine.
  • the molecule can also be detectably labeled using
  • fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the molecule using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the molecule also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent- tagged molecule is then determined by detecting the presence of luminescence that arises during the course of chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a “detection step” may use any of a variety of known methods to detect the presence of nucleic acid (e.g., methylated DNA) or polypeptide.
  • the types of detection methods in which probes can be used include Western blots, Southern blots, dot or slot blots, and Northern blots.
  • an effective amount is meant an amount of a compound, alone or in a combination, required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian decides the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • the term "purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • purified can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • modifications for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • substantially pure is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it.
  • the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%), by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.
  • hematopoietic stem cell an immature cell that can develop into all types of blood cells, including white blood cells, red blood cells, and platelets.
  • isolated nucleic acid is meant a nucleic acid that is free of the genes which flank it in the naturally-occurring genome of the organism from which the nucleic acid is derived.
  • the term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a synthetic cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion
  • Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones.
  • the isolated nucleic acid is a purified cDNA or RNA polynucleotide.
  • Isolated nucleic acid molecules also include messenger ribonucleic acid
  • an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it.
  • the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • marker any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
  • module alter (increase or decrease). Such alterations are detected by standard art-known methods such as those described herein.
  • normal amount refers to a normal amount of a complex in an individual known not to be diagnosed with disease, e.g., SDS.
  • the amount of the molecule can be measured in a test sample and compared to the "normal control level," utilizing techniques such as reference limits, discrimination limits, or risk defining thresholds to define cutoff points and abnormal values.
  • the "normal control level” means the level of one or more proteins (or nucleic acids) or combined protein indices (or combined nucleic acid indices) typically found in a subject known not to be suffering from disease, e.g., SDS. Such normal control levels and cutoff points may vary based on whether a molecule is used alone or in a formula combining other proteins into an index.
  • the level that is determined may be the same as a control level or a cut off level or a threshold level, or may be increased or decreased relative to a control level or a cut off level or a threshold level.
  • the control subject is a matched control of the same species, gender, ethnicity, age group, smoking status, body mass index (BMI), current therapeutic regimen status, medical history, or a combination thereof, but differs from the subject being diagnosed in that the control does not suffer from the disease in question or is not at risk for the disease.
  • BMI body mass index
  • the level that is determined may be an increased level.
  • the term "increased" with respect to level refers to any % increase above a control level.
  • the increased level may be at least or about a 1% increase, at least or about a 5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about a 80% increase, at least or about a 85%) increase, at least or about a 90% increase, or at least or about a 95% increase, relative to a control level.
  • the level that is determined may be a decreased level.
  • the term "decreased" with respect to level refers to any % decrease below a control level.
  • the decreased level may be at least or about a 1%) decrease, at least or about a 5% decrease, at least or about a 10% decrease, at least or about a 15%) decrease, at least or about a 20% decrease, at least or about a 25% decrease, at least or about a 30% decrease, at least or about a 35% decrease, at least or about a 40% decrease, at least or about a 45% decrease, at least or about a 50% decrease, at least or about a 55% decrease, at least or about a 60% decrease, at least or about a 65% decrease, at least or about a 70% decrease, at least or about a 75% decrease, at least or about a 80% decrease, at least or about a 85% decrease, at least or about a 90% decrease, or at least or about a 95% decrease, relative to a control level.
  • Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but typically exhibit substantial identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • pharmaceutically acceptable carrier includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
  • the carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose;
  • starches such as corn starch and potato starch
  • cellulose, and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate
  • powdered tragacanth malt
  • gelatin talc
  • excipients such as cocoa butter and suppository waxes
  • oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil
  • glycols such as propylene glycol
  • polyols such as glycerin, sorbitol, mannitol and polyethylene glycol
  • esters such as ethyl oleate and ethyl laurate
  • agar buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
  • protein or “polypeptide” or “peptide” is meant any chain of more than two natural or unnatural amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring or non-naturally occurring polypeptide or peptide, as is described herein.
  • post-translational modification e.g., glycosylation or phosphorylation
  • Primer set means a set of oligonucleotides that may be used, for example, for PCR.
  • a primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about” that particular value in addition to the value itself.
  • data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9.
  • a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.
  • reduces is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
  • sample refers to a biological sample obtained for the purpose of evaluation in vitro.
  • the sample or patient sample preferably may comprise any body fluid or tissue.
  • the bodily fluid includes, but is not limited to, blood, plasma, serum, lymph, breast milk, saliva, mucous, semen, vaginal secretions, cellular extracts, inflammatory fluids, cerebrospinal fluid, feces, vitreous humor, or urine obtained from the subject.
  • the sample is a composite panel of at least two of a blood sample, a plasma sample, a serum sample, and a urine sample.
  • the sample comprises blood or a fraction thereof (e.g., plasma, serum, fraction obtained via leukopheresis).
  • Preferred samples are whole blood, serum, plasma, or urine.
  • a sample can also be a partially purified fraction of a tissue or bodily fluid.
  • a reference sample can be a "normal" sample, from a donor not having the disease or condition fluid, or from a normal tissue in a subject having the disease or condition.
  • a reference sample can also be from an untreated donor or cell culture not treated with an active agent (e.g., no treatment or administration of vehicle only).
  • a reference sample can also be taken at a "zero time point" prior to contacting the cell or subject with the agent or therapeutic intervention to be tested or at the start of a prospective study.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 60%>, at least 70%, at least 80%), at least 85%>, at least 90%, at least 95%, or at least 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • subject as used herein includes all members of the animal kingdom prone to suffering from the indicated disorder.
  • the subject is a mammal, and in some aspects, the subject is a human.
  • companion animals such as dogs and cats as well as livestock such as cows, horses, sheep, goats, pigs, and other
  • a subject "suffering from or suspected of suffering from” a specific disease, condition, or syndrome e.g., SDS
  • Subjects suffering from, and suspected of suffering from, a specific disease, condition, or syndrome are not necessarily two distinct groups.
  • susceptible to or “prone to” or “predisposed to” or “at risk of developing” a specific disease or condition refers to an individual who based on genetic, environmental, health, and/or other risk factors is more likely to develop a disease or condition than the general population.
  • An increase in likelihood of developing a disease may be an increase of about 10%, 20%, 50%, 100%, 150%, 200%, or more.
  • treating and “treatment” as used herein refer to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an adverse condition, disorder, or disease, so as to effect a reduction in severity and/or frequency of symptoms, eliminate the symptoms and/or their underlying cause, and/or facilitate improvement or remediation of damage. It is appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • a composition of the invention is administered orally or systemically.
  • Other modes of administration include rectal, topical, intraocular, buccal, intravaginal, intracisternal, intracerebroventricular, intratracheal, nasal, transdermal, within/on implants, or parenteral routes.
  • parenteral includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations.
  • Compositions comprising a composition of the invention can be added to a physiological fluid, such as blood. Oral administration can be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule.
  • Parenteral modalities may be preferable for more acute illness, or for therapy in patients that are unable to tolerate enteral administration due to gastrointestinal intolerance, ileus, or other concomitants of critical illness. Inhaled therapy may be most appropriate for pulmonary vascular diseases (e.g., pulmonary hypertension).
  • Kits or pharmaceutical systems may be assembled into kits or pharmaceutical systems for use in the methods described herein.
  • Kits or pharmaceutical systems according to this aspect of the invention comprise a carrier means, such as a box, carton, tube, having in close confinement therein one or more container means, such as vials, tubes, ampoules, bottles, syringes, or bags.
  • the kits or pharmaceutical systems of the invention may also comprise associated instructions for using the kit.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
  • FIG. 1 A-FIG. ID is a series of t-distributed Stochastic Neighbor Embedding (tSNE) plots showing that supervised dimensionality reduction identifies lineage commitment of CD34 + cells.
  • FIG. 1 A-FIG. ID is a series of t-distributed Stochastic Neighbor Embedding (tSNE) plots showing that supervised dimensionality reduction identifies lineage commitment of CD34 + cells.
  • FIG. 1A is a tSNE plot of hematopoietic lineage commitment derived from an empirically- defined gene expression signature of the cells based on donor identify. Shown here
  • IB is a tSNE plot of hematopoietic lineage commitment derived from an empirically-defined gene expression signature of the cells based on mRNA expression of selected signature genes. Color indicates Transcript-Per-Million (TPM)>1 for the indicated stem- (orange), myeloid- (blue), erythroid- (green), or lymphoid- (red) enriched mRNA. The presence of two colors indicates co- expression. Grey indicates TPM ⁇ 1 for all four factors.
  • FIG. 1C is a tSNE plot of hematopoietic lineage commitment derived from an empirically-defined gene expression signature of the cells based on mRNA expression of lineage-restricted genes reported elsewhere.
  • FIG. ID is a tSNE plot of hematopoietic lineage commitment derived from an empirically-defined gene expression signature of the cells based on immunophenotypes. Color indicates membership in a gated immunophenotypic subset as shown in FIG. 5A-FIG. 5B. Grey indicates cells that were ungated or sorted without indexing. Numerical axes derived from tSNE are arbitrary and therefore not shown.
  • FIG. 2B is a tSNE plot and histogram representing the alteration of cellular architecture of early hematopoiesis in SDS.
  • HSC hematopoietic stem cells
  • MLP multipotent progenitor cells
  • MLP multilymphoid progenitor cells
  • CMP common myloid progenitor cells
  • GMP granulocyte-monocyte progenitor cells
  • MEP megakaryocyte-erythroid progenitor cells
  • FIG. 3A-FIG. 3C is a histogram with a pie chart inset, Venn diagram, a violin plot and combination histogram/line graph showing that TGFP signaling is selectively activated in SDS stem and multipotent progenitor cells.
  • FIG. 3 A is a histogram that shows differentially expressed genes were identified among all SDS versus normal cells and within each cluster. To aid biological interpretation, this gene set was filtered to focus on genes with False Discovery Rate (FDR) adjusted p-value ⁇ .05 and log2(fold change) >
  • FDR False Discovery Rate
  • FIG. 3C on the left is a split violin plot showing the summed expression of 25 upregulated TGFp targets and 52 down- regulated TGFp targets in SDS HSC/MPP. On the right of FIG.
  • FIG. 3C is a combination bar/line graph showing Log2 fold changes (primary axis, bars) and ⁇ -values (secondary axis, lines) for the gene sets plotted in FIG. 3B. Significance was determined by two- way ANOVA, with Holm-Sidak's multiple comparisons test.
  • FIG. 4A-FIG. 4F is a series of photomicrographs, box and whisker plots, a diagram and a heat map showing that TGFP pathway activation through TGFpRl suppresses hematopoiesis in SDS bone marrow (BM) progenitor cells.
  • FIG. 4A is a series of images showing images showing 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) and phospho-SMAD2 staining of primary BM CD34+ cells from normal donor bone marrow and SDS bone marrow, either untreated or treated with AVID200.
  • FIG. 4A is a series of images showing images showing 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) and phospho-SMAD2 staining of primary BM CD34+ cells from
  • FIG. 4E is a diagram showing the role of TGFP signaling in SDS bone marrow failure.
  • TGFpi and/or TGFP3 ligands (targets of AVID200 inhibitor) activate signaling through the TGFpRl receptor (target of SD208 inhibitor) on SDS HSC/MPP.
  • TGFp ligands are primarily derived from a CD34 " cell type in bone marrow because TGFP ligand mRNAs were not detected in CD34 + hematopoietic stem/progenitor cells (HSPC).
  • FIG. 4F is a heatmap showing expression of extracellular proteins annotated to a TGFp network that was enriched among dysregulated proteins in SDS patient plasma. Asterisks indicate TGFP family ligands.
  • FIG. 5A-FIG. 5D is a table, area graph, bar graph, and heatmap showing derivation of lineage commitment gene expression signature.
  • FIG. 5A is a table showing immunotypes.
  • FIG. 5C is a bar graph showing the results of hematopoietic colony forming assays demonstrating enrichment of mixed colonies from HSC and MPP gates, myeloid colonies from CMP and GMP gates, and erythroid colonies from the MEP gate.
  • FIG. 5A-FIG. 5D is a table, area graph, bar graph, and heatmap showing derivation of lineage commitment gene expression signature.
  • FIG. 5A is a table showing immunotypes.
  • FIG. 5B is a series of area graphs that show a representative gating scheme used to purify CD34 +
  • 5D is a heatmap showing a 79 gene signature derived from sequencing 100 cells purified from each gate. Expression values reflect the average expression of each gene across two biological and two technical replicates per subset. High expression of erythroid genes such as GATA1 and Krueppel-like factor 1 (KLF1) in the MPP subset is likely due to the recently reported enrichment of MEP in the CD34 + CD38 mid CD45RA " CD135- population (Sanada et al., 2016 Blood, 2016 128(7), 923-33), which was gated as MPP under the sorting strategy adapted from Laurenti (Laurenti et al., 2013 Nature Immunology, 14: 756-763). Immunophenotypic MPPs did not cluster with GATA1 -expressing MEP, as shown in FIG. ID.
  • FIG. 6 is a tSNE plot showing that SDS GMP deficiency is present in the absence of symptomatic neutropenia.
  • a tSNE plot of hematopoietic lineage commitment was derived from an empirically-derived gene expression signature, and colored based on SDS diagnosis and active neutropenia (absolute neutrophil count ⁇ 1500/ul).
  • FIG. 7A-FIG. 7B is a series of photographs and a whisker plot.
  • FIG. 7A is a series of representative, full-well images from methylcellulose colony forming assays performed on primary bone marrow mononuclear cells from SDS patients and a normal donor in the presence or absence of 1 uM SD208.
  • FIG. 5B is a whisker plot showing the number of colonies formed by normal donor and SDS patient BM-derived mononuclear cells with increasing concentrations of SD208, normalized to the 0 uM treatment. Significance was determined by two-way
  • FIG. 8 is a web diagram showing a dysregulated protein network including TGFP3 and associated factors in SDS patient plasma. Significant networks were assembled from
  • the embodiments disclosed herein are based upon the surprising discovery that activated transforming growth factor beta (TGFP) signaling in early hematopoietic progenitors promotes bone marrow failure in Shwachman-Diamond Syndrome (SDS). As described herein, despite the basic cellular function of SBDS in ribosomal subunit joining and mitotic spindle stabilization (Menne et al., 2007 Nature Genetics, 39:486-495;
  • scRNA-seq single cell RNA (ribonucleic acid) sequencing
  • TGFP inhibitors (AVID200 and SD208) increased hematopoietic colony formation of SDS patient BM.
  • TGFP3 and other TGFP pathway members were elevated in SDS patient blood plasma. The data presented herein establishes the TGFp pathway as a biomarker and therapeutic target in SDS, and translates insights from single cell biology into a therapeutic intervention.
  • Described herein are advanced single cell technologies that were leveraged to perform the first direct analysis of primary human SDS hematopoietic progenitors. Whereas most single cell transcriptomic studies have focused on dissecting and characterizing cell types (Villani et al., 2017 Science 356; Tirosh et al., 2016 Science, 352: 189-196; Kumar et al., 2014 Nature, 516:56- 61; Darmanis et al., 2015 Proceedings of the National Academy of Sciences, 112), the results presented herein demonstrate the power of single cell transcriptomics to uncover a disease mechanism in rare cells.
  • TGFP Fanconi Anemia
  • Described herein is a broader role for TGFP in a mechanistically distinct BM failure syndrome.
  • TGFP inhibitors are already in clinical trials to treat myelodysplastic syndrome, cancer, and pulmonary fibrosis, among others (Herbertz et al., 2015 Drug Des. Devel. Then, 9:4479-4499, incorporated herein by reference).
  • the results presented herein demonstrate that TGFpi/3 inhibition by an agent, such as AVID200, is an effective therapy across clinically-heterogeneous SDS patients and different marrow failure disorders.
  • RNA sequencing to identify that specific targets genes of the TGFp signaling pathway are dysreglulated in SDS hematopoietic stem cells and multipotent progenitors, but not in lineage-committed hematopoietic progenitors.
  • inhibition of TGFP signaling extracellularly e.g., by trapping TGFpi/3 ligands with AVID200
  • intracellularly e.g., by blocking TGFpRl kinase activity with SD-208
  • TGFp family ligands (TGFP3, GDF15, and BMP1) are elevated in patient plasma. These data suggest that ligand-dependent stimulation of TGFp signaling in a rare subset of SDS hematopoietic progenitors causes SDS bone marrow failure. Thus, the results presented herein indicate that the TGFp pathway is a new therapeutic target and biomarker for SDS.
  • SDS patients suffer from bone marrow failure, exocrine pancreatic dysfunction, skeletal anomalies and increased risk of acute myeloid leukemia.
  • the only curative treatment for the hematologic complications of SDS is a hematopoietic stem cell transplant, but outcomes were limited by the high risk of regimen-related toxicities.
  • the data described herein suggest that therapeutic inhibition of TGFp ameliorates hematological symptoms in SDS patients.
  • Therapeutic modalities include TGFP inhibitors that are already in clinical use or development, or new inhibitors with selective and targeted activities.
  • TGFp pathway components or other genes that are dysregulated in patient serum enable a simple blood diagnostic test to complement genetic and clinical diagnoses. This invention allows for the development of specialized inhibitors with optimal dosing, delivery, and specificity to treat pediatric SDS patients.
  • Bone marrow failure refers to the decreased production of one or more major hematopoietic cell lineages, which ultimately leads to diminished or absent hematopoietic precursors in the bone marrow as well as attendant cytopenias (Moore C and Krishnan K 2017 Bone Marrow Failure, StatPearls). Bone marrow failure may be acquired or inherited.
  • IBMF Inherited bone marrow failure
  • IBMFSs The most common inherited bone marrow failure syndromes are Fanconi anemia (FA), dyskeratosis congenita (DC), Shwachman-Diamond syndrome (SDS), congenital amegakaryocytic thrombocytopenia (CAMT), Blackfan-Diamond anemia (BDA), and reticular dysgenesis (RD).
  • FA Fanconi anemia
  • DC dyskeratosis congenita
  • SDS Shwachman-Diamond syndrome
  • AKT congenital amegakaryocytic thrombocytopenia
  • BDA Blackfan-Diamond anemia
  • RD reticular dysgenesis
  • Shwachman-Diamond syndrome (SDS; also known as Shwachman-Bodian-Diamond syndrome (SBDS), pancreatic insufficiency, or bone marrow dysfunction) is a rare multisystemic syndrome characterized by chronic neutropenia, pancreatic exocrine insufficiency associated with steatorrhea and growth failure, skeletal dysplasia with short stature, and an increased risk of bone marrow aplasia or leukemic transformation. More specifically, SDS is a rare and clinically- heterogeneous bone marrow (BM) failure syndrome caused by mutations in the SBDS gene, which encodes a ribosomal protein involved in ribosomal biogenesis and other cellular processes.
  • BM bone marrow
  • Mutations in the SBDS gene might be caused by gene conversion, which occurs when an intact SBDS gene and its non-functional pseudogene aberrantly recombine at meiosis, leading to an incorporation of pseudogene-like sequences into the "good copy" of the SBDS gene.
  • the function of the BM is to produce new blood cells, which include red and white blood cells, as well as platelets for blood clotting.
  • the BM does not make some or all of the blood cell types. Specifically, patients with SDS do not regenerate an adequate number of neutrophils, which is a condition known as neutropenia. This decrease in white blood cell production makes SDS patients vulnerable to infections such as pneumonia, ear infections, and skin infections. Indeed, the most common anomaly in SDS is usually intermittent, moderate neutropenia that often causes recurrent infections. Some patients also experience mild anemia and thrombocytopenia.
  • SDS SDS-derived neurodegenerative disease .
  • cutaneous e.g., eczema or ichthyosis
  • dental anomalies e.g., dental anomalies
  • psychomotor retardation Mild to severe intellectual disability causes learning difficulties in approximately 50% of patients. Hematologic manifestations may be complicated by bone marrow aplasia, acute myeloid leukemia or a myelodysplastic syndrome. In the neonatal period, there are generally no symptoms observed; however, some cases were reported with pancytopenia, respiratory distress, and severe spondylometaphyseal dysplasia.
  • MDS myelodysplastic syndrome
  • AML acute myeloid leukemia
  • Diagnosis of SDS can be performed in a variety of ways, such as through blood testing, stool collection, bone marrow biopsy, and genetic testing for mutations in the SBDS gene.
  • An initial diagnosis of SDS is typically based upon clinical, laboratory, and radiologic findings.
  • SDS is generally diagnosed based on evidence of exocrine pancreatic dysfunction and neutropenia.
  • Blood analysis of an individual with SDS shows neutropenia (absolute neutrophil count ⁇ 1500/mL) that can be associated with mild to moderate thrombocytopenia (e.g., a platelet count below 50,000/mm 3 ), moderate anemia, and a rise in fetal hemoglobin.
  • exocrine pancreatic insufficiency can be detected by serum analysis showing low levels of pancreatic isoamylase and/or trypsinogen, stool analysis showing low fecal elastase, and magnetic resonance imaging (MRI) revealing a characteristic pancreatic aspect with fat degeneration (MRI could be normal until the age of 5).
  • Imagery also allows detection, usually after the age of 5, of metaphyseal anomalies and abnormal growth plate development. Bone marrow smears usually reveal varying degrees of hypocellularity with dysgranulopoieisis or dyserythropoieisis. Skeletal abnormalities and short stature are characteristics that would support a diagnosis of SDS. Diagnosis is confirmed by genetic testing, e.g., for mutations in the SDS gene.
  • the treatment can be a combination of oral pancreatic enzyme replacements, fat soluble vitamins, hematopoietic stem cell transplantation, growth hormone, and AML chemotherapeutic drugs (Shwachman-Diamond syndrome, rarediseases.info.nih.gov/diseases).
  • pancreatic exocrine dysfunction may be treated with pancreatic enzyme supplementation
  • neutropenia may be treated with granulocyte-colony stimulating factor (G-CSF) to boost peripheral neutrophil counts. Severe hematological complications require hematopoietic stem cell transplantation.
  • G-CSF granulocyte-colony stimulating factor
  • hematopoiesis Because mature blood cells have a finite life span, they are continuously replaced in a process called hematopoiesis.
  • the BM is the site of hematopoiesis in humans, where
  • Hematopoietic stem cells promote blood cell differentiation and proliferation.
  • Hematopoietic stem cells are immature cells that can develop into all types of blood cells, including white blood cells, red blood cells, and platelets.
  • Hematopoietic stem cells are found in the bone marrow. During the differentiation process, hematopoietic stem cells progress through various maturational stages, from stem cells to multipotent progenitor cells, to finally terminating at lineage committed cells. As differentiation progresses, the multipotent progenitor cells respond to differentiation signals to lose their self-renewal properties. Blood cells fall into two distinct multipotent progenitor lineages: lymphoid, which include T-Cells, B-Cells, and natural killer cell or myloid, which include megakaryocytes, erythrocytes, granulocytes, and macrophages (Kondo and Motonari, 2010 Immunol. Rev., 238:37-46).
  • lymphoid which include T-Cells, B-Cells
  • natural killer cell or myloid which include megakaryocytes, erythrocytes, granulocytes, and macrophages
  • hematopoiesis in the BM is regulated by hematopoietic cytokines, which can "influence blood cell progenitor survival, proliferation, differentiation commitment, maturation, and functional activation.” (Metcalf and Donald, 2008 Blood, 111 :485-491).
  • methods of gene expression profiling can be divided into two large groups: methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides.
  • Methods known in the art for the quantification of messenger RNA (mRNA) expression in a sample include northern blotting and in situ hybridization, Ribonuclease
  • RNAse protection assays
  • RNA Sequencing RNA-seq
  • RT-PCR reverse transcription polymerase chain reaction
  • antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA- protein duplexes.
  • Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).
  • SAGE Serial Analysis of Gene Expression
  • MPSS massively parallel signature sequencing
  • RT-PCR is used to compare mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between closely related mRNAs, and/or to analyze RNA structure.
  • a first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into complementary DNA (cDNA), followed by amplification in a PCR reaction.
  • cDNA complementary DNA
  • extracted RNA is reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif, USA), following the manufacturer's instructions.
  • the cDNA is then used as template in a subsequent PCR amplification and quantitative analysis using, for example, a TaqMan RTM (Life Technologies, Inc., Grand Island, N.Y.) assay.
  • TGFp nucleic acid molecule a polynucleotide encoding a TGFp polypeptide.
  • An exemplary TGFp nucleic acid molecule is provided at NCBI Accession No. X02812, version X02812.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 1):
  • Transforming Growth Factor ⁇ (TGFP) polypeptide is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAA36738, version AAA36738.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 2):
  • TGFP3 nucleic acid molecule Transforming Growth Factor ⁇ 3 (TGFP3) nucleic acid molecule
  • TGFP3 nucleic acid molecule is meant a polynucleotide encoding a TGFP3 polypeptide.
  • An exemplary TGFP3 nucleic acid molecule is provided at NCBI Accession No. NM_003239, version NM_003239.4, incorporated herein by reference, and reproduced below (SEQ ID NO: 3):
  • Transforming Growth Factor ⁇ 3 (TGFP3) polypeptide is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No.
  • GDF15 nucleic acid molecule a polynucleotide encoding a GDF15 polypeptide.
  • An exemplary GDF15 nucleic acid molecule is provided at NCBI Accession No. NM_004864, version NM_004864.3, incorporated herein by reference, and reproduced below (SEQ ID NO: 5): 1 ctgaggccca gaaatgtgcc ctagctttac taggagcgccccacctaaa gatcctcccccccc
  • GDF15 GDF15 polypeptide
  • EAW84694 version EAW84694.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 6):
  • BMP1 nucleic acid molecule By “Bone Morphogenic Protein 1 (BMP1) nucleic acid molecule” is meant a polynucleotide encoding a BMP1 polypeptide.
  • An exemplary BMP1 nucleic acid molecule is provided at NCBI Accession No. NM_001199, version NM_001199.3, incorporated herein by reference, and reproduced below (SEQ ID NO: 7):
  • BMP1 polypeptide By “Bone Morphogenic Protein 1 (BMP1) polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAI01764, version AAI01764.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 8):
  • SBDS polypeptide By “Shwachman-Bodian -Diamond Syndrome (SBDS) polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. EAX07906, version EAX07906.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 10):
  • G-CSF nucleic acid molecule granulocyte-colony stimulating factor (G-CSF) nucleic acid molecule
  • G-CSF granulocyte-colony stimulating factor
  • An exemplary GCSF nucleic acid molecule is provided at NCBI Accession No. X03656, version X03656.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 11):
  • G-CSF granulocyte-colony stimulating factor
  • CAA27290 version CAA27290.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 12):
  • Differential gene expression can also be identified, or confirmed using a microarray technique.
  • polynucleotide sequences of interest including cDNAs and oligonucleotides
  • the arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest.
  • the source of mRNA typically is total RNA isolated from human tumors or tumor cell lines and corresponding normal tissues or cell lines. Thus, RNA is isolated from a variety of primary tumors or tumor cell lines. If the source of mRNA is a primary tumor, mRNA is extracted from frozen or archived tissue samples.
  • PCR-amplified inserts of cDNA clones are applied to a substrate in a dense array.
  • the microarrayed genes, immobilized on the microchip, are suitable for hybridization under stringent conditions.
  • fluorescently labeled cDNA probes are generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest.
  • Labeled cDNA probes applied to the chip hybridize with specificity to loci of DNA on the array. After washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a charge-coupled device (CCD) camera. Quantification of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.
  • CCD charge-coupled device
  • dual color fluorescence is used. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously.
  • the miniaturized scale of the hybridization can afford a convenient and rapid evaluation of the expression pattern for large numbers of genes.
  • such methods can have sensitivity required to detect rare transcripts, which are expressed at fewer than 1000, fewer than 100, or fewer than 10 copies per cell.
  • such methods can detect at least approximately two-fold differences in expression levels (Schena et al., Proc. Natl. Acad. Sci. USA 93(2): 106-149 (1996)).
  • microarray analysis is performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.
  • RNA sequencing also called whole transcriptome shotgun sequencing (WTSS)
  • WTSS whole transcriptome shotgun sequencing
  • NGS next-generation sequencing
  • RNA-Seq is used to analyze the continually changing cellular transcriptome. See, e.g., Wang et al., 2009 Nat Rev Genet, 10(1): 57-63, incorporated herein by reference. Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries.
  • RNA-Seq Prior to RNA-Seq, gene expression experiments were done with hybridization-based microarrays. Issues with microarrays include cross-hybridization artifacts, poor quantification of lowly and highly expressed genes, and needing to know the sequence of interest. Because of these technical issues, transcriptomics transitioned to sequencing-based methods. These progressed from Sanger sequencing of Expressed Sequence Tag libraries, to chemical tag-based methods (e.g., serial analysis of gene expression), and finally to the current technology, NGS of cDNA (notably RNA-Seq).
  • AVID200 is a TGF- ⁇ inhibitor for the treatment of anemia associated with myelodysplastic syndromes (Thwaites et al., Blood 2017, 130: 2532, incorporated herein by reference).
  • SD-208 is a selective TGF-PRl inhibitor, the compound of which is provided below.
  • TGFP inhibitors include LY2157299 (Galunisertib; 4-(2-(6-methylpyridin-2-yl)-5,6- dihydro-4H-pyrrolo[l,2-b]pyrazol-3-yl)quinoline-6-carboxamide)), TEW-7197 (TEW7197; EW- 7197; Vactosertib), Bortezomib (Velcade®; LDP 341; MLN341; PS-341; [(lR)-3-Methyl-l- [[(2S)-l-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronic Acid; FDA- approved), P144 (Disitertide), Pirfenidone (Es), Esulfenidone (Es)
  • LY333531 (Ruboxistaurin), and LY2109761 the structure of each of which TGFP inhibitor is provided below. LY2157299 Monohvdrate
  • LY2157299 in combination with enzalutamide is being examined for use in treatment of prostate cancer.
  • LY2157299 in combination with paclitaxel and carboplatin is being examined for treatment of carcinosarcoma and ovarian cancer.
  • LY2157299 in combination with capecitabine and fluorouracil is being examined for treatment of rectal adenocarcinoma.
  • LY2157299 in combination with paclitaxel is being examined for treatment of estrogen receptor negative, HER2/Neu negative, progesterone receptor negative, recurrent breast carcinoma, stage IV breast cancer, and triple-negative breast carcinoma.
  • LY2157299 monohydrate in combination with radiation therapy is being examined for treatment of metastatic breast cancer.
  • LY2157299 in combination with nivolumab is being examined for treatment of solid tumor, non-small cell lung cancer recurrent, and hepatocellular carcinoma recurrent.
  • TEW-7197 is being examined for treatment of advanced stage solid tumors.
  • Bortezomib is being examined for treatment of bronchiolitis obliterans.
  • P144 is being examined for treatment of skin fibrosis.
  • Pirfenidone is being examined for treatment of diabetic nephropathy and albuminuria.
  • TEW-7197 in combination with pomalidomide is being examined for treatment of multiple myeloma.
  • LY333531 is being examined for treatment of diabetic nephropathy
  • compositions or agents described herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline.
  • routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient.
  • Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a therapeutic identified herein in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin.
  • the amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms. Generally, amounts will be in the range of those used for other agents used in the treatment of SDS, although in certain instances lower amounts will be needed because of the increased specificity of the compound.
  • the administration of a compound or a combination of compounds for the treatment of SDS may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing SDS.
  • the compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition.
  • the composition may be provided in a dosage form that is suitable for parenteral (e.g.,
  • compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R.
  • Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models.
  • the dosage may vary from between about 1 ⁇ g compound/Kg body weight to about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg body weight to about 1000 mg/Kg body weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body weight.
  • this dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 mg/Kg body weight.
  • doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body.
  • the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight.
  • this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.
  • compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration.
  • controlled release formulations which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with the thymus; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target SDS by using carriers or chemical derivatives to deliver
  • controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level. Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question.
  • controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings.
  • the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner.
  • Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
  • the pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, nontoxic pharmaceutically acceptable carriers and adjuvants.
  • injection, infusion or implantation subcutaneous, intravenous, intramuscular, intraperitoneal, or the like
  • suitable delivery devices or implants containing conventional, nontoxic pharmaceutically acceptable carriers and adjuvants.
  • compositions for parenteral use may be provided in unit dosage forms (e.g., in single- dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below).
  • the composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
  • the composition may include suitable parenterally acceptable carriers and/or excipients.
  • the active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release.
  • composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
  • the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection.
  • the suitable active SDS therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle.
  • acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution.
  • the aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).
  • a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol.
  • Controlled release parenteral compositions may be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions.
  • the active drug may be incorporated in biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.
  • Biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate), poly(2- hy droxy ethyl -L-glutam- nine) and, poly(lactic acid).
  • Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies.
  • Materials for use in implants can be nonbiodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof).
  • biodegradable e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof.
  • methods of the present invention for clinical aspects are combined with other agents effective in the treatment of SDS, such as oral pancreatic enzyme replacements, fat soluble vitamins, hematopoietic stem cell transplantation, growth hormone, or AML chemotherapeutic drugs. More generally, these other compositions would be provided in a combined amount effective to ameliorate hematological symptoms in SDS patients. This process may involve administering a TGFP inhibitor concurrently with another agent.
  • the present inventive therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agent and present invention are applied separately to the individual, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the agent and inventive therapy would still be able to exert an advantageously combined effect on the cell.
  • kits or pharmaceutical systems for use in ameliorating SDS.
  • Kits or pharmaceutical systems according to this aspect of the invention comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampoules, or bottles.
  • the kits or pharmaceutical systems of the invention may also comprise associated instructions for using the agents of the invention.
  • Ethylenediaminetetraacetic acid EDTA
  • BSA Bovine Serum Albumin
  • Mononuclear cells were collected from the interface, washed once, pelleted for 5 min at 1200 rpm and 20°C, and resuspended at 40 ⁇ per 107,397 cells in MACS buffer + 1 ⁇ /ml RNaseOUT (Thermo Fisher Scientific, Waltham, MA, USA).
  • CD34 + cells were positively selected on an AutoMACS instrument using the Indirect CD34 MicroBead Kit (Miltenyi, Bergisch Gladbach, Germany), and singulated on the CI Instrument (Fluidigm, San Francisco, CA, USA).
  • cDNA libraries were prepared using the SMARTer ultra Low RNA Kit (Clontech, Mountain View, CA, USA).
  • protocol conditions were modified to ascertain immunophenotypes from single cells in accordance with the newest available methods.
  • red blood cells were lysed with ammonium chloride (Stem Cell Technologies, Vancouver, CA).
  • Mononuclear cells were pelleted for 5 min at 1200 rpm and 20°C, washed twice, and resuspended in PBS + 1 ⁇ /ml RNaseOUT. Cells were stained as described below.
  • Single CD34 + cells were sorted into 5 ⁇ Turbo Capture Lysis (TCL) buffer (Qiagen, Hilden, Germany) in 96 well plates using a FACS Aria II instrument (BD, Franklin Lakes, NJ, USA) on index mode. Two technical replicates of 100 cells from each gated CD34 + subset - HSC, MPP, MLP, CMP, GMP, MEP - were sorted into 5 ⁇ TCL buffer in separate 96 well plates.
  • cDNA libraries were prepared using the SMART-Seq v4 ultra Low RNA Kit (Clontech). Libraries from all samples were sequenced on a HiSeq 2500 Instrument (Illumina, San Diego, CA) to a read depth of ⁇ 3 M paired-end, 25 bp reads per single cell, or -12 M paired- end, 25 bp reads per 100 cells.
  • the antibodies used were: brilliant violet 421-anti-CD90 (BD 562556, 1 :20), alexa fluor 488- anti-CD34 (Biolegend, San Diego, CA 343518, 1 :20), brilliant violet 71 l-anti-CD38 (BD
  • Live/dead staining was performed immediately prior to sorting using Zombie Aqua Fixable Viability Dye (Biolegend). Cells were sorted on a FACSAria II instrument (BD), and data analysis was performed in FlowJo vlO.0.8.
  • Paired-end reads were mapped to the hg38 human transcriptome (Gencode v24) using STAR v2.4.2a (Dobin et al., 2013 Bioinformatics, 29: 15-21). Aligned reads are being made available through the database of Genotypes and Phenotypes (dbGaP) (BioProject ID:
  • PRJNA316220 with updated information as soon as it becomes available.
  • Gene expression levels were quantified as transcript-per-million (TPM) in RSEM32 (Li, B. & Dewey, C.N., 2011 BMC Bioinformatics, 12:323). Cells with at least 1000 expressed genes (defined by TPM>1) and genes expressed in at least 50 single cells were kept. This resulted in 11094 genes in 583 single cells. The same set of 11094 genes was analyzed to derive lineage signature genes from 100 cell libraries made from fluorescence-activated cell sorting (FACS)-purified CD34 + subsets.
  • FACS fluorescence-activated cell sorting
  • TPM values were divided by 10 to better reflect the complexity of single cell libraries which was estimated to be -100,000 transcripts.
  • the data were log2 transformed (log2(TPM/10 +1)).
  • PCA Principal Component Analysis
  • PCs principal components
  • t-SNE Stochastic Neighbor Embedding
  • the tSNE coordinates were used for partitioning around medoids (PAM), a
  • BM-derived mononuclear cells were cutured for 30-32 h in StemSpan SFEM II (Stem Cell Technologies) supplemented with 100 ng/mL of Stem Cell Factor (SCF), Thyroid Peroxidase (TPO), FMS-like tyrosine kinase 3 ligand (Flt3L) and 20 ng/mL of interleukin-3 (IL- 3) (PreproTech, Rocky Hill, NJ).
  • SCF Stem Cell Factor
  • TPO Thyroid Peroxidase
  • FMS-like tyrosine kinase 3 ligand FMS-like tyrosine kinase 3 ligand
  • IL- 3 interleukin-3
  • the 25,000-50,000 cells were spun onto coverslips (ES0117580, Azer Scientific, Morgantown, PA) using a cytospin instrument (Thermo Shandon) at 380 rpm for 5 min; fixed with 4% Paraformaldehyde (PFA) in IX PBS for 10 min at room temperature (RT); washed 2X with IX PBS; permeabilized with 0.3% TritonX in IX PBS solution for 10 min at RT; washed 2X with IX PBS; blocked in 10% fetal bovine serum (FBS), 0.1% nonyl phenoxypolyethoxylethanol ( P40) in IX PBS for lh at RT; incubated with 1 :250 anti-p-smad2 (Invitrogen, 44-244G) in blocking solution for 14-16 h at 4°C; washed 3X with 0.1% P40 in IX PBS at RT for 10 min; incubated with 1 : 1,000 diluted anti-rabbit IgG-Alexa
  • BM-derived mononuclear were cultured for 24h in StemSpan SFEM II (Stem Cell Technologies) supplemented with 100 ng/mL of SCF, TPO, Flt3L and 20 ng/mL of IL-3 (PreproTech, Rocky Hill, NJ). Cells were resuspended at 10,000 cells/mL for control and 20,000 cells/mL for SDS in the presence or absence of 0, 0.25, 0.5, 1, or 5 ⁇ SD208 (Tocris, Bristol, UK), and incubated for lhr at 37°C/5% C02.
  • the 200 ⁇ _, of cell suspension was mixed with 3 mL of Methocult H4434 (Stem Cell Technologies), and 1 mL was plated in triplicate in a SmartDish 6-well plate (Stem Cell Technologies). After 14 days of growth at 37°C/5% C02, colonies were manually counted by two independent, blinded investigators.
  • SOMAscan® SomaLogic, ⁇ ⁇ , CO was performed on 50 ⁇ of EDTA-plasma from six patients and six normal controls at the BIDMC Genomics, Proteomics, Bioinformatics and Systems Biology Center. Samples were prepared and run using the SOMAscan Assay Kit for Human Plasma, 1.3k (cat. # 900-00011), according to the manufacturer's protocol. Five pooled controls and one no-protein buffer control provided in the kit were run in parallel with the samples. Median normalization and calibration of the data was performed according to the standard quality control protocols at SomaLogic. All samples passed the established quality control criteria. Proteins with p-values ⁇ 0.01 were analyzed. Benjamini-Hochberg adjusted p- values are reported in Table 3.
  • FIG 2A statistical significance was determined by the chi-squared test, and the frequency of cells in each cluster was compared between SDS and normal.
  • FIG. 2B, FIG. 3C, FIG. 4D, FIG. 7B statistical significance was determined by two-way ANOVA with Holm- Sidak's multiple correction test in GraphPad Prism 7.
  • FIG. 2B the frequency of cells was compared between SDS and normal cells within each cluster.
  • FIG. 3C log2 expression was compared between SDS and normal cells within each cluster.
  • FIG. 4D and FIG. 7B relative colony number was compared between each drug dose and the 0 ⁇ treatment.
  • FIG. 4B and FIG. 4C statistical significance was determined by one-way ANOVA with Holm-Sidak's multiple correction test in GraphPad Prism 7; SDS samples were compared to normal samples that were stained and imaged concurrently.
  • Example 2 Supervised dimensionality reduction maps lineage commitment of CD34 + cells.
  • G-CSF Granulocyte-colony stimulating factor
  • hematopoietic lineage commitment (and not other unrelated variables), a supervised analysis was performed using an empirically-derived gene set. Specifically, FACS-purified HSC, MPP, common myeloid progenitors (CMPs), multilymphoid progenitors (MLPs), GMP, and megakaryocyte-erythroid progenitors (MEPs) were sequenced from normal BM (Laurenti et al., 2013 Nature Immunology, 14:756-763), and identified a 79-gene signature that distinguished these cell types (FIG. 5 A-FIG. 5D). Principal component analysis was performed on cells from normal donors and SDS patients, and significant principal components were visualized using tSNE (FIG. 1 A-FIG.
  • FIG. 1 A-FIG. ID Table 1 (Van der Maaten, L., Hinton, G., 2008 Journal of Machine Learning Research, 2579-2605). For simplicity, SDS cells are masked in FIG. 1 A-FIG. ID. Cells from four normal donors were interspersed in a configuration that suggested population structure related to lineage commitment (FIG. 1 A). Cells are colored based on (FIG. 1 A) donor identity, (FIG. IB) mRNA expression of selected signature genes, (FIG. 1C) mRNA expression of lineage-restricted genes reported elsewhere (Laurenti et al., 2013 Nature Immunology, 14: 756- 763), and (FIG. ID) immunophenotypes.
  • EVI1, IRF8, GATA1, and MME lineage-predictive signature genes. These genes are specifically associated with stem- myeloid-, erythroid-, and lymphoid- restricted expression patterns, respectively (Velten et al., 2017 Nat. Cell Biol., 19:271-281). Most cells expressed only one of these four genes, and expression of each gene was concentrated in a distinct region of the tSNE map (FIG. IB). Similar results were obtained using genes that were not present in the 79-gene signature (FIG. 1C). To confirm patterns of lineage commitment determined by mRNA expression, indexed surface marker intensities on a subset of normal cells were examined.
  • FIG. 2A shows the same map as in FIG. 1 A-FIG. ID, with cells from SDS patients unmasked. SDS and normal cells were intermixed, but their distribution and relative frequencies differed ( ⁇ 2 p ⁇ 0.0001). These changes were quantified using k-means clustering. Five clusters were defined based on maximum silhouette value, and named for the most enriched immunophenotypic subpopulation within the cluster (FIG. 2A). CMP, MLP/CLP, GMP and MEP each designated a distinct cluster whereas HSC and MPP were enriched in the same cluster.
  • TGFp signaling is selectively activated in SDS stem and multipotent progenitors
  • HSC/MPP or CMP FOG. 3 A
  • HSC/MPP and CMP are the primarily affected cell types in SDS, but the affected genes are distinct between cell types.
  • TGFp induces context-dependent effects on cell growth, survival, inflammation, and extracellular matrix.
  • TGFpi and TGFP3 have potent growth inhibitory effects on HSC (Hatzfeld et al., 1991 J Exp. Med., 174:925-929; Scandura et al., 2004 Proc. Natl. Acad. Sci., 101 : 15231-15236; Challen et al., 2010 Cell Stem Cell, 6:265-278).
  • Example 5 TGFP pathway activation through TGFpRl suppresses hematopoiesis in SDS BM progenitors
  • BM cells from SDS patients exhibit impaired hematopoietic colony formation in vitro (Dror, Y. & Freedman, M.H., 1999 Blood, 94:3048-3054) (FIG. 7A).
  • primary BM mononuclear cells were cultured from SDS patients and normal donors (Table 2) in methylcellulose supplemented with AVID200 and SD208, which inhibits TGFpRl kinase activity (Gold et al., 2012 N
  • TGFP3 was significantly upregulated in SDS patient plasma, along with several other factors that were annotated to a network of TGFp-associated factors (FIG. 4F, FIG. 8). As described herein, these and other dysregulated plasma proteins that were common across clinically-heterogeneous patients serve as diagnostic biomarkers for SDS (Table 3).
  • SDS is a multi -organ system disorder including hematologic, gastrointestinal (GI), neurocognitive, and skeletal manifestations. GI and neurologic complications are usual causes of morbidity for patients with SDS and are treated with supportive care. The usual causes of mortality are bone marrow failure or leukemia. Hematopoietic stem cell (HSC) transplant offers curative treatment for SDS; however, patients are at increased risk of regimen-related toxicities and are not cured of GI, neurological, or other co-morbidities. Moreover, some patients lack a suitable transplant donors. Prior to the invention described herein, there was an urgent unmet need for non-transplant treatments for SDS.
  • TGFpi and TGFP3 exert potent growth inhibitory effects on HSCs (Hatzfeld et al., 1991 J Exp Med, 174(4): p. 925-9; Scandura et al., 2004, Proc Natl Acad Sci U S A, 101(42): 15231- 6; Challen et al., 2010 Cell Stem Cell, 6(3): 265-78).
  • the TGFp3 pathway is a therapeutic target for SDS (FIG. 4F; FIG. 7A; and FIG. 7B.
  • aptamer-based proteomic screening of SDS patients' blood plasma identified increased levels of TGFP3.
  • Pharmacologic inhibition of TGFp improved hematopoietic colony formation of SDS patient-derived bone marrows.
  • the results presented herein implicate the TGFp signaling pathway as a new target for SDS therapy.
  • SDS is an underdiagnosed, multi-system disorder caused by autosomal recessive mutations in the SBDS gene (Lindsley et al., 2017 N Engl J Med, 376(6): 536-547; Myers et al., 2014 The Journal of Pediatrics, 164: 866-70).
  • the major causes of mortality are bone marrow failure, MDS, and AML.
  • HSC transplant offers potentially curative therapy for hematologic complications; however, survival is reduced by the high risk of transplant regimen-related toxicities and potential short-term and long-term effects on organ function. Furthermore, transplant does not improve additional co-morbidities associated with SDS such as exocrine pancreatic dysfunction and neurocognitive abnormalities.
  • the data presented herein implicates the TGFp pathway as a new target for SDS therapy.
  • TGFP inhibitors are in clinical development for other disorders (Herbertz et al., 2015 Drug Des Devel Ther, 9: 4479-99) including pulsed therapy in oncology clinical trials. By contrast, TGFP inhibitors would require chronic administration for bone marrow failure therapy. The broad spectrum of TGFP functions obviates long-term use of TGFP inhibitors, particularly in children. Described herein are experiments that define the cellular and molecular targets of TGFP pathway inhibition.
  • TGFP inhibitors on CD34 + cells knocking down SBDS is assessed.
  • the identity, dose and timing of effective TGFP inhibition on SDS hematopoiesis is determined and RNA-seq is used to define the specific cell types and genes affected by efficient TGFp inhibition.
  • Second, HSC-specific targets of TGFp inhibition are modulated and SDS
  • hematopoiesis is re-assessed.
  • the results presented herein necessitate expansion of TGFp inhibitors already in clinical development or the development of formulations that more precisely deliver TGFp inhibitors to specific targets in SDS hematopoiesis.
  • TGFP signaling in hematopoietic progenitors promotes bone marrow failure in SDS patients
  • inhibiting TGFP signaling improves SDS hematopoiesis.
  • TGFp signaling exerts multiple effects across organ systems and is essential for development. Defining the cellular and molecular targets of TGFP inhibitors that improve SDS hematopoiesis informs therapeutic strategies to develop specific pharmacologic agents.
  • hematopoiesis (1) normal-donor CD34 + cells knocking down SBDS; and (2) SDS patient- derived induced pluripotent stem cells (iPSC) that can be induced to differentiate along hematopoietic lineages (Park et al., 2008 Cell, 134(3): 1-10).
  • Primary SDS patient samples are available through the North American SDS Registry, which includes a clinically-annotated repository of blood and bone marrow samples from over 100 genetically-characterized SDS patients collected over 12+ years.
  • TGFP inhibitors (SD208 and Galunisertib) are dose-escalated on normal-donor CD34 + cells knocking down SBDS.
  • RNA-seq of colonies demonstrating the greatest improvement in hematopoiesis is performed.
  • the cellular and molecular targets of TGFp inhibition are validated in SDS patient-derived iPSC by ectopically expressing genes down- regulated by TGFP inhibitors or by knocking down genes up-regulated by TGFP inhibitors and then retesting hematopoiesis.
  • HSC transplant is the only curative treatment for hematologic symptoms of SDS, but prior to the invention described herein, outcomes are limited by high sensitivity of SDS patients to regimen-related toxicities. Accordingly, described herein is the development of non-transplant therapies to treat SDS. Elucidation of the relevant cellular and molecular targets informs medicinal chemistry approaches to tailor the pharmacologic inhibitors which improves efficacy and minimizes toxicities. These agents improve or prevent hematological complications and also ameliorate GI and neurologic symptoms.
  • the TGFp pathway is a new therapeutic target to treat bone marrow failure. Additional experiments elucidate whether inhibition of TGFp also exerts a protective effect against leukemia development in these patients with cancer predisposition.
  • Described herein is the expanded use of TGFp inhibitors for bone marrow failure syndromes.
  • Example 7 Precision targeting of TGFp signaling in hematopoietic stem cells to treat bone marrow failure in Shwachman-Diamond Syndrome
  • BM failure is an ideal match for single cell approaches because they involve rare cell dysfunction in a complex, protean environment. Described herein is an examination of the molecular pathogenesis of BM failure in patients with Shwachman-Diamond Syndrome (SDS) at single cell resolution.
  • SDS Shwachman-Diamond Syndrome
  • SDS is a rare genetic disorder caused by mutations in the SBDS gene, which encodes a co-factor for ribosome biogenesis (Huang, J.N. & Shimamura, 2010 Current Opinion in
  • AML myelodysplasia and acute myeloid leukemia
  • HSC hematopoietic stem cell
  • BM dysfunction is surprisingly complex. Most patients exhibit generalized BM hypocellularity, but the first and most severely affected hematopoietic lineages vary. Neutropenia and myelodysplasia are common, with
  • a single cell perspective will shed new light on the pathogenesis of inherited BM failures
  • SDS is related to several other inherited BM failure syndromes that also carry increased risk for AML (e.g. Fanconi anemia (FA), dyskeratosis congenita, severe congenital neutropenia) (Ruggero, D. & Pandolfi, P.P., 2003 Nature Reviews. Cancer, 3 : 179-192; Ruggero, D. & Shimamura, 2014 Blood, 124: 2784-2792; Savage, S.A. & Dufour, C. 2017 Semin Hematol, 54: 105-114).
  • results presented herein provide a paradigm to link defects in individual cells to complex disease phenotypes.
  • RNA-sequencing Single cell RNA-sequencing (scRNA-seq)
  • HSPC hematopoietic stem and progenitor cells
  • scRNA-seq single cell RNA-sequencing
  • TGFp target genes were not activated in lineage-committed
  • TGFP acts on many different cells, but the downstream effectors and phenotypic outcomes vary depending on cell type and functional state.
  • TGFp activation in rare HSCs contributes to SDS BM failure, the pathway in normal and SDS BM cells was inhibited.
  • TGFP inhibitors promoted hematopoietic colony formation of SDS BM cells, but had no effect on normal BM cells (FIG. 7B; FIG. 4D), suggesting that TGFp inhibitors selectively rescue SDS hematopoiesis by attenuating activated signaling that occurs specifically in the context of SDS HSCs.
  • TGFp acts selectively on subsets of SDS HSCs, and inhibiting TGFp targets that are activated in specific subsets of SDS HSCs will maximize therapeutic benefit.
  • the mechanisms underlying the heterogeneous activation of TGFP signaling in rare SDS HSCs were unclear. The only way to define these mechanisms is through single cell analysis.
  • TGFP targets in functional subsets of SDS HSCs.
  • the data described herein supports a clinical trial using TGFP inhibitors to treat BM failure.
  • oncogenic risks associated with these drugs limit their long term therapeutic potential (Feagins, L.A. 2010 Inflamm Bowel Dis, 16: 1963-1968; Hong et al., 2010 World J Gastroenterol, 16: 2080-2093).
  • Precise targeting of TGFp effectors in specific HSC subsets can prevent oncogenic effects of global TGFp inhibition.
  • this protocol requires:
  • results presented herein comprehensively define human HSC heterogeneity at unprecedented single cell resolution, and identify the specific proteins and mRNAs within specific HSC subsets that can be targeted to precisely inhibit the pathogenic function of TGFp in SDS.
  • single cell transcriptomics pipeline for fresh HSPC has been designed, which 1) preserves natural biology by minimizing ex vivo manipulations; and 2)
  • TGFp activation was defined as a molecular defect in SDS that specifically affects rare HSCs.
  • a 2 nd generation pipeline is implemented to drill deeply into the affected HSC population and uncover the underlying mechanisms.
  • Key additions to the 2 nd generation pipeline include 1) the ability to analyze fresh or frozen samples; 2) single cell proteomics using CyTOF; and 3) an updated analysis workflow including transcriptional signatures that reflect key aspects of HSC function al heterogeneity, and new algorithms for single cell clustering and visualization.
  • scRNAseq mononuclear cells are stained for HSC surface markers and TGFp receptors, and smart-seq2 cDNA libraries are generated from up to 384 index-sorted HSC per sample.
  • CyTOF staining is performed with a panel of up to 40 metal-conjugated antibodies against HSC surface markers, TGFp receptors, SBDS and related proteins (e.g., eIF6, EFL1), and TGFP signal transducers (e.g., SMADs and transcriptional co-activators).
  • TGFp signaling activity in single cells is deduced based on the combined status of protein mediators and transcriptional targets.
  • viS E23 is used to spatially arrange cells in two dimensions based on
  • TGFp mediators and effectors transcriptional signatures of HSC function.
  • the expression level or degree of correlation for TGFp mediators and effectors is then overlaid using color as a third dimension.
  • Other single cell visualization tools that may be implemented include principal component analysis, SPADE24, or ARIAD E25. It is expected that proteomic and transcriptional hallmarks of TGFP signaling will map to specific subset(s) of SDS HSCs. Thus, the target cell(s), and the molecular targets within these cells, are identified and characterized for therapeutic inhibition of TGFp signaling in SDS.
  • results presented herein comprehensively map human HSC heterogeneity, providing new insight into a cell population with critical roles in hematologic disease and malignancy.
  • the BM chip contains a matrix of co-cultured BM stromal cells and HSPCs adjacent to a vascular endothelial cell-lined chamber with cytokine-supplemented medium.
  • Germline SBDS mutations that occur in patients are mimicked by performing stable SBDS knockdown in HSPC, BM stromal cell, and vascular endothelial cell parent cultures prior to co- culturing them on BM chips. Alternatively, chips are populated with extra cells that are preserved from fresh patient BM samples. The efficiency of hematopoiesis in normal versus SDS chips in the presence or absence of TGFP inhibitors is assessed by flow cytometry and microscopy. Furthermore, the contributions of different cell types to TGFP-dependent SDS phenotypes is assessed using single cell profiling methods as described above.
  • results presented herein provide key preclinical data rationalizing precision targeting of the TGFp pathway in rare cells as a safer and more efficacious long-term therapy for SDS BM failure.
  • results presented herein define therapeutic strategies to specifically treat a molecular defect that occurs at the root of the hematopoietic tree in SDS. This innovative approach stops the intractable and life-threatening cascade of complex and variable hematologic symptoms in SDS patients.
  • results presented herein lie at the intersection of basic and translational cancer research, and transforms SDS patient care.

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Abstract

La présente invention concerne des compositions et des méthodes de traitement des symptômes du syndrome de Shwachman-Diamond.
PCT/US2018/042913 2017-07-19 2018-07-19 Inhibition de tgf-bêta pour traiter des symptômes hématologiques du syndrome de shwachman-diamond WO2019018662A1 (fr)

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CN111321207A (zh) * 2020-02-15 2020-06-23 中国医学科学院血液病医院(中国医学科学院血液学研究所) 一种先天性骨髓衰竭性疾病基因分型的检测方法
KR20210054659A (ko) * 2019-11-05 2021-05-14 중앙대학교 산학협력단 슈와크만-보디안-다이아몬드 증후군 유전자의 발현 억제제를 유효성분으로 포함하는 유방암 치료제 효율 증진용 조성물
CN113584159A (zh) * 2021-09-01 2021-11-02 翌圣生物科技(上海)股份有限公司 用于sbds基因突变检测的引物组及基因扩增方法
CN115282280A (zh) * 2022-08-12 2022-11-04 中国科学技术大学 TGF-β1信号抑制剂的新用途

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KR20210054659A (ko) * 2019-11-05 2021-05-14 중앙대학교 산학협력단 슈와크만-보디안-다이아몬드 증후군 유전자의 발현 억제제를 유효성분으로 포함하는 유방암 치료제 효율 증진용 조성물
KR102337860B1 (ko) 2019-11-05 2021-12-10 중앙대학교 산학협력단 슈와크만-보디안-다이아몬드 증후군 유전자의 발현 억제제를 유효성분으로 포함하는 유방암 치료제 효율 증진용 조성물
CN111321207A (zh) * 2020-02-15 2020-06-23 中国医学科学院血液病医院(中国医学科学院血液学研究所) 一种先天性骨髓衰竭性疾病基因分型的检测方法
CN113584159A (zh) * 2021-09-01 2021-11-02 翌圣生物科技(上海)股份有限公司 用于sbds基因突变检测的引物组及基因扩增方法
CN115282280A (zh) * 2022-08-12 2022-11-04 中国科学技术大学 TGF-β1信号抑制剂的新用途

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